A kind of Marinomonas pontica and its application

Abstract

The present invention discloses a Luteimonas aestuarii and its application. The preservation number of the Luteimonas aestuarii R1 is CGMCC No. 31499. Luteimonas aestuarii R1 can efficiently degrade starch in the condensate water of the space station under the space station environment, assisting in the recycling of the condensate water, and providing a technical basis for the further research and optimization of the space station environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Luteimonas aestuarii and its application. Background Art

[0002] Since the space station is far from the earth and material transportation is difficult, recycling the waste generated by astronauts during their production and life in the space station is an effective method to solve the large amount of waste in the space station while ensuring the daily needs of astronauts. The water resource recycling in the space station mainly involves the condensation of atmospheric moisture and the purification and recycling of water such as urine and domestic wastewater. Among them, the treatment of urine and domestic wastewater and the purification and recycling of water 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 on-orbit survival of astronauts. Through the environmental control and life support subsystem, the recycling of substances such as oxygen and water can be realized. Currently, more than 95% of the water used by on-orbit astronauts is obtained through regeneration means. For example, the patent with the publication number CN105130076B discloses a method for recycling and treating source-separated urine in a space station that makes full use of the characteristics of the space environment, using the strong sunlight and radiation rays in the space station to deodorize, disinfect, and degrade pollutants in urine. The patent with the publication number CN117228768A uses membrane distillation technology, coupling the high water recovery rate of the distillation method and the mild operating conditions of the membrane technology, and designs a highly efficient, compact, and low-power space station waste liquid purification and recovery device. The patent with the publication number CN112321051A organically integrates membrane distillation, reverse osmosis, evaporation bags, and supercritical water oxidation technology to achieve the comprehensive treatment and reuse of space station wastewater.

[0004] However, when astronauts consume food daily, food residues will be produced. These residues will dissolve into the sweat droplets volatilized by astronauts and then enter the condensed water through the water treatment system, resulting in the presence of starch components in the condensed water. Due to the high temperature at the outlet of the condensed water, the amylase produced by general microorganisms under high temperature and the space station environment cannot efficiently degrade starch, and there is currently no report on the research of starch treatment in space station condensed water. Summary of the Invention

[0005] To solve the above technical problems and achieve the full recycling and utilization of space station condensed water, the present invention provides the following technical solutions.

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

[0007] Tide xanthomonas R1 has the characteristics of high temperature resistance and tolerance to the space station environment, and can secrete amylase to degrade starch in the space station wastewater.

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

[0009] Preferably, the viable count of tide xanthomonas R1 in the bacterial agent ≥ 4.65×10 8 cfu / mL.

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

[0011] In a fourth aspect, the present invention provides a preparation method of the bacterial agent described in the second aspect, comprising the following steps:

[0012] Activating the strain of tide xanthomonas R1, then inoculating it into an LB liquid medium, and obtaining a culture of tide xanthomonas R1 through shaking culture, and preparing a liquid bacterial agent or a solid bacterial agent containing tide xanthomonas R1 by adjusting the viable count of effective viable bacteria of tide xanthomonas R1.

[0013] Preferably, the bacterial agent further comprises excipients, such as adsorbents and strain protectants.

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

[0015] Preferably, the time for shaking culture of tide xanthomonas R1 is 15 - 20 h, for example: 15 h, 16 h, 17 h, 18 h, 19 h, 20 h.

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

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

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

[0019] Preferably, the concentration of the starch ≤ 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 application of the Luteimonas aestuarii R1 described in the first aspect or the bacterial agent described in the second aspect in the preparation of starch degradation-related products.

[0021] Advantages of the present invention:

[0022] The Luteimonas aestuarii R1 of the present invention can efficiently degrade starch in the condensate water of the space station under the space station environment, assisting in the recycling of condensate water, and providing a technical basis for the further research and optimization of the space station environment. Description of the drawings

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

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

[0025] Figure 3 Shown are the comparison results of the starch degradation abilities of different strains.

[0026] Deposit of cultures for patent procedures:

[0027] The Luteimonas aestuarii R1 of the present invention;

[0028] Deposit date: July 31, 2024;

[0029] Depositary institution: China General Microbiological Culture Collection Center (CGMCC);

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

[0031] Deposit number: CGMCC No. 31499;

[0032] Taxonomic name: Luteimonas aestuarii. Detailed implementation manners

[0033] The technical solutions of the present invention will be further described below in conjunction with examples and drawings. The advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the examples are merely 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 meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0035] Isolation and Identification of Luteimonas aestuarii Strain R1 in Example 1

[0036] 1.1 Strain Isolation

[0037] The sample source was the condensate water of the space station. Different sampling carriers were selected to sample at the designated positions. After returning to the ground, first, contact cultivation was carried out on the surface of the sampling carrier with LB solid medium contact discs, and then the sampling carrier was shaken and eluted with PBS solution and centrifuged. The centrifuged bacterial solution was used for spreading cultivation on LB solid medium. The two culture dishes were placed in an incubator at 36 °C for 48 h, and single colonies with different morphologies were picked for purification and identification.

