Cold-resistant bacillus subtilis for pond culture under low-temperature condition

Abstract

The invention provides cold-resistant bacillus subtilis for pond culture under a low-temperature condition. The preservation number of the cold-resistant bacillus subtilis is CGMCC (China General Microbiological Culture Collection Center) No.36068. The cold-resistant bacillus subtilis strain provided by the invention has remarkable comprehensive application advantages, can keep higher growth activity and metabolic capability in a low-temperature environment, and continuously plays a water body purification function. Ammonia nitrogen and nitrite nitrogen in a water body can be efficiently degraded, the environment of the aquatic water body is remarkably improved by promoting the nitrogen circulation process, and harm caused by nitrogen pollution is reduced. The strain is high in safety, does not cause poison or stress reaction to bred animals even under the condition of high-concentration use, and has good ecological safety and environmental friendliness.

Description

Technical Field

[0001] This invention belongs to the field of beneficial microbial screening and application technology, specifically relating to a cold-resistant *Bacillus subtilis* strain for pond culture under low-temperature conditions. Background Technology

[0002] In low-temperature (water temperature <18℃) pond aquaculture systems, water quality management in early spring faces unique challenges. The low temperature significantly weakens the enzyme activity of traditional nitrifying bacteria, leading to the accumulation of ammonia and nitrite nitrogen, which can form a complex toxicity that directly threatens cultured organisms. Currently available probiotic products are mostly screened based on ambient temperature environments, often exhibiting low survival rates and insufficient metabolic activity under low-temperature conditions, making them difficult to effectively colonize and perform their intended functions in actual aquaculture water bodies. Therefore, there is an urgent need to directly isolate and screen native environmental control strains from low-temperature aquaculture environments (such as bottom sediment and biofilm) that can adapt to low temperatures and simultaneously remove ammonia and nitrite nitrogen efficiently. These strains can maintain high metabolic activity under low-temperature conditions, achieving in-situ efficient biological denitrification during low-temperature periods, providing a reliable microbial control method for the steady-state management and sustainable development of low-temperature aquaculture water bodies. Summary of the Invention

[0003] The purpose of this invention is to provide a cold-resistant Bacillus subtilis for pond culture under low-temperature conditions, which can be used to treat ammonia nitrogen and nitrite nitrogen in water under low-temperature conditions, thereby making up for the shortcomings of the prior art.

[0004] This invention first provides a cold-resistant Bacillus subtilis (Bt) Peribacillus frigoritolerans Strain JQ-24 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 accession number CGMCC No. 36068.

[0005] This invention also provides an application of the JQ-24 strain in the degradation of ammonia nitrogen in water; The present invention also provides another use for the JQ-24 strain, which is its application in the degradation of nitrite nitrogen in water bodies; The present invention also provides another use of the JQ-24 strain, which is to promote the secretion of proteases and lipases in fish fry to improve digestive performance.

[0006] The present invention also provides a method for degrading ammonia nitrogen and / or nitrite nitrogen in water, which involves using the above-mentioned JQ-24 strain to treat the water. The water body mentioned, as a specific example, is a low-temperature water body used for pond aquaculture.

[0007] The present invention also provides a method for treating water under low temperature conditions, wherein the method comprises adding the above-mentioned cold-resistant Bacillus subtilis strain to the water.

[0008] The cold-resistant *Bacillus subtilis* strain provided by this invention has significant comprehensive application advantages. It maintains high growth activity and metabolic capacity in low-temperature environments, continuously exerting its water purification function. It can efficiently degrade ammonia nitrogen and nitrite nitrogen in water, significantly improving the aquaculture water environment and reducing the harm caused by nitrogen pollution by promoting the nitrogen cycle. This strain has high safety; even at high concentrations, it will not cause toxicity or stress reactions in farmed animals, demonstrating good ecological safety and environmental friendliness. Attached Figure Description

