Spanish glycothrix 9 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a Spanish glycothrix strain 9 and application thereof. According to the invention, a new strain of chemoautotrophic Spanaensis 9 with carbon sequestration capability is found by research, and the preservation number of the new strain of chemoautotrophic Spanaensis 9 is CGMCC (China General Microbiological Culture Collection Center) No.33978. The invention also discloses a preparation method of the new strain of chemoautotrophic Spanaensis 9. The strain can convert inorganic carbon (CO2 or carbonate) into organic carbon, is not limited by illumination, increases the SOC content of naked sand in a short time, and lays a foundation for field planting and survival of other plants.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a strain of Saccharothrix hispanica 9 and applications thereof. Background Art

[0002] Under the combined effects of chronic drought stress and poor nutrient conditions, soil organic carbon (SOC) reserves in desert ecosystems are significantly constrained. The sparse vegetation in these regions results in a lack of plant-derived organic carbon input, hindering the effective accumulation of soil carbon. Notably, biological soil crusts (BSCs), a key biological component of desert surface cover, demonstrate a unique ecological function in enhancing surface SOC content. These complexes are formed by autotrophic and heterotrophic organisms, such as microorganisms, algae, lichens, and mosses, tightly binding to surface soil particles through the secretion of cementing substances such as extracellular polysaccharides (EPS), mycelium, and rhizoids. Based on the dominant species, they can be divided into three major groups: algal crusts, lichen crusts, and moss crusts. Currently, cyanobacteria are the most widely used to enhance desert soil SOC content. Cultivating algal crusts indoors and spraying them outdoors can effectively increase desert soil SOC.

[0003] Despite this, cyanobacteria also have certain limitations due to their specific light, water, and nutrient requirements. First, during the initial stages of BSC formation, shifting sand easily covers the algal crust surface, reducing light capture by 40%–65%, thereby hindering cyanobacteria's photosynthesis and carbon fixation. Second, water plays a crucial role in the formation and development of BSCs. When the surface soil moisture content falls below 3.5%, photoautotrophic activity is completely inhibited, causing exponential decline in cyanobacterial biomass and even death. Third, cyanobacterial colonization is highly dependent on soil substrate conditions. Initially, low levels of organic matter, total nitrogen, and total phosphorus in the soil are unfavorable for cyanobacterial colonization and survival. In contrast, the oligotrophic adaptation mechanisms (C:N:P requirement ratio of 100:3:0.3) and desiccation tolerance (survival threshold moisture content >1.5%) of chemoautotrophic microorganisms favor their survival in desert habitats. Studies have shown that before the formation of BSCs, chemoautotrophic microorganisms can act as pioneer species to promote the formation of soil aggregates by secreting EPS, which can increase the water holding capacity of the surface microenvironment by 15% to 20% and input 2.8 to 4.6 g C·m - ²·a - ¹ of initial organic carbon, creating the necessary microenvironment for subsequent cyanobacteria colonization.

[0004] Therefore, it is necessary to develop more chemoautotrophic microorganisms. Summary of the Invention

[0005] One of the objects of the present invention is to provide an autotrophic microorganism capable of fixing inorganic carbon.

[0006] The present invention provides a strain of Saccharothrix hispanica ( Saccharothrix espanaensis ) 9, its deposit number is CGMCC No.33978.

[0007] Saccharothrix hispanica of the present invention Saccharothrix espanaensis 9 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 Saccharothrips hispanica. Saccharothrix espanaensis , the deposit number is CGMCC No. 33978.

[0008] The present invention discovered a chemoautotrophic Spanish Saccharothrix 9 with carbon fixation ability, which can hydrolyze starch and cellulose, can grow without relying on any exogenous organic matter, is not restricted by light, can increase the SOC content in bare sand in a short period of time, improve desert soil, and provide the prerequisites for the later colonization and crust development of algae, etc., which is of great significance to improving the effect of desertification control.

