Acetobacter xylinum and application thereof
By ARTP mutagenesis and acclimatization of xystosanthesia, the obtained strain P2-A1 can efficiently use xylose to ferment bacterial cellulose, solving the problem of low xylose utilization efficiency in the prior art, and significantly improving the yield of bacterial cellulose and the recycling rate of production resources.
Patent Information
- Application Number
- CN202510094254.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-05-06
AI Technical Summary
In the prior art, xylose is used in low efficiency and consumes very little, which affects the yield and production rate of bacterial cellulose, and is difficult to improve the recycling rate of production resources.
After screening by ARTP mutagenesis treatment, a xystosomega strain P2-A1 was obtained which efficiently utilized xylose. This strain can efficiently use xylose to ferment bacterial cellulose.
The efficiency of the strain's utilization of xylose was significantly improved, the yield of bacterial cellulose was improved, and the recycling rate of production resources was enhanced. The increase in the yield of bacterial cellulose reached 93.59%.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of strain breeding, and in particular to a xylacetobacter and an application thereof. Background Art
[0002] Gluconacetobacter xylinus is a Gram-negative aerobic bacterium with unique ecological functions, such as decomposing wood fiber and producing disease resistance, and is widely used in agriculture, environmental protection and industry. In the industrial field, Gluconacetobacter xylinus can produce bacterial cellulose (BC) through fermentation. Bacterial cellulose is a high molecular weight cellulose with unique physical and chemical properties, such as high water absorption, high mechanical strength, high purity, good biocompatibility and plasticity.
[0003] In the process of producing refined cotton from bamboo pulp, hemicellulose is produced as a byproduct. Hemicellulose can be removed by chemical or enzymatic methods to improve the purity of cellulose. The removed hemicellulose can be further hydrolyzed and converted into xylose. In the prior art, when bacterial cellulose is produced by fermentation with xyloacetobacter, glucose, sucrose, glycerol, etc. are mainly used as carbon sources, and xylose is also used as a carbon source, but the utilization efficiency of xylose is low and the consumption is very small, which greatly affects the yield and production rate of bacterial cellulose.
[0004] In the process of producing bacterial cellulose by fermentation with Acetobacter xylosus, how to improve the utilization efficiency of xylose, thereby increasing the recycling rate of production resources and promoting a sustainable bioeconomy is an issue worthy of research and exploration. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a xylacetobacter in view of the deficiencies of the prior art.
[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned xylacetobacter in fermentation to produce bacterial cellulose.
[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0008] A strain of Gluconacetobacter xylinus, classified and named Gluconacetobacter xylinus, with strain number P2-A1, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 4, 2024, with the deposit number CGMCC No.32895.
[0009] The xyloseacetobacter is obtained by screening through ARTP mutagenesis treatment and then domesticating in a domestication medium containing different xylose concentrations.
[0010] Specifically, the conditions of the ARTP mutagenesis are as follows: under the conditions of a power supply of 120 W, a gas flow rate of 10.0 SLM, and an irradiation distance of 2 mm, the mutagenesis time is set to 0 to 200 s (45 s, 60 s, 75 s, 90 s, 105 s, 120 s, 135 s, 150 s, 180 s, 200 s) for ARTP mutagenesis treatment.
[0011] Specifically, in the screening, the formula of the screening plate culture medium is: 10-40 g / L xylose, 5-20 g / L yeast powder, 10-40 g / L peptone, and 10-40 g / L agar powder.
[0012] Specifically, after ARTP mutagenesis treatment and screening, a total of 30 candidate mutagenesis strains, namely A1 to A30, were obtained; after primary screening, A1 and A27 mutagenesis strains were selected for rescreening.
[0013] Further, after repeated screening, the mutagenic strain A1 was selected for subsequent domestication.
[0014] The acclimation medium containing different xylose concentrations has a formula of: xylose 10-30 g / L, yeast powder 5 g / L, ammonium sulfate 3 g / L, corn steep liquor 10 g / L, KH2PO4 0.5 g / L, K2HPO4·3H2O 0.5 g / L, MgSO4·7H2O0.5 g / L, pH 5.0.