[0009] Figure 1 : A diagram showing the growth morphology of a cold-resistant Bacillus subtilis strain; Figure 2 Phylogenetic analysis diagram of cold-resistant Bacillus subtilis strains; Figure 3 : Effect of pH on the growth rate of cold-resistant Bacillus subtilis strains; Figure 4 Graph showing the effect of temperature on the growth of cold-resistant *Bacillus subtilis*; Figure 5 : Effect of salinity on the growth of cold-resistant Bacillus subtilis; Figure 6 : Effect of metal ions on the growth of cold-resistant Bacillus subtilis; Figure 7 : Effect of pH on nitrogen reduction performance of cold-resistant Bacillus subtilis; Figure 8 : Effect of temperature on nitrogen reduction performance of cold-resistant Bacillus subtilis; Figure 9 : Effect of metal ions on the nitrogen-reducing performance of cold-resistant Bacillus subtilis; Figure 10 Safety assessment of strain JQ-24 in hemolysis and antibiotic susceptibility tests.

[0010] Figure 11 Evaluation diagram of how strain JQ-24 promotes the secretion of proteases and lipases in fish fry to improve digestive performance. Detailed Implementation

[0011] The basic culture medium used in the embodiments of the present invention is LB medium. The ammonia nitrogen degradation test medium (C:N=15:1) is: NH4Cl 0.05453 (NH4+-N: 20mg / L), glucose 0.54g, pure water 1L, pH 8.0; the nitrite nitrogen degradation test medium (C:N=15:1) is: NaNO2 0.0985g (NO2--N: 20mg / L), glucose 0.7488g, pure water 1L, pH 8.0.

[0012] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0013] Example 1: Isolation, purification and screening of bacterial strains Weigh 3 g of bottom mud from aquaculture ponds in Bosten Lake area, Xinjiang, and place the sample in a sterilized Erlenmeyer flask. Add 50 mL of sterilized 0.9% physiological saline and seal the flask with sealing film. Place the Erlenmeyer flask containing the soil in a 30 ℃ shaking incubator and treat it at 200 rpm for 30 min. After treatment, remove the Erlenmeyer flask and let it stand for 10 min. Transfer 1 mL of the supernatant to a sterilized centrifuge tube to obtain the bottom mud suspension stock solution.

[0014] The sediment suspension was diluted to 10 solutions using 0.9% physiological saline. -1 10 -2 10 -3 10 -4 Soil suspension dilution. Water samples from aquaculture ponds were treated in the same way as the sediment suspension. The stock solution and dilution were plated on LB agar and incubated at 30 °C for 48 h. Single colonies were picked based on color, size, and morphology and streaked for purification. The purified strains were then preserved in glycerol.

[0015] The preserved bacterial strain was inoculated onto LB solid medium and streaked repeatedly. After incubation at 30 °C for 24 h, pure single colonies were obtained. These pure single colonies were then inoculated into test tubes containing LB liquid medium and incubated at 28 °C and 180 r / min in a constant temperature shaking incubator for 48 h. The culture was then centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and the culture was resuspended in physiological saline. This process was repeated three times to form a bacterial suspension of a specific concentration.

[0016] The concentrations of ammonia nitrogen and nitrite nitrogen in the initial degradation test medium were determined. Then, the bacterial suspension was inoculated into the degradation test medium at a ratio of 5%, with three replicates for each strain. The culture was carried out in a constant temperature shaking incubator at 28 ℃ and 180 r / min for 48 h. The bacterial suspension was centrifuged at 8000 r / min, and the supernatant was collected after 10 min. The concentrations of ammonia nitrogen and nitrite nitrogen were determined using the indophenol blue method and the dextrose coupling method, and the degradation rates of ammonia nitrogen and nitrite nitrogen were calculated.

[0017] Forty-one bacterial strains were isolated from sediment and water samples, and their ammonia nitrogen degradation effects are shown in Table 1.

