CN108998398B - Method for culturing acetic acid bacteria by adopting micro waterfall type trapezoidal closed culture device - Google Patents
Method for culturing acetic acid bacteria by adopting micro waterfall type trapezoidal closed culture device Download PDFInfo
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Abstract
本发明公开了一种采用微瀑布式梯形密闭培养装置培养醋酸菌的方法，以巴氏醋杆菌Acetobacter pasteurium为菌株，以每100mL蒸馏水中加入6～8g葡萄糖、1～3mL无水乙醇、3～5g酵母提取物、1～3g牛肉膏、0.2～0.3g柠檬酸钠与柠檬酸质量比为1:1的混合物、5～7.5g氯化钠、2.5～5g乙酸钠、0.75～1.25g吐温‑20、pH值为6～7的溶液为快培液态培养基，采用微瀑布式梯形密闭培养装置高密度培养醋酸菌，可培养得到大量菌体。本发明所用设备成本低、机械化程度高、人力投入少、培养效果好，可实现全封闭连续化自动化培养，在培养结束后一次性导出菌悬液既可得到成品。本装置形成的液体循环回路，提高了原料的利用率与菌浓度，最大限度的提高了设备利用率。
The invention discloses a method for culturing acetic acid bacteria by adopting a micro-waterfall trapezoidal closed culture device. Acetobacter pasteurium is used as a strain, and 6-8 g of glucose, 1-3 mL of anhydrous ethanol, 3-3 mL of absolute ethanol are added per 100 mL of distilled water. 5g yeast extract, 1～3g beef extract, 0.2～0.3g mixture of sodium citrate and citric acid mass ratio of 1:1, 5～7.5g sodium chloride, 2.5～5g sodium acetate, 0.75～1.25g Tween ‑20. The solution with a pH value of 6-7 is a liquid medium for rapid cultivation, and a micro-waterfall trapezoidal closed cultivation device is used to cultivate acetic acid bacteria at a high density, which can cultivate a large amount of bacteria. The equipment used in the invention has low cost, high degree of mechanization, less manpower input and good cultivation effect, and can realize fully enclosed continuous automatic cultivation. The liquid circulation loop formed by the device improves the utilization rate of raw materials and bacterial concentration, and maximizes the utilization rate of equipment.
Description
Technical Field
The invention belongs to the technical field of acetic acid bacteria culture, and particularly relates to a method for quickly culturing acetic acid bacteria by adopting a micro waterfall type trapezoidal closed acetic acid bacteria culture device.
Background
The main reason for high-density culture of microorganisms is that many microbial products are accumulated in the cells of the microorganisms, and if a large amount of microbial products are obtained, the microorganisms need to be produced industrially in a centralized and large-scale manner. With the intensive research, the physiological characteristics of many important microorganisms in industrial production are well studied, and together with the continuous development of bioengineering technology, high-density culture of microorganisms is applied to many production. High density culture refers to obtaining a higher cell density, and generally refers to a culture mode in which a higher cell density is obtained than that obtained by conventional culture, and is called high density culture. It belongs to a relative concept, and all treatment modes capable of obviously increasing the thallus concentration can be called high-density culture. From a microscopic perspective, the consumption of nutrients in a culture solution and the accumulation of the metabolic length of the growth of microorganisms in the culture process are key to finally cause the cell growth to stop, and the microorganism high-density culture technology advocates a circular sustainable continuous culture mode, aims to improve the utilization rate of equipment through continuous culture, and maintains the microorganisms at a higher growth rate through controlling the concentration of the nutrients and the concentration of metabolites so as to achieve high-rate and high-concentration culture.
The general high-density culture modes of microorganisms mainly comprise: and (3) dialysis culture, namely, a culture mode of effectively separating metabolites in a culture medium by using a membrane separation technology and simultaneously enabling nutrient substances to enter the culture medium through a membrane so as to achieve the purposes of supplementing the nutrient substances and eliminating the metabolites on the basis of not losing cells. And fed-batch culture, namely a culture mode that nutrients with low concentration are added in the initial culture process, and new nutrient components are gradually added in the culture process to enable the cells to further grow and metabolize until the culture is finally finished. Of these, fed-batch culture studies are the most mature and widely used. But at present, the design and modification of equipment for high-density culture of acetic acid bacteria and other related researches are rare.
Disclosure of Invention
In order to overcome the problems, the invention provides a method for quickly culturing acetic acid bacteria, which has the advantages of low equipment cost, high mechanization degree, less manpower input, good culture effect and high speed.
The technical scheme adopted for solving the technical problems comprises the following steps:
1. culture of Acetobacter strains
Inoculating the strain subjected to Acetobacter pasteurianus protease activation culture into a domestication culture medium for domestication culture at 30 +/-1 ℃ for 48 hours, wherein the domestication culture medium is prepared by adding 2-6 g of glucose, 0.5-2 g of yeast extract, 0.5-2 g of peptone, and/or peptone,0.3～0.4g Na2HPO4·2H2O, 0.1-0.2 g of sodium citrate and 1-2 g of agar, and adding 1-3 mL of absolute ethanol and 0-3 mL of glacial acetic acid after sterilization; repeating the domestication culture for 0-4 times according to the method, and gradually and equivalently increasing glacial acetic acid added into each 100mL of distilled water in the domestication culture medium by 0-1 mL; after the acclimatization culture is finished, separating out dominant acetic acid bacteria to carry out primary amplification culture and secondary amplification culture to obtain a seed culture solution.
