Texassothrix 8 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a Texassothrix sp. 8 and application of the Texassothrix sp. 8. According to the invention, a novel chemoautotrophic carbon sequestration bacterium, namely Saccharothrix texassis 8, is found through research, and the preservation number of the Saccharothrix texassis 8 is CGMCC (China General Microbiological Culture Collection Center) No.33977. CO2 in the atmosphere can be fixed, inorganic carbon can be converted into organic carbon, the SOC content of bare desert sand can be increased within a short time, and the compound can be used as a pioneer species to improve desert soil and has important significance on desert ecological restoration.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a Saccharothrix texas 8 strain and applications thereof. Background Art

[0002] Desert ecosystems, characterized by drought stress and nutrient-deficient habitats, significantly limit the input of plant-derived organic carbon, resulting in low soil organic carbon (SOC) reserves. As a key component of desert ecosystems, biological soil crusts (BSCs) can effectively increase surface SOC content. BSCs are complexes formed by autotrophic organisms such as algae, mosses, and carbon-fixing microorganisms, and heterotrophic organisms such as bacteria and fungi, cemented to surface soil particles through secretions such as mycelium, rhizophores, and polysaccharides. Based on species composition, BSCs can be divided into algal crusts, lichen crusts, and moss crusts. Studies have found that, in addition to algae and mosses, BSCs also harbor abundant photoautotrophic and chemoautotrophic carbon-fixing microbial communities with considerable carbon sequestration potential. Compared to photoautotrophic carbon-fixing bacteria, chemoautotrophic carbon-fixing bacteria are not restricted by light and have lower requirements for water and soil substrates. They can act as pioneer species, converting inorganic carbon into organic carbon, thereby improving desert soils and providing the prerequisites for later algal colonization and crust formation. Therefore, isolating and culturing chemoautotrophic strains in BSCs and investigating their effects on desert soil improvement is of great significance to desert ecological restoration. Summary of the Invention

[0003] One of the purposes of the present invention is to provide a new bacterial strain capable of converting inorganic carbon into organic carbon and its application.

[0004] The present invention provides a strain of Saccharothrix texas ( Saccharothrix texasensis ) 8, its deposit number is CGMCC No.33977.

[0005] Saccharothrix texas of the present invention Saccharothrix texasensis 8 was deposited on March 26, 2025, at the General Microbiology Center of China Culture Collection Administration (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China), and was classified as Saccharothrix texas Saccharothrix texasensis , the deposit number is CGMCC No. 33977.

[0006] The Saccharothrix texas 8 of the present invention is a chemoautotrophic bacterium with carbon fixation ability. It is not restricted by light and can grow without relying on any exogenous organic matter. It has low requirements for water and soil matrix. It can increase the SOC content of bare sand in desert areas in a short period of time. It can act as a pioneer species to convert inorganic carbon into organic carbon, thereby improving desert soil and providing prerequisites for the later colonization of algae and the succession of crusts.

[0007] The present invention also provides a bacterial agent containing the above-mentioned Saccharothrix texas ( Saccharothrix texasensis )8.

[0008] The bacterial agent of the present invention can be a liquid bacterial agent or a solid bacterial agent, and can include other strains with the same or different functions.

[0009] The present invention also provides the above-mentioned Saccharothrix texas ( Saccharothrix texasensis )8 or the use of bacterial agents in converting inorganic carbon into organic carbon.

[0010] In the application of the present invention, the inorganic carbon is carbon dioxide and / or carbonate.

[0011] The present invention also provides the above-mentioned Saccharothrix texas ( Saccharothrix texasensis )8 or the application of bacterial agents in improving soil organic carbon.

[0012] In the application of the present invention, the soil comes from a desert area.

[0013] The present invention also provides the above-mentioned Saccharothrix texas ( Saccharothrix texasensis )8 or the application of bacterial agents in enhancing the activity of carbon fixation enzymes.

