Fermentation medium, fermentation method and application

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a fermentation culture medium and a fermentation method and application thereof.Based on the total volume of the fermentation culture medium, the fermentation culture medium comprises 4-5 g / L of a carbon source, and the carbon source comprises malt syrup and sodium acetate; 35-39 g / L of a nitrogen source, wherein the nitrogen source comprises corn steep liquor and yeast powder; 1-3g / L of inorganic nutritive salt, 5-9g / L of trace elements and a nutrient additive with the volume mass fraction of 0.05-0.2%. The optimized culture medium is used for carrying out fermentation culture on a strain, so that on one hand, the fermentation time for microorganisms to reach a stable phase is effectively shortened; on the other hand, the number of effective bacterial colonies of the fermentation liquor is nearly doubled and can reach 2.3 * 10 < 10 > CFU / ml at most.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a fermentation medium and a Pseudomonas fermentation method and application. Background Art

[0002] Biological treatment technology is the leading and core technology in the current wastewater treatment field. It is safe, efficient and low-cost, and plays an important role in the field of industrial wastewater treatment. However, chemical wastewater is the focus and difficulty of wastewater biological treatment. For example, wastewater from industries such as petrochemicals, coking, and pharmaceuticals contains a large amount of highly toxic organic substances such as phenols, polycyclic aromatic hydrocarbons, and antibiotics, which seriously inhibit the growth and activity of microorganisms and reduce the efficiency of wastewater biological treatment.

[0003] At present, physical and chemical processes are usually used for pretreatment of difficult-to-degrade organic wastewater. The treatment cost is high, the operation is difficult, and secondary pollution is easy to occur. The conventional biological treatment process has low microbial abundance, poor tolerance, and low degradation efficiency for treating highly toxic organic matter, resulting in low efficiency, long cycle, and poor effect in the biological treatment of such wastewater. Refractory pollutants require the coordinated metabolism of different degrading bacteria to degrade quickly, but in the process of degradation, with the succession of colony types, the degradation efficiency of pollutants may be greatly reduced, which is not conducive to large-scale treatment. Recombining the degradation genes of different pollutants, combining the metabolic pathways of pollutants of different strains, and constructing efficient bacteria with special degradation functions can effectively improve the degradation ability of microorganisms. There is a lot of application space and development potential for constructing efficient bacteria for pollutant treatment.

[0004] After reasonable transformation, genetically engineered bacteria can simultaneously realize the advantages of multiple environmental microorganisms for pollutant treatment, among which chassis microorganisms are particularly important. Chassis microorganisms are the host for metabolic reactions, and their good biological properties and performance characteristics are conducive to the effective expression of exogenous genes. By splicing the excavated gene elements into chassis microorganisms, the purpose of rationally constructing efficient and stress-resistant bacteria can be achieved. This study uses Pseudomonas, which is of the same genus in taxonomy, as a target for high-density fermentation culture, and constructs an efficient fermentation method to meet the needs of high-throughput strain performance verification and fermentation process development capabilities, thereby providing necessary guarantees for the industrialization of synthetic biology chassis microorganisms. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a fermentation medium and a fermentation method and use to solve the problems in the prior art.

[0006] To achieve the above-mentioned object and other related objects, the present invention first provides a fermentation medium, based on the total volume of the fermentation medium, the fermentation medium comprises:

[0007] 4-5 g / L of carbon source, wherein the carbon source comprises maltose syrup and sodium acetate;

[0008] 35-39 g / L of nitrogen source, wherein the nitrogen source comprises corn steep liquor and yeast powder;

[0009] 1-3 g / L of inorganic nutrient salts, 5-9 g / L of trace elements and 0.05-0.2% of the volume mass fraction of nutrient additives.

[0010] The present invention also provides use of the fermentation medium in fermenting and culturing Pseudomonas.

[0011] The present invention also provides a fermentation method of Pseudomonas, which comprises the following steps:

[0012] I) First stage culture: inoculating the seed liquid into the fermentation medium to carry out the first stage fermentation culture;

[0013] II) Second stage culture: feeding by feeding method and continuing culture until the end of fermentation.

[0014] As described above, a fermentation medium, a fermentation method and uses of the present invention have the following beneficial effects:

[0015] 1) The raw materials are widely available: the present invention uses industrial-grade glucose, maltose syrup, sodium acetate, crude glycerol, corn steep liquor, ammonium sulfate, yeast powder, humic acid and the like as the main fermentation raw materials for fermentation culture, and obtains good fermentation effect. The carbon source used by the strain is widely available, the price is relatively cheap, and it is relatively stable, which can maintain the stability of fermentation culture and save costs; the optimal carbon-nitrogen ratio of the strain fermentation culture is determined, and the fermentation yield is improved;

[0016] 2) The fermentation culture method is stable: the shake flask seed liquid culture cycle is about 14-18h, OD: 3.0-5.0, fermentation culture inoculation amount: 3%-5%, and the batch fermentation culture cycle is about 24h. Many repeated tests have achieved good results. At the same time, after the strains fermented before and after optimization were stored in a 4°C refrigerator for 30 days, the retention rate of live bacteria obtained by the new fermentation method was about 13% higher than the same period last year;

[0017] 3) Improvement of strain fermentation efficiency: The Pseudomonas was batch fermented with the original fermentation medium, and the culture cycle was about 25 to 28 hours. After the fermentation culture entered the stable period, the colony count of the fermentation liquid (stock solution) was about 0.8 to 1.2*10 10 CFU / ml. The optimized culture medium and fermentation control parameters of the Pseudomonas fermentation culture were improved, and the strain was fermented and cultured using the optimized new fermentation method. First, the fermentation time for the microorganism to reach the stable period was effectively shortened; second, the effective colony count of the fermentation liquid was nearly doubled, reaching a maximum of 2.3*10 10CFU / ml.

