PSEUDOMONAS GRAMINIS STRAIN CAPABLE OF DEGRADING CELLULOSE AT LOW TEMPERATURE AND USE THEREOF

Abstract

A Pseudomonas graminis strain capable of degrading cellulose at a low temperature and use thereof. The accession number of the Pseudomonas graminis strain is CGMCC NO.: 18751. The strain has relatively strong capability of degrading cellulose at both a low temperature of 4° C. and a condition of 10° C., and the cellulose degradation capacities are basically the same. Although the cellulose degradation capacity is weakened at 30° C., the degradation amplitude is not large, and meanwhile, a KY240 strain also has potassium and inorganicphosphorus degradation capacity, and is expected to be used for preparing a bacterial fertilizer for degrading cellulose, promoting straw decomposition and improving soil fertility, particularly improving the amount of potassium and phosphorus available for plants in soil.

Claims

1. A Pseudomonas graminis strain preserved in China General Microbiological Culture Collection Center (CGMCC) of Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, with the accession number of CGMCC No.: 18751.

2. A microbial preparation comprising the Pseudomonas graminis strain according to claim 1.

3. The microbial preparation according to claim 2, wherein it is used in at least one of the following aspects: degrading an organic matter containing cellulose; promoting straw decomposition; degrading potassium and inorganicphosphorus; and preparing microbial fertilizer.

4. Use of the Pseudomonas graminis strain according to claim 1, comprising: degrading an organic matter containing cellulose; promoting straw decomposition; degrading potassium and inorganicphosphorus; or preparing microbial fertilizer.

5. A method for promoting cellulose degradation, comprising culturing a mixture of the Pseudomonas graminis strain according to claim 1 and cellulose.

6. The method according to claim 5, wherein the culture temperature is 0-40° C.

7. A method for degrading potassium, comprising culturing a mixture of the Pseudomonas graminis strain according to claim 1 and a potassium-containing mineral.

8. The method according to claim 7, wherein the potassium-containing mineral is selected from potassium feldspar, plagioclase, microcline, illite, vermiculite, montmorillonite, silicate and mica.

9. A method for degrading inorganicphosphorus, comprising culturing a mixture of the Pseudomonas graminis strain according to claim 1 and a phosphorus-containing mineral.

10. The method according to claim 9, wherein the phosphorus-containing mineral is selected from tricalcium phosphate, rock phosphate powder, fluorapatite, chloroapatite, hydroxyapatite, goyazite, vivianite, lignite, peat soil, wavellite, variscite and strengite.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1 shows the homologous comparison analysis (Neighbor-Join) of the KY240 strain and Pseudomonas graminis.

[0064] FIG. 2 shows the morphology of the KY240 strain under a microscope (10×100);

[0065] FIG. 3 shows the result of degradation of cellulose by the KY240 strain at 4° C.;

[0066] FIG. 4 shows the result of degradation of cellulose by the KY240 strain at 10° C.;

[0067] FIG. 5 shows the result of degradation of cellulose by the KY240 strain at 30° C.;

[0068] FIG. 6 shows the determination result of the potassium degrading capability of the KY240 strain;

[0069] FIG. 7 shows the determination result of the inorganicphosphorus degrading capability of the KY240 strain;

[0070] FIG. 8 shows the determination result of living bacteria count of the strains 92068 and KY240 at 30° C.;

[0071] FIG. 9 shows the determination result of living bacteria count of the strains 92068 and KY240 at 4° C.;

[0072] FIG. 10 shows the decomposition of wheat straws by the KY240 strain and the Bacillus subtilis 92068 strain at a low temperature of 4° C.

DESCRIPTION OF THE EMBODIMENTS

[0073] After massive screening on soil from all parts of China, the inventor have screened out a strain from nearly 300,000 microbial strains, the strain can grow stably and rapidly and degrade cellulose stably at a low temperature of 4° C., and can also degrade cellulose at medium-high temperatures, and the strain is named as KY240. Furthermore, the strain has multiple functions of degrading mineral potassium and inorganicphosphorus.

Determination of 16sDNA and Physiological Morphology Analysis of KY240 Strain:
Determination of 16sDNA of Strain

Extraction of Bacterial DNA by CTAB Method

[0074] 1) A single colony was inoculated in 5 mL R2A, and cultured at 30° C. overnight.

[0075] 2) 1 mL of a seed culture solution was inoculated into 100 ml of a R2A solution, and cultured at 37° C. and 220 r/min for 16 hours.

[0076] 3) The culture was centrifuged at 5,000 r/min for 10 minutes, and the supernatant was discarded.

[0077] 4) The precipitate was added with 10 mL TE for centrifugal washing, and then the bacteria were dissolved with 10 mL TE, mixed well, and stored at −20° C. for later use.