[0015] Specifically, the domestication is carried out using the MMC (microbial microdroplet culture system) system.
[0016] Specifically, after ARTP mutagenesis screening and domestication, the strain's efficiency in utilizing xylose was significantly improved, and an ARTP mutagenesis domesticated strain P2-A1 was obtained.
[0017] The morphological characteristics of the ARTP-induced domesticated strain P2-A1 are as follows: the single colony is round, protruding, opaque, beige, and has a smooth and moist surface. When examined under a microscope, the P2-A1 bacteria are short rod-shaped, with a single size of 1.22-1.49 μm×0.69-0.91 μm, and mostly appear in pairs or chains.
[0018] The application of the xyloseacetobacter in the fermentation to produce bacterial cellulose is also within the scope of protection of the present invention.
[0019] Wherein, the xyloseacetobacter produces bacterial cellulose by fermenting and consuming any one or a combination of xylose, molasses and glucose.
[0020] A method for producing bacterial cellulose by fermenting the xylinacetobacillus is provided, wherein the seed liquid of the xylinacetobacillus is inoculated into a fermentation medium, and bacterial cellulose is produced by static fermentation or dynamic fermentation.
[0021] Wherein, the inoculation amount is 10-20% v / v.
[0022] The formula of the fermentation medium is: molasses 10-20 g / L, xylose 5-20 g / L, ammonium sulfate 2-5 g / L, corn steep liquor 5-40 g / L, KH2PO4 0.5-3 g / L, K2HPO4·3H2O 0.5-3 g / L, MgSO4·7H2O 0.5-5 g / L, zinc sulfate 0.01-0.2 g / L, malic acid 1-10 g / L, the solvent is water, and the pH is adjusted to 5.5;
[0023] or,
[0024] Xylose 15-40 g / L, ammonium sulfate 2-5 g / L, corn steep liquor 5-40 g / L, KH2PO4 0.5-3 g / L, K2HPO4·3H2O 0.5-3 g / L, MgSO4·7H2O 2 g / L, zinc sulfate 0.01-0.2 g / L, malic acid 1-10 g / L, the solvent is water, and the pH is adjusted to 5.5.
[0025] The static fermentation is carried out under the following conditions: static culture at 26-32°C for 4-14 days.
[0026] The conditions of the dynamic fermentation are as follows: culturing at 26-32° C. and 120-180 rpm for 2-5 days.
[0027] A microbial preparation producing bacterial cellulose is also within the scope of protection of the present invention.
[0028] Wherein, the microbial preparation contains the xylacetobacter.
[0029] Beneficial effects:
[0030] After ARTP mutagenesis treatment and screening, the present invention is domesticated with an acclimation medium containing different xylose concentrations to obtain a xylose-efficient acetobacter. The strain is classified and named as Gluconacetobacter xylinus, with the strain number P2-A1. It has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 4, 2024, with the deposit number CGMCC No.32895. The xylose-acetobacter can efficiently utilize xylose to ferment and produce bacterial cellulose, improve the utilization efficiency of xylose obtained by hydrolysis of hemicellulose, a by-product of bamboo pulp production of refined cotton, and improve the recycling rate of production resources. Compared with the original strain, the original strain has almost no consumption of xylose, and the bacterial cellulose yield of xylose-acetobacter P2-A1 has increased by 93.59%. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.
[0032] Figure 1 This is the colony growth of the original strain ATCC 700178 on the screening plates at different mutagenesis time gradients.
[0033] Figure 2 This is a 24-well plate fermentation verification diagram of 30 single colony strains. The first row from left to right is A1 to A6; the second row from left to right is A7 to A12; the third row from left to right is A13 to A18; the fourth row from left to right is A19 to A24; the fifth row from left to right is A25 to A30.
[0034] Figure 3 This is a diagram showing the shake flask verification results of the mutagenic strains A1 and A27.
