A marine strain of *Roseola molaris* for saline-alkali soil improvement and its application

Abstract

This invention provides a *Roseolariomoraxella salina* strain for saline-alkali soil improvement, with accession number CGMCC No. 36067. The *Roseolariomoraxella salina* strain provided by this invention exhibits significant comprehensive application advantages. This strain demonstrates excellent survival and growth capabilities under saline-alkali stress, effectively reducing salinity and alkalinity in saline-alkali soils and thus improving the saline-alkali environment. Whole-genome analysis shows that this strain carries multiple functional genes related to osmotic regulation and ion transport, providing a molecular basis for its salinity-reducing ability. Adding citric acid or betaine can further enhance its salinity-reducing performance, and its biomass is significantly increased after optimization of fermentation conditions. This strain shows promising application prospects in saline-alkali soil improvement and saline-alkali aquaculture environment management.

Description

Technical Field

[0001] This invention belongs to the field of beneficial microbial screening and application technology, specifically relating to a marine Ruseolaria rosenbergii strain for saline-alkali soil improvement and its application. Background Technology

[0002] Soil salinization is a significant limiting factor restricting the development and utilization of saline-alkali land resources and the sustainable development of agriculture. Saline-alkali land is widely distributed in my country, covering a large total area, mainly concentrated in the Northeast, Northwest, and North China regions. Saline-alkali soils typically exhibit characteristics such as high salinity, high alkalinity, high pH, ​​and complex ionic composition, primarily manifested in the presence of soluble salts (such as Na+). + Cl — CO3 2— HCO — The content of these substances is high, and the ratio of ions is unbalanced. These physicochemical properties can lead to the destruction of soil aggregate structure, decreased permeability, and severe soil compaction. At the same time, they inhibit soil microbial activity and reduce the availability of nutrients such as nitrogen, phosphorus, and potassium, thereby significantly limiting plant root development and normal crop growth, ultimately affecting agricultural yield and ecosystem stability.

[0003] Currently, measures to improve saline-alkali soils mainly include physical methods (such as salt removal and leaching), chemical methods (such as applying gypsum and acid conditioners), and biological methods. Among these, microbial remediation technology has gradually become an important research direction for saline-alkali land management due to its advantages such as low cost, environmental friendliness, strong sustainability, and low risk of secondary pollution. Related studies have shown that some functional microorganisms can improve soil structure, regulate ion balance, and promote nutrient transformation by secreting organic acids, extracellular polysaccharides, enzymes, and other substances, thereby alleviating saline-alkali stress to a certain extent.

[0004] However, existing microbial inoculants still have significant limitations. First, most strains are currently derived from ordinary farmland or laboratory culture environments, not from saline-alkali ecosystems. Their survival ability under extreme conditions such as high salt, high alkalinity, and high osmotic pressure is weak, making it difficult to establish long-term colonization in saline-alkali soils. Second, saline-alkali soil environments are highly complex and heterogeneous, with significant differences in salt types, pH ranges, and ionic composition across different regions, making it difficult for single-function strains to function stably in such variable environments. Furthermore, the functional expression of microorganisms in actual soil is significantly affected by environmental factors, and existing inoculants often exhibit the problem of "good laboratory results but unstable field performance."

[0005] More importantly, screening functional strains with excellent salinity-reducing capabilities from saline-alkali soils presents significant technical challenges. On one hand, the saline-alkali environment exerts intense selection pressure on microorganisms, limiting the number of long-term surviving bacterial communities, whose metabolic characteristics are complex and whose functional expression varies considerably. On the other hand, even if salt- and alkali-tolerant strains are isolated, their actual effects on reducing soil salinity, regulating pH, or improving soil structure are often limited, making it difficult to simultaneously meet the requirements of "high adaptability" and "high functionality." Furthermore, significant functional differences exist between different strains, leading to problems such as low efficiency, poor reproducibility, and an incomplete functional evaluation system during the screening process.

