Tidal vine xanthomonas sp. And application thereof

Abstract

The invention discloses a Luteomonas solanacearum and application of the Luteomonas solanacearum. The preservation number of the Luteomonas solanacearum R1 is CGMCC (China General Microbiological Culture Collection Center) No.31499. The invention also discloses a preparation method of the Luteomonas solanacearum and application of the Luteomonas solanacearum. The xanthomonas tidal fusarium R1 can efficiently degrade starch in space station condensate water in a space station environment, the condensate water can be recycled in an auxiliary manner, and a technical basis is provided for further research and optimization of the space station environment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to tidal Xanthomonas and applications thereof. Background Art

[0002] Since the space station is far away from the earth and the transportation of materials is difficult, recycling the waste generated by the production and life of astronauts in the space station is an effective way to solve the problem of large amounts of waste in the space station while ensuring the daily needs of astronauts. The water resource recovery of the space station mainly involves the condensation of atmospheric water and the purification and recycling of urine, domestic wastewater and other water. Among them, the treatment and recycling of urine and domestic wastewater are the most critical.

[0003] The environmental control and life support subsystem can create an environment similar to that on the ground in the spacecraft to ensure the survival of astronauts in orbit. The environmental control and life support subsystem can realize the recycling of oxygen, water and other substances. At present, more than 95% of the water used by astronauts in orbit is obtained through regeneration means. For example, the patent with publication number CN105130076B discloses a recovery and treatment method for urine separated from space station sources that fully utilizes the characteristics of the space environment, and uses the strong sunlight and radiation rays in the space station to deodorize, disinfect and degrade pollutants in urine. The patent with publication number CN117228768A uses membrane distillation technology, couples the high water recovery rate of distillation method with the mild operating conditions of membrane technology, and designs an efficient and low-power space station waste liquid purification and recovery device. The patent with publication number CN112321051A organically integrates membrane distillation, reverse osmosis, evaporation bag, and supercritical water oxidation technology to achieve comprehensive treatment and reuse of space station wastewater.

[0004] However, when astronauts eat food on a daily basis, they produce food residues, which dissolve into the evaporative sweat droplets of the astronauts and then enter the condensed water through the water treatment system, resulting in starch components in the condensed water. Due to the high temperature at the condensed water outlet, the amylase produced by general microorganisms under high temperature and space station environment cannot efficiently degrade starch. At present, there are no reports on the treatment of starch in the condensed water of the space station. Summary of the invention

[0005] In order to solve the above-mentioned technical problems and realize the full recycling and utilization of condensed water in the space station, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a tidal Luteimonas R1, wherein the tidal Luteimonas R1 is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC No.31499 and a deposit date of July 31, 2024.

[0007] Tidal Xanthomonas R1 is resistant to high temperatures and the space station environment, and can secrete amylase to degrade starch in space station wastewater.

[0008] In a second aspect, the present invention provides a bacterial agent, which comprises the tidal Xanthomonas R1 described in the first aspect.

[0009] Preferably, the number of viable bacteria of Xanthomonas tidalis R1 in the bacterial agent is ≥4.65×10 8 cfu / mL.

[0010] In a third aspect, the present invention provides a composition comprising the bacterial agent described in the second aspect.

[0011] In a fourth aspect, the present invention provides a method for preparing the bacterial agent according to the second aspect, comprising the following steps:

[0012] The method comprises activating the tidal Xanthomonas R1 strain, then inoculating it into LB liquid culture medium, obtaining a culture of tidal Xanthomonas R1 through shaking culture, and obtaining a liquid bacterial agent or solid bacterial agent containing tidal Xanthomonas R1 through adjusting the effective viable bacterial count of tidal Xanthomonas R1.

[0013] Preferably, the bacterial agent also includes auxiliary materials, such as adsorbents and bacterial strain protectants.

[0014] Preferably, the temperature of the shaking culture of tidal Xanthomonas R1 is 35-40°C, for example: 35°C, 36°C, 37°C, 38°C, 39°C, 40°C.