[0018] The deposit information of Pseudomonas aeruginosa of the present invention is as follows:

[0019] Deposit number: CGMCC NO.27611;

[0020] Classification and nomenclature: Pseudomonas aeruginosa;

[0021] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0022] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China;

[0023] Date of preservation: June 12, 2023. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It shows the fermentation schematic diagram after overall optimization of the fermentation method 5.8 in the present invention. DETAILED DESCRIPTION

[0025] The present invention uses Pseudomonas aeruginosa (deposit number: CGMCC27611) independently screened by our company as the test strain, selects different types of nutrients for testing based on the widely used basic culture medium for Pseudomonas, and cultivates and amplifies the microorganisms through different seed inoculation amounts, combinations and ratios of different components of the fermentation culture medium, feeding methods, and optimization of fermentation control parameters (pH, DO, temperature, etc.), thereby achieving the development of regulated fermentation conditions in the direction of higher cell yields.

[0026] The present invention first provides a fermentation medium, based on the total volume of the fermentation medium, the fermentation medium comprises:

[0027] 4-5 g / L of carbon source, wherein the carbon source comprises maltose syrup and sodium acetate;

[0028] 35-39 g / L of nitrogen source, wherein the nitrogen source comprises corn steep liquor and yeast powder;

[0029] 1-3 g / L of inorganic nutrient salts, 5-9 g / L of trace elements and 0.05-0.2% of the volume mass fraction of nutrient additives.

[0030] In certain embodiments of the present invention, among the carbon sources, the amount of carbon provided by maltose syrup is greater than the amount of carbon provided by sodium acetate.

[0031] In certain embodiments of the present invention, in the carbon source, the ratio of the amount of carbon provided by maltose syrup to the amount of carbon provided by sodium acetate is (2-3):1.

[0032] In certain embodiments of the present invention, among the nitrogen sources, the amount of nitrogen provided by corn steep liquor is more than 4 times the amount of nitrogen provided by yeast powder.

[0033] In certain embodiments of the present invention, the amount of nitrogen provided by the corn steep liquor is 4 to 5 times the amount of nitrogen provided by the yeast powder.

[0034] In certain embodiments of the present invention, the ions of the inorganic nutrient salt are selected from Na + , K + Mg 2+ One or more of .

[0035] In certain embodiments of the present invention, the ions of the inorganic nutrient salt are provided by NaCl, KCl, and MgCl2.

[0036] In a specific embodiment of the present invention, based on the total volume of the fermentation medium, the inorganic nutrient salts include NaCl, KCl and MgCl2, the working concentration of NaCl is 0.3-0.7 g / L, the working concentration of KCl is 0.1-0.3 g / L, and the working concentration of MgCl2 is 0.8-1.2 g / L.

[0037] In certain embodiments of the present invention, the trace element is selected from Fe 3+ 、Zn 2+ , Ca 2+ , Cu 2+ , Mn 2+ , EDTA or more.

[0038] In certain embodiments of the present invention, the trace element ions are FeCl3·6H2O, ZnSO4·7H2O, CaCl 2、 Provided by CuCl2·2H2O and MnSO4·H2O.

[0039] In a specific embodiment of the present invention, based on the total volume of the fermentation medium, the trace elements include FeCl3·6H2O, ZnSO4·7H2O, CaCl 2、CuCl2·2H2O, MnSO4·H2O and EDTA, the working concentration of FeCl3·6H2O is 4-8g / L, the working concentration of ZnSO4·7H2O is 0.4-0.8g / L, the working concentration of CaCl2 is 0.1-0.3g / L, the working concentration of CuCl2·2H2O is 0.1-0.3g / L, the working concentration of MnSO4·H2O is 0.1-0.5g / L, and the working concentration of EDTA is 0.4-0.8g / L.

[0040] In certain embodiments of the present invention, the nutritional additive is humic acid.

[0041] The present invention also provides use of the fermentation medium in fermenting and culturing Pseudomonas.

[0042] In certain embodiments of the present invention, the Pseudomonas is Pseudomonas aeruginosa.

[0043] In certain embodiments of the present invention, the deposit number of the Pseudomonas aeruginosa strain is CGMCC27611.

[0044] The present invention also provides a fermentation method of Pseudomonas, which comprises the following steps:

[0045] I. First stage culture: inoculating the seed liquid into the fermentation medium to carry out the first stage fermentation culture;

[0046] II. Second stage of cultivation: feed is added by flow addition and cultivation is continued until the end of fermentation.

[0047] In certain embodiments of the present invention, the Pseudomonas is Pseudomonas aeruginosa.

[0048] In certain embodiments of the present invention, the deposit number of the Pseudomonas aeruginosa strain is CGMCC27611.