[0006] Therefore, how to efficiently screen microbial strains from saline-alkali soil that have both good environmental adaptability and significant salinity reduction function, and achieve their stable colonization and continuous effect in complex saline-alkali environments, remains a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a marine Rhodesia molariae strain for improving saline-alkali soil and its application, namely a probiotic strain that can improve saline-alkali soil and improve the aquaculture environment in saline-alkali water.

[0008] The present invention first provides a strain of Rossellomorea aquimaris JQ-14, which was deposited on September 25, 2025 at the China General Microbiological Culture Collection Center located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the accession number CGMCC No. 36067.

[0009] The present invention also provides an application of the aforementioned marine Ruseolium molluscum, which is in the application of reducing salinity in the environment;

[0010] The present invention also provides another use of the aforementioned marine Ruseolium molluscum, which is in reducing the alkalinity of the environment;

[0011] The environment described, as a specific example, is saline-alkali soil or saline-alkali water.

[0012] In another aspect, the present invention provides an article for improving saline-alkali soil and improving the aquaculture environment in saline-alkali water, comprising the above-mentioned live bacteria of *Roseola molasses*.

[0013] The present invention also provides a method for improving saline-alkali soil and improving the aquaculture environment in saline-alkali water. The method involves treating the soil with the aforementioned *Roseola molasses*.

[0014] In another aspect, the present invention also provides a method for culturing the aforementioned marine Moraxella rosenbergii, wherein the inoculum size is 3.46%, the salinity is 9.83‰, and the pH is 7.97.

[0015] The *Roseolario Moraxella salina* strain provided by this invention possesses significant comprehensive application advantages. This strain exhibits excellent survival and growth capabilities under saline-alkali stress, effectively reducing salinity and alkalinity in saline-alkali soils and significantly improving saline-alkali environments. Whole-genome analysis reveals that this strain carries multiple functional genes related to osmotic regulation and ion transport, providing a molecular basis for its salinity-reducing ability. Adding citric acid or betaine can further enhance its salinity-reducing performance, and its biomass is significantly increased after optimization of fermentation conditions. This strain shows promising application prospects in saline-alkali soil improvement and saline-alkali aquaculture environment management. Attached Figure Description

[0016] Figure 1 : The salt-alkali reduction effect of the strain;

[0017] Figure 2 : Diagram of the growth morphology of the strain;

[0018] Figure 3 Phylogenetic tree analysis diagram of the strains;

[0019] Figure 4 Diagram showing the salinity tolerance of strain JQ-14;

[0020] Figure 5 : Diagram showing the alkalinity tolerance of strain JQ-14;

[0021] Figure 6 : Effect of exogenous added substances on the salt-alkali reduction function of strain JQ-14;

[0022] Figure 7 : GO annotation analysis diagram of strain JQ-14;

[0023] Figure 8 KEGG annotation analysis diagram of strain JQ-14;

[0024] Figure 9 : Actual application effect diagram of strain JQ-14;

[0025] Figure 10 : Effect of exogenous additives on the growth of JQ-14 strain;

[0026] Figure 11 Fermentation condition response surface plot of strain JQ-14. Detailed Implementation

[0027] The basic culture medium used in this embodiment of the invention is LB medium, but other commonly used culture media can also be selected.

[0028] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0029] Example 1: Isolation, purification and screening of bacterial strains

[0030] Weigh 3 g of saline-alkali soil sample and place it in a sterile Erlenmeyer flask. Add 50 mL of sterile 0.9% physiological saline and seal the flask with sealing film. Place the Erlenmeyer flask in a 30℃ shaking incubator and shake at 200 rpm for 30 min. After removing it and letting it stand for 10 min, transfer 10 mL of the supernatant to a sterile centrifuge tube; this is the soil suspension stock solution. Take 200 μL of the soil suspension stock solution and dilutions of 10, 100, and 1000 times, respectively, and spread them on LB agar plates. Invert the plates in a 30℃ incubator and incubate for 3 days, observing the colony growth.

[0031] Single colonies of varying morphology, size, and color were selected and further purified using the streak plating method until a single strain was obtained. Each single colony was inoculated into LB broth and activated by incubation at 30°C and 180 rpm for 24 h with shaking. The activated bacterial solution was then mixed with a preservative (glycerol and 0.9% physiological saline in a 4:1 ratio) at a volume ratio of 5:2 to prepare glycerol-containing bacteria, which were stored at -80°C for later use.

