Klebsiella and application thereof

Abstract

The invention provides a Klebsiella strain and application thereof. The Klebsiella sp. HDJT1 is screened from a mixed compost sample of sugar beet pulp, sugar beet soil and sludge, and is preserved in the China General Microbiological Culture Collection Center on December 24, 2024, and the preservation number is CGMCC NO.33184. The Klebsiella sp. HDJT1 is applied to degradation of sugar beet pulp, degradation of sugar beet soil, degradation of sugar beet pulp and degradation of sugar beet soil. Comprising the following steps: sugar beet pulp pretreatment, strain activation, fermentative degradation, degradation product detection and degradation effect analysis. The Klebsiella HDJT1 screened by the invention has the advantages that the sugar beet pulp cellulose can be efficiently degraded, the environmental adaptability is excellent, and the Klebsiella HDJT1 grows well under the culture conditions that the temperature is 30 DEG C and the pH value is 7.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a Klebsiella strain and use thereof. BACKGROUND

[0002] Cellulose resources are the largest renewable resources on earth. More than 90% of the products of photosynthesis on earth are lignocellulosic materials every year, and the efficient degradation thereof depends on the cellulase system secreted by microorganisms. However, most of the cellulose resources are only used as fuel or landfilled, which not only increases the processing cost, wastes land resources, but also pollutes the environment and leads to the loss of rich biomass resources. Cellulose is a linear high-molecular polymer composed of glucose units connected by β-1, 4 glycosidic bonds, and the molecular chain forms a highly crystalline microfibril structure through hydrogen bonds and is wrapped in a matrix composed of hemicellulose and lignin. This complex structure makes it difficult for natural cellulose to be directly utilized, and its degradation needs to break through the double restrictions of physical and chemical barriers. Compared with the high energy consumption and environmental pollution of chemical degradation method, the biological degradation method has the advantages of mild conditions, specific products, etc., but has the bottlenecks of low degradation efficiency and poor adaptability of strains.

[0003] Sugar beet pulp is an agricultural byproduct rich in cellulose, and its main components are hemicellulose 26-32%, cellulose 22-24%, uronic acid 21.5-23%, lignin 1-2%, protein 7-8%, ash 7.5-12%, and 0.5% residual sucrose. Due to the complex structure of sugar beet pulp, it is difficult to degrade, and its effective utilization is limited, so it is of important practical significance to study the function of its degrading bacteria. The research on cellulose-degrading bacteria started early at home and abroad, covering a variety of microorganisms from fungi to bacteria, revealing the diversity of enzyme system composition of degrading bacteria and its degradation mechanism, but the research on the isolation, identification and application of sugar beet pulp degrading bacteria is still insufficient. Therefore, it is of important application value to optimize the screening and culture conditions of sugar beet pulp cellulose-degrading bacteria. At present, there is a lack of efficient and adaptable cellulose-degrading bacterial strains on the market, and their application in complex environments still faces many challenges. SUMMARY

[0004] Technical problems to be solved: In view of the above technical problems, the purpose of the present application is to provide a Klebsiella and its use. The Klebsiella HDJT1 is screened from a mixed sludge composting system, which has been preserved in the China General Microbiological Culture Collection Center on December 24, 2024, with the preservation number CGMCC NO.33184, and the classification name is Klebsiella michiganensis. The endo-beta-1,4-glucanase activity and beta-glucosidase activity of the screened Klebsiella HDJT1 are relatively high, and it has a significant cellulose degradation capacity. It grows well in a near neutral environment with a temperature close to 30 DEG C and a pH value of 7.0.

[0005] Technical scheme: Klebsiella HDJT1, which has been preserved in the China General Microbiological Culture Collection Center on December 24, 2024, with the preservation number CGMCC NO.33184.

[0006] Further, the Klebsiella HDJT1 is screened from a beet residue, beet soil and sludge mixed compost sample, wherein the beet residue and beet soil are from the Hulan campus of Harbin University of Heilongjiang, the beet soil refers to the soil in the beet field which is not penetrated by the beet root system, and the five-point sampling method is used; the sludge is from the intersection of Hejiagou and Songhua River.