[0035] Figure 4 Plate images of the original strain ATCC 700178, strain A1, and strain P2-A1.
[0036] Figure 5 Microscopic images of the original strain ATCC 700178, strain A1 and strain P2-A1.
[0037] Figure 6 The yield of bacterial cellulose (BC) under different fermentation medium formulations. DETAILED DESCRIPTION
[0038] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.
[0039] In the following examples, the original strain was purchased from the American Type Culture Collection, and the strain collection number is ATCC 700178.
[0040] Example 1: ARTP mutagenesis of the original strain
[0041] (1) The original strain ATCC 700178 was activated and inoculated into a seed culture medium, and then 50 μL of cellulase was added and cultured at 30°C and 150 rpm for 2 days to obtain a seed solution;
[0042] Wherein, the formula of the seed culture medium is: 20g / L glucose, 10g / L yeast powder, and 20g / L peptone.
[0043] (2) Take 100 mL of the seed solution prepared in step (1), centrifuge at 4000 r / min for 10 min, wash the cells three times with sterile saline, and finally resuspend to 10 mL with sterile saline (concentrated 10 times);
[0044] (3) The concentrated bacterial solution was mixed with 10% v / v glycerol to obtain a premixed solution, and 10 μL was evenly coated on the center of the slide. Under room temperature and temperature pressure, the power supply was 120 W, the gas flow rate was 10.0 SLM, and the irradiation distance was 2 mm. The mutagenesis time was set to 0 to 200 s (45 s, 60 s, 75 s, 90 s, 105 s, 120 s, 135 s, 150 s, 180 s, 200 s) for ARTP mutagenesis treatment.
[0045] After the mutagenesis is completed, the slides are placed in sterile EP tubes containing 0.5 mL of physiological saline and shaken in an oscillator to elute to obtain a new premix. 200 μL of the new premix with different mutagenesis time gradients is taken and spread on the screening plate culture medium, and counted after culturing at 30°C for 3 to 5 days.
[0046] The formula of the screening plate culture medium is: 20 g / L xylose, 10 g / L yeast powder, 20 g / L peptone, and 20 g / L agar powder.
[0047] (4) Preliminary screening: Based on the growth of colonies on each gradient plate ( Figure 1 ), 50 single colonies with good growth, full colonies and large shapes were selected from the screening plate with a mutagenesis time of 135-200s and transferred to the screening plate for activation, and then 30 strains with faster growth and better results were selected and placed in a 24-well plate containing fermentation medium, and cultured at 30°C for 10 days for initial fermentation screening verification. Figure 2 These are photos of fermentation of single colony strains in 24-well plates. From left to right, they are A1 to A30, representing 1 to 30 selected strains, respectively.
[0048] The formula of the fermentation medium is as follows: 10 g / L glucose, 10 g / L xylose, 10 g / L yeast powder, 4 g / L ammonium sulfate, 20 g / L corn steep liquor, 1 g / L KH2PO4, 1 g / L K2HPO4·3H2O, 1 g / L MgSO4·7H2O, 1.2 g / L citric acid, 10 g / L trisodium citrate, 5 mL / L ethanol (added after seeding), the solvent is water, and the pH is adjusted to 5.5.
[0049] After the initial fermentation screening was completed, the wet weight, glucose consumption, and xylose consumption of each bacterial strain were measured and calculated. The results are shown in Table 1.
[0050] As can be seen from Table 1, after liquid chromatography determination, all the glucose in the fermentation medium was consumed. However, there were differences in the utilization of xylose. The mutant strain A27 had the best xylose utilization effect, consuming about 4.3g / L; followed by the mutant strain A1, consuming 3.68g / L; the mutant strains A6, A25, A26, and A28 had poor xylose utilization effects. Because the system was too small, the dry weight could not be accurately detected. According to the wet weight results, it was found that A1 and A27 not only had obvious xylose utilization effects, but also had the highest wet weight.