[0018] Table 1: Ammonia nitrogen degradation rate of screened strains serial number Initial ammonia nitrogen concentration (mg / L) Final ammonia nitrogen concentration (mg / L) Degradation rate serial number Initial ammonia nitrogen concentration (mg / L) Final ammonia nitrogen concentration (mg / L) Degradation rate 14 21.5 12.69 40.96% 37 19.79 15.10 23.72% 20 21.5 12.69 40.96% 4 28.02 21.67 22.66% 6 28.02 17.1 38.97% 2 28.02 21.73 22.45% 9 28.02 17.38 37.97% 1 28.02 22.3 20.41% 8 28.02 17.5 37.54% 31 19.79 15.95 19.38% 3 28.02 17.61 37.15% 25 19.96 16.93 15.20% 5 28.02 17.84 36.33% 39 19.79 16.98 14.18% 17 21.5 14.24 33.78% 38 19.79 17.16 13.31% 12 21.5 14.87 30.85% 21 19.96 17.61 11.76% 33 19.79 13.84 30.08% 40 19.79 17.61 11.00% 32 19.79 13.90 29.79% 23 19.96 18.01 9.75% 19 21.5 15.10 29.78% 26 19.96 18.30 8.32% 15 21.5 15.15 29.52% 28 19.96 18.36 8.03% 16 21.5 15.33 28.72% 24 19.96 18.53 7.17% 7 28.02 20.19 27.94% 35 19.79 18.41 6.95% 36 19.79 14.52 26.61% 22 19.96 18.81 5.74% 13 21.5 15.84 26.33% 30 19.96 18.81 5.74% 10 28.02 20.76 25.91% 27 19.96 19.16 4.02% 11 21.5 15.95 25.79% 41 19.79 19.39 2.04% 18 21.5 16.07 25.26% 29 19.96 19.73 1.15% 34 19.79 14.92 24.58% Table 2 shows the nitrite nitrogen degradation efficiency of 41 bacterial strains. Among the screened strains, most strains had an ammonia nitrogen degradation rate between 0% and 1%, while strain 20 had a degradation efficiency of 5.59%. The nitrogen reduction effect of strain 20 was further verified by combining the degradation efficiency of ammonia nitrogen and nitrite nitrogen.

[0019] Table 2: Nitrite degradation rate of different strains serial number Initial concentration of nitrite (mg / L) Final concentration of nitrite nitrogen (mg / L) Degradation rate serial number Initial concentration of nitrite (mg / L) Final concentration of nitrite (mg / L) Degradation rate 20 19.93 18.55 6.89% 2 19.51 19.51 0.00% 30 19.65 18.55 5.59% 3 19.51 19.61 0.00% 4 19.51 18.60 4.69% 5 19.51 19.93 0.00% 14 19.93 19.28 3.22% 8 19.51 19.74 0.00% 12 19.93 19.42 2.53% 10 19.51 19.65 0.00% 16 19.93 19.42 2.53% 11 19.93 20.43 0.00% 18 19.93 19.47 2.30% 17 19.93 20.06 0.00% 24 19.65 19.28 1.86% 21 19.65 20.43 0.00% 36 19.38 19.10 1.42% 23 19.65 19.79 0.00% 7 19.51 19.24 1.41% 25 19.65 19.74 0.00% 38 19.38 19.15 1.18% 27 19.65 20.06 0.00% 22 19.65 19.42 1.16% 29 19.65 19.88 0.00% 26 19.65 19.42 1.16% 31 19.38 19.42 0.00% 39 19.38 19.19 0.94% 32 19.38 19.47 0.00% 28 19.65 19.47 0.93% 33 19.38 19.56 0.00% 15 19.93 19.74 0.92% 34 19.38 19.79 0.00% 6 19.51 19.38 0.70% 35 19.38 20.20 0.00% 13 19.93 19.79 0.69% 37 19.38 19.56 0.00% 1 19.51 19.42 0.47% 40 19.38 19.47 0.00% 9 19.51 19.47 0.23% 41 19.38 20.57 0.00% 19 19.93 19.88 0.23% The nitrogen reduction effect of strain 20 (nitrite-degrading bacteria) was re-screened and verified, and the nitrite nitrogen concentration in the ammonia nitrogen degradation medium was measured to quantify the nitrite nitrogen produced during the ammonia nitrogen degradation process. The ammonia nitrogen degradation results are shown in Table 3, with a degradation rate of 21.80% for strain 20. The nitrite nitrogen degradation effect is shown in Table 4, with a degradation rate of 4.86% for strain 20. Among the screened strains, strain 20 showed better nitrite nitrogen degradation performance and produced lower levels of ammonia nitrogen.