[0049] In certain embodiments of the present invention, the fermentation culture temperature is 35-40°C.

[0050] In a preferred embodiment of the present invention, the fermentation culture temperature is 35°C.

[0051] In certain embodiments of the present invention, the pH during the fermentation culture process is maintained at 7.0-8.0.

[0052] In a preferred embodiment of the present invention, the pH during the fermentation culture process is maintained at 7.0-7.5.

[0053] In certain embodiments of the present invention, the dissolved oxygen concentration of the fermentation culture is 40% to 70%.

[0054] In a preferred embodiment of the present invention, the dissolved oxygen concentration of the fermentation culture is 50% to 60%.

[0055] In certain embodiments of the present invention, the ventilation rate during the fermentation culture process is 1 L / min to 3 L / min.

[0056] In certain embodiments of the present invention, the stirring speed during the fermentation culture process is 200 rpm to 400 rpm.

[0057] In certain embodiments of the present invention, in step I), the inoculation amount is 3% to 5%, specifically referring to the percentage of the volume of the seed liquid relative to the volume of the fermentation medium.

[0058] In certain embodiments of the present invention, in step I), the duration of the first stage of cultivation is such that when the dissolved oxygen concentration in the fermenter is higher than a set value, the first stage of cultivation is terminated.

[0059] In certain embodiments of the present invention, in step II), feeding is performed according to the following method:

[0060] II1) calculating the total added carbon and nitrogen amount: the carbon and nitrogen amount in the fermentation medium is recorded as the first carbon and nitrogen amount, while keeping the nitrogen amount in the fermentation medium unchanged, the carbon amount in the medium is increased until the carbon-nitrogen ratio in the medium is 4 to 8:1, and the carbon and nitrogen amount when the carbon-nitrogen ratio is 4 to 8:1 is recorded as the second carbon and nitrogen amount, and 1.25 to 2.5 times the second carbon and nitrogen amount is the total added carbon and nitrogen amount;

[0061] II2) Subtract the first carbon-nitrogen amount from the total added carbon-nitrogen amount calculated in step II1) to calculate the remaining carbon-nitrogen amount, which is the carbon-nitrogen amount added by the supplementary feed.

[0062] In certain embodiments of the present invention, in step II1), the ratio of carbon to nitrogen in the second carbon to nitrogen content is 4-5:1.

[0063] In certain embodiments of the present invention, in step II1), the total amount of carbon and nitrogen added is 1.5 to 2 times the second amount of carbon and nitrogen.

[0064] In certain embodiments of the present invention, in step II1), the ratio of carbon to nitrogen in the total added carbon to nitrogen amount is 4 to 5:1.

[0065] In certain embodiments of the present invention, in step II), when the dissolved oxygen concentration is higher than 40% to 70%, the feeding is started to initiate the second stage of cultivation.

[0066] In a preferred embodiment of the present invention, in step II), when the dissolved oxygen concentration is higher than 50% to 60%, the feeding process is started to initiate the second stage of cultivation.

[0067] In certain embodiments of the present invention, in step II), the carbon source fed in the feed comprises maltose syrup and sodium acetate.

[0068] Further, the ratio of the amount of carbon provided by the maltose syrup to the amount of carbon provided by the sodium acetate is the same as that in the fermentation medium.

[0069] Furthermore, the carbon source concentration of the feed is 34-53 g / L.

[0070] In certain embodiments of the present invention, in step II), the nitrogen source added by flow addition includes corn steep liquor and yeast powder.

[0071] Furthermore, the ratio of the nitrogen amount provided by the corn steep liquor to the nitrogen amount provided by the yeast powder is the same as that in the fermentation medium.

[0072] Furthermore, the carbon source concentration of the feed is 28-39 g / L.

[0073] In certain embodiments of the present invention, in step II), when the dissolved oxygen concentration drops to 40% to 70%, the feeding is stopped, and the fermentation culture process includes multiple feedings until the remaining carbon and nitrogen are replenished.

[0074] In a preferred embodiment of the present invention, in step II), when the dissolved oxygen concentration drops to 50% to 60%, the feeding is stopped.

[0075] In certain embodiments of the present invention, the total fermentation culture period of the first stage culture and the second stage culture is 20 to 24 hours.

[0076] The present invention also includes sampling and testing relevant indicators every 2 hours after the strain enters the logarithmic phase, and adjusting the feed amount according to the test indicators and changes in pH and dissolved oxygen concentration to prevent abnormal growth caused by insufficient or excessive nutrition, dissolved oxygen, etc. of the strain during the fermentation process.

[0077] The present invention also includes stopping the tank, discharging materials, and shutting down the fermentation control system, air compressor, and refrigerator when the bacterial strain fermentation enters a stable period; and performing relative index analysis on the collected fermentation liquid. The bacterial liquid that is not used temporarily can be refrigerated and stored at 4°C.

[0078] In certain embodiments of the present invention, in step I), the following steps are further included before the seed liquid is inoculated into the fermentation medium:

[0079] 1) Rejuvenation of bacterial strains;

[0080] 2) Seed culture;

[0081] 3) Preparation before fermentation.

[0082] In certain embodiments of the present invention, in step 1), the method for rejuvenation of the strain is a conventional method in the art.