2. Device disinfection
Under the closed condition, the micro-waterfall type trapezoid closed culture device is fumigated for 24 hours by using a glacial acetic acid aqueous solution with the mass fraction of 95%, and is irradiated for 2 hours by using a 55W ultraviolet sterilizing lamp, wherein the using amount of the glacial acetic acid aqueous solution with the mass fraction of 95% in each cubic meter of closed space is 100-200 mL.
3. Circulating culture of acetic acid bacteria
Under the aseptic condition, inoculating the seed culture solution in the step 1 with a fast-culture liquid culture medium, allowing the obtained culture solution to flow into a culture tank from a liquid injection port, allowing the culture solution to flow into the step-shaped guide plate at the lowermost layer from the step-shaped guide plate at the uppermost layer by layer, allowing the culture solution to flow into a heating tank from a liquid outlet at the bottom of the culture tank, and allowing the heated culture solution to circularly flow into the culture tank through a peristaltic pump for culture, wherein the circulating flow rate of the culture solution is 500-800 mL/min, and the culture temperature is 25-35 ℃; and after culturing for 5-9 days, closing the constant flow pump and the heating tank, recovering the bacterial suspension in the culture tank, and collecting thalli after centrifugation to obtain the acetic acid bacteria.
The fast culture liquid medium is prepared by adding 6-8 g of glucose, 1-3 mL of absolute ethyl alcohol, 3-5 g of yeast extract, 1-3 g of beef extract, 0.2-0.3 g of a mixture of sodium citrate and citric acid in a mass ratio of 1:1, 5-7.5 g of sodium chloride, 2.5-5 g of sodium acetate and 0.75-1.25 g of Tween-20 into per 100mL of distilled water, wherein the pH value is 6-7.
The micro waterfall type trapezoidal closed culture device comprises: the top of culture tank is provided with annotates the liquid mouth, and top-down has set gradually 4 at least upper surface open-ended rectangular channels in the culture tank, is provided with the stairstepping guide plate in every rectangular channel, and the stairstepping guide plate of adjacent rectangular channel is crisscross to be set up, and the end of stairstepping guide plate is provided with the water conservancy diversion hole, and the bottom of culture tank is provided with the liquid outlet, and the liquid outlet passes through the pipeline and adds the entry looks UNICOM of hot groove, and the export of hot groove passes through the pipeline and adds the liquid mouth looks UNICOM at liquid mouth looks UNICOM through constant flow pump and culture tank top.
In the micro waterfall type trapezoid closed culture device, the rectangular groove preferably has a length of 80-120 cm, a width of 10-20 cm and a height of 6-12 cm; preferably, the number of steps of the stepped guide plate is 10-30, the height difference between adjacent steps is 1-12 mm, further preferably, the number of steps of the stepped guide plate is 15-25, and the height difference between adjacent steps is 3-6 mm; preferably, the diameter of the flow guide hole is 1-3 cm.
In the step 3, the inoculation amount of the seed culture solution is preferably 10% to 14%.
In the step 3, the fast culture liquid medium is preferably prepared by adding 7g of glucose, 2mL of absolute ethyl alcohol, 4g of yeast extract, 2g of beef extract, 0.25g of a mixture of sodium citrate and citric acid in a mass ratio of 1:1, 6.25g of sodium chloride, 3.75g of sodium acetate and 1g of Tween-20 into every 100mL of distilled water, and the pH value is 6.8.
In the step 3, the circulation flow rate of the culture solution is preferably 530-550 mL/min, and the culture temperature is preferably 30 ℃.
Compared with the prior art, the invention has the following advantages:
1. the invention adopts Acetobacter pasteurianus to be applied to the acetic acid bacteria B-1 strain cultured in high density, and determines the culture medium and culture conditions for the high density culture of the B-1 strain; when the strain is fermented for 7 days in the optimal fermentation culture medium, the DCW of the acetobacter strain B-1 can reach 7.09g/L, and compared with the scheme before optimization, the concentration of the obtained strain is improved by 11.5 times.
2. The invention has the advantages of low equipment cost, high degree of mechanization, less manpower input and good culture effect. The device utilizes the three-dimensional ladder structure in the ladder-shaped guide plate to promote the growth of the acetic acid bacteria by fully utilizing the advantages of the culture medium and the equipment in the culture process. The device can realize full-closed continuous automatic culture in the culture production process, and can obtain finished products by once leading out bacterial suspension after the culture is finished. The liquid circulation loop formed by the device improves the utilization rate and the bacteria concentration of raw materials, can realize the generation of larger production efficiency in a smaller space, and improves the equipment utilization rate to the maximum extent.
Drawings
FIG. 1 is a schematic structural view of a micro-waterfall type trapezoidal closed culture apparatus of the present invention.
Detailed Description
The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.
Example 1
1. Culture of Acetobacter strains
Inoculating the strain subjected to Acetobacter pasteurianus protease activation culture into a domestication culture medium at 30 +/-1 ℃ for domestication culture for 48 hours, wherein the activation culture medium is prepared by adding 10g of glucose, 1g of yeast extract and 2g of CaCO into per 100mL of distilled water31.5g of agar, and the activated culture condition is that the culture is carried out for 5 days at 30 ℃, and the acclimatized culture medium is obtained by adding 4g of glucose, 1g of yeast extract, 1g of peptone and 0.34g of Na into every 100mL of distilled water2HPO4·2H2O, 0.15g of sodium citrate and 1.5g of agar, sterilizing, adding 2mL of absolute ethanol and 0.5mL of glacial acetic acid to prepare the product, and culturing for 3d under the acclimation culture condition at 30 ℃; repeating acclimatization culture for 4 times according to the method, and gradually and equivalently increasing glacial acetic acid added into each 100mL of distilled water in the acclimatization culture medium by 0.5 mL; after the acclimatization culture is finished, separating out dominant acetic acid bacteria to carry out primary amplification culture and secondary amplification culture to obtain a seed culture solution.