[0038] Strains OR243819, OR243821, OR243822, OR243825, and R1 were initially isolated. The 16S rRNA of the five strains of bacteria was compared for similarity on the NCBI website. Among them, the 16S rRNA of strain R1 had a low similarity on the NCBI website. Therefore, strain R1 was sent for whole-genome sequencing, and it was predicted that strain R1 might have the effect of degrading starch. After the iodine solution-starch medium experiment, it was found that a clear zone appeared around the colony.

[0039]

[0040]

[0041] 1.2 Strain Identification

[0042] Species identification was carried out based on the 16S RNA gene of strain R1 (see Table 1). After identification, the taxonomic name of strain R1 was 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] Construct a phylogenetic tree 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 condensate

[0080] Dissolve the reagents in Table 2 in deionized water to prepare simulated condensate (hereinafter referred to as condensate).

[0081] Table 2 Preparation of condensate

[0082]

[0083]

[0084] 2.2 Preparation of bacterial liquid

[0085] Respectively pipette 10 μL of the bacterial liquid of strains OR243819, OR243821, OR243822, OR243825 and R1 from the glycerol tubes and inoculate them into 7.5 mL of sterile LB liquid medium, and shake and culture them in a shaker incubator at 36 °C for 17 h. Use sterile LB liquid medium as the control group, and the OD values of each strain measured at an absorbance of 600 nm are 0.434, 0.429, 0.342, 0.366 and 0.351 respectively.

[0086] 2.3 Extraction of crude enzyme solution

[0087] Dispense the bacterial liquid into 1.5 mL centrifuge tubes, centrifuge at 3500 rpm for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube to obtain the crude enzyme solution.

[0088] 2.4 Strain starch degradation ability test experiment

[0089] 1% starch-condensate: Weigh 1 g of soluble starch and dissolve it in 100 mL of the above-mentioned condensate.

[0090] Standard curve (as Figure 2 shown): From a concentration of 0.7% to 0% concentration, prepare a starch-condensate for each 0.1% concentration interval. Pipette 1 mL of the solution of each concentration into a 1.5 mL centrifuge tube and place it in a 45 °C metal bath together with the experimental group.

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

[0092] Control group: To eliminate the influence on the OD value of the experimental group after adding the crude enzyme solution, pipette 1 mL of condensate water into a 1.5 mL centrifuge tube, add 25 μL of crude enzyme solution to it, and place it in a 45°C metal bath.

[0093] Absorbance detection: For each concentration of the standard curve, the 5 experimental groups and the control group are all set with three parallel replicates during detection. Use a 96 - well plate as the container, and respectively load 50 μL of the reacted liquid of each experimental group and the control group into each well of the 96 - well plate. Finally, uniformly add 150 μL of iodine solution to the wells, and detect the absorbance of the mixed liquid at 660 nm. Take the starch concentration in the condensate water as the abscissa and OD 660 as the ordinate to draw a standard curve (see Figure 2 ). Calculate the starch concentration after degradation, the absolute amount of starch degradation, and the starch degradation rate of each group according to the following formulas, and the results are shown in Table 3.

[0094] Starch concentration after degradation (%) = (OD value of the 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 results of starch degradation abilities of different strains

[0098]

[0099] As can be seen from Table 3 and Figure 3 it can be seen that strain R1 degrades 1.22 mg of starch within 5 minutes. Compared with the other four strains, the starch degradation rate of strain R1 is greater, and its starch degradation ability is significantly higher than that of other strains.

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

[0101] Determine the starch degradation ability of strain R1 according to the method of Example 2, with the differences as follows:

[0102] 1. When strain R1 was cultured for 17 h during the preparation of the bacterial solution, the effective viable count of R1 in the bacterial solution was detected to be 4.65×10 8 cfu / mL, and the OD value measured at an absorbance of 600 nm was 0.351.

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

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

[0105] Table 4 Test results of starch degradation in each group

[0106]

[0107]

[0108] Note: Groups 1 - 9 respectively represent experimental groups 1 - 9, and CK represents the 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 min, 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 situation 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 have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various 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 Thalassomonas loyana, characterized in that: The Luteimonas aestuarii R1 is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No. 31499 and the deposit date of July 31, 2024.

2. A bacterial agent, characterized in that: The bacterial agent contains the Luteimonas aestuarii described in claim 1.

3. The microbial agent according to claim 2, characterized in that: The viable count of *Luteimonas aestuarii* R1 in the microbial agent ≥ 4.65×10 8 cfu / mL.

4. A composition, characterized in that: The composition contains the Luteimonas aestuarii described in claim 1.

5. Use of the Luteimonas aestuarii described in claim 1 or the bacterial agent described in any one of claims 2-3 in degrading starch.

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

7. The application according to claim 6, wherein: The wastewater of the space station is the condensate water of the space station.

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

9. Use of the Luteimonas aestuarii described in claim 1 or the bacterial agent described in any one of claims 2-3 in preparing starch degradation-related products.

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