[0051] Table 1 Characterization of wet weight and residual xylose of different bacterial colony mutagenesis strains
[0052]
[0053] (5) Rescreening: After fully activating the A1 and A27 mutant strains and the original strain obtained by the initial screening of the 24-well plate, shake flask fermentation verification was performed. The specific steps are as follows: After the strains are activated, they are inoculated into 500 mL conical flasks containing 150 mL fermentation medium at a 10% v / v inoculation rate, and after static culture at 30°C for 10 days, the bacterial cellulose yield and the remaining xylose in the medium are measured.
[0054] The results are shown in Table 2 and Figure 3 As shown in the figure, it was determined that the consumption of xylose by A1 and A27 could reach more than 4 g / L, among which the yield of A1 was more significantly improved, while the original strain consumed almost no xylose. Therefore, the mutagenized strain A1 was selected for subsequent domestication.
[0055] Table 2 Shake flask fermentation verification
[0056] Strain number Remaining xylose after fermentation g / L Yield g / L original 9.72 2.93 A1 5.01 5.07 A27 5.62 4.13
[0057] Example 2: Domestication of mutagenic strains
[0058] 1. Pre-experimental fermentation verification
[0059] A1 was used as the starting strain. After the strain was activated, it was inoculated into 500 mL conical flasks containing 150 mL fermentation medium at a rate of 10% v / v. After static culture at 30°C for 10 days, the bacterial cellulose yield and the remaining xylose in the medium were determined.
[0060] Among them, the formula of the fermentation medium is: xylose 10-40g / L, yeast powder 10g / L, ammonium sulfate 4g / L, corn steep liquor 20g / L, KH2PO4 1g / L, K2HPO4·3H2O 1g / L, MgSO4·7H2O 2g / L, citric acid 1.2g / L, trisodium citrate 10g / L, ethanol 5mL / L (added after seeding), the solvent is water, and the pH is adjusted to 5.5.
[0061] Table 3 Preliminary fermentation verification
[0062] Initial xylose concentration g / L Remaining xylose after fermentation g / L Yield g / L 10 4.72 3.08 20 15.53 2.59 30 28.12 0.91 40 38.32 0.25
[0063] The results are shown in Table 3. Preliminary experiments with different xylose concentrations showed that when the xylose concentration was ≥30 g / L, cell growth stagnated and there was almost no yield. Therefore, 10 to 30 g / L xylose was selected as the concentration range for subsequent adaptive evolution.
[0064] 2. Domestication of mutagenic strains
[0065] Basic culture medium: xylose 10 g / L, yeast powder 5 g / L, ammonium sulfate 3 g / L, corn steep liquor 10 g / L, KH2PO4 0.5 g / L, K2HPO4·3H2O 0.5 g / L, MgSO4·7H2O 0.5 g / L, pH 5.0.
[0066] Acclimation medium: xylose 10-30 g / L, yeast powder 5 g / L, ammonium sulfate 3 g / L, corn steep liquor 10 g / L, KH2PO4 0.5 g / L, K2HPO4·3H2O 0.5 g / L, MgSO4·7H2O 0.5 g / L, pH 5.0.
[0067] Taking A1 as the starting strain, 30 independent droplet systems containing A1 mutagenic strain and basal culture medium were generated in the MMC (microbial micro-droplet culture system). During the 30-day acclimation period, 10-30 g / L of xylose was selected as the adaptive acclimation concentration range, and the 30 droplet systems were transferred to acclimation medium containing different xylose concentrations (10 g / L, 14 g / L, 18 g / L, 22 g / L, 26 g / L and 30 g / L) in sequence, OD=600 nm was set, OD detection cycle was 6 h, time was selected as the passage mode, passage was performed every 40 h, and the xylose concentration was increased to a new level every 3 passages, and continuous passage and acclimation were performed at a temperature of 30 ° C and static. Each concentration of acclimation medium is an independent acclimation cycle, and each cycle lasts for 5 days. During the acclimation process, the online detection function of the MMC system was used to monitor the growth of the strain. After the acclimation period, the growth data of all droplet systems were collected to analyze the effects of different xylose concentrations on strain growth. The results showed that at a xylose concentration of 30 g / L, the strains in droplets 1, 2, and 8 grew well.