2. Device disinfection
Under the closed condition, the micro-waterfall type trapezoid closed culture device is fumigated for 24 hours by using a glacial acetic acid aqueous solution with the mass fraction of 95%, and is irradiated for 2 hours by using a 55W ultraviolet sterilizing lamp, wherein the using amount of the glacial acetic acid aqueous solution with the mass fraction of 95% in each cubic meter of closed space is 100-200 mL.
As shown in fig. 1, the micro waterfall type trapezoidal closed culture apparatus of the present embodiment is composed of a constant flow pump 1, a heating tank 2, a culture tank 3, a rectangular tank 4, and a stepped guide plate 5. The top of the culture tank 3 is provided with a liquid injection port b, 4 rectangular grooves 4 with openings on the upper surfaces are stacked in the culture tank 3 from top to bottom, and the rectangular grooves 4 are 100cm long, 15cm wide and 10cm high. Processing has stairstepping guide plate 5 in every rectangular channel 4, and stairstepping guide plate 5's ladder number is 20, and the difference in height between the adjacent ladder is 3mm, and stairstepping guide plate 5 in the adjacent rectangular channel 4 is crisscross to be set up. The tail end of the stepped guide plate 5 is provided with a guide hole a, and the diameter of the guide hole a is 1.5 cm. The bottom of the culture tank 3 is provided with a liquid outlet which is communicated with the inlet of the heating tank 2 through a pipeline, and the outlet of the heating tank 2 is communicated with the liquid inlet at the top of the culture tank 4 through a pipeline and a constant flow pump 1.
3. Circulating culture of acetic acid bacteria
Under the aseptic condition, inoculating 12% of the seed culture solution in the step 1 into a fast-culture liquid culture medium, wherein 7g of glucose, 2mL of absolute ethyl alcohol, 4g of yeast extract, 2g of beef extract, a mixture of 0.25g of sodium citrate and citric acid in a mass ratio of 1:1, 6.25g of sodium chloride, 3.75g of sodium acetate and 1.0g of tween-20 are added into each 100mL of distilled water, and the pH value is 6.8; then, the obtained culture solution flows into the culture tank 3 from the liquid injection port b, flows into the stepped guide plate 5 at the lowest layer by layer from the stepped guide plate 5 at the uppermost layer, then flows into the heating tank 2 through the liquid outlet at the bottom of the culture tank 3, and the heated culture solution circularly flows into the culture tank 3 through the constant flow pump 1 to be cultured, wherein the circulating flow rate of the culture solution is 533mL/min, and the culture temperature is 30 ℃; after 7 days of culture, the constant flow pump 1 and the heating tank 2 are closed, the bacterial suspension in the culture tank 3 is recovered, and thalli are collected after centrifugation, thus obtaining the acetic acid bacteria suspension with the DCW concentration of 7.72 g/L.
In order to determine the process conditions of the present invention, the inventors conducted a number of research experiments, as follows:
a. test strains
Acetobacter pasteurianus passarianus (purchased from China general microbiological culture Collection center, and the strain number is 1.41); LB acetic acid bacteria, purchased from Shanxi Bao chicken Dingli Biotech Co., Ltd; 13 acetic acid bacteria separated from tomato vinegar, persimmon vinegar, apricot vinegar and red date vinegar in laboratory are used for the experiment, and the total of 15 acetic acid bacteria are shown in table 1.
TABLE 1 Acetobacter origin and nomenclature
b. Culture medium
Acetic acid bacteria activation culture medium: 10% of glucose, 1% of yeast extract and 2% of CaCO31.5% agar.
Acetic acid bacteria domestication culture medium: 4% glucose, 1% yeast extract, 1% peptone, 1.5% agar, 0.34% Na2HPO4·2H2O, 0.15% sodium citrate, 2% (V/V) absolute ethanol (added after the culture medium is cooled to below 60 ℃ after being sterilized). The media used below were sterilized, and the dishes used were 12cm in diameter.
c. Strain activation
Dissolving Acetobacter pasteurianus lyophilized powder and LB acetic acid bacteria dry powder with normal saline, inoculating on activated culture medium, and culturing at 30 deg.C for 5 d.
d. Preparation of seed culture solution
Inoculating activated strain and 13 acetic acid bacteria separated from laboratory fermented tomato vinegar, persimmon vinegar, apricot vinegar and red date vinegar into acclimation culture medium, performing amplification culture, and culturing at 30 deg.C for 3 d.
e. Surface static culture
Taking 1mL of the seed culture solution, respectively inoculating into a culture dish with a diameter of 12cm and containing 70mL of fast culture liquid culture medium, and culturing at 30 ℃ for 7 d.
f. Analytical method
Biomass determination: and (4) measuring by a colorimetric method. The OD value of the bacterial suspension is measured by using an ultraviolet-visible spectrophotometer at the wavelength of 600 nm. And drawing a standard curve by taking the light absorption value OD as an abscissa and taking the cell dry weight DCW as an ordinate.