[0020] Table 3: Ammonia Nitrogen Degradation Effect of Secondary Screening

[0021] Table 4: Degradation effect of nitrite nitrogen by secondary screening

[0022] The morphological characteristics of the screened strains are as follows: Figure 1 As shown, after 48 hours of cultivation on LB solid medium, the mature colonies of the strain were orange, round, with smooth edges, opaque, and with a smooth surface. BLAST alignment analysis of the strain's 16S rRNA sequence revealed that strain JQ-24 clustered with *Bacillus subtilis*. Figure 2 ).

[0023] 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 strain JQ-24 clustered with *Bacillus subtilis* (a cold-resistant strain). Figure 2 It was named *Bacillus thuringiensis* (Central-resistant *Bacillus*). Peribacillus frigoritolerans Strain JQ-24 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 accession number CGMCC No. 36068.

[0024] Example 2: Optimization of fermentation conditions for strains LB medium was used as the basal medium to explore the optimal fermentation conditions under different factors.

[0025] Temperature: Set four temperature gradients: 8 ℃, 12 ℃, 16 ℃, and 28 ℃, while keeping other conditions consistent.

[0026] Salinity: The salinity was determined by the amount of sodium chloride added to the culture medium in parts per thousand. Three salinity levels were set: 0, 5, and 10, while keeping other conditions consistent.

[0027] pH: Set five initial pH values: 5.0, 6.0, 7.0, 8.0, and 9.0, while keeping other conditions consistent.

[0028] Metal ions: Based on the strain's response to K + Mg 2+ Ca 2+ Fe 2+ and Mn 2+ To account for differences in utilization conditions, K₂HPO₄·3H₂O was added at a mass fraction of 0.5%, MgSO₄·7H₂O and CaCl₂ at a mass fraction of 0.05%, and FeSO₄·7H₂O and MnCl₂ at a mass fraction of 0.0005%, while keeping other conditions consistent, to determine the optimal fermentation metal ions.

[0029] OD values ​​of bacterial cultures were measured at 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h. 600 The optimal fermentation conditions were determined based on the OD value, with three replicates per group.

[0030] The effect of pH on bacterial growth is shown in [reference]. Figure 3 .from Figure 3 It was found that the growth of the strains stopped after 48 hours of cultivation at pH 5 to 9. Among them, strain 20 showed significantly inhibited growth at pH 5, but grew better at pH 7 and pH 8.

[0031] The effect of temperature on strain growth is shown in Figure 4 ,from Figure 4 It can be seen that strain 20 has strong adaptability and grows relatively quickly under all low-temperature conditions of 16℃. From 24 hours onwards, there was no significant difference in growth among the strains under the four temperature conditions. Therefore, the strain can adapt well to low-temperature environments.

[0032] The effect of salinity on the growth of the strain is shown in Figure 5 ,from Figure 5 It can be seen that strain JQ-24 has strong adaptability and grows rapidly under all salinity conditions. However, the bacterial OD at 48h and 72h is relatively low under 10‰ salinity conditions. 600The values ​​were significantly higher than those of other groups, indicating that the strain grew best under salinity conditions of 10‰.

[0033] The effect of metal ions on strain growth is shown in Figure 6 ,from Figure 6 It can be seen that K was 24 hours ago. + The growth-promoting effect on the strain was significantly higher than that of other groups. However, the addition of Mn at 72h... 2+ It has a significant promoting effect on the growth of the strain.

[0034] Example 3: Evaluation of Nitrogen Degradation Performance at Low Temperatures The bacterial strain was activated in LB medium. After 48 hours of culture, the culture solution was centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and the culture was resuspended in physiological saline. This process was repeated three times to form a bacterial suspension of a certain concentration. The bacterial suspension was then inoculated into degradation test medium at a ratio of 5%, with three replicates for each strain. Single-factor experiments were used to analyze the nitrogen reduction effect of the strain under different environmental factors, exploring the effects of different temperatures, pH values, and metal ions on the nitrogen reduction effect. A single environmental factor was fixed, and the remaining experimental conditions were: temperature 28 ℃, pH 8, centrifugation speed 180 r / min, and C / N ratio 15. After 48 hours of culture, the bacterial solution was centrifuged at 8000 r / min for 10 min, and the supernatant was collected to determine the concentrations of ammonia nitrogen and nitrite nitrogen, evaluating the degradation effect of ammonia nitrogen and nitrite nitrogen. The various detection conditions are as follows: 1) pH: 5.0, 6.0, 7.0, 8.0, and 9.0; 2) Temperature: 8 ℃, 12 ℃, 16 ℃, 28 ℃; 3) Metal ions: Add K₂HPO₄ at a mass fraction of 0.5%. 4∙ Add 0.05% MgSO₄ to 3H₂O. 4∙ 7H2O and CaCl2 were added to FeSO4 at a concentration of 0.0005%. 4∙ 7H2O and MnCl2.