[0068] After the No. 1, 2, and 8 droplet strains were activated on the screening plate, they were inoculated into 500 mL conical flasks containing 150 mL fermentation medium at a 10% v / v inoculation rate, and cultured in a shaking incubator at 150 rpm and 30°C for 10 days. The dry weight of the strains, bacterial cellulose production, and residual xylose in the culture medium were determined. The results are shown in Table 4. The xylose utilization rates of No. 1, 2, and 8 strains were improved to varying degrees. Among them, the xylose utilization capacity of No. 2 strain increased to 6 g / L after 10 days of fermentation, and the production reached 6.20 g / L. The No. 2 strain was named P2-A1.
[0069] Table 4 Shake flask fermentation verification
[0070] Strain number Residual xylose g / L Total dry weight g Yield g / L 1 4.18 0.85 5.67 2 3.92 0.93 6.20 8 4.46 0.74 4.93
[0071] Example 3: Characterization of growth characteristics of strain P2-A1
[0072] 1. P2-A1 morphology and structure characterization
[0073] The strain P2-A1, strain A1 and original strain ATCC 700178 were inoculated on the screening plates respectively and cultured at 30℃ for 2-3 days. The morphology of the three strains was observed. Figure 4 As shown, the single colony of P2-A1 is round, protruding, opaque, beige, and has a smooth and moist surface; compared with the original strain ATCC 700178, the colony is easier to pick up.
[0074] The three strains were further examined under a microscope and found that ( Figure 5), P2-A1 bacteria are short rod-shaped, with a single size of 1.22-1.49μm×0.69-0.91μm, mostly appearing in pairs or chains, while ATCC700178 and A1 are mostly single, with a few appearing in pairs.
[0075] 2. Collection at the Collection Center
[0076] The mutagenized and domesticated strain P2-A1 was sent to the preservation center for preservation, classified and named Gluconacetobacter xylinus, strain number P2-A1, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 4, 2024, with the preservation number CGMCC No.32895, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
[0077] Example 4: Improvement of fermentation medium formulation
[0078] After the mutagenesis and domestication strain P2-A1 was activated on the screening plate, it was inoculated into the seed culture medium in Example 1 and cultured at 30°C and 180r / min for 16 to 24 hours until obvious flocs appeared. Then, according to the inoculation amount of 10% v / v, it was inoculated into 500mL shaking bottles containing 150mL of different fermentation medium (control fermentation medium, improved fermentation medium 1, and improved fermentation medium 2), and cultured at 30°C for 4 to 14 days. 18 parallel samples were made for each fermentation medium, and 3 bottles were taken out on the 4th, 6th, 8th, 10th, 12th, and 14th days, respectively, to detect the yield of bacterial cellulose (BC).
[0079] The formula of the control fermentation medium is as follows: 10 g / L glucose, 10 g / L xylose, 10 g / L yeast powder, 4 g / L ammonium sulfate, 20 g / L corn steep liquor, 1 g / L KH2PO4, 1 g / L K2HPO4·3H2O, 1 g / L MgSO4·7H2O, 2 g / L citric acid, 1.2 g / L trisodium citrate, 10 g / L ethanol, 5 mL / L (added after seeding), the solvent is water, and the pH is adjusted to 5.5.
[0080] The formula of the improved fermentation medium 1 is: 20 g / L xylose, 5 g / L ammonium sulfate, 30 g / L corn steep liquor, 1 g / L KH2PO4, 1 g / L K2HPO4·3H2O, 2 g / L MgSO4·7H2O, 0.1 g / L zinc sulfate, 5 g / L malic acid, the solvent is water, and the pH is adjusted to 5.5.