And (3) measuring the content of acetic acid and ethanol: gas chromatography was used. Diluting acetic acid bacteria fermentation liquor by 100 times by using n-butanol (chromatographic purity) as a solvent, and then carrying out sample determination, wherein the parameters of a gas chromatography instrument are as follows: GC-2010PLUS AF 230V shimadzu gas chromatograph equipped with FID detector and Labsolutions workstation (shimadzu, japan), column: HP-INNOWAX capillary column, column ID: 327817H, length 30.0m, inner diameter 0.32mm, film thickness 0.25 μm. Reagent: glacial acetic acid (chromatographically pure), absolute ethanol (chromatographically pure), n-butanol (chromatographically pure). Chromatographic conditions are as follows: column temperature: the equilibration temperature is 60 ℃, and the equilibration time is 3 min. The starting temperature was 60 deg.C (1 min hold), and the temperature was increased to 120 deg.C (1 min hold) at a rate of 10 deg.C/min. Detector temperature: at 210 ℃. The gasification temperature: at 210 ℃. Sample introduction mode: and (4) splitting. Sample introduction time: for 1 min. Carrier gas: high purity nitrogen, pressure 77.4kPa, flow 76.7mL/min, split ratio 28: 1. And (4) flow program: hydrogen flow rate: 40 mL/min. Air flow rate: 400 mL/min. Tail blowing: 30 mL/min. Sample introduction: 1 μ L. The determination method comprises the following steps: and (4) an external standard method.
1. Strain screening and acetic acid acclimation test
Surface static culture was performed on 15 acetic acid bacterial strains under optimal culture conditions for 3d, and cell growth and acetic acid production of each acetic acid bacterial strain were investigated. The concentration of glacial acetic acid in the acclimation culture medium is gradually increased to 2%, and the cell growth condition of each acetic acid bacterial strain is observed. The results are shown in Table 2.
Table 2 comparison of cell growth and acid productivity of each strain with that of the strains grown under high acidity conditions
Note: "-" indicates that the strain failed to grow under the conditions.
In conclusion, LB-1 acetic acid bacteria in the fermentation liquor have the strongest acid-producing capability, but the biomass is low. Compared with LB-1 acetic acid bacteria, Acetobacter pasteurianus strain B-1 is easy to culture, still maintains higher activity when the concentration of glacial acetic acid is 2 percent, and has stronger acid production capacity. Therefore, the acclimatized Acetobacter pasteurianus strain with the glacial acetic acid concentration of 2% is selected as a starting strain for research.
2. Screening of carbon source in high-density culture of acetic acid bacteria
2.1 Effect of Single carbon Source on growth and acid-producing ability of B-1 cells of Acetobacter Strain
Adding 70mL of water into a 150mL triangular flask, and adding 1% yeast extract, 1% peptone and 0.34% Na2HPO4·2H2O, 0.15% sodium citrate, single carbon source set as: glucose, lactose, malt extract powder, sucrose, mannose, mannitol, absolute ethyl alcohol, glycerol, fructose, maltose and xylose, wherein the concentration of the glucose, the lactose, the malt extract powder, the sucrose, the mannose, the mannitol, the absolute ethyl alcohol, the glycerol, the fructose, the maltose and the xylose is 4% (solid is calculated by mass and liquid is calculated by volume), and the culture medium is transferred into a culture dish with the diameter of 12cm while the culture medium is hot after sterilization. After the medium was cooled to room temperature, 1mL of the seed culture was inoculated and cultured at 30 ℃ for 7 days. After the culture is finished, the influence of a single carbon source on the growth and acid (acetic acid) production capacity of cells of the acetobacter strain B-1 is measured, and the result is shown in a table 3.
TABLE 3 growth and acid productivity of B-1 cells of Acetobacter strains under a single carbon source
Note: "-" indicates that the strain does not produce acid under these conditions.
As is clear from Table 3, glucose had the most prominent effect on the growth of Acetobacter aceti strain B-1, and maltose and fructose were the second ones. The most significant acid production effect on acetic acid bacterial strain B-1 is absolute ethanol. Therefore, the carbon source in the fast culture liquid medium is glucose and absolute ethyl alcohol.
2.2 Effect of glucose concentration on growth of B-1 cells of Acetobacter strains
Adding 70mL of water into a 150mL triangular flask, and adding 1% yeast extract, 1% peptone and 0.34% Na2HPO4·2H2O, 0.15% sodium citrate, and 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% glucose as single carbon source were added, and the effect of glucose concentration on the growth of acetic acid bacterial strain B-1 cells was investigated in the same 2.1 test procedure, and the results are shown in Table 4.
TABLE 4 Effect of glucose on the growth of B-1 cells of Acetobacter strains
As can be seen from Table 4, the glucose concentration significantly affected the growth of B-1 cells of Acetobacter strains. When the glucose concentration was 6%, DCW of Acetobacter strain B-1 reached a maximum of 1.461 g/L. Therefore, a glucose concentration of 6% was chosen as the central level for the subsequent optimization experiments.
2.3 Effect of ethanol concentration on growth of B-1 cells of Acetobacter strains
Adding 70mL of water into a 150mL triangular flask, and adding 1% yeast extract, 1% peptone and 0.34% Na2HPO4·2H2O and 0.15% sodium citrate, after sterilization, adding 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% of absolute ethanol as a single carbon source after the culture medium is cooled to below 60 ℃, and researching the influence of the ethanol concentration on the growth of the acetic acid bacteria strain B-1 by the same 2.1 test operation, wherein the results are shown in Table 5.
TABLE 5 Effect of ethanol concentration on growth of B-1 cells of Acetobacter strains
As can be seen from Table 5, the biomass was changed by the change in the ethanol concentration. When the concentration of the ethanol is 2 percent, the biomass of the acetobacter strain B-1 reaches the highest value of 1.21 g/L; as the ethanol concentration continued to increase, the biomass began to decrease. Therefore, an ethanol concentration of 2% was chosen as the central level for the subsequent optimization experiments.