[0078] 5) 3.5 mL of the bacterial suspension was taken, added with 184 μL of 10% SDS, mixed well, added with 37 μL of 10 mg/mL proteinase K, mixed well, and incubated at 37° C. for 1 hour.

[0079] 6) It was added with 740 μL of 5 mol/L NaCl, then added with 512 μL of CTAB/NaCl, mixed well, and incubated at 65° C. for 10 min.

[0080] 7) It was added with equal volume of chloroform/isoamylol, mixed well, and centrifuged at 10,000 r/min for 5 minutes, and the supernatant was retained.

[0081] 8) The supernatant was added with equal volume of phenol: chloroform: isoamylol (25: 24: 1), mixed well, and centrifuged at 10,000 r/min for 5 minutes, and the supernatant was retained.

[0082] 9) The supernatant was added with 0.6 times the volume of isopropanol, mixed well, and centrifuged at 10,000 r/min for 5 minutes, the DNA precipitate was collected and centrifuged, and the DNA precipitate was subjected to centrifugal washing with 70% ethanol.

[0083] 10) The DNA was dissolved with 1 mL TE, added with RNaseA at a final concentration of 20 μg/mL, and stored at 4° C.

Amplification and Sequencing

[0084] PCR amplification of 16S rDNA was conducted with 16S rDNA universal primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO.: 1)) and 1492r (5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO.: 2)). PCR reaction conditions: pre-denaturation at 94° C. for 30 s; denaturation at 94° C. for 30 s, annealing at 52° C. for 30 s and extending at 72° C. for 60 s, and repeating these for 35 cycles. The PCR products were subjected to 1.5% agarose gel electrophoresis, which was followed by recovery, purification and sequencing (by Beijing meiyimei biotechnology Co., Ltd.). According to the obtained 16S rDNA sequence, the homologous sequences were searched by Blast in GenBank, and subjected to homologous sequence analysis and comparison, so as to establish a phylogenetic tree.

[0085] 16S sequencing result of KY240 strain

[0086] The 16sDNA sequencing result of the KY240 strain was:

TABLE-US-00001 (SEQ ID NO.: 3) ACCGTCCTCCCGAAGGTTAGACTAGCTACTTCTGGTGCAACCCACTCCCA TGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACA TTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAG ACTGCGATCCGGACTACGATCGGTTTTCTGGGATTAGCTCCACCTCGCGG CTTGGCAACCCTCTGTACCGACCATTGTAGCACGTGTGTAGCCCAGGCCG TAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCAC CGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGGACAA GGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGA CGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGGCACCAATCCATC TCTGGAAAGTTCATTGGATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCT TCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCAT TTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGT TAGCTGCGCCACTAAAAGCTCAAGGCTTCCAACGGCTAGTTGACATCGTT TACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTC GCACCTCAGTGTCAGTATGAGCCCAGGTGGTCGCCTTCGCCACTGGTGTT CCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCT CTGCCCTACTCTAGCTTGCCAGTTTTGGATGCAGTTCCCAGGTTGAGCCC GGGGATTTCACATTCAACTTAACAAACCACCTACGCGCGCTTTACGCCCA GTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCAC AGAGTTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAACAGCAAGGTATT CGCTTACTGCCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCT TCTTCACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATA TTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGT GTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTGGTGAGCC ATTACCTCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGCAAG GCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCGTC CCTTTCGAGACGTTGTCCCCCACTACCAGGCAGATTCCTAGGCATTACTC ACCCGTCCGCCGCTGAATCAGAGAGCAAGCTCTCTTCATCCGCTCGACTT GC.

[0087] The sequencing result showed that the KY240 strain had a high degree of homology greater than 99.97% with Pseudomonas graminis (FIG. 1 was comparative analysis of homology between the KY240 strain and Pseudomonas graminis).

Morphological Observation of KY240 Strain

Morphological Observation of Strain

[0088] The screened strain was inoculated onto a R2A plate and cultured at 30° C. for 2 d. The size, shape, color, gloss, viscosity, bulge shape, transparency, edge characteristics and presence or absence of spores of the colony were observed.

Morphology Observation Results of Strain

[0089] It was observed that when the KY240 strain (Pseudomonas graminis, Pseudomonas) was cultured and grown on the R2A medium for 2 d, the colony had a round shape, was yellow-green and opaque, had a smooth and moist surface, a regular edge, a halo and a convex center, and upon determination with a microscope, it had a diameter of about 2-3 μm, was round, shiny, sticky, slightly raised and had smooth and neat edges (FIG. 2, the scale length in the figure was 10 μm)

Determination of Cellulose Degrading Capability of KY240 Strain

Determination of Cellulose Degrading Capability of KY240 Strain at Different Temperatures

[0090] The KY240 strain, which had been cultured on the R2A medium for 2 d, was inoculated into a CMC medium, and Bacillus subtilis (the strain 92068) was set as a positive control. The KY240 strain and the 92068 strain were simultaneously and respectively cultured at 4° C. and 10° C. for 7 d, and cultured at 0° C. for 2 days, and then they were fumigated with an iodine solution, and determined for the diameter of a CMC degradation halo in mm.