[0083] Specifically, the steps of rejuvenation of the strain are as follows: using a freeze-dried tube strain stored at an ultra-low temperature of -80°C in a biosafety cabinet, picking 3 to 5 small magnetic beads to a solid LB plate in the biosafety cabinet, oscillating the small magnetic beads to coat the plate, and placing the plate in a biochemical incubator at 35°C to 40°C for culturing; after the activation, continue to pick a single bacteria streak LB plate culture medium for rejuvenation culture (repeat 2 to 3 times), transfer the strain after the rejuvenation culture to a slant LB culture medium, and store in cold storage after the culture is completed.

[0084] In certain embodiments of the present invention, in step 2), the seed culture method is a conventional culture method in the art.

[0085] Specifically, take a test tube slant culture stored at 4°C, scrape a ring of bacterial moss and inoculate it into the seed culture medium under the protection of the flame of an alcohol lamp in a biosafety cabinet. After the inoculation is completed, place the seed bottle in a shaker at 35°C to 40°C and 200-00rpm to culture until the late logarithmic stage.

[0086] In certain embodiments of the present invention, in step 3), the preparation before fermentation comprises the following steps:

[0087] 31) Clean and disinfect the fermentation tank; specifically, prepare a 1% dilute alkali solution, add about 70% of the volume of the tank body, soak for about 1 hour, wash with clean water until the pH value of the tank body is neutral, continue to add 1% dilute acid solution to soak for about 40 minutes, and wash with clean water until the pH value of the tank body is about neutral;

[0088] 32) Prepare the fermentation system; specifically, start the air compressor, refrigerator, and fermentation control system; connect and calibrate the pH and dissolved oxygen electrodes;

[0089] 33) preparing culture medium and feed and sterilizing; specifically, preparing fermentation culture medium, acid, alkali, carbon source and nitrogen source feed bottles, adding fermentation culture medium into fermentation tank, and sealing or sealing the air filter element and feed port of the fermentation tank air inlet, respectively, and transferring them into the autoclave for sterilization at a sterilization temperature of 115-121° C. and a sterilization time of 20-30 min;

[0090] 34) Connect to the fermentation control system; after sterilization, connect the fermentation tank to the fermentation control system, introduce sterile air to maintain pressure, start stirring, and adjust the speed; connect temperature, pH, and dissolved oxygen electrodes, and adjust the fermentation culture temperature to cool down.

[0091] In certain embodiments of the present invention, in step II), tank washing is further included after the second stage of fermentation culture is completed.

[0092] Specifically, ① remove the pH and dissolved oxygen electrodes, clean the bacteria on the surface of the tank with deionized water, and then screw them into the protective cover containing electrode protection liquid for protection;

[0093] ② Disassemble the feeding needle and feeding bottle, clean the feeding needle and silicone tube, and clean the feeding bottle;

[0094] ③ Wash the tank after fermentation with water, add a certain amount of 1% alkali solution to clean and disinfect it after cleaning, and wash it with clean water until it is approximately neutral.

[0095] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0096] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0097] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0098] Any technology not specifically disclosed in the present invention may adopt existing technology.

[0099] 1. Bacteria rejuvenation and purification stage

[0100] 1. Materials and equipment

[0101] 1.1 Source of Pseudomonas for testing

[0102] The Pseudomonas used in this experiment was Pseudomonas aeruginosa screened independently, with a preservation number of CGMCC No.27611.

[0103] 1.2 Activation and original fermentation medium

[0104] ① Solid LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, agar powder 15 g / L, pH: 7.0±0.5, 121℃, sterilization for 20 min.

[0105] ②Seed culture medium: tryptone 20g / L, yeast powder 5g / L, NaCl 0.5g / L, KCl 0.19g / L, MgCl20.95g / L, glucose 3.6g / L, trace elements 2mL / L, pH 7.0±0.5, 121℃, sterilization for 20min.

[0106] ③Basic fermentation medium: tryptone 20g / L, yeast powder 5g / L, NaCl 0.5g / L, KCl 0.19g / L, MgCl20.95g / L, glucose 10g / L, trace elements 2mL / L, pH 7.0±0.5, 121℃, sterilization for 20min.

[0107] Trace element solution: FeCl3·6H2O 6g / L, ZnSO4·7H2O 0.58g / L, CaCl2 0.2g / L, CuCl2·2H2O0.2g / L, MnSO4·H2O 0.3g / L, EDTA 0.5g / L.

[0108] 1.3 Equipment

[0109] Biological safety cabinet, constant temperature shaker, constant temperature incubator, UV-visible spectrophotometer, high-pressure steam sterilizer, electronic analytical balance, high-speed refrigerated centrifuge, biological microscope, 5L triple fermentation tank.

[0110] 1.4 Evaluation Method

[0111] Bacterial solution OD detection: After the culture is completed, select 600nm (OD 600 ) was used as the measuring wavelength and the OD value of the bacterial culture solution was determined by ultraviolet spectrophotometry.

[0112] Colony count: After the bacteria enter the logarithmic phase, samples are taken every 2 hours to test relevant indicators.

[0113] 2. Rejuvenation process of strains

[0114] Use the freeze-dried tube strains stored at -80℃ in the biosafety cabinet, pick 3 to 5 small magnetic beads to the solid LB plate in the biosafety cabinet, shake the small magnetic beads to coat the plate, and place it in the biochemical incubator at 35℃ for culture. Repeat the above steps more than three times to achieve the purpose of activation or purification. The activated strains are stored in the slant for later use.

