Non-sterilized fermentation medium for clostridium tyrobutyricum and fermentation method

Abstract

The clostridium tyrobutyricum is clostridium tyrobutyricum L319 with the preservation number of GDMCC 62289, and the culture medium takes one or two of urea or formamide as a sole nitrogen source or takes phosphite as a sole phosphorus source. The culture medium does not need to be sterilized, the efficient low-cost non-sterilization fermentation production process of the clostridium tyrobutyricum is achieved, the production cost is further reduced, butyric acid can be produced by the clostridium tyrobutyricum in a non-sterilization system, and the advantage proportion of the clostridium tyrobutyricum can reach 95%.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to a non-sterile fermentation process culture medium and a fermentation method for Clostridium tyrobutyricum. Background Art

[0002] Microbial fermentation is considered a green and sustainable way to produce compounds. More and more microorganisms that are specific and efficient in producing certain compounds have been discovered or modified. However, these microorganisms often need a specific community to function. Contamination from other bacteria will greatly reduce the production efficiency of the compound and even destroy the overall function of the microbial fermentation production line. For this reason, microbial fermentation currently mainly requires the addition of antibiotics and high temperature and high pressure to maintain the purity of the strain during the fermentation process. However, the addition of antibiotics has the potential risks of reduced half-life effects and the generation of mutant strains due to abuse, while the high temperature and high pressure sterilization conditions will increase the cost of production and also bring safety risks to the treatment.

[0003] To avoid the above-mentioned hidden dangers, non-sterile fermentation is an effective fermentation process idea. It provides one or multiple extreme conditions (temperature, pH, osmotic pressure, inhibitors) at the same time, or configures carbon sources, nitrogen sources, and phosphorus sources that can be used by unconventional strains, or endows microorganisms through metabolic engineering to inhibit the growth of other contaminating microorganisms without affecting the growth of the main fermentation strain and the fermentation of production compounds.

[0004] Clostridium tyrobutyricum is an obligate anaerobic Clostridium that can naturally produce short-chain fatty acids (acetic acid and butyric acid). Existing technologies have obtained fermentation strains of Clostridium tyrobutyricum that can efficiently synthesize butyric acid, butyl butyrate, butanol and other high-value compounds through metabolic engineering.

[0005] The traditional fermentation process of Clostridium tyrobutyricum requires sterilization to control the singleness and high efficiency of the strain, which brings huge sterilization costs, including the purchase and maintenance of sterilization equipment, electricity and water costs. Therefore, the development of a non-sterile fermentation process of Clostridium tyrobutyricum can reduce costs and improve the economic value of producing fermentation chemicals with Clostridium tyrobutyricum. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the existing technologies, the object of the present invention is to provide a non-sterile fermentation medium and a fermentation method for Clostridium tyrobutyricum.

[0007] The present invention realizes a high-efficiency and low-cost non-sterile fermentation production process of Clostridium tyrobutyricum by introducing unconventional nitrogen sources and phosphorus sources, thereby further reducing production costs.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A non-sterile fermentation medium of Clostridium tyrobutyricum, wherein the Clostridium tyrobutyricum is Clostridium tyrobutyricum L319 with the preservation number of GDMCC 62289,

[0010] The culture medium uses one or both of urea and formamide as the sole nitrogen source or uses phosphite as the sole phosphorus source.

[0011] The culture medium can use urea or formamide as the sole nitrogen source and phosphite as the sole phosphorus source, or can use urea or formamide as the sole nitrogen source and any available phosphorus source, or can use phosphite as the sole phosphorus source and any available nitrogen source.

[0012] Preferably, the formula of the culture medium is:

[0013] 5~10g / L glucose, 1~2 g / L sodium bicarbonate, a nitrogen source, 0.8~3 g / L sodium chloride, 0.2~0.5 g / L magnesium sulfate heptahydrate, 0.5~1.2 g / L L-cysteine ​​hydrochloride, a phosphorus source, 0.1~0.2 g / L potassium chloride, 0.01~0.05 g / L anhydrous calcium chloride, a metal ion solution and a vitamin solution, and the pH of the culture medium is 6.0~6.5.