[0014] In the application of the present invention, the carbon-fixing enzyme is ATP-citrate lyase and / or ribulose-1,5-bisphosphate carboxylase.

[0015] The present invention also provides a carbon fixer, which includes the above-mentioned Saccharothrix texas ( Saccharothrix texasensis ) 8 or bacterial agents.

[0016] The carbon fixer of the present invention can fix CO2 in the atmosphere.

[0017] The beneficial effects of the present invention are at least: The present invention provides a new chemoautotrophic carbon-fixing bacterium, which can fix CO2 in the atmosphere, convert inorganic carbon into organic carbon, and increase the SOC content of desert bare sand in a short period of time. It can serve as a pioneer species to improve desert soil and is of great significance to desert ecological restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1It is in the form of colonies.

[0019] Figure 2 is a phylogenetic tree.

[0020] Figure 3 The carbon fixation enzyme activity test results of the strain.

[0021] Figure 4 The TOC content test results of the bacterial liquid are shown in Figure 2.

[0022] Figure 5 These are the test results for SOC content in sterilized bare sand and non-sterilized bare sand after cultivation.

[0023] Figure 6 The results of strain growth test are shown in Figure 2. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available or prepared according to conventional methods in the art.

[0026] Example 1 Sample collection Samples for this experiment were collected in July 2023 at the National Positioning Observation and Research Station for the Maowusu Sandy Land Ecosystem in Yanchi, Ningxia. Within the station, vegetation and BSCs are interspersed, with BSC coverage reaching 80% in the intervegetative spaces. To isolate as many carbon-fixing strains as possible, areas with abundant algal, lichen, and moss crusts were selected. Ten 1 m × 1 m plots were set up in an S-shaped pattern within each BSC-covered area, with each plot spaced at least 50 m apart. Within each plot, soil samples from the crust layer were collected using a five-point sampling method. Crust samples from five locations within the same plot were combined into a single sample, for a total of 30 samples. Sampling equipment was disinfected before sampling. After sampling, samples were stored at 4°C and promptly brought back to the laboratory. After sieving through a 2 mm sieve, subsequent isolation experiments were immediately performed.

[0027] Test culture medium The carbon-free inorganic culture medium (per liter) contains: 1 g MnSO₄, 0.5 g Na₂HPO₄, 0.5 g KH₂PO₄, 1 g MgSO₄, 0.2 g CaCl₂, 1 g NaHCO₃, 0.5 g NH₄Cl, 1 g KNO₃, 0.4 g NaCl, and 2 mL of trace element solution. The trace element solution (per liter) contains: 0.3 g FeCl₃, 0.3 g FeSO₄·7H₂O, 0.15 g MnSO₄·H₂O, 0.14 g ZnSO₄, and 0.2 g CoCl₂. The trace elements are sterilized by passing through a 0.22 μm filter. The pH is 7.5, and 20 g agar (not added to the liquid culture medium) is added. Sterilize at 121°C for 20 min. This medium is used for screening carbon-fixing strains and determining carbon-fixing enzyme activity and carbon content in the culture solution.

[0028] Each liter of Luria-Bertani (LB) medium includes: 5 g yeast extract, 10 g NaCl, 10 g tryptone, pH 7.2-7.4, 20 g agar (not added to liquid culture medium), sterilized at 121°C for 15 min, and used for bare sand culture experiments and strain growth determination.

[0029] Isolation, purification and subculture of carbon-fixing strains Weigh 10 g of sample and add it to a triangular flask filled with 90 mL of sterile water. Add a small amount of sterilized glass beads and shake it at 30°C and 150 r / min for about 20 min to make a suspension. Perform gradient dilution. -3 Dilute the concentration, aspirate 100 μL of the sample suspension, and spread it onto a carbon-free, inorganic solid medium. Set aside an equal amount of sterile water as a control, with three replicates per group. Place the plate in a dark incubator at 30°C and incubate upside down for 5 days. Based on the morphology of the colonies on the plate, select individual colonies and streak them onto fresh carbon-free, inorganic medium for purification. Repeat this process until the plate is visually pure.