[0115] 3. Seed cultivation

[0116] Take the test tube slant culture stored at 4°C, scrape a ring of bacterial moss and inoculate it into the seed culture medium under the protection of the alcohol lamp flame in the biosafety cabinet. After the inoculation is completed, place the seed bottle in a shaker at 35°C and 200 rpm to culture until the late logarithmic stage.

[0117] 4. Preparation before fermentation:

[0118] Clean and disinfect the fermentation tank; specifically, prepare a 1% dilute alkali solution, add about 70% of the volume of the tank body and soak for about 1 hour, wash with clean water until the pH value of the tank body is neutral, continue to add 1% dilute acid solution and soak for about 40 minutes, and wash with clean water until the pH value of the tank body is about neutral;

[0119] Prepare the fermentation system; specifically, start the air compressor, refrigerator, and fermentation control system; connect and calibrate the pH and dissolved oxygen electrodes;

[0120] The culture medium and feed are prepared and sterilized; specifically, fermentation culture medium, acid, alkali, carbon source and nitrogen source feed bottles are prepared, fermentation culture medium is added to the fermentation tank, and the air filter element and feed port of the fermentation tank are sealed or sealed, and the fermentation tank is moved into the autoclave for sterilization at a sterilization temperature of 120°C and a sterilization time of 30 minutes;

[0121] Connect to the fermentation control system; after sterilization is completed, connect the fermentation tank to the fermentation control system, introduce sterile air to maintain pressure, start stirring, and adjust the speed; connect temperature, pH, and dissolved oxygen electrodes, and adjust the fermentation culture temperature to cool down.

[0122] 5. Fermentation method optimization process

[0123] Based on the basic fermentation medium mentioned in the Pseudomonas literature and in 1.2③ above, the optimal inoculation amount of Pseudomonas fermentation culture was tested; the optimal pH, temperature, and dissolved oxygen during the fermentation culture process were tested;

[0124] Industrial-grade corn starch, crude glycerol, maltose syrup, sodium acetate and glucose were used to replace the original carbon source raw materials, and the effects of different carbon source compositions and different carbon-nitrogen ratios on the fermentation culture of Pseudomonas were studied respectively. By optimizing the carbon source composition of the culture medium, the carbon-nitrogen ratio and fermentation control parameters, a stable and efficient fermentation culture process of Pseudomonas was finally determined.

[0125] 5.1 Study on fermentation control parameters

[0126] Take the test tube slant bacteria stored at 4℃, scrape a ringlet of bacterial moss in the biosafety cabinet and inoculate it into 100mL seed culture medium, culture it at 35℃ and 200rpm shaking to the late logarithmic stage, and inoculate it into shake flask basic fermentation medium for fermentation culture. Set different inoculation amounts, pH gradients, and temperature gradients, and compare the growth characteristics of Pseudomonas in the fermentation process to obtain the optimal conditions. The relevant detection indicators are shown in Table 1.

[0127] Table 1 Data related to the experiment of different culture parameters of Pseudomonas

[0128]

[0129]

[0130] It can be seen from Table 1 that the optimum inoculation amount for the fermentation culture of the Pseudomonas species is 3% to 5%; the optimum pH control range is 7.0 to 7.5; and the optimum temperature is 35°C to 40°C.

[0131] 5.2 Study on dissolved oxygen (DO) control in fermentation

[0132] The optimal dissolved oxygen control test for Pseudomonas was explored in a 5L triple fermentation tank. The inoculation amount of the strain was determined to be 4%, the pH was controlled at 7.0-7.5, the temperature was controlled at 35°C, and different DO gradients were set. The optimal dissolved oxygen conditions after the fermentation culture of Pseudomonas entered the logarithmic phase were obtained by comparison. The OD values ​​of different culture cycles were detected to determine the optimal culture dissolved oxygen concentration. The detection indicators are shown in Table 2.

[0133] Table 2 Effect of different dissolved oxygen on fermentation of Pseudomonas

[0134]

[0135] It can be seen from Table 2 that the optimal dissolved oxygen control range for the Pseudomonas is 50% to 60%, the lowest dissolved oxygen control should not be lower than 40%, and the highest should not exceed 70%.

[0136] 5.3 Study on the influence of different carbon sources in fermentation medium on fermentation

[0137] On the basis of the above fermentation parameter optimization, only the type of carbon source in the basic fermentation medium described in 1.2③ was adjusted, and glycerol, maltose syrup, glycerol, sodium acetate and some combinations were used to replace equal amounts of carbon. A 5L triple fermenter was used for testing to verify the effects of fermentation media with different carbon sources on Pseudomonas fermentation culture. The relevant detection indicators are shown in Table 3.

[0138] Table 3 Related indicators of fermentation culture with different carbon source compositions and ratios

[0139]

[0140]

[0141] As can be seen from the table above, the fermentation culture of Pseudomonas spp. with different proportions of glucose, maltose syrup, sodium acetate, and maltose syrup + sodium acetate can achieve ideal results, and the number of bacterial colonies in the culture liquid is greater than 1.0*10 10 cfu / ml.