[0014] Preferably, the formula of the metal ion solution is: 1~2 g / L nitrilotriacetic acid, 1~3 g / L MgSO4, 30~50g / L MnSO4, 0.1~1.0 g / L NaCl, 10~100.0 mg / L FeSO4, 10~100.0 mg / L CaCl2, 10~100.0mg / L CoCl2, 10~100.0 mg / L ZnSO4, 1~10.0 mg / L CuSO4, 1~10.0 mg / L AlK(SO4)2, and 1~10.0mg / L Na2MoO4.

[0015] Preferably, the formula of the vitamin solution is: 2~20 mg / L biotin, 2~20 mg / L folic acid, 10~100 mg / L pyridoxine hydrochloride, 5~50 mg / L thiamine hydrochloride, 5~50 mg / L riboflavin, 5~50 mg / L niacin, 5~50 mg / L calcium pantothenate, 0.1~1 mg / L cyanocobalamin, 5~50 mg / L p-aminobenzoic acid, and 5~50 mg / L lipoic acid.

[0016] Preferably, the concentration of N in the culture medium is 18-130 mM. More preferably, the nitrogen source in the culture medium is a mixture of urea and formamide, and the concentration of urea is 20 mM and the concentration of formamide is 50 mM.

[0017] Preferably, the concentration of P element in the culture medium is 1-200 mM. Most preferably, the concentration of P element in the culture medium is 50 mM.

[0018] The present invention further provides a method for producing butyric acid by fermentation using the non-sterile fermentation medium, comprising:

[0019] (1) Activation of bacterial strains;

[0020] (2) Seed culture;

[0021] (3) Anaerobic fermentation: inoculating the seed liquid into the non-sterile fermentation medium at an inoculation amount of 3-8% (v / v), and fermenting and culturing at 37° C. under anaerobic conditions for 50-60 h to obtain butyric acid;

[0022] The strain is Clostridium tyrobutyricum L319 with the preservation number of GDMCC 62289.

[0023] Preferably, the bacterial strain activation is as follows: after streaking the bacterial strain on a plate, the activation culture is carried out in an anaerobic incubator at 37° C. for 20 to 36 hours, and then the bacterial strain is inoculated into RCM culture medium and statically cultured for 12 to 18 hours.

[0024] The formula of the RCM culture medium is: 10 g / L peptone, 10 g / L beef extract powder, 5 g / L glucose, 5 g / L NaCl, 3 g / L yeast powder, 3 g / L anhydrous sodium acetate, 1 g / L soluble starch, 0.5 g / L L-cysteine ​​hydrochloride, pH 6.5.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention uses culture medium components with unconventional nitrogen and phosphorus sources to achieve normal growth of Clostridium tyrobutyricum strains.

[0027] (2) The culture medium of the present invention can achieve non-sterile fermentation, and the dominant proportion of Clostridium tyrobutyricum can reach 95%.

[0028] (3) The application of the non-sterile fermentation medium and fermentation method of the present invention can enable Clostridium tyrobutyricum to produce 2.2 g / L butyric acid in a non-sterile system.

[0029] (4) The culture medium of the present invention does not require sterilization, thus realizing a highly efficient and low-cost non-sterile fermentation production process of Clostridium tyrobutyricum, further reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The growth conditions of Clostridium tyrobutyricum in Example 1 in culture medium with different concentrations of urea as the sole nitrogen source.

[0031] Figure 2 This is the dominant proportion of Clostridium tyrobutyricum in Example 1 in the culture medium with 20 mM urea as the only nitrogen source.

[0032] Figure 3 The growth conditions of Clostridium tyrobutyricum in Example 1 in a culture medium with different concentrations of formamide as the sole nitrogen source.