[0030] In order to obtain a strain with strong adaptability, stable genetic characteristics and not prone to mutation, the above-mentioned purified strain was subcultured in a carbon-free inorganic solid culture medium, and the strains with a significantly weakened growth trend during the subculture were discarded. A total of 9 subcultures were performed in this experiment (during the subculture, the last three generations were cultured under continuous light conditions, the temperature remained unchanged, and the growth rate and morphological characteristics were consistent with those in the absence of light). The 10th generation of the target strain was obtained, and the morphology of the young single colonies was observed.

[0031] 16S rDNA sequencing and identification of carbon-fixing strains Genomic DNA was extracted from the strain using the spin column bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.). PCR amplification was performed using the extracted bacterial total DNA as a template. The upstream and downstream sequences of the universal primers were 27 F (SEQ ID No. 1): 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492 R (SEQ ID No. 2): 5'-CGGTTACCTTGTTACGACTT-3', respectively. The PCR system and reaction cycles were set according to the kit (Sangon). PCR products were verified by 1% agarose gel electrophoresis and sent to the company (Reboxing) for sequencing. Sequencing results were then compared against the NCBI database using BLAST, and a phylogenetic tree was constructed.

[0032] Finally, a strain of Saccharothrix texas was obtained ( Saccharothrix texasensis ) 8, and deposited it with the deposit number CGMCC No.33977.

[0033] Activity assay of carbon-fixing enzymes (ATP-citrate lyase (ACLY) and ribulose-1,5-bisphosphate carboxylase (RubisCO)) in strains The strain (Saccharothrix texaxensis 8) was inoculated in a carbon-free, inorganic liquid medium and cultured in a shaking incubator at 30°C, 150 rpm, and darkness. A blank control (CK) was also established, with three replicates per group. To prevent prolonged incubation from affecting enzyme activity, incubation was terminated upon color change (indicating the stationary phase). This experiment lasted for a total of 20 days. After the incubation period, 100 μL of the control solution was plated onto LB medium for incubation to ensure no contamination during the shaking incubation process. The enzyme activity of the relevant bacterial solution was then measured using a kit (Sangon).

[0034] Determination of carbon content in bacterial suspension The bacterial solution cultured in the above-mentioned carbon-free inorganic liquid medium for 20 days was filtered through a 0.45 μm filter membrane and diluted with sterile deionized water. The sample was placed in a total organic carbon analyzer (Shimadzu TOC-LCPH model) to measure the total carbon (TC) and total inorganic carbon (TIC) contents of the bacterial solution. The total organic carbon (TOC) content of the bacterial solution was then calculated by subtraction.

[0035] Bare sand culture experiment of carbon-fixing bacteria To better investigate the carbon sequestration capacity of the strain in bare sand and its potential for application, this experiment employed sterilized and unsterilized bare sand. Bare sand sampling was consistent with the BSC sampling described above. After thoroughly mixing all bare sand samples into a single sample, 18 100-g samples were weighed and placed in 250-mL Erlenmeyer flasks. Nine flasks containing bare sand were used for sterilized culture experiments, while the remaining nine were used for unsterilized culture experiments. The bare sand sterilization process was as follows: After sealing the flasks with sterile sealing film and newspaper, the flasks were sterilized in an autoclave at 121°C for 4 hours, followed by overnight cooling. After repeating the sterilization three times, the flasks were allowed to cool to ensure complete sterilization and no condensation remained.