[0142] 5.4 Study on the effect of different carbon-nitrogen ratios of culture medium on fermentation

[0143] The basic fermentation medium was optimized using a 5L fermenter. The glucose content in the basic fermentation medium described in 1.2③ was adjusted, and different carbon-nitrogen ratios were set to verify the optimal carbon-nitrogen ratio for the fermentation culture of Pseudomonas. The relevant detection indicators are shown in Table 4.

[0144] Table 4 Effects of different carbon-nitrogen ratios on Pseudomonas fermentation culture

[0145] serial number Carbon-nitrogen ratio OD Remark 1# 1 12.1 2# 2 16.3 3# 2.6～2.8 24.0 Control group 4# 4 28.9 5# 5 30.5 6# 6 30.1 7# 7 29.2 8# 8 27.5

[0146] It can be seen from the above table that when the carbon-nitrogen ratio in the fermentation medium is 5, the OD value of Pseudomonas fermentation culture to the stable period is the highest; when the carbon-nitrogen ratio is in the range of 4 to 8, the OD value of the fermentation liquid of this strain is higher than that of the control group. Considering the cost factor, a carbon-nitrogen ratio of 4 to 5 is more economical and reasonable. The number of colonies in the fermentation liquid of the 3# test group is 1.0*10 10 cfu / ml; the number of colonies in the fermentation liquid of the 5# test group was 1.3*10 10 cfu / ml.

[0147] 5.5 Study on the effect of different nitrogen source combinations in culture medium on fermentation

[0148] Ammonium chloride, ammonium sulfate, corn steep liquor, urea, soybean cake powder, and yeast powder were selected as the main nitrogen source raw materials for the fermentation medium, replacing the trypsin and yeast powder of the basic fermentation medium described in the original 1.2③, and a 5L fermenter was used for testing. The nitrogen content of the fermentation medium was replaced with an equal amount of nitrogen based on the nitrogen content of trypsin and yeast powder in the basic fermentation medium, and the effects of different nitrogen source types and combinations on the fermentation culture of the Pseudomonas were verified. The fermentation medium was prepared according to the optimized carbon source composition and the optimal carbon-nitrogen ratio, and the Pseudomonas was cultured with the optimized fermentation control parameters. The relevant detection indicators are shown in Table 5.

[0149] Table 5 Related indicators of fermentation culture with different nitrogen source compositions and ratios

[0150]

[0151]

[0152] It can be seen from the above table that in the fermentation culture test with inorganic nitrogen sources ammonium chloride and ammonium sulfate as the only nitrogen source, ammonium sulfate has a slightly better effect and a shorter fermentation cycle; yeast powder, corn slurry, urea and soybean cake powder are used as the only nitrogen sources for fermentation culture, and yeast powder and corn slurry have better fermentation effects; the fermentation effect is ideal when ammonium sulfate + yeast powder (≤3:1), ammonium sulfate + corn slurry (≤3:1) and corn slurry + yeast powder (≥3:1) are used as nitrogen source combinations.

[0153] 5.6 Research on fermentation micronutrient additives

[0154] Humic acid was added as a trace nutrient to the basic fermentation medium described in the original 1.2③, and different addition concentrations were set. A small-scale fermentation test was carried out with the optimized medium and control parameters to verify the effect of adding humic acid on the fermentation effect of the strain. The test results are shown in Table 6.

[0155] Table 6 Effects of different humic acid contents on bacterial fermentation

[0156]

[0157] It can be seen from the table above that adding a certain amount of humic acid can effectively promote the fermentation culture effect of this strain. The optimal addition amount is 0.05% to 0.1%.

[0158] 5.7 Study on the effects of feed supplement and total carbon and nitrogen addition on fermentation

[0159] On the basis of the above improvements, the effect of the total amount of carbon source + nitrogen source (carbon-nitrogen ratio of 5:1) added to the culture medium on fermentation was verified. While ensuring that the total volume of the fermentation liquid in each test group was consistent, the total amount of carbon and nitrogen added in the control group was calculated according to the following steps:

[0160] 1) The seed fermentation medium described in 1.2② is used as the base material, wherein the carbon source in the seed culture medium, i.e., glucose, is replaced by an equal amount of carbon with maltose syrup + sodium acetate (carbon ratio of 3:1), and the nitrogen source in the seed culture medium, i.e., tryptone + yeast powder, is replaced by an equal amount of nitrogen with corn steep liquor + yeast powder (nitrogen ratio of 4:1), and the first carbon and nitrogen amount is calculated;

[0161] The specific calculation steps are as follows:

[0162] The concentration of glucose in the original 1.2② seed culture medium is 3.6g / L, and the carbon content in glucose is 40%. Therefore, the carbon content in 3.6g / L glucose is 1.44g / L.

[0163] Original 1.2② In the seed culture medium, tryptone is 20g / L, yeast powder is 5g / L, the nitrogen content in tryptone is 13%, the nitrogen content in yeast powder is 8%, then the nitrogen concentration in the seed culture medium is 3g / L;

[0164] Then the carbon content provided by maltose syrup + sodium acetate (carbon ratio of 3:1) is guaranteed to be 1.44g / L, and the nitrogen content provided by corn syrup + yeast powder (nitrogen ratio of 4:1) is guaranteed to be 3g / L for equal carbon and nitrogen replacement;

[0165] The carbon content in maltose syrup is 28.5%, and the carbon content in sodium acetate is 29.3%;

[0166] Assume that in a 1L system, the mass of maltose syrup is xg and the mass of sodium acetate is yg, then

[0167] 0.285x+0.293y=1.44 (1)

[0168] 0.285x÷0.293y=3 (2)

[0169] Combining equations (1) and (2), we can obtain: x = 3.79gy = 1.23g

[0170] The concentration of the maltose syrup + sodium acetate mixture is 5.02 g / L.