[0015] Preferably, the shaking culture time of tidal Xanthomonas R1 is 15-20 hours, for example: 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours.

[0016] In a fourth aspect, the present invention provides use of the tidal Xanthomonas R1 described in the first aspect or the bacterial agent described in the second aspect in degrading starch.

[0017] Preferably, the starch is starch in space station wastewater.

[0018] Preferably, the wastewater is space station condensate water.

[0019] Preferably, the concentration of the starch is ≤1.0%, for example: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%.

[0020] In a fifth aspect, the present invention provides the use of the tidal Xanthomonas R1 described in the first aspect or the bacterial agent described in the second aspect in the preparation of starch degradation related products.

[0021] Beneficial effects of the present invention:

[0022] The tidal Xanthomonas R1 of the present invention can efficiently degrade starch in the condensed water of the space station under the space station environment, assist in the recycling of the condensed water, and provide a technical basis for further research and optimization of the space station environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shown is the phylogenetic tree of strain R1;

[0024] Figure 2 Shown is the standard curve for amylase activity assay;

[0025] Figure 3 Shown is the comparison of starch degradation ability of different strains.

[0026] Culture deposits for patent procedures:

[0027] The tidal Xanthomonas R1 of the present invention;

[0028] Deposit date: July 31, 2024;

[0029] Depository: China General Microbiology Center (CGMCC);

[0030] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code: 100101;

[0031] Deposit number: CGMCC No.31499;

[0032] Classification name: Luteimonas aestuarii. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments are only exemplary and do not limit the scope of the present invention.

[0034] It should be noted that the experimental methods used in the following examples are all conventional methods in the art unless otherwise specified. Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention relates.

[0035] Example 1 Isolation and Identification of Xanthomonas tidalis R1

[0036] 1.1 Strain Isolation

[0037] The source of the sample is condensed water from the space station. Different sampling carriers are selected for sampling at designated locations. After returning to the ground, the LB solid culture medium contact plate is first used for contact culture on the surface of the sampling carrier, and then the sampling carrier is shaken, eluted and centrifuged with PBS solution. The bacterial liquid after centrifugation is used for LB solid culture medium coating culture. The two culture dishes are placed in a 36°C incubator for 48 hours, and single colonies with different morphologies are picked for purification and identification.

[0038] Strains OR243819, OR243821, OR243822, OR243825 and R1 were initially isolated, and the 16S rRNA of Pentaphyllum was compared on the NCBI website. The 16S rRNA of R1 had a low similarity on the NCBI website, so the whole genome of R1 was tested, and it was predicted that R1 might have the effect of degrading starch. After the iodine solution-starch culture medium experiment, it was found that transparent circles did appear around the colonies.

[0039]

[0040]

[0041] 1.2 Strain identification

[0042] The strain R1 was identified based on its 16s RNA gene (see Table 1), and the strain R1 was named Luteimonas aestuarii.

[0043] Table 1

[0044]

[0045] The 16s RNA gene sequence of strain R1 is as follows:

[0046] AGAGTTTGATCCTGGCTCAGAGTGAACGCTGGCGGCAGGCCTAACAC

[0047] ATGCAAGTCGAACGGCAGCACAGGGGAGCTTGCTCCCTGGGTGGCGAGTG

[0048] GCGGACGGGTGAGGAATACATCGGAATCTGCCCTATCGTGGGGGATAACGT

[0049] AGGGAAACTTACGCTAATACCGCATAAGACCTTCGGGTGAAAGAGGGGGA

[0050] TCGCAAGACCTCTCGCGATTGGATGAGCCGATGTCGGATTAGCTAGTTGGC

[0051] GGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGA

[0052] TCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCA

[0053] GTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGG

[0054] GTGAAGAAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGAAAGAAAACCT

[0055] TCCGGTTAATACCCGGAGGGAATGACGGTACCCAAAGAATAAGCACCGGCT

[0056] AACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGG

[0057] AATTACTGGGCGTAAAGCGTGCGTAGGTGGTTCGTTAAGTCTGATGTGAAA

[0058] GCCCTGGGCTCAACCTGGGAATTGCATTGGATACTGGCGGGCTAGAGTGCG

[0059] GTAGAGGGTAGTGGAATTCCTGGTGTAGCAGTGAAATGCGTAGAGATCAGG

[0060] AGGAACATCCGTGGCGAAGGCGACTGCCTGGACCAGCACTGACACTGAGG

[0061] CACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCC

[0062] CTAAACGATGCGAACTGGATGTTGGGTTCAACTAGGAACTCAGTATCGAAG

[0063] CTAACGCGTTAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACT

[0064] CAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATT

[0065] CGATGCAACGCGCAGAACCTTACCTGGCCTTGACATCCACGGAACTTTCCA

[0066] GAGATGGATTGGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGT

[0067] CGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC

[0068] CCTTGTCCTTAGTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACCGCC

[0069] GGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTT

[0070] ACGGCCAGGGCTACACACGTACTACAATGGGAAGGACAGAGGGCTGCAAA

[0071] CCCGCGAGGGCAAGCCAATCCCAGAAACCTTCTCTCAGTCCGGATCGGAG

[0072] TCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGCAGATCAGCA

[0073] TTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA

[0074] TGGGAGTTTGTTGCACCAGAAGCAGGTAGCCTAACCGCAAGGAGGGCGCT

[0075] TGCCACGGTGTGGCCGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAT

[0076] CGGAAGGTGCGGCTGGATCACCT(SEQ ID NO:1)

[0077] The phylogenetic tree was constructed based on the 16s RNA gene of strain R1 (see Figure 1 )

[0078] Example 2 Determination of starch degradation ability of different strains

[0079] 2.1 Preparation of simulated condensed water

[0080] The reagents in Table 2 were dissolved in deionized water to prepare simulated condensed water (hereinafter referred to as condensed water).

[0081] Table 2 Preparation of condensed water

[0082]

[0083]

[0084] 2.2 Preparation of bacterial culture

[0085] 10 μL of bacterial solution of strains OR243819, OR243821, OR243822, OR243825 and R1 were taken from the glycerol tubes and inoculated into 7.5 mL of sterile LB liquid medium, and cultured in a shaking incubator at 36°C for 17 hours. The sterile LB liquid medium was used as the control group, and the OD values ​​of each strain were measured at 600 nm absorbance to be 0.434, 0.429, 0.342, 0.366 and 0.351, respectively.

[0086] 2.3 Extraction of crude enzyme solution

[0087] The bacterial solution was dispensed into 1.5 mL centrifuge tubes, centrifuged at 3500 rpm for 10 min, and the supernatant was transferred to a new 1.5 mL centrifuge tube to obtain a crude enzyme solution.

[0088] 2.4 Experimental study on the starch degradation ability of strains

[0089] 1% starch-condensed water: weigh 1 g of soluble starch and dissolve it in 100 mL of the above condensed water.

[0090] Standard curve (such as Figure 2 As shown): from 0.7% concentration to 0% concentration, starch-condensed water was prepared at every 0.1% concentration, and 1 mL of solution of each concentration was pipetted into a 1.5 mL centrifuge tube and placed in a 45°C metal bath together with the experimental group.

[0091] Experimental group: Take 5 1.5mL centrifuge tubes, and pipette 1mL of 0.7% starch-condensed water into each 1.5mL centrifuge tube. Then place the 5 1.5mL centrifuge tubes in a 45℃ metal bath for insulation, and add 25μL of crude enzyme solution to each centrifuge tube after 5 minutes to form 5 experimental groups.

[0092] Control group: In order to eliminate the influence of crude enzyme solution on the OD value of the experimental group, 1 mL of condensed water was taken into a 1.5 mL centrifuge tube, and 25 μL of crude enzyme solution was added thereto, and the tube was placed in a 45°C metal bath.