[0032] The preserved bacterial culture was removed from the refrigerator, thawed, and streaked onto a solid culture medium. Single colonies were picked and inoculated into LB liquid medium, and cultured in an incubator with shaking (180 rpm) for 3 days. After the culture was completed, the fermentation broth was centrifuged at 3500 rpm for 10 minutes, and the supernatant was collected to determine the salt-lowering effect. LB liquid medium without inoculation was used as a control. Chloride ion concentration was measured at the beginning and end of the culture. The measurement method was as follows: 1 mL of supernatant was placed in a 50 mL Erlenmeyer flask, 4 mL of pure water and 0.3 mL of potassium chromate indicator were added, and titrated with silver nitrate standard solution (0.141 mol / L) until the solution changed from yellow to light brick red, which was the endpoint. For bacteria with good salt-lowering effect, an alkali-lowering test was performed using LB liquid medium (initial pH 9.0). After culturing at 180 rpm for 48 hours in an incubator with shaking, the culture was centrifuged, and the pH of the supernatant was measured to determine the alkali-lowering effect. The results showed that JQ-14 reduced salt content by 3.95% ± 0.24% and reduced alkali content by 17.19% ± 0.45%. Figure 1 ).

[0033] Example 2: Taxonomic identification and salt and alkali tolerance test of the strain

[0034] After 48 hours of incubation on LB plates, mature colonies of JQ-14 are white, round, with smooth edges, opaque, and with a smooth surface. Figure 2).

[0035] The 16S rDNA gene sequence of the screened strains was determined, and homology sequence alignment was performed using NCBI BLAST. A phylogenetic tree was then constructed using MEGA7 based on the neighbor-joining method for strain identification. The results showed that JQ-14 clustered with *Moraxella rosenbergii* (seawater rosenbergii). Figure 3 The strain was named Rossellomorea aquimaris JQ-14 and deposited on September 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC) located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36067.

[0036] LB solid culture media containing different concentrations of NaCl were prepared, with final NaCl concentrations set at 1%, 2%, and 3%. Simultaneously, the pH of the LB medium (1% NaCl) was adjusted using 1 mol / L NaOH and 1 mol / L HCl solutions to prepare solid plates with pH values ​​of 7.0, 8.0, and 9.0. JQ-14 was activated and inoculated onto these plates. After incubation at 30℃ for 24 h, colony growth was observed to assess the salt and alkali tolerance of JQ-14.

[0037] The results showed that JQ-14 grew well under three salinity conditions of 1%, 2%, and 3% NaCl, without any obvious inhibition. Figure 4 Meanwhile, this strain can grow stably within a pH range of 7 to 9. Figure 5 These results demonstrate that JQ-14 possesses the ability to survive and grow in saline-alkali stress environments.

[0038] Example 3: Effects of adding different substances on the salt-lowering and alkali-reducing function of JQ-14

[0039] The effects of adding exogenous substances glycine, betaine, glutamic acid, proline, and citric acid (0.5 g / L) individually to basal LB liquid medium (NaCl 1%) on the salt-lowering function of JQ-14 were determined. K⁺ (0.5% w / v K₂HPO₂·3H₂O) and Ca⁺ were added individually to basal LB liquid medium (NaCl 1%). 2+ (0.05% w / v CaCl2), Mg 2+ (0.05% w / vMgSO4·7H2O), Fe 2+ (0.0005% w / v FeSO4·7H2O) and Mn 2+ (0.0005% w / v MnCl2) was used to determine the effect on the alkali-reducing function of JQ-14.

[0040] The results showed that adding citric acid or betaine alone could significantly improve the salt reduction rate of JQ-14. Figure 6 Compared with the control group, the salt reduction rate increased by 69.8% in the citric acid-added group and by 70.9% in the betaine-added group. Regarding alkali reduction performance, the addition of metal ions to the culture medium did not significantly promote JQ-14.