[0007] Further, the optimal culture temperature of the Klebsiella HDJT1 is 30±1 DEG C, the optimal culture pH value is 7.0±0.5, and the culture medium is SDY medium.

[0008] Use of Klebsiella HDJT1 in producing cellulase.

[0009] Use of Klebsiella HDJT1 in efficiently degrading beet residue.

[0010] Further, the use of the Klebsiella HDJT1 in efficiently degrading beet residue includes but is not limited to producing soluble reducing sugar and biological hydrogen.

[0011] Use of Klebsiella HDJT1 in improving soil nutrients.

[0012] Further, the use of the high-efficiency cellulose-degrading strain in efficiently degrading beet residue includes the following steps: S1. Crushing or cooking treatment of the desugared beet residue to increase its surface area or change its physical and chemical properties, so as to facilitate better degradation of cellulose by the strain; S2. Inoculating the Klebsiella HDJT1 strain into the SDY medium for activation culture, so as to restore its activity and proliferation, adjust the concentration of the bacterial liquid, and obtain the activated Klebsiella HDJT1 bacterial liquid; S3. The activated Klebsiella HDJT1 bacterial solution is inoculated into the inorganic salt liquid medium containing sugar beet residues, and fermentation degradation is carried out at a temperature of 30 DEG C and a pH value of 7.0, and stirring is carried out during the fermentation process to promote the growth and cellulose degradation of the strain.

[0013] Further, the SDY medium formula in the step S2 is peptone 10 g / L, yeast powder 10 g / L, and glucose 40 g / L.

[0014] Further, the activated Klebsiella HDJT1 bacterial solution concentration in the step S2 is OD 600 =1.0.

[0015] Further, the activated Klebsiella HDJT1 bacterial solution in the step S3 is 2% of the volume of the inorganic salt liquid medium containing sugar beet residues, that is, 1 mL of the activated bacterial solution treated by the step S2 is added into 50 mL of the inorganic salt medium.

[0016] Further, the fermentation medium containing sugar beet residues in the step S3 is an inorganic salt liquid medium, and the formula is potassium phosphate dibasic trihydrate (K2HPO4·3H2O) 1.0 g / L, sodium chloride (NaCl) 1.0 g / L, ammonium chloride (NH4Cl) 1.0 g / L, ferric chloride hexahydrate (FeCl3·6H2O) 0.01 g / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.4 g, anhydrous calcium chloride (CaCl2) 0.075 g, and 0.5 g of the sugar beet residues treated by the step S1 is added into 50 mL of the inorganic salt liquid medium.

[0017] Further, the improvement of soil nutrients mainly includes the improvement of soil alkali-hydrolyzable nitrogen and effective sulfur content. Beneficial effects

[0018] The Klebsiella HDJT1 strain adopted in the present application is screened from a mixed sludge compost sample, and has been preserved in the China General Microbiological Culture Collection Center on December 24, 2024, with a preservation number of CGMCC NO.33184, has the characteristics of high-efficiency degradation of sugar beet residue cellulose and easy cultivation, and grows well in a suitable temperature near 30 DEG C and a near-neutral environment with a pH value of 7.0.

[0019] The Klebsiella HDJT1 strain screened in the present application has the biosynthesis ability of alcohol and organic acid such as ethanol, acetic acid and lactic acid, and has certain potential in carbohydrate metabolism.

[0020] The Klebsiella HDJT1 strain can produce nitrogen fixation and dissimilatory nitrate reduction related genes, and can effectively improve the nitrogen cycle of soil microorganisms and the alkali-hydrolyzable nitrogen content.