[0009] The present invention also provides a bacterial agent containing the above-mentioned Spanish Saccharothrix ( Saccharothrix espanaensis )9.

[0010] 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.

[0011] The present invention also provides the above-mentioned Spanish Saccharothrix ( Saccharothrix espanaensis ) 9 or the application of bacterial agents in enhancing the activity of carbon fixation enzymes.

[0012] In the application of the present invention, the carbon-fixing enzyme is ATP-citrate lyase and / or ribulose-1,5-bisphosphate carboxylase.

[0013] The present invention also provides the above-mentioned Spanish Saccharothrix ( Saccharothrix espanaensis ) 9 or the use of bacterial agents in hydrolyzing starch and / or cellulose.

[0014] The present invention also provides the above-mentioned Spanish Saccharothrix ( Saccharothrix espanaensis )9 or the use of bacterial agents in converting inorganic carbon into organic carbon.

[0015] In the application of the present invention, the inorganic carbon is CO2 and / or carbonate.

[0016] The present invention also provides the above-mentioned Spanish Saccharothrix ( Saccharothrix espanaensis ) 9 or the application of bacterial agents in increasing soil organic carbon.

[0017] In the application of the present invention, the soil comes from a desert area.

[0018] The present invention also provides a carbon fixer, which includes the above-mentioned Spanish Saccharothrips ( Saccharothrix espanaensis ) 9 or bacterial agents.

[0019] The beneficial effects of the present invention are at least: The present invention provides a new chemoautotrophic Spanish Saccharothrix 9 with carbon fixation ability, which can convert inorganic carbon (CO2 or carbonate) into organic carbon through redox reactions, and can increase the SOC content of bare sand in a short period of time, laying the foundation for the colonization and survival of other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is in the form of colonies.

[0021] Figure 2 is a phylogenetic tree.

[0022] Figure 3 The carbon fixation enzyme activity test results of the strain.

[0023] Figure 4 The TOC content test results of the bacterial liquid are shown in Figure 2.

[0024] Figure 5 This is the test result of starch hydrolysis ability.

[0025] Figure 6 This is the test result of cellulose hydrolysis ability.

[0026] Figure 7 These are the test results for SOC content in sterilized bare sand and non-sterilized bare sand after cultivation.

[0027] Figure 8 The results of strain growth test are shown in Figure 2. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Each liter of nutrient medium includes: 10 g of peptone, 5 g of NaCl, 3 g of beef extract powder, pH 7.1-7.5, 20 g of agar (not added to liquid culture medium), sterilized at 121°C for 15 min, and used for determining the starch hydrolysis ability of the strain.

[0034] Each liter of sodium carboxymethylcellulose culture medium includes: 10 g sodium carboxymethylcellulose, 2.5 g Na2HPO4, 1.5 g KH2PO4, 2.5 g peptone, 0.5 g yeast extract, pH 7.0-7.4, 20 g agar (not added to liquid culture medium), sterilized at 121°C for 25 min, for the determination of the strain's cellulose hydrolysis ability.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Finally, a Spanish Saccharothrips strain ( Saccharothrix espanaensis) 9, and deposited it with the deposit number CGMCC No.33978.

[0039] Activity assay of carbon-fixing enzymes (ATP-citrate lyase (ACLY) and ribulose-1,5-bisphosphate carboxylase (RubisCO)) in strains The strain (Saccharothrips hispanica 9) 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 culture, ensuring no contamination during the shaking incubation process. The enzyme activity of the relevant bacterial solution was then measured using a kit (Sangon).

[0040] 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.