[0033] Figure 4 This is the dominant proportion of Clostridium tyrobutyricum in Example 1 in the culture medium with 50 mM formamide as the only nitrogen source.

[0034] Figure 5 The growth conditions of Clostridium tyrobutyricum in Example 1 in a culture medium with different concentrations of sodium phosphite as the sole phosphorus source.

[0035] Figure 6 These are the strain detection results of non-sterile fermentation of Clostridium tyrobutyricum in different culture media in Example 1.

[0036] Figure 7 The growth conditions of Clostridium tyrobutyricum in the culture medium with the combined nitrogen source containing different concentrations of urea as the sole nitrogen source in Example 2, wherein F represents the concentration of formamide and U represents the concentration of urea.

[0037] Figure 8 This is the dominant proportion of Clostridium tyrobutyricum in Example 2 in the culture medium with 20 mM urea as the only nitrogen source.

[0038] Fig. 9 The growth conditions of Clostridium tyrobutyricum in the culture medium containing different concentrations of combined nitrogen source and phosphorus source in Example 3 are shown in FIG. 1 , wherein Pt represents the concentration of sodium phosphite, F represents the concentration of formamide, and U represents the concentration of urea.

[0039] Fig.10 This is the dominant proportion of Clostridium tyrobutyricum in Example 3 in the culture medium with 20 mM urea as the only nitrogen source. DETAILED DESCRIPTION

[0040] Example 1

[0041] (1) Cultivation of Clostridium tyrobutyricum: The strain used is the preserved Clostridium tyrobutyricum (Accession No.: GDMCC 62289), which is a strict anaerobic bacterium. Related operations need to be performed in an anaerobic environment (anaerobic clean bench). Unless otherwise specified, the culture conditions are 37°C and static culture.

[0042] Take out the cryopreserved tube containing Clostridium tyrobutyricum from the -80℃ refrigerator and thaw it. Use a disposable inoculation loop to dip an appropriate amount of bacterial solution to streak the plate. Place the plate upside down in an anaerobic incubator and culture it for 20-36 hours. Pick the single colony grown on the plate into 1 mL RCM liquid culture medium (10 g / L peptone, 10 g / L beef extract powder, 5 g / L glucose, 5 g / L NaCl, 3 g / L yeast powder, 3 g / L anhydrous sodium acetate, 1 g / L soluble starch, 0.5 g / L L-cysteine ​​hydrochloride, pH 6.5) and culture it statically for 12-18 hours. When OD 600 After reaching 1.5-2.0, transfer to a serum bottle containing 30 mL RCM liquid culture medium at a 3% inoculation volume, culture to the late logarithmic period (at this time, obvious bubbles are generated in the serum bottle), mix the bacterial solution and 30% glycerol solution in a 1:1 ratio in a cryopreservation tube, and store in a -80℃ refrigerator. The activated bacterial solution can be stored in a 4℃ refrigerator for about 2 days.

[0043] (2) Verification and optimization experiments of nitrogen and phosphorus sources:

[0044] Conventional inorganic salt liquid culture medium (control group): 10 g / L glucose, 2 g / L sodium bicarbonate, 1 g / L ammonium chloride, 0.8 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 0.5 g / L L-cysteine ​​hydrochloride, 0.16 g / L sodium dihydrogen phosphate, 0.1 g / L potassium chloride, 0.02 g / L anhydrous calcium chloride, metal ion solution (1.5 g / L nitrilotriacetic acid, 3.0 g / LMgSO4, 50.0 g / L MnSO4, NaCl 1.0 g / L, FeSO4 100.0 mg / L, CaCl2 100.0 mg / L, CoCl2 100.0 mg / L, ZnSO4 100.0 mg / L, CuSO4 10.0 mg / L, AlK(SO4)2 10.0 mg / L, Na2MoO4 10.0 mg / L), vitamin solution (20 100 mg / L biotin, 20 mg / L folic acid, 100 mg / L pyridoxine hydrochloride, 50 mg / L thiamine hydrochloride, 50 mg / L riboflavin, 50 mg / L niacin, 50 mg / L calcium pantothenate, 1 mg / L cyanocobalamin, 50 mg / L p-aminobenzoic acid, 50 mg / L lipoic acid), pH 6.5.