[0036] Take 8 young cells of Saccharothrix texas (cultured for 2 days) in LB liquid medium, and confirm the number by the "Thoma method" of blood cell counting plate. 8 The CFU / mL standard was used, and the bacteria were diluted with sterile deionized water. 5 mL of the bacterial solution was applied to sterilized and non-sterilized bare sand respectively. To eliminate the specific effect, a laboratory-stored Escherichia coli without carbon fixation ability ( Escherichia coli ), with a blank medium control, and three replicates per group. To maintain initial soil moisture conditions and optimize microbial activity, each sample was infused with 8 mL of sterile deionized water (adjusting the bare sand soil moisture content to approximately 60% of its field capacity) and gently shaken for uniform distribution. All procedures were performed in a clean bench to ensure no bacterial contamination. The prepared samples were incubated in a 30°C incubator in the dark for 30 days. SOC content was then determined using the potassium dichromate external heating method.

[0037] Determination of carbon-fixing bacterial strain expansion and growth Using a disposable inoculating loop, inoculate one loopful of Saccharothrips texas strain 8 into 20 mL of LB liquid medium and culture in a shaking incubator. A blank control should also be included. Three replicates should be performed for each group. Shake incubator conditions should be set at 30°C, 150 rpm, and darkness. The bacterial concentration should be measured every two hours using a microplate reader until the strain reaches the end of growth phase. A growth curve should be plotted.

[0038] The bacterial liquid in the LB liquid medium at the stable period was centrifuged to remove the supernatant, washed twice with sterile water to remove the residual culture medium, sealed with sealing film, placed in an oven to dry, and then the dry weight of the bacteria was measured.

[0039] Experimental results ① Morphological characteristics: white, curly, concave, rough. Colony morphology see Figure 1 .

[0040] ②16S sequencing results are as follows:

[0041] Phylogenetic tree Figure 2 .

[0042] ③The results of the carbon fixation enzyme activity test of the strain are shown in Figure 3 In the figure, different capital letters indicate significant differences in ACLY activity, and different lowercase letters indicate significant differences in RubisCO activity.

[0043] ④The results of TOC content test of bacterial solution are shown in Figure 4 The letters above the bars in the figure are different, indicating significant differences.

[0044] ⑤ The results of SOC content test of sterilized bare sand and non-sterilized bare sand after culture are shown in Figure 5 CK-1 is an E. coli control, and CK-2 is a blank culture medium control. Different capital letters in the figure indicate significant differences in the SOC of sterilized bare sand, and different lowercase letters indicate significant differences in the SOC of non-sterile bare sand.

[0045] ⑥Strain growth test results are shown in Figure 6 .

[0046] According to the above results, the Saccharothrix texas 8 strain of the present invention can fix CO2 in the atmosphere, convert inorganic carbon into organic carbon, and increase the SOC content of bare desert sand in a short period of time.

[0047] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A strain of Saccharothrix texas ( Saccharothrix texasensis ) 8, characterized in that, The deposit number is CGMCC No.33977.

2. A bacterial agent, characterized in that Containing the Saccharothrix texas described in claim 1 ( Saccharothrix texasensis )8.

3. The Saccharothrix texasii of claim 1 ( Saccharothrix texasensis ) 8, or use of the bacterial agent according to claim 2 in converting inorganic carbon into organic carbon.

4. The use according to claim 3, characterized in that The inorganic carbon is carbon dioxide and / or carbonate.

5. The Saccharothrix texasii of claim 1 ( Saccharothrix texasensis ) 8, or use of the bacterial agent according to claim 2 in increasing soil organic carbon.

6. The use according to claim 5, characterized in that The soil originates from a desert area.

7. The Saccharothrix texasii of claim 1 ( Saccharothrix texasensis ) 8, or use of the bacterial agent according to claim 2 in enhancing the activity of carbon fixation enzymes.

8. The use according to claim 7, characterized in that The carbon-fixing enzyme is ATP-citrate lyase and / or ribulose-1,5-bisphosphate carboxylase.

9. A carbon fixer, characterized in that Including the texas sugar string mushroom of claim 1 ( Saccharothrix texasensis ) 8, or the bacterial agent according to claim 2.

10. The carbon fixer according to claim 9, characterized in that Can fix CO2 in the atmosphere.

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