2.4 Effect of Complex carbon sources on growth and acid productivity of B-1 cells of Acetobacter strains
Adding 70mL of water into a 150mL triangular flask, and adding 1% yeast extract, 1% peptone and 0.34% Na2HPO4·2H2O, 0.15% sodium citrate, 5% and 1%, 5% and 2%, 5% and 3%, 6% and 1%, 6% and 2%, 6% and 3%, and 7% respectively based on the ratio of glucose to absolute ethyl alcoholAnd 1%, 7% and 2%, 7% and 3% as composite carbon sources, the effect of the mass ratio of glucose to ethanol on the growth and acid production ability of acetic acid bacteria strain B-1 cells was investigated in the same 2.1 test procedures, and the results are shown in Table 6.
TABLE 6 Effect of Complex carbon sources on the growth and acid-producing ability of cells of Acetobacter strain B-1 (n ═ 3)
Note: the combined index is DCW value + acetic acid content value
As can be seen from Table 6, glucose and absolute ethanol as a complex carbon source have significant effects on the growth and acid production capacity of B-1 cells of the acetobacter strain. Wherein DCW representing cell growth is basically maintained between 1.20 and 1.80g/L, which directly proves that the composite carbon source has more prominent influence on the cell growth of the acetobacter strain B-1. Experiments prove that when the adding proportion of the glucose and the absolute ethyl alcohol is respectively 7 percent and 2 percent, the biomass of the acetobacter strain B-1 reaches a higher value of 1.79g/L, the acetic acid is produced by 1.78g/100mL, and the highest value of the comprehensive index reaches 3.57. Therefore, the adding proportion of glucose and absolute ethyl alcohol is respectively 7 percent and 2 percent, and the glucose and the absolute ethyl alcohol are used as the composite carbon source for culturing the acetic acid bacteria strain B-1.
3. Screening of nitrogen source in high-density culture of acetic acid bacteria
3.1 Effect of Single Nitrogen Source on growth and acid production Capacity of B-1 cells of Acetobacter Strain
Adding 70mL water into 150mL triangular flask, adding 7% glucose, 2% anhydrous ethanol, and 0.34% Na2HPO4·2H2O, 0.15% sodium citrate, and separately added: peptone, yeast extract, beef extract and tryptone are 1 percent respectively, and the culture medium is transferred into a culture dish with the diameter of 12cm while the culture medium is hot after sterilization. After the medium was cooled to room temperature, 1mL of the seed culture was inoculated and cultured at 30 ℃ for 7 days. After the culture was completed, the influence of a single nitrogen source on the growth and acid-producing ability of cells of Acetobacter strain B-1 was determined, and the results are shown in Table 7.
TABLE 7 growth and acid productivity of B-1 cells of Acetobacter strains under a single carbon source
As can be seen from Table 7, the yeast extract significantly promoted the growth of Acetobacter aceti strain B-1, and beef extract and yeast extract were inferior. Meanwhile, no proper nitrogen source has a remarkable influence on the acid production of the acetobacter strain B-1. Therefore, the nitrogen source in the fast culture liquid medium is selected from yeast extract and beef extract.
3.2 Effect of Yeast extract concentration on growth of B-1 cells of Acetobacter strains
Adding 70mL water into 150mL triangular flask, adding 7% glucose, 2% anhydrous ethanol, and 0.34% Na2HPO4·2H2O, 0.15% sodium citrate, and 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% yeast extract as a single nitrogen source were added, and the effect of yeast extract concentration on the growth of acetic acid bacteria strain B-1 cells was investigated in the same 2.1 test procedure, and the results are shown in Table 8.
TABLE 8 influence of Yeast extract concentration on the growth of acetic acid bacteria strain B-1 cells
As can be seen from Table 8, the effect of the concentration of yeast extract on the growth of B-1 cells of the Acetobacter strain was significant. When the concentration of the yeast extract is 3.5 percent, the DCW index of the acetobacter strain B-1 reaches the highest value of 3.46 g/L; then, the concentration of the yeast extract tends to be gentle, which indicates that the yeast extract has no promotion effect on the growth of the thalli after reaching a certain concentration. Therefore, a yeast extract concentration of 3.5% was chosen as the central level for the subsequent optimization experiments.
3.3 Effect of beef extract concentration on growth of B-1 cells of Acetobacter strains
Adding 70mL water into 150mL triangular flask, adding 7% glucose, 2% anhydrous ethanol, and 0.34% Na2HPO4·2H2O, 0.15 percent of sodium citrate, and 0.5 percent, 1 percent, 1.5 percent, 2 percent of sodium citrate are respectively added,Beef extract of 2.5%, 3%, 3.5%, 4% was used as a single nitrogen source, and the effect of beef extract concentration on the growth of B-1 cells of Acetobacter strains was investigated in the same 2.1 test procedures, and the results are shown in Table 9.
TABLE 9 Effect of beef extract concentration on growth of B-1 cells of Acetobacter strains
As can be seen from Table 9, the effect of beef extract concentration on the growth of acetic acid bacteria strain B-1 cells was significant. When the concentration of the beef extract is 2%, the DCW of the acetobacter strain B-1 reaches the peak value of 2.90 g/L; and then begins to fall. Therefore, a beef extract concentration of 2% was selected as the central level for the subsequent optimization experiments.
3.4 Effect of Complex Nitrogen Source on growth and acid production Capacity of Acetobacter Strain B-1 cells
Adding 70mL water into 150mL triangular flask, adding 7% glucose, 2% anhydrous ethanol, and 0.34% Na2HPO4·2H2O and 0.15% sodium citrate, 3% and 1.5%, 3% and 2%, 3% and 2.5%, 3.5% and 1.5%, 3.5% and 2%, 3.5% and 2.5%, 4% and 1.5%, 4% and 2%, 4% and 2.5% respectively according to the adding ratio of the yeast extract to the beef extract are taken as composite carbon sources, and the influence of the mass ratio of the yeast extract to the beef extract on the cell growth and acid production capacity of the acetic acid bacteria strain B-1 is researched in the same 2.1 test operation, and the result is shown in a table 10.