CMC degrading capability=the diameter of the CMC degradation halo in mm+X

[0091] Note: X was a weighting coefficient, which was −1, 0, 1 and 2 accordingly according to the transparency of the degradation halo of the strain. The number 2 represented that the degradation halo was completely transparent; the number 1 represented that the degradation halo was subtransparent; and 0 represented that the degradation halo was opaque. However, there were traces of hydrolysis on the surface of the culture medium, and thus the degradation halo was basically cannot be observed by human eyes, but after the colony was washed with water, there are weak traces of hydrolysis at the place where the bacteria were inoculated, and −1 represented that there was no any hydrolysis activity. This method was also applied to test the potassium degrading capability and inorganicphosphorus degrading capability of bacteria. Determination results of cellulose degrading capability of KY240 strain

[0092] The determination results were as shown in Table 1 and FIGS. 3-5. The results showed that the cellulose degrading capability of the KY240 strain was obviously higher than that of the control Bacillus subtilis strain 92068 at 4° C. and 10° C.

TABLE-US-00002 TABLE 1 Cellulose degrading capability of strains on the CMC medium at different temperatures Cellulose degrading capability Culture 92068 (CK) KY240 temperature Bacillus subtilis Pseudomonas graminis  4° C. <5 32.2 10° C. <5 31.5 30° C. 39.6 22.8

[0093] In view of the above, Bacillus subtilis (92068) could only degrade cellulose efficiently at 30° C. Compared with Bacillus subtilis (92068), KY240 had a strong cellulose degrading capability both at a low temperature of 4° C. and a condition of 10° C., and the cellulose degrading capabilities of it were basically the same. Although the cellulose degrading capability of it was decreased at the condition of 30° C., the decrease amplitude was not large. It could be seen from this that KY240 was a microbial strain with a wide working range, which could degrade cellulose at a low temperature (4° C.-10° C.) and could also degrade cellulose stably and efficiently at 30° C.

Determination of Multifunctionality of KY240 Strain

Determination of Potassium Degrading Capability of KY240 Strain

[0094] The KY240 strain which had been cultured on the R2A medium for 2 days, was inoculated into a potassium medium (10.0 g of glucose, 0.5 g of yeast powder, 1.0 g of ammonium sulfate, 2.0 g of disodium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 1.0 g of calcium carbonate, 15.0 g of agar powder, 1.0 g of potassium feldspar, and 1,000 mL of water), and Bacillus subtilis (strain 92068) was set as a positive control. They were cultured at room temperature, the colonies were washed off with water, and the diameters of potassium degradation halos were determined in mm. potassium degrading capability=the diameter of the potassium degradation halo in mm+X

[0095] Note: X was a weighting coefficient, which was −2, −1, 0, 1 and 2 accordingly according to the transparency of the potassium degradation halo of the strain.

Determination Results of the Potassium Degrading Capability of KY240 Strain

[0096] The results were as shown in Table 2 and FIG. 6. The results showed that the potassium degrading capability of the KY240 strain was obviously higher than that of the control Bacillus subtilis strain 92068.

TABLE-US-00003 TABLE 2 Potassium degrading capability of strains on a potassium medium 92068 (CK) KY240 Strain Bacillus subtilis Pseudomonas graminis Capability of 10.7 36.2 degrading potassium

Determination of Inorganicphosphorus Degrading Capability of KY240 Strain

[0097] The KY240 strain which had been cultured on the R2A medium for 2 days, was inoculated into a inorganicphosphorus medium (10.0 g of glucose, 0.5 g of yeast extract, 0.5 g of ammonium sulfate, 0.02 g of potassium chloride, 0.02 g of sodium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.0001 g of manganese sulfate, 0.0001 g of ferric sulfate, 10.0 g of agar powder, 5.0 g of tricalcium phosphate, and 1000 mL of water), and Bacillus subtilis (strain 92068) was set as a positive control. They were cultured at room temperature, the colonies were washed off with water, and the diameters of phosphorus degradation halos were determined in mm.

inorganicphosphorus degrading capability=the diameter of the inorganicphosphorus degradation halo in mm+X

[0098] Note: X was a weighting coefficient, which was −2, −1, 0, 1 and 2 accordingly according to the transparency of the phosphorus degradation halo of the strain.