[0035] The effect of pH on the nitrogen-reducing performance of strains, such as Figure 7 As shown in the figure, pH significantly affects the nitrogen reduction performance of the strain. The JQ-24 strain showed the best ammonia nitrogen degradation efficiency (49.02%) at pH 9 and the best nitrite nitrogen degradation efficiency (28.22%) at pH 7.

[0036] The effect of temperature on the nitrogen-reducing performance of strains, such as Figure 8 As shown, low temperature had no significant effect on the degradation of ammonia nitrogen and nitrite nitrogen by strain JQ-24.

[0037] The effect of metal ions on the nitrogen-reducing performance of strains, such as Figure 10 As shown. K + and Mg 2+ It significantly promotes the ammonia nitrogen degradation effect of the strain, especially in Mg 2+ Under the specified conditions, JQ-24 bacteria showed good ammonia nitrogen degradation efficiency, reaching 48.43%. For nitrite nitrogen, the addition of K... + It can significantly promote the degradation rate of nitrite nitrogen in strain JQ-24, reaching 26.98%.

[0038] After obtaining ammonia and nitrite degrading strains based on the initial screening results, their fermentation conditions were optimized and their nitrogen reduction performance was evaluated. The results showed that the strains grew best at pH 7-8 with the addition of K⁺ or Mn²⁺.

[0039] The above results indicate that strain JQ-24 has a good degradation effect on ammonia nitrogen and nitrite nitrogen under low temperature and high alkalinity conditions.

[0040] Example 4: Safety assessment of strain JQ-24 in hemolysis and antibiotic susceptibility tests The safety of strain JQ-24 was evaluated through hemolysis and antibiotic susceptibility tests, aiming to provide a reference for the safety of the obtained strain in seedling cultivation.

[0041] Freshly activated, cold-resistant *Bacillus subtilis* strains were inoculated onto the center of blood agar plates. *Escherichia coli* DH5α (known for its non-hemolytic activity) was used as a negative control, and *Vibrio parahaemolyticus* as a positive control. The plates were incubated at the optimal low temperature (15°C) for 5–7 days to investigate the effect of temperature on hemolysin expression. After incubation, the type of hemolysis around the colonies was observed and recorded: completely transparent β-hemolytic rings, partially hemolyzed, grass-green α-hemolytic rings, or unchanged γ-hemolysis.

[0042] Research results are as follows Figure 10 The results showed that strain JQ-24 exhibited γ-hemolysis on fish blood plates under low temperature conditions, meaning it did not show any hemolysis.

[0043] The tested cold-resistant *Bacillus cereus* was cultured at 15°C to the logarithmic growth phase and prepared into a homogeneous bacterial suspension with a turbidity of 0.5 McFarland using sterile physiological saline. A sterile cotton swab was used to apply the bacterial suspension three times evenly to the surface of a Mueller-Hinton agar plate, which was then allowed to dry. Using sterile forceps, selected antibiotic discs (such as penicillin G tablets, oxacillin, ampicillin, and other commonly used aquatic antibiotics, as well as control drugs) were equidistantly affixed to the plate surface. The plate was then incubated at 15°C in the dark for 72 hours. After incubation, the diameter of the inhibition zone around each disc was precisely measured using calipers, and the results were recorded as sensitive (S), intermediate (I), or resistant (R) to assess the antibiotic susceptibility spectrum of this cold-resistant *Bacillus cereus*. The drug susceptibility test results showed that strain JQ-24 was moderately sensitive to 9 antibiotics, including minocycline, and sensitive to 6 antibiotics, including ceftriaxone.