[0045] The other components were the same, except that ammonium chloride in the inorganic salt liquid culture medium was replaced with urea, that is, urea was used as the only nitrogen source, and the concentration gradient was set to 20 mM, 40 mM, 80 mM, and 120 mM.

[0046] The other components were the same, except that ammonium chloride in the inorganic salt liquid culture medium was replaced with formamide, that is, formamide was used as the only nitrogen source, and the concentration gradient was set to 50 mM, 100 mM, and 150 mM.

[0047] The other components were the same, except that the sodium dihydrogen phosphate in the inorganic salt liquid culture medium was replaced with sodium phosphite, and the concentration gradient was set to 20 mM, 50 mM, 70 mM, 90 mM, 100 mM, and 200 mM.

[0048] The activated seed liquid was inoculated into the above-mentioned culture media respectively, and fermented and cultured at 37°C under anaerobic conditions for 50-60 hours, and the growth and butyric acid production of Clostridium tyrobutyricum were determined.

[0049] The strain proportion was calculated by diluting the fermentation broth and applying it to the RCM plate, taking a single colony on the plate for 16sRNA sequencing, and obtaining the strain identification results to calculate the dominant proportion of Clostridium tyrobutyricum.

[0050] The growth of Clostridium tyrobutyricum with urea as the only nitrogen source is shown in Figure 1 As shown in the figure, the addition of 80 mM and 120 mM urea significantly inhibited the growth of Clostridium tyrobutyricum, and 120 mM urea even completely inhibited it; while the addition of 20 mM and 40 mM urea promoted the growth of Clostridium tyrobutyricum. When 20 mM urea was added, the maximum biomass of Clostridium tyrobutyricum increased by 78.8% compared with the control group (marked 0 mM in the figure). In the detection of miscellaneous bacteria, such as Figure 2 As shown, compared with the control group (conventional inorganic salts), the proportion of Clostridium tyrobutyricum obtained in the culture medium with 20 mM urea as the only nitrogen source increased from 15% to 66%, effectively improving the dominant growth performance of Clostridium tyrobutyricum under non-sterilization conditions.

[0051] Growth of Clostridium tyrobutyricum with formamide as the sole nitrogen source Figure 3 As shown in the figure, unlike the addition of urea, the three concentrations of formamide promoted the growth of Clostridium tyrobutyricum to varying degrees; among them, under the condition of adding 50 mM formamide, the maximum biomass increased by 51.6% compared with the control group (marked 0 mM in the figure). Based on this analysis, Clostridium tyrobutyricum has a high degree of utilization of formamide. In addition, compared with the control group (conventional inorganic salts), the proportion of strains ( Figure 4 ), the proportion of Clostridium tyrobutyricum increased from 15% to 53%. The results showed that an appropriate amount of formamide can increase the dominant proportion of Clostridium tyrobutyricum in non-sterile fermentation.

[0052] The growth of Clostridium tyrobutyricum with sodium phosphite as the only phosphorus source is shown in Figure 5As shown in the figure, the addition of 100 mM and 200 mM phosphite significantly inhibited the growth of Clostridium tyrobutyricum. The addition of 50 mM, 70 mM, and 90 mM phosphite increased the maximum biomass of Clostridium tyrobutyricum by 14.7%, 19.1%, and 40.0%, respectively. The results of the detection of foreign bacteria showed that ( Figure 6 ) Clostridium tyrobutyricum can achieve a dominant strain ratio of 78% by non-sterile fermentation in a culture medium with phosphite as the only phosphorus source.