[0041] Example 4: Bacterial whole genome analysis

[0042] 50 mL of fermentation broth from the strain in logarithmic growth phase was collected by centrifugation at 14000 g for 5 min at 24℃, and whole-genome sequencing was performed. DNA was extracted using the MagPure bacterial DNA extraction kit (D6361-02, Magen, China). DNA concentration was determined using Qubit 4.0 software (Thermo, Q33226). DNA integrity was assessed by 1% agarose gel electrophoresis. Qualified libraries were sequenced on the MGI DNBSEQ-T7 sequencing platform. Raw sequencing data were first processed using Fastp for basic statistical and quality assessment, followed by quality control filtering to obtain highly reliable data. Then, the second-generation sequencing data were assembled using SPAdes software to obtain preliminary contig sequences. To further refine the assembly results, gaps in the contigs were filled using the GapFiller tool. Finally, Pilon was used to correct the assembled sequences to correct base recognition errors and small-scale insertion / deletion variations. Finally, the sequences are further annotated and processed using NCBI's PGAP or Pr tools to ensure the integrity and reliability of the data.

[0043] In the GO database ( Figure 7 The top three functions of JQ-14 annotated genes are cellular processes, metabolic processes, and membrane processes. The top three functions with the highest abundance in JQ-14 gene KEGG annotations are all concentrated in metabolic pathways. Figure 8The genes identified in JQ-14, in order of frequency, are carbohydrate metabolism, metabolic overview, and amino acid metabolism. The main desalination-related genes screened in JQ-14 include potassium ion transport systems (kdpA, kdpB, kdpC, kdpD, kdpE), glycine-betaine / proline transport systems (proV, proW, proX), osmotic protection substance transport systems (opuA, opuBD, opuC), trehalose synthesis (otsA), betaine synthesis (betB), and acetic acid synthesis (ldh, ackA, pta). Other desalination-related genes in the JQ-14 genome include clcB (chloride channel protein), corA (cobalt / magnesium transporter), and nhaP2 (K... + / H + (anti-transporter) and nhaA (Na) + / H + (Reverse transport protein), gltA2 (citric acid synthase), aceB (malate synthase).

[0044] Example 5: Practical Application Effects of JQ-14

[0045] The experiment consisted of two treatments: a control group (CK, no bacterial strain added) and group 14 (JQ-14 added). Each group had three replicates, for a total of six small buckets. The saline-alkali soil used in the test was taken from saline-alkali paddy fields in Tangshan, Hebei Province. After removing impurities, it was air-dried naturally. At the beginning of the experiment, the mixed saline-alkali soil was divided into 5 L buckets, with each bucket containing 600 g.

[0046] JQ-14 was streaked, and single colonies were picked and inoculated into liquid culture medium. The cultures were then incubated at 30°C with shaking at 180 rpm for 48 h. The OD values ​​of the bacterial culture were then calculated. 600 The value was diluted to 1, and then 14 groups of 100 mL diluted bacterial cultures were centrifuged and resuspended in deionized water for later use.

[0047] The experiment lasted 25 days, with treatments administered every 5 days. The control group received 100 mL of deionized water, while the 14 groups received 100 mL of bacterial suspension. Spraying combined with stirring was used to ensure thorough mixing of the bacterial suspension with the soil. After treatment, the containers were placed in a 24℃ incubator, and weighed every two days, with deionized water added to maintain a soil moisture content of 30%. Soil organic matter, conductivity, pH, exchangeable sodium percentage (ESP), and total salt content were measured at the beginning and end of the experiment to evaluate the improvement effect of different bacterial suspension treatments on saline-alkali soils. The OR group represented the initial soil sample's physicochemical properties. Soil organic matter was measured using the potassium persulfate oxidation method. 20.00 g of air-dried soil sample (passed through a 2 mm sieve) was weighed into a shaking flask, 100 mL of carbon dioxide-free water was added, and the mixture was shaken at 20℃ for 30 min, allowed to stand for 30 min, filtered, and the conductivity was measured using an EZ-9909A filter. Take another 10.0 g soil sample, add 25 mL of carbon dioxide-free water, stir magnetically for 2 min, let stand for 30 min, and then measure the pH. ESP was determined by ethanol-ammonium chloride extraction-atomic absorption spectrometry, and total salt content was determined by drying method.