[0044] Table 5: Antimicrobial susceptibility test results of strain JQ-24 Antibiotics Drug concentration in paper Inhibition zone diameter (mm) of strain JQ-24 Judgment Result Penicillin G tablets 10U / piece 0 R Oxyphenidyl 1μ / piece 0 R Ampicillin 10μ / piece 16.31 S Carbenicillin 100μ / piece 20.15 S Piperacillin 100μ / piece 0 R Cephalexin 30μ / piece 0 R cefazolin 30μ / piece 17.48 S Cefadroxil 30μ / piece 11.03 I Cefuroxime 30μ / piece 15.30 S Ceftazidime 30μ / piece 20.19 S Ceftriaxone 30μ / piece 19.41 S cefoperazone 75μ / piece 13.40 I Amikacin 30μ / piece 13.58 I Gentamicin 10μ / piece 14.69 I Kanamycin 30μ / piece 13.59 I Neomycin 30μ / piece 14.53 I tetracycline 30μ / piece 11.74 I Doxorubicin 30μ / piece 0 R Minocycline 30μ / piece 12.28 I Erythromycin 15μ / piece 11.13 I Note: R indicates drug resistance, I indicates moderate sensitivity, and S indicates sensitivity. Example 5: Evaluation of the effect of JQ-24 strain on promoting the secretion of proteases and lipases in fish fry to improve digestive performance This aquaculture experiment aimed to verify the effects of cold-resistant *Bacillus subtilis* on the activity of digestive enzymes in fish at a live in vivo level. Healthy, uniformly sized target juvenile fish were randomly divided into four groups: the NC group (no bacterial solution added), the L-20 group (low concentration of JQ-24 strain: 1×10⁻⁶), and the L-20 group (low concentration of JQ-24 strain: 1×10⁻⁶). 4 CFU / mL), M-20 group (medium concentration JQ-24 strain group: 1×10 5 CFU / mL) and H-20 group (high concentration of JQ-24 strain group: 1×10 6 (CFU / mL). Ten fish fry were placed in a 10 L tank in each group, with three replicates per group. The culture experiment lasted for 8 weeks. At the middle (week 4) and end of the experiment, samples were randomly taken from each group to collect the contents of the foregut and midgut of the fish. The activities of protease and lipase were measured to evaluate the effect of strain JQ-24 on enhancing the activity of endogenous protease and lipase in the fish intestine.

[0045] The results show that ( Figure 11 The JQ-24 strain can effectively promote the secretion of endogenous proteases and lipases in the intestines of fish fry. Specifically, a concentration of 1×10⁻⁶ is recommended. 6 The treatment with a CFU / mL concentration (H-20 group) showed the most significant effect. These results preliminarily confirm that strain JQ-24 potentially enhances digestive capacity by promoting the secretion of proteases and lipases in fish fry.

Claims

1. A cold-tolerant Paenibacillus sp. characterized in that, The preservation number of the cold-tolerant Paenibacillus is CGMCC No. 36068.

2. The cold-tolerant Paenibacillus of claim 1 is applied to degrade ammonia nitrogen in water.

3. The cold-tolerant Paenibacillus of claim 1 is applied to degrade nitrite nitrogen in water.

4. The cold-tolerant Paenibacillus of claim 1 is applied to promote the secretion of protease and lipase by fish fry to improve the digestive performance.

5. The use according to claim 4, wherein the compound is ###0002### The application is the application of the cold-tolerant Paenibacillus as a feed additive.

6. A method for degrading ammonia nitrogen and / or nitrite nitrogen in a water body, characterized in that, The method is to use the cold-tolerant Paenibacillus of claim 1 to treat water.

7. The body of water of claim 6, wherein, The water is pond culture water.

8. A method of treating a body of water under hypothermic conditions, characterized by, The method is to add the cold-tolerant Paenibacillus strain of claim 1 to water. The application is the application of the cold-tolerant Paenibacillus as a feed additive. The method is to use the cold-tolerant Paenibacillus of claim 1 to treat water. The water is pond culture water. The method is to add the cold-tolerant Paenibacillus strain of claim 1 to water.

Images