[0171] The nitrogen content in corn steep liquor is 7% to 8%, and the nitrogen content in yeast powder is 7% to 8%;

[0172] Assuming that the nitrogen content of corn syrup is 8% and the nitrogen content of yeast powder is 8%, and in a 1L system, the mass of corn syrup is ag and the mass of yeast powder is bg, then

[0173] 0.08a+0.08b=3 (3)

[0174] 0.08a÷0.08b=4 (4)

[0175] Combining equations (3) and (4), we can obtain: a = 30g b = 7.5g

[0176] The concentration of the corn steep liquor + yeast powder mixture is 37.5 g / L.

[0177] 2) Then only the carbon source is added until the ratio of carbon to nitrogen in the culture medium is 5:1. The amount of carbon and nitrogen when the ratio of carbon to nitrogen is 5:1 is recorded as the total amount of carbon and nitrogen added in the control group and recorded as M. The specific calculation steps are as follows:

[0178] Assume that in a 1L system, the carbon amount in the base material is 1.44g and the nitrogen amount is 3g, then the first total carbon and nitrogen amount is 4.44g, recorded as A; only increase the carbon source to a carbon-nitrogen ratio of 5:1. When the carbon-nitrogen ratio is 5:1, the carbon amount is 15g, and the total amount of carbon and nitrogen added in the control group is 18g.

[0179] Taking the total amount of carbon and nitrogen added in the control group as the benchmark, the effects of the total amount of carbon and nitrogen added in the experimental group on the fermentation of the strain were calculated. The total amount of carbon and nitrogen added in the experimental group was 1.25 times (1.25M), 1.5 times (1.5M), 1.75 times (1.75M), 2 times (2M) and 2.5 times (2.5M) of the control group respectively.

[0180] Based on the seed culture medium after equal replacement of carbon and nitrogen, the remaining carbon and nitrogen were supplemented by feeding. The remaining carbon and nitrogen amount was the total carbon and nitrogen amount calculated in each group minus the first carbon and nitrogen amount, and then the OD was measured. 600 The relevant test indicators are shown in Table 7. Feeding by feeding is beneficial to the stable control of dissolved oxygen during fermentation and also reduces the inhibitory effect of the osmotic pressure of the fermentation liquid on the strains to a certain extent.

[0181] Table 7: Effects of different feed amounts on Pseudomonas fermentation culture

[0182]

[0183] It can be seen from the above table that when the total amount of carbon and nitrogen added is 1.75 times that of the control group, the fermentation culture effect of Pseudomonas is the best.

[0184] 5.8 Fermentation method to optimize overall effect

[0185] The optimized fermentation method was used to carry out a comparative test of fermentation culture of Pseudomonas. The volume of the fermentation medium was 3L, and the fermentation medium included: 5g / L of a mixture of maltose syrup and sodium acetate (carbon ratio of 3:1), 37.5g / L of a mixture of corn steep liquor and yeast powder (nitrogen ratio of 4:1), 0.05% humic acid by volume mass fraction, 0.5g / L of NaCl, 0.19g / L of KCl, 0.95g / L of MgCl2, 6g / L of FeCl3·6H2O, 0.58g / L of ZnSO4·7H2O, 0.2g / L of CaCl2, 0.2g / L of CuCl2·2H2O, 0.3g / L of MnSO4·H2O, and 0.5g / L of EDTA.

[0186] Combined with the calculation method in 5.7, calculate the total amount of carbon and nitrogen added in the 3L system. The specific calculation steps are as follows:

[0187] The amount of carbon contained in the fermentation medium is 1.44×3=4.32g;

[0188] The amount of nitrogen contained in the fermentation medium is 3×3=9g;

[0189] The first carbon and nitrogen total amount is 4.32+9=13.32g;

[0190] Only the amount of carbon is increased, and the total amount of the second carbon and nitrogen when the carbon-nitrogen ratio is 5:1 is calculated to be 9×5+9=54g;

[0191] From 5.7, we can know that the fermentation culture effect is best when the total amount of carbon and nitrogen added is 1.75 times the total amount of carbon and nitrogen in the second batch, and the total amount of carbon and nitrogen added is 94.5g.

[0192] The total added carbon and nitrogen amount minus the first carbon and nitrogen amount in the fermentation medium, the remaining carbon and nitrogen amount was fed by feeding, and the remaining carbon and nitrogen amount was 81.18g.

[0193] When the dissolved oxygen concentration is higher than 50% to 60%, the first stage of fermentation culture is ended and the feeding is started. The concentration of the mixture of maltose syrup and sodium acetate in the carbon source of the feeding is 40 g / L, and the concentration of the mixture of corn slurry and yeast powder in the nitrogen source is 32 g / L. When the dissolved oxygen concentration of the fermenter is lower than 50% to 60%, the feeding is stopped. Starting the feeding and stopping the feeding are one feeding cycle. In this embodiment, multiple feeding cycles are included until the remaining carbon and nitrogen are used up and the carbon and nitrogen ratio in the final fermenter is ensured to meet 5:1.