[0048] Adding JQ-14 to the soil had no significant effect on soil organic matter content. Figure 9 Regarding soil salinity, adding JQ-14 alone significantly reduced soil electrical conductivity and total salt content. For soil pH, adding JQ-14 alone significantly reduced soil pH. Furthermore, adding JQ-14 alone significantly reduced soil ESP. In conclusion, JQ-14 can significantly reduce soil salinity and alkalinity in practical applications.

[0049] Example 6: Optimization of Fermentation Conditions

[0050] By setting pH gradients of 5.0, 6.0, 7.0, 8.0, and 9.0, and by adding different metal ions (K+, K ... + Mg 2+ Ca 2+ Fe 2+ Mn 2+ Treatment groups (K₂HPO₄·3H₂O 0.5%, MgSO₄·7H₂O and CaCl₂ 0.05% each, FeSO₄·7H₂O and MnCl₂ 0.0005%) were cultured under identical conditions to preliminarily determine the suitable pH range and metal ion species. Subsequently, a Box-Behnken response surface methodology was used to optimize three key factors: inoculum size (A: 2%, 4%, 6%), salinity (B: NaCl addition of 0‰, 5‰, 10‰), and initial pH (C: 6, 7, 8). 600The values ​​were used as response values, and a three-factor, three-level central composite experiment was designed using Design Expert 13.0 software. Each group had three replicates. Optimal fermentation conditions were predicted, and the effects were validated based on these predicted optimal fermentation conditions.

[0051] JQ-14 exhibited good growth within a pH range of 6.0–8.0, therefore, the pH level was optimized in subsequent response surface methodology using three levels: 6.0, 7.0, and 8.0. Mg was added. 2+ The OD value of the fermentation broth in the treatment group after 48 h was significantly higher than that in other groups, indicating that Mg²⁺ has a promoting effect on the growth of JQ-14. Figure 10 Therefore, subsequent response surface methodology experiments uniformly used LB medium supplemented with 0.05% MgSO4·7H2O as the basal medium. Based on the response surface analysis results ( Figure 11 The interaction among inoculum size, salinity, and pH significantly affected the biomass of JQ-14, with a relatively steep three-dimensional response surface indicating a strong interaction effect among the factors. Based on the established regression model, the optimal culture conditions for JQ-14 were predicted to be an inoculum size of 3.46%, a salinity of 9.83‰, and a pH of 7.97. Under these conditions, the predicted OD... 600 The value is 0.656, which was verified by the actual measured OD value in the experiment. 600 The value was 0.668, which is close to the predicted value. Compared with 0.600 before optimization, the biomass increased by about 11.33%. After response surface methodology optimization, the biomass of JQ-14 was significantly improved compared with that before optimization.

Claims

1. A marine *Roseolariomoraxella* species, characterized in that, The preservation number of the marine *Roseola molluscum* is CGMCC No. 36067.

2. The application of the marine molluscum roselliae according to claim 1 in reducing environmental salinity.

3. The application of the marine Moraxella rosenbergii strain according to claim 1 in reducing the alkalinity of the environment.

4. The application as described in claim 2 or 3, characterized in that, The environment described is saline-alkali soil or saline-alkali water.

5. A product for improving saline-alkali soil and the aquaculture environment in saline-alkali water, characterized in that, The product contains live cells of *Roseola molasses* as described in claim 1.

6. The article of claim 5, characterized in that, The product in question is a bacterial solution.

7. A method for improving saline-alkali soil and enhancing the aquaculture environment in saline-alkali water, characterized in that, The method involves treating the bacteria described in claim 1 using *Roseola molasses*.

8. A method for culturing *Roseolario Moraxella salina* as described in claim 1, characterized in that, The method described herein includes an inoculum size of 3.46%, a salinity of 9.83‰, and a pH of 7.97.

Images