TABLE 10 Effect of Complex Nitrogen sources on the cell growth and acid productivity of Acetobacter strain B-1 (n ═ 3)
Note: the combined index in the table is DCW value + acetic acid content value.
As can be seen from Table 10, the yeast extract and beef extract as a complex nitrogen source significantly affected the growth and acid production ability of the cells of the acetic acid bacterial strain B-1. Wherein DCW representing cell growth is basically maintained between 3.07 and 5.10 g/L. Experiments prove that when the adding proportion of the yeast extract to the beef extract is 4% and 2%, the biomass of the acetobacter strain B-1 reaches a higher value of 5.10g/L, the acetic acid is produced by 1.67g/100mL, and the highest value of the comprehensive index reaches 6.77. Therefore, the yeast extract and the beef extract are added in a proportion of 4 percent and 2 percent to serve as a compound nitrogen source for culturing the acetic acid bacteria strain B-1.
4. Screening of buffer salt in high-density culture of acetic acid bacteria
4.1 Effect of buffer salt combinations on growth and acid productivity of B-1 cells of Acetobacter strains
Adding 70mL of water into a 150mL triangular flask, adding 7% of glucose, 2% of absolute ethyl alcohol, 4% of yeast extract and 2% of beef extract, and respectively adding: k2HPO4、KH2PO4、NaH2PO4、Na2HPO4Citric acid and sodium citrate (added after being compounded in a mass ratio of 1:1 in pairs respectively, the total amount of the added components is 0.2 percent), and the culture medium is transferred into a culture dish with the diameter of 12cm while the culture medium is hot after sterilization. After the medium was cooled to room temperature, 1mL of the seed culture was inoculated and cultured at 30 ℃ for 7 days. After the culture was completed, the effect of the buffer salts on the growth and acid productivity of B-1 cells of Acetobacter strains was determined, and the results are shown in Table 11.
TABLE 11 Effect of buffer salt combinations on the growth and acid productivity of B-1 cells of Acetobacter strains
As can be seen from Table 11, the buffer salt combination had a significant effect on the growth of Acetobacter strain B-1. Wherein, the sodium citrate and the citric acid are the most suitable buffer salt combination for the growth of the acetobacter strain B-1. Therefore, sodium citrate and citric acid are selected as the buffer salt combination in the fast culture liquid medium.
4.2 Effect of buffer salt concentration on growth of Acetobacter strains B-1 cells
70mL of water is added into a 150mL triangular flask, 7% of glucose, 2% of absolute ethyl alcohol, 4% of yeast extract and 2% of beef extract are added, a combination of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% and 4% of sodium citrate and citric acid in a mass ratio of 1:1 is respectively added as buffer salt, and the influence of the buffer salt on the growth of the cells of the acetobacter strain B-1 is researched by the same test operation of 2.1, and the result is shown in Table 12.
TABLE 12 Effect of buffer salt concentration on growth of B-1 cells of Acetobacter strains
As can be seen from Table 12, the sodium citrate and citrate buffer salt concentrations had a significant effect on the growth of cells of Acetobacter fabarum strain B-1. When the concentration of the buffer salt is 0.25%, the DCW of the test strain reaches the peak value of 5.509 g/L; the biomass then begins to decline. Therefore, 0.25% was selected as the optimum buffer salt concentration for the culture of Acetobacter strain B-1.
5. Screening of inorganic salts and growth factors in high-density culture of acetic acid bacteria
5.1 inorganic salts and growth factor preference test
Inorganic salts and growth factors have extremely critical influence on the synthesis of target products and the growth metabolism of microorganisms, but because of excessive test factors, the expected effect of screening is difficult to achieve by the traditional single-factor test. In the experiment, 17 inorganic salts and growth factors are selected as influencing factors, and the code number and the coding level of each factor are shown in Table 13.
TABLE 13 Plackett-Burman test design level and coding
The results of 20 experiments according to the Plackett-Burman design using 17 inorganic salts and growth factors as the study factors, the response values of which are the cell growth and acid-producing ability of Acetobacter strain B-1, are shown in Table 14.
TABLE 14 Plackett-Burman test results
The DCW of the acetic acid bacteria strain B-1 in the table above was subjected to linear regression, and by analysis of variance and significance test, the model (see formula 2-1) was found to be very significant (P)<0.01) and the key factors affecting the cell biomass of the strain are: NaCl (P)<0.01)、MgSO4·7H4O、CaCl4LiCl and CoQ10。
DCW(g/L)＝13.29+0.81X1-0.67X4+0.38X6-1.27X7-0.58X11+0.41X14-0.49X17-0.58X18
Formula 2-1
The acetic acid content average value of the acid production capacity of the acetic acid bacteria strain B-1 in the table is subjected to linear regression, and through analysis of variance and significance test, the model (shown as a formula 2-2) is very significant (P)<0.01) and obtaining the key factors influencing the acid production capacity of the bacterial strain as follows: sodium acetate (P)<0.01), Tween-20 (P)<0.01)、ZnSO4·7H2O and CaCl2. Acetic acid content of 1.75-0.031X1-0.11X3+0.045X4+0.023X11-0.026X12-0.063X15+0.055X17+0.031X19+0.062X20
Formula 2-2
5.2 Single factor assay for optimal concentration of inorganic salts and growth factors
According to the Plackett-Burman test, 5 levels of NaCl, sodium acetate and Tween-20 are respectively set, and single-factor tests are carried out by taking the cell growth and acid production capacity of the acetobacter strain B-1 as indexes, selecting NaCl concentrations of 2.5, 3.75, 5, 6.25 and 7.5g/L, sodium acetate concentrations of 2.5, 3.75, 5, 6.25 and 7.5g/L and Tween-20 concentrations of 0.5, 0.75, 1, 1.25 and 1.5 g/L. The effect of inorganic salt and growth factor concentration on the growth of acetic acid bacteria strain B-1 cells is studied by the same test operation as 2.1, and the results are shown in tables 15-17.