[0053] Example 2

[0054] According to the results of Example 1, the ammonium chloride in the inorganic salt liquid culture medium was replaced with 50 mM formamide, and 20 mM, 40 mM, 60 mM and 80 mM urea were added as a combined nitrogen source, respectively.

[0055] The culture was carried out according to the method of Example 1, with conventional inorganic salt culture medium as the control group. The results are as follows Figure 7 As shown in the figure, the combination of 50 mM formamide and 20 mM urea had the least effect on the growth of Clostridium tyrobutyricum, which was consistent with the optimal addition amount of formamide and urea alone. Figure 8 ) was also mentioned to be 76% higher, indicating that this unconventional nitrogen source can effectively improve the strain advantage of Clostridium tyrobutyricum in non-sterile fermentation.

[0056] Example 3

[0057] Phosphite medium formula: 5 g / L glucose, 1 g / L sodium bicarbonate, 1 g / L ammonium chloride, 3 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, 1.2 g / L L-cysteine ​​hydrochloride, 50 mM sodium phosphite, 0.2 g / L potassium chloride, 0.01 g / L anhydrous calcium chloride, metal ion solution (1 g / L nitrilotriacetic acid, 1.0 g / L MgSO4, 30.0 g / LMnSO4, NaCl 0.1 g / L, FeSO4 10.0 mg / L, CaCl2 10.0 mg / L, CoCl2 10.0 mg / L, ZnSO4 10.0 mg / L, CuSO4 1.0 mg / L, AlK(SO4)2 1.0 mg / L, Na2MoO4 1.0 mg / L), vitamin solution (2 mg / L biotin, 2 mg / L folic acid, 10 5 mg / L pyridoxine hydrochloride, 5 mg / L thiamine hydrochloride, 5 mg / L riboflavin, 5 mg / L niacin, 5 mg / L calcium pantothenate, 0.1 mg / L cyanocobalamin, 5 mg / L p-aminobenzoic acid, 5 mg / L lipoic acid), pH 6.0.

[0058] Urea medium formula: 10 g / L glucose, 2 g / L sodium bicarbonate, 20 mM urea, 0.8 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 0.5 g / L L-cysteine ​​hydrochloride, 1 mM sodium dihydrogen phosphate, 0.1 g / L potassium chloride, 0.05 g / L anhydrous calcium chloride, metal ion solution (2 g / L nitrilotriacetic acid, 3.0 g / L MgSO4, 50.0 g / L MnSO4, NaCl 1.0 g / L, FeSO4 100.0 mg / L, CaCl2 100.0 mg / L, CoCl2 100.0 mg / L, ZnSO4 100.0 mg / L, CuSO4 10.0 mg / L, AlK(SO4)2 10.0 mg / L, Na2MoO4 10.0 mg / L), vitamin solution (20 100 mg / L biotin, 20 mg / L folic acid, 100 mg / L pyridoxine hydrochloride, 50 mg / L thiamine hydrochloride, 50 mg / L riboflavin, 50 mg / L niacin, 50 mg / L calcium pantothenate, 1 mg / L cyanocobalamin, 50 mg / L p-aminobenzoic acid, 50 mg / L lipoic acid), pH 6.5.

[0059] The activated seed liquid was inoculated into the above-mentioned culture media respectively, and cultured according to the method of Example 1. The results showed that the above-mentioned culture media all achieved a dominant strain ratio of more than 70%.

[0060] Example 4

[0061] According to the results of Examples 1 and 2, the ammonium chloride in the inorganic salt liquid culture medium was replaced with 50 mM formamide and 20 mM urea, and the sodium dihydrogen phosphate in the inorganic salt liquid culture medium was replaced with 50 mM phosphite. The culture was carried out according to the method of Example 1, and the conventional inorganic salt culture medium was used as the control group to observe the growth of Clostridium tyrobutyricum. Fig. 9 shown.