Determination Results of Inorganicphosphorus Degrading Capability of KY240 Strain

[0099] The results were as shown in Table 3 and FIG. 7. The results showed that the inorganicphosphorus degrading capability of the KY240 strain was obviously higher than that of the control Bacillus subtilis strain 92068.

TABLE-US-00004 TABLE 3 inorganicphosphorus degrading capability of strains on an inorganicphosphorus medium 92068 (CK) KY240 Strain Bacillus subtilis Pseudomonas graminis Inorganicphosphorus <5 27.2 degrading capability

Determination of Growth of KY240 Strain at 30° C./4° C.

[0100] The screened strains were inoculated into 50 mL of a R2A liquid medium (0.50 g of yeast powder, 0.50 g of peptone, 0.50 g of tryptone, 0.50 g of glucose, 0.50 g of soluble starch, 0.30 g of dipotassium hydrogen phosphate, 0.30 g of sodium pyruvate, 0.05 g of magnesium sulfate, and 1000 mL of water), and subjected to shake-flask culture at 30° C./4° C. and 200 rpm overnight. The R2A liquid medium was used as the positive control. OD.sub.600 nm of the strain was determined, and the strain was re-inoculated in 100 mL of the R2A liquid medium at the quantified OD.sub.600 nm=0.05, and the living bacteria counts of the strain at (30° C.: 4 h, 8 h, 12 h, 24 h)/(4° C.: 0 h, 24 h, 48 h, 72 h) was determined respectively. The bacterial solution at each time point was taken and diluted to a proper dilution, and then plated onto a petri dish. Each dilution was made in triplicates, and the petri dish was cultured in a 30° C. incubator to calculate the average number of colonies. A curve graph was depicted with the time/h as the abscissa and the effective colony number as the ordinate. The Bacillus subtilis strain 92068 was set as the positive control.

Result

Determination of Living Bacteria Count of KY240 Strain at 30° C.

[0101] The experimental results were as shown in FIG. 8. It could be seen from FIG. 8 that at 30° C., the number of the KY240 strain reached 38 million/mL at 12 h, and the strain 92068, which was a high-temperature growth strain, reached a peak of 120 million/mL at 8 h, which was 3 times that of the KY240 strain, and the KY240 strain could continue to grow after 12 h.

Determination of Living Bacteria Count of KY240 Strain at 4° C.

[0102] The experimental results were as shown in FIG. 9. It could be seen from FIG. 9 that the KY240 strain could grow rapidly and stably at 4° C. and reached 940 million/mL at 72 h, in contrast the strain 92068 did not grow at 72 h.

Determination of Wheat Straw Decomposition Capability of KY240 Strain at the Condition of 4° C.

[0103] Wheat straws were taken as the substrate. 3 g of them were weighed into each test tube, and each test tube was added with a small and equal amount of liquid R2A to make it wet, added with 1 mL of the bacteria solution, shaken uniformly, cultured at a low temperature of 4° C. for decomposition, and timely and appropriately irrigated with equal amount of the bacteria solution according to its growth requirements, and the decomposition of the wheat straws were observed every week. The R2A liquid medium on which no bacteria was inoculated was set as the blank control CK1, and the R2A liquid medium inoculated with the Bacillus subtilis 92068 was set as the positive control CK2.

Experimental Results of Wheat Straw Decomposition Capability of KY240 Strain at the Condition of 4° C.

[0104] The experimental results were as shown in FIG. 10. The results showed that at a low temperature (4° C.), the decomposed degree and speed of the wheat straws decomposed by the KY240 strain were significantly higher than those of the wheat straws decomposed by the Bacillus subtilis strain 92068.

[0105] Conclusion: the KY240 strain could degrade cellulose at a low temperature of 4° C., and upon sequencing analysis of the 16sDNA of the strain, the strain was highly homologous to Pseudomonas graminis. Compared with a commercial strain Bacillus subtilis 92068, the KY240 strain has stronger capability of degrading cellulose at a low temperature of 4° C. and a condition of 10° C., and the cellulose degradation capacities are basically the same. Although the cellulose degradation capacity is weakened at 30° C., the degradation amplitude is not large, and meanwhile, the KY240 strain also has potassium and inorganicphosphorus degradation capacity, which has a promotion effect on the decomposition of wheat straws, and the effect is obviously superior to that of the commercial strain 92068. Therefore, KY240 is a functional microbial strain with both a wide working range and multiple functions, which can both degrade cellulose at a low temperature (4° C.) and medium-high temperatures (10° C.-30° C.), and has the capability of degrading both potassium and inorganicphosphorus, and has a promotion effect on the decomposition of wheat straws. It is expected to be used for preparing bacterial fertilizer for degrading cellulose, promoting straw decomposition, and improving soil fertility, especially increasing the amount of potassium and phosphorus in soil available to plants.