[0081] The formula of the improved fermentation medium 2 is: molasses (calculated by reducing sugar concentration) 10g / L, xylose 10g / L, ammonium sulfate 5g / L, corn steep liquor 30g / L, KH2PO4 1g / L, K2HPO4·3H2O 1g / L, MgSO4·7H2O 2g / L, zinc sulfate 0.1g / L, malic acid 5g / L, the solvent is water, and the pH is adjusted to 5.5.
[0082] The results are as follows Figure 6 As shown in the figure, compared with the control fermentation medium, the fermentation rate of the improved fermentation medium 1 is slower, but the final yield is not significantly different. After fermentation with the improved fermentation medium 2, the fermentation rate is significantly improved, and the yield can reach 6g / L after 8 days of fermentation. Therefore, the improved fermentation medium 2 is selected as the final fermentation medium.
[0083] Example 5: Shake flask dynamic culture
[0084] The original strain and the induced domesticated strain P2-A1 were used as target strains for dynamic culture in shake flasks. The plate activation and seed culture process were the same as in Example 4. The seed liquids of the two strains were inoculated into the fermentation medium (improved fermentation medium 2) at an inoculation rate of 10% v / v, respectively, and cultured for about 2.5 days at 30°C and a rotation speed of 160rpm. The results showed that the xylose consumption concentration of the induced domesticated strain P2-A1 reached 4.23g / L and the yield reached 3.02g / L; while the original strain had almost no xylose consumption, with a yield of 1.56g / L, and the yield of the induced domesticated strain P2-A1 increased by 93.59%.
[0085] The present invention provides a strain of xylose acetobacter and its application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.
Claims
1. A strain of Gluconacetobacter xylinus, classified and named Gluconacetobacter xylinus, with strain number P2-A1, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 4, 2024, with the deposit number CGMCC No.32895.
2. The xylacetobacter according to claim 1, characterized in that The xyloseacetobacter is obtained by screening through ARTP mutagenesis treatment and then domesticating in a domestication medium containing different xylose concentrations.
3. Use of the xyloseacetobacter described in claim 1 in fermentation to produce bacterial cellulose.
4. The use according to claim 3, characterized in that: The xyloseacetobacter produces bacterial cellulose by fermenting and consuming any one or a combination of xylose, molasses and glucose.
5. A method for producing bacterial cellulose by fermenting Acetobacter xylosus according to claim 1 or 2, characterized in that: The seed liquid of xylinacetobacillus according to claim 1 or 2 is inoculated into a fermentation medium, and bacterial cellulose is produced by static fermentation or dynamic fermentation.
6. The method according to claim 5, characterized in that The inoculation has an inoculation amount of 10-20% v / v.
7. The method according to claim 5, characterized in that The formula of the fermentation medium is: molasses 10-20 g / L, xylose 5-20 g / L, ammonium sulfate 2-5 g / L, corn steep liquor 5-40 g / L, KH2PO4 0.5-3 g / L, K2HPO4·3H2O 0.5-3 g / L, MgSO4·7H2O 0.5-5 g / L, zinc sulfate 0.01-0.2 g / L, malic acid 1-10 g / L, the solvent is water, and the pH is adjusted to 5.5; or, Xylose 15-40 g / L, ammonium sulfate 2-5 g / L, corn syrup 5-40 g / L, KH2PO4 0.5-3 g / L, K2HPO4·3H2O 0.5-3 g / L, MgSO4·7H2O 2 g / L, zinc sulfate 0.01-0.2 g / L, malic acid 1-10 g / L, the solvent is water, and the pH is adjusted to 5.0-5.
5.
8. The method according to claim 5, characterized in that The static fermentation is carried out under the following conditions: static culture at 26-32° C. for 4-14 days.
9. The method according to claim 5, characterized in that The dynamic fermentation conditions are as follows: culturing at 26-32° C. and 120-180 rpm for 2-5 days.
10. A microbial preparation for producing bacterial cellulose, characterized in that: The microbial preparation contains the xylacetobacter described in claim 1.