[0041] Determination of starch and cellulose hydrolysis ability of carbon-fixing strains Take 10 μL of the above bacterial solution in the logarithmic growth phase and inoculate it into the relevant functional culture medium. Acinetobacter calcoaceticus ), the negative bacteria in the cellulose hydrolysis test were aminowolan Methylobacterium ( Methylobacterium aminovorans )) and uninoculated as controls, with 4 replicates per group. After culturing for 4 days in starch and cellulose media (the above-mentioned nutrient medium and sodium carboxymethyl cellulose medium), relevant reagents (starch hydrolysis: Lugol's iodine solution, cellulose hydrolysis: 0.1% Congo red solution and 1 mol / L NaCl solution) were added dropwise. R ( D / d , D is the diameter of the transparent circle, d The hydrolysis capacity was determined by the size of the colony (where ∠ is the diameter of the colony).

[0042] 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.

[0043] Take 9 young Saccharothrix spp. in LB liquid medium (cultured for 12 hours), 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. To eliminate the specific effect, a laboratory-stored Escherichia coli strain 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 injected 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, followed by determination of SOC content using the potassium dichromate external heating method.

[0044] Determination of carbon-fixing bacterial strain expansion and growth Using a disposable inoculating loop, inoculate one loopful of Saccharothrips hispanica strain 9 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.

[0045] 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.

[0046] Experimental results ① Morphological characteristics: white, whorl-shaped, rough, with neat edges. Colony morphology is shown in Figure 1 .

[0047] ②16S sequencing results are as follows:

[0048] Phylogenetic tree Figure 2 .

[0049] ③The results of the carbon fixation enzyme activity test of the strain are shown in Figure 3 Among them, different capital letters indicate significant differences in ACLY activity, and different lowercase letters indicate significant differences in RubisCO activity.

[0050] ④The results of TOC content test of bacterial solution are shown in Figure 4 Note: TOC results are embedded in TC, and different lowercase letters indicate significant differences in TOC contents.

[0051] ⑤ The results of the test on starch hydrolysis ability of carbon-fixing strains are shown in Figure 5 Among them, number 15 represents negative bacteria.

[0052] Hydrolysis cycle of Saccharothrix hispanica 9 R= 3.00 ± 0.11.

[0053] ⑥ The results of the test on the cellulose hydrolysis ability of carbon-fixing strains are shown in Figure 6 Among them, number 2 represents negative bacteria.

[0054] Hydrolysis cycle of Saccharothrix hispanica 9 R= 3.00 ± 0.05.

[0055] ⑦ The results of SOC content test of sterilized bare sand and non-sterilized bare sand after culture are shown in Figure 7 CK-1 is the E. coli control, and CK-2 is the 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-sterilized bare sand.

[0056] ⑧Strain growth test results are shown in Figure 8 .

[0057] 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 hispanica ( Saccharothrix espanaensis ) 9, characterized in that, The deposit number is CGMCC No.33978.

2. A bacterial agent, characterized in that Containing the Spanish Saccharothrix according to claim 1 ( Saccharothrix espanaensis )9.

3. The Spanish Saccharothrix according to claim 1 ( Saccharothrix espanaensis ) 9, or use of the bacterial agent according to claim 2 in enhancing the activity of carbon fixation enzymes.

4. The use according to claim 3, characterized in that The carbon-fixing enzyme is ATP-citrate lyase and / or ribulose-1,5-bisphosphate carboxylase.

5. The Spanish Saccharothrix according to claim 1 ( Saccharothrix espanaensis ) 9, or use of the bacterial agent according to claim 2 in hydrolyzing starch and / or cellulose.

6. The Spanish Saccharothrix according to claim 1 ( Saccharothrix espanaensis ) 9, or use of the bacterial agent according to claim 2 in converting inorganic carbon into organic carbon.

7. The use according to claim 6, characterized in that The inorganic carbon is CO2 and / or carbonate.

8. The Spanish Saccharothrix of claim 1 ( Saccharothrix espanaensis ) 9, or use of the bacterial agent according to claim 2 in increasing soil organic carbon.

9. The use according to claim 8, characterized in that The soil originates from a desert area.

10. A carbon fixer, characterized in that Including the Spanish saccharothrix according to claim 1 ( Saccharothrix espanaensis ) 9, or the bacterial agent according to claim 2.

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