TABLE 15 Effect of sodium chloride concentration on growth of acetic acid bacteria strains B-1 cells
TABLE 16 Effect of sodium acetate concentration on growth of acetic acid bacteria strains B-1 cells
TABLE 17 Effect of Tween-20 concentration on growth of Acetobacter strain B-1 cells
As can be seen from tables 15-17, the concentrations of sodium chloride, sodium acetate and Tween-20 have significant influence on the growth of the acetic acid bacteria strain B-1 cells. When the concentration of the sodium chloride is 6.25g/L, the biomass of the acetobacter strain B-1 reaches the maximum value of 6.54 g/L; as the sodium chloride concentration continued to increase, the biomass began to decline. Therefore, the concentration of sodium chloride is selected to be 6.25 g/L. When the concentration of sodium acetate is 3.75g/L, the biomass of the acetobacter strain B-1 reaches the maximum value of 6.70 g/L; as the sodium acetate concentration continued to increase, the biomass began to decrease. Therefore, the concentration of sodium acetate was selected to be 3.75 g/L. When the concentration of the Tween-20 is 1g/L, the biomass of the acetobacter strain B-1 reaches the highest value of 6.78 g/L; when the concentration of the Tween-20 is continuously increased, the biomass tends to be balanced, which indicates that the Tween-20 with high concentration has no promotion effect on the growth of the thalli of the acetobacter strain B-1. Therefore, the concentration of the Tween-20 is 1 g/L.
6. Acetic acid bacteria fermentation initial condition optimization test
6.1 Effect of initial pH on growth of acetic acid bacterial strains B-1 cells
The initial pH values of the fast-culture liquid culture media are respectively adjusted to 6.4, 6.8, 7.2, 7.6 and 8.0, the influence of the initial pH values on the growth of the cells of the acetobacter strain B-1 is studied, and the results are shown in Table 18.
TABLE 18 Effect of initial pH on growth of acetic acid bacteria strain B-1 cells
As can be seen from Table 18, the initial pH of the culture broth significantly affected the growth of the cells, and the initial pH was weakly acidic to facilitate the growth of the cells of Acetobacter strain B-1, so the initial pH of the culture broth was selected to be 6.8.
6.2 Effect of initial inoculum size on growth of B-1 cells of Acetobacter strains
The initial pH of the fast-culture liquid medium was adjusted to 6.8, the medium was inoculated according to different inoculum sizes and cultured at 30 ℃ for 7 days, and the effect of the inoculum size on cell growth was studied, the results are shown in Table 19.
TABLE 19 Effect of initial inoculum size on growth of B-1 cells of Acetobacter strains
As can be seen from Table 19, DCW also shows a parabolic trend with the change of the inoculation amount. When the inoculum size was 12%, the DCW peaked. It can be seen that the inoculation amount is optimal when 12% is selected.
6.3 Effect of culture temperature on growth of Acetobacter strains B-1 cells
In order to investigate the effect of the culture temperature on DCW of Acetobacter fabarum strain B-1, the culture operations were performed at 22, 26, 30, 34, and 38 ℃ respectively in this experiment, and the results are shown in Table 20.
TABLE 20 Effect of culture temperature on growth of B-1 cells of Acetobacter strains
As can be seen from Table 20, DCW tended to increase and decrease with the increase of the culture temperature, and reached the highest DCW at a culture temperature of 30 ℃. Therefore, 30 ℃ is selected as the optimum culture temperature of the acetobacter strain B-1.
7. Factors influencing growth of acetic acid bacteria in high-density fast culture device
7.1 Effect of the height differential between adjacent steps in a stepped baffle on the growth of B-1 cells of Acetobacter strains
In order to study the effect of the height difference between the adjacent steps in the stepped baffle on the growth of the cells of the acetobacter strain B-1, the experiment was conducted with 20 steps in the stepped baffle, and the culture operation was conducted with the height difference between the two adjacent steps of 2mm, 3mm, 4mm, 5mm, and 6mm, and the results are shown in Table 21.
TABLE 21 Effect of height differences between adjacent steps in step fluidic plates on growth of B-1 cells of Acetobacter strains
As can be seen from Table 21, the height difference between adjacent steps in the stepped fluidic plate is either too low or too high to facilitate the growth of B-1 cells of Acetobacter strains. With the increase of the height difference between the adjacent steps, the biomass of the Acetobacter aceti strain B-1 is increased and then tends to be balanced, and when the height difference between the adjacent steps is 3mm, the biomass reaches the highest value. Therefore, 3mm is selected as the height difference between the most suitable adjacent steps in the stepped guide plate.
7.2 Effect of culture broth circulation Rate on growth of B-1 cells of Acetobacter strains
In order to study the effect of the circulation rate of the culture solution on the growth of the cells of the acetobacter strain B-1, the culture operation was performed at the flow rates of 400, 533, 667, 800 and 1200mL/min with the constant flow pump 5 selected in this experiment, and the results are shown in Table 22.