[0194] The remaining parameters are as follows: inoculation volume 3% to 5%; pH: 7.0 to 7.5; temperature 37°C. The growth characteristics of different culture times during the fermentation culture process were clarified, and the microbial concentrations before and after the method optimization were compared. The relevant growth indicators are shown in Table 8.

[0195] Table 8 Growth of strains during the culture period before and after optimization of the fermentation method

[0196]

[0197] As can be seen from the table above, in the process of fermentation culture of Pseudomonas by the basic fermentation method in the literature, the culture enters the stable period after about 22 hours, the OD value of the fermentation liquid is the highest at 24 hours, and the ideal fermentation culture cycle is about 22 to 24 hours. The fermentation culture of Pseudomonas was carried out with the optimized fermentation medium and fermentation control parameters, and the culture entered the stable period after about 20 hours, the OD value of the fermentation liquid is the highest at 22 hours, and the ideal fermentation culture cycle is about 20 to 24 hours. After the fermentation method is optimized, compared with the best fermentation result before optimization, the microbial concentration in the same fermentation cycle increased by about 66.7% to 91.7%, and the effect was significant.

[0198] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. A fermentation medium, characterized in that Based on the total volume of the fermentation medium, the fermentation medium includes: 4 to 5 g / L of a carbon source, wherein the carbon source includes maltose syrup and sodium acetate; 35-39 g / L of nitrogen source, wherein the nitrogen source comprises a mixture of corn steep liquor and yeast powder; 1-3 g / L of inorganic nutrient salts, 5-9 g / L of trace elements and 0.05-0.2% of the volume mass fraction of nutrient additives.

2. The fermentation medium according to claim 1, characterized in that Includes one or more of the following features: 1) In the carbon source, the amount of carbon provided by maltose syrup is greater than the amount of carbon provided by sodium acetate; 2) In the nitrogen source, the amount of nitrogen provided by corn steep liquor is more than 4 times the amount of nitrogen provided by yeast powder; 3) The ions of the inorganic nutrient salt are selected from Na + , K + Mg 2+ One or more of; 4) The trace elements are selected from Fe 3+ 、Zn 2+ , Ca 2+ , Cu 2+ , Mn 2+ , EDTA or more; 5) The nutritional additive is humic acid.

3. Use of the fermentation medium according to any one of claims 1 to 2 in fermentation and cultivation of Pseudomonas.

4. A fermentation method of Pseudomonas, characterized in that: The fermentation method comprises the following steps: I. First stage culture: inoculating the seed liquid into the fermentation medium of claim 1 to carry out the first stage fermentation culture; II. Second stage culture: feeding in a flow addition manner and continuing the fermentation culture until the fermentation is completed.

5. The fermentation method according to claim 4, characterized in that The fermentation conditions are selected from any one or more of the following: 1) The fermentation temperature is 35-40°C; 2) The pH during the fermentation process is maintained at 7.0 to 8.0; 3) The dissolved oxygen concentration of the fermentation culture is 40% to 70%; 4) The ventilation rate during the fermentation culture process is 1 L / min to 3 L / min; 5) The stirring speed during the fermentation culture process is 200 rpm to 400 rpm.

6. The fermentation method according to claim 4, characterized in that In step I), the inoculation amount is 3% to 5%.

7. The fermentation method according to claim 4, characterized in that In step II), feed is performed as follows: II1) calculating the total added carbon and nitrogen amount: the carbon and nitrogen amount in the fermentation medium is recorded as the first carbon and nitrogen amount, while keeping the nitrogen amount in the fermentation medium unchanged, the carbon amount in the medium is increased until the carbon-nitrogen ratio in the medium is 4 to 8:1, and the carbon and nitrogen amount when the carbon-nitrogen ratio is 4 to 8:1 is recorded as the second carbon and nitrogen amount, and 1.25 to 2.5 times the second carbon and nitrogen amount is the total added carbon and nitrogen amount; II2) Subtract the first carbon-nitrogen amount from the total added carbon-nitrogen amount calculated in step II1) to calculate the remaining carbon-nitrogen amount, which is the carbon-nitrogen amount added by the supplementary feed.

8. The fermentation method according to claim 5, characterized in that In step II), any one or more of the following conditions are included: II3) When the dissolved oxygen concentration of the fermentation culture is higher than 40% to 70%, the feeding is started to start the second stage of culture; II4) When the dissolved oxygen concentration is higher than the set value, start the feeding process, and stop the feeding process when the dissolved oxygen concentration is lower than the set value. II5) The carbon source of the feed includes maltose syrup and sodium acetate; II6) The concentration of the carbon source in the feed is 34-53 g / L; II7) The nitrogen source of the feed includes corn steep liquor and yeast powder; II8) The nitrogen source concentration of the feed addition is 28-39 g / L.

9. The fermentation method according to claim 8, characterized in that The fermentation culture process includes multiple feedings until the remaining carbon and nitrogen are replenished.

10. The fermentation method according to claim 4, characterized in that The total fermentation culture period of the first stage culture and the second stage culture is 20 to 24 hours.

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