[0093] Absorbance detection: Three parallel replicates were set for each concentration of the standard curve, the five experimental groups, and the control group. A 96-well plate was used as a container. 50 μL of the reaction liquid of each experimental group and the control group was loaded into each well of the 96-well plate. Finally, 150 μL of iodine solution was added to the wells to detect the absorbance of the mixed liquid at 660 nm. The starch concentration in the condensed water was used as the horizontal axis, and OD 660 Draw a standard curve for the ordinate (see Figure 2 The starch concentration after degradation, the absolute amount of starch degradation and the starch degradation rate of each group were calculated according to the following formula. The results are shown in Table 3.

[0094] Starch concentration after degradation (%) = (OD value of experimental group - 0.0833) ÷ 536.54 × 100% (Formula 1)

[0095] Absolute amount of starch degradation (mg) = (initial concentration - concentration after degradation) × 1000 (Formula 2)

[0096] Starch degradation rate (mg·mL -1 ·min -1 ) = absolute amount of starch degradation × 1 (mg·L -1 )÷reaction time (min) (Formula 3)

[0097] Table 3 Comparison of starch degradation ability of different strains

[0098]

[0099] From Table 3 and Figure 3 It can be seen that strain R1 degraded 1.22 mg of starch within 5 minutes. Compared with the other four strains, R1 had a higher starch degradation rate and its starch degradation ability was significantly higher than that of other strains.

[0100] Example 3 Determination of starch degradation ability of strain R1

[0101] The starch degradation ability of strain R1 was determined according to the method of Example 2, with the following differences:

[0102] 1. During the preparation of bacterial solution, when strain R1 was cultured for 17 hours, the number of effective live bacteria of R1 in the bacterial solution was detected to be 4.65×10 8 cfu / mL, and the OD value of the bacterial solution was measured at 600nm absorbance to be 0.351.

[0103] 2. Experimental groups in the test of starch degradation ability of strain R1: Take 9 1.5mL centrifuge tubes, and draw 1mL of 0.7% starch-condensed water into each 1.5mL centrifuge tube. Then place the 9 1.5mL centrifuge tubes in a 45℃ metal bath for insulation, and add 25μL crude enzyme solution to one of the tubes every 5min, forming 9 experimental groups with reaction times of 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, and 45min, respectively.

[0104] The results of starch concentration after degradation, absolute amount of starch degradation and starch degradation rate of each group are shown in Table 4.

[0105] Table 4 Starch degradation test results of each group

[0106]

[0107]

[0108] Note: Groups 1-9 represent experimental groups 1-9, CK represents control group

[0109] The results in Table 4 show that when the reaction temperature is 45°C and the substrate concentration is 0.7%, in the first 5 minutes, the crude enzyme solution of strain R1 can degrade 1.22 mg / mL of starch, and the degradation rate is the highest. As time increases, the starch degradation rate gradually decreases until it stabilizes. The starch degradation of strain R1 shows that strain R1 can effectively degrade starch and has excellent starch degradation ability.

[0110] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A tidal Xanthomonas sp., characterized in that: The Luteimonas aestuarii R1 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 31499 and the deposit date July 31, 2024.

2. A bacterial agent, characterized in that: The bacterial agent comprises the tidal Xanthomonas according to claim 1.

3. The bacterial agent according to claim 2, characterized in that: The number of viable bacteria of Xanthomonas tidalis R1 in the bacterial agent is ≥4.65×10 8 cfu / mL.

4. A composition, characterized in that: The composition comprises the tidal Xanthomonas according to claim 1.

5. Use of the tidal Xanthomonas according to claim 1 or the bacterial agent according to any one of claims 2-3 in degrading starch.

6. The use according to claim 5, characterized in that: The starch is starch in space station wastewater.

7. The use according to claim 6, characterized in that: The space station wastewater is space station condensate water.

8. The use according to claim 6, characterized in that: The starch concentration in the space station wastewater is ≤1.0%.

9. Use of the tidal Xanthomonas according to claim 1 or the bacterial agent according to any one of claims 2-3 in the preparation of starch degradation related products.

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