[0062] The final strain proportion also shows that Fig.10 As shown, Clostridium tyrobutyricum can achieve a 95% dominant strain under a specific unconventional combination of phosphorus and nitrogen sources, which meets the strain dominance requirements of the fermentation process.

[0063] The butyric acid yields obtained from the culture medium used in the control group and Examples 1 to 4 were measured respectively, and the results are shown in Table 1.

[0064] Table 1 Fermentation performance of non-sterile fermentation of Clostridium tyrobutyricum

[0065]

Claims

1. A non-sterile fermentation medium for Clostridium tyrobutyricum, characterized in that: The Clostridium tyrobutyricum is Clostridium tyrobutyricum L319 with the deposit number of GDMCC 62289. The culture medium uses one or both of urea and formamide as the sole nitrogen source or uses phosphite as the sole phosphorus source.

2. The non-sterile fermentation medium according to claim 1, characterized in that The formula of the culture medium is: 5~10g / L glucose, 1~2 g / L sodium bicarbonate, a nitrogen source, 0.8~3 g / L sodium chloride, 0.2~0.5 g / L magnesium sulfate heptahydrate, 0.5~1.2 g / L L-cysteine ​​hydrochloride, a phosphorus source, 0.1~0.2 g / L potassium chloride, 0.01~0.05 g / L anhydrous calcium chloride, a metal ion solution and a vitamin solution, and the pH of the culture medium is 6.0~6.

5.

3. The non-sterile fermentation medium according to claim 2, characterized in that The formula of the metal ion solution is: 1~2 g / L nitrilotriacetic acid, 1~3 g / L MgSO4, 30~50 g / L MnSO4, 0.1~1.0 g / L NaCl, 10~100.0 mg / L FeSO4, 10~100.0 mg / L CaCl2, 10~100.0 mg / L CoCl2, 10~100.0 mg / L ZnSO4, 1~10.0 mg / L CuSO4, 1~10.0 mg / L AlK(SO4)2, and 1~10.0 mg / L Na2MoO4.

4. The non-sterile fermentation medium according to claim 2, characterized in that The formula of the vitamin solution is: 2~20 mg / L biotin, 2~20 mg / L folic acid, 10~100 mg / L pyridoxine hydrochloride, 5~50 mg / L thiamine hydrochloride, 5~50 mg / L riboflavin, 5~50 mg / L niacin, 5~50 mg / L calcium pantothenate, 0.1~1 mg / L cyanocobalamin, 5~50 mg / L p-aminobenzoic acid, and 5~50 mg / L lipoic acid.

5. The non-sterile fermentation medium according to claim 1, characterized in that The concentration of N element in the culture medium is 18-130 mM.

6. The non-sterile fermentation medium according to claim 1, characterized in that The nitrogen source in the culture medium is a mixture of urea and formamide.

7. The non-sterile fermentation medium according to claim 6, characterized in that The concentration of urea in the culture medium is 20 mM, and the concentration of formamide is 50 mM.

8. The non-sterile fermentation medium according to claim 1, characterized in that The concentration of the P element in the culture medium is 1-200 mM, preferably, the concentration of the P element in the culture medium is 50 mM.

9. A method for producing butyric acid by fermentation using the non-sterile fermentation medium according to any one of claims 1 to 8, characterized in that: include: (1) Activation of bacterial strains; (2) Seed culture; (3) Anaerobic fermentation: inoculating the seed liquid into the non-sterile fermentation medium at an inoculation rate of 3-8% v / v, and fermenting at 37° C. under anaerobic conditions for 50-60 h to obtain butyric acid; The strain is Clostridium tyrobutyricum L319 with the preservation number of GDMCC 62289.

10. The method according to claim 9, characterized in that The bacterial strain activation is as follows: after streaking the bacterial strain on a plate, the bacterial strain is activated and cultured in an anaerobic incubator at 37° C. for 20 to 36 hours, and then inoculated into an RCM culture medium and statically cultured for 12 to 18 hours.

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