[0021] Klebsiella HDJT1 also has the function of assimilating sulfate and participating in the biosynthesis of sulfur-containing amino acids (such as cysteine and methionine), which plays an important role in improving soil sulfur availability. Klebsiella helps the circulation of sulfur compounds in the soil by reducing sulfate to sulfide and incorporating it into these amino acids. This process not only replenishes the sulfur pool, but also makes sulfur more easily absorbed by plants and other soil organisms, thereby increasing the total sulfur content in the soil ecosystem. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A phylogenetic tree of strain HDJT1; Figure 2 Effects of different temperatures on the growth of Klebsiella HDJT1; Figure 3 Effects of different pH values on the growth of Klebsiella HDJT1; Figure 4 Biological function property determination results of Klebsiella HDJT1, wherein figure (a) is a carboxymethylcellulose sodium-congo red solid culture medium plate; figure (b) is an SDY solid culture medium plate; and figure (c) is a crystal violet staining body type microscopic effect drawing; Figure 5 Bioinformatics prediction of carbon, nitrogen and sulfur metabolic pathways of Klebsiella HDJT1, wherein figure (a) is a potential regulatory network of carbon metabolism; figure (b) is a potential regulatory network of nitrogen metabolism; and figure (c) is a potential regulatory network of sulfur metabolism pathway, and the size of the circle represents the relative abundance of each metabolic pathway; Figure 6 A figure showing the amount of hydrogen produced per liter of fermentation broth by Klebsiella HDJT1 at different time points under different amounts of sugar beet residue added in Example 5; Figure 7 A figure showing the alkali hydrolysis nitrogen content in the soil attached to the surface of the blocky sugar beet residue after 7 days of enzymatic hydrolysis by Klebsiella HDJT1 in Example 5; Figure 8 A figure showing the production of available sulfur accompanied by the reduction of sulfide during the degradation of cellulose by Klebsiella HDJT1 in Example 5; wherein figure (a) is a figure showing the reduction of sulfide / accumulation of available sulfur accompanied by the enzymatic hydrolysis of carboxymethylcellulose sodium by HDJT1; and figure (b) is a figure showing the reduction of sulfide / accumulation of available sulfur accompanied by the enzymatic hydrolysis of salicylic acid by HDJT1. DETAILED DESCRIPTION

[0023] The present application proposes a Klebsiella strain and its use. In order to make the purpose, technical scheme and effect of the present application more clear and explicit, the present application will be further described in detail below in conjunction with examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. Example 1

[0024] Strain screening and identification, including the following steps: S1. Screening of strains: Take 1 g of soil sample and place it in a test tube containing 300 mL of enrichment medium, mix well, and incubate in a constant temperature shaking incubator at 32°C and 120 r / min for 3 days. Dilute the bacterial solution with sterile water, take 50 μL of the bacterial solution at dilutions of 10 -3 , 10 -4 , 10 -5 , and plate them on the screening medium, with 3 parallel plates for each dilution. Incubate in a constant temperature incubator at 37°C for 3 days, until obvious colonies grow. Pick single colonies with obvious differences in characteristics, and plate them on the screening medium (CMC-Na-Congo red plate medium) using the plate streak method, and continue incubation in a constant temperature incubator at 37°C. Repeat this step until pure bacteria are obtained.

[0025] The formulations of the enrichment medium and the screening medium are shown in Table 1. Each medium is made up to 1 L with deionized water, stirred and boiled for more than 1 min to completely dissolve, divided into test tubes, autoclaved at 121°C for 15 min, and shaken when the medium cools to 50°C. Pour the plates and store at 25°C for use;

[0026] S2. Identification of strains: Inoculate strain HDJT1 in liquid LB medium and incubate in a constant temperature incubator at 32°C for 12-24 h to the logarithmic growth phase. Centrifuge the bacterial solution at 5000 rpm for 5 min, discard the supernatant, and collect the bacterial pellet. Resuspend the bacterial pellet in CTAB lysis buffer and add proteinase K, and incubate in a 70°C water bath for 1 h to lyse the cells. Add anhydrous ethanol and isopropanol, mix well, and let stand for 15 min to precipitate the DNA. Carefully pipette the supernatant, transfer the precipitate to a new centrifuge tube, add sodium chloride solution, and let stand at room temperature for 10 min. Add anhydrous ethanol again, centrifuge to collect the DNA precipitate, wash with 70% ethanol, air dry, and dissolve in sterile water to obtain genomic DNA. Use universal primers for bacterial 16S rDNA (27F, 1492R) to send the purified PCR product to Beijing Liuhewadake Gene Technology Co., Ltd. for bidirectional sequencing.