TABLE 22 Effect of broth circulation Rate on growth of B-1 cells of Acetobacter strains
As can be seen from Table 22, too low or too high a circulation rate of the culture medium is not favorable for the growth of B-1 cells of the Acetobacter strain. Along with the increase of the circulation speed of the culture solution, the biomass of the acetic acid bacteria strain B-1 tends to increase first and then decrease, and when the circulation speed of the culture solution is 533mL/min, the biomass reaches the highest value. Therefore, 533mL/min is selected as the optimal culture solution circulation speed of the acetobacter strain B-1.
Claims (6)
1. A method for culturing acetic acid bacteria by adopting a micro waterfall type trapezoid closed culture device is characterized by comprising the following steps:
(1) culture of Acetobacter strains
Acetobacter pasteurianusAcetobacter pasteuriumInoculating the activated and cultured strain into a domestication culture medium for domestication and culture at the temperature of 30 +/-1 ℃ for 48 hours, wherein the domestication culture medium is prepared by adding 2-6 g of glucose, 0.5-2 g of yeast extract, 0.5-2 g of peptone and 0.3-0.4 g of Na into per 100mL of distilled water2HPO4•2H2O, 0.1-0.2 g of sodium citrate and 1-2 g of agar, and adding 1-3 mL of absolute ethanol and 0-3 mL of glacial acetic acid after sterilization; repeating the domestication culture for 0-4 times according to the method, and gradually and equivalently increasing glacial acetic acid added into each 100mL of distilled water in the domestication culture medium by 0-1 mL; after the acclimatization culture is finished, separating out dominant acetic acid bacteria to carry out primary amplification culture and secondary amplification culture to obtain a seed culture solution;
(2) device disinfection
Under a closed condition, fumigating a micro waterfall type trapezoid closed culture device for 24 hours by using a glacial acetic acid aqueous solution with the mass fraction of 95%, and simultaneously irradiating for 2 hours by using a 55W ultraviolet sterilizing lamp, wherein the using amount of the glacial acetic acid aqueous solution with the mass fraction of 95% in each cubic meter of closed space is 100-200 mL;
the micro waterfall type trapezoidal closed culture device comprises: the top of the culture tank (3) is provided with a liquid injection port (b), at least 4 rectangular tanks (4) with open upper surfaces are sequentially arranged in the culture tank (3) from top to bottom, a step-shaped guide plate (5) is arranged in each rectangular tank (4), the step-shaped guide plates (5) in adjacent rectangular tanks (4) are arranged in a staggered manner, the tail ends of the step-shaped guide plates (5) are provided with guide holes (a), the bottom of the culture tank (3) is provided with a liquid outlet, the liquid outlet is communicated with the inlet of the heating tank (2) through a pipeline, and the outlet of the heating tank (2) is communicated with the liquid injection port at the top of the culture tank (3) through a constant flow pump (1) through a pipeline; the number of the steps of the stepped guide plate (5) is 10-30, and the height difference between adjacent steps is 1-12 mm;
(3) circulating culture of acetic acid bacteria
Under the aseptic condition, inoculating the seed culture solution in the step (1) with a fast-culture liquid culture medium, wherein the inoculation amount of the seed culture solution is 10-14%, the obtained culture solution flows into the culture tank (3) from the liquid injection port (b), flows into the stepped guide plate (5) at the lowermost layer by layer from the stepped guide plate (5) at the uppermost layer, then flows into the heating tank (2) through the liquid outlet at the bottom of the culture tank (3), the heated culture solution circularly flows into the culture tank (3) through the constant flow pump (1) for culture, the circulating flow rate of the culture solution is 500-800 mL/min, and the culture temperature is 25-35 ℃; after culturing for 5-9 days, closing the constant flow pump (1) and the heating tank (2), recovering the bacterial suspension in the culture tank (3), and collecting thalli after centrifugation to obtain acetic acid bacteria;
the fast culture liquid medium is prepared by adding 6-8 g of glucose, 1-3 mL of absolute ethyl alcohol, 3-5 g of yeast extract, 1-3 g of beef extract, a mixture of 0.2-0.3 g of sodium citrate and citric acid in a mass ratio of 1:1, 5-7.5 g of sodium chloride, 2.5-5 g of sodium acetate and 0.75-1.25 g of Tween-20 into per 100mL of distilled water, wherein the pH value is 6-7.
2. The method for culturing acetic acid bacteria by adopting the micro waterfall type trapezoid closed culture device as claimed in claim 1, wherein: the rectangular groove (4) is 80-120 cm long, 10-20 cm wide and 6-12 cm high.
3. The method for culturing acetic acid bacteria by adopting the micro waterfall type trapezoid closed culture device as claimed in claim 1, wherein: the number of steps of the stepped guide plate (5) is 15-25, and the height difference between adjacent steps is 3-6 mm.
4. The method for culturing acetic acid bacteria by adopting the micro waterfall type trapezoid closed culture device as claimed in claim 1, wherein: the diameter of the diversion hole (a) is 1-3 cm.
5. The method for culturing acetic acid bacteria by adopting the micro waterfall type trapezoid closed culture device as claimed in claim 1, wherein: in the step (3), 7g of glucose, 2mL of absolute ethyl alcohol, 4g of yeast extract, 2g of beef extract, 0.25g of a mixture of sodium citrate and citric acid in a mass ratio of 1:1, 6.25g of sodium chloride, 3.75g of sodium acetate and 1g of tween-20 are added into every 100mL of distilled water, and the pH value is 6.8.
6. The method for culturing acetic acid bacteria by adopting the micro waterfall type trapezoid closed culture device as claimed in claim 1, wherein: in the step (3), the circulation flow rate of the culture solution is 530-550 mL/min, and the culture temperature is 30 ℃.
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