[0027] Compare the obtained sequencing results with the NCBI Blast tool, search the NCBI 16S rRNA database, and determine the phylogenetic relationship and taxonomic status of the strain Figure 1Based on the comparison results, the genus of strain HDJT1 was determined, and homology analysis with known sequences confirmed that it belongs to the genus Klebsiella. The screened strain was inoculated onto slant agar and stored at 4°C, or preserved long-term using the glycerol tube storage method.

[0028]

[0029] Table 2 shows that, through BLAST comparison, Klebsiella HDJT1 and Klebsiella grimontii The 16S rRNA sequence showed high homology (99.725%) with... Klebsiella oxytoca The sequence homology was 98.698-99.18, and... Klebsiella aerogenes The sequence homology was 98.627–98.766, indicating that HDJT1 shares extremely high similarity with these strains in the 16S rRNA sequence. Therefore, this strain was named Klebsiella HDJT1. Klebsiella sp. HDJT1), whose gene sequence has been uploaded to NCBI GenBank, accession number PQ774857.1. Example 2

[0030] The culture of Klebsiella HDJT1 strain includes the following steps: Single colonies were picked, diluted with sterile deionized water to form a suspension, and then evenly inoculated into fresh SDY liquid medium. The culture was then incubated at different temperatures (30℃, 35℃, 40℃, 45℃, and 50℃) for 48 h, with OD values ​​measured every 12 h. 600 . Example 3

[0031] The culture of Klebsiella HDJT1 strain includes the following steps: Single colonies were picked and inoculated into fresh SDY liquid medium, and activated by shaking at 30°C for 24 h. The activated bacteria were then inoculated into SDY liquid medium at different pH values ​​at a volume ratio of 0.2%. The pH was adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0 with sulfuric acid and sodium hydroxide, respectively, and incubated at 30°C for 48 h. The OD of the bacterial culture was measured every 12 h. 600 . Example 4

[0032] The bacteria liquid with OD600=1 was inoculated into the SDY medium at a volume ratio of 2% for activation culture at 30°C, 120 rpm for 72 h. The activated bacteria liquid was inoculated into the enzyme production medium at a volume ratio of 2% for enzyme production fermentation in the enzyme production medium at 30°C, 120 rpm for 120 h. The fermentation liquid was centrifuged in a 4°C 5000 rpm centrifuge for 10 min, and the supernatant was the crude enzyme liquid for enzyme activity determination. Performance test:

[0033] (1) Cellulase production performance observation An enrichment medium solid plate containing 1.5% agar was prepared, sterilized, and poured into a sterile plate while hot and cooled to solidify. The Klebsiella HDJT1 strain was inoculated into the SDY liquid medium and cultured at 30°C for 24 h for activation. The activated bacteria liquid was diluted to 10 5 CFU / mL, and uniformly coated on the screening medium plate using a sterile spreader. The inoculated plate was placed in a 30°C constant temperature incubator and cultured for 72 h. After the culture ended, Congo red stain (0.1% w / v) was added for staining for 15 min, the excess dye was poured off, 1M NaCl solution was added for decolorization for 15 min, the NaCl solution was gently poured off, and the formation of transparent circles around the colonies was observed.

[0034] (2) Colony characteristic observation: An SDY solid medium containing 1.5% agar was prepared, sterilized, and poured into a sterile plate and cooled to solidify. The Klebsiella HDJT1 strain was inoculated into the liquid medium and cultured at 32°C for 24 h for activation. The activated bacteria liquid was diluted to 10 5 CFU / mL, and uniformly coated on the SDY solid medium using a sterile spreader. The inoculated plate was placed in a 32°C constant temperature incubator and cultured for 24 h. After the culture ended, the morphology, color, and size of the colonies were observed.

[0035] (3) Biofilm formation ability observation: An LB liquid medium was prepared, sterilized, and divided into sterile test tubes. The Klebsiella HDJT1 strain was inoculated into the SDY liquid medium and cultured at 32°C for 24 h for activation. The activated bacteria liquid was diluted to 10 5CFU / mL, inoculated into LB liquid medium, 32°C for 72 h, without shaking the medium during the culture, so that the strains adhere to the test tube wall to form a biofilm. After the end of the culture, the supernatant was gently poured off, and the test tube wall was washed twice with sterile distilled water to remove unattached bacteria. Crystal violet staining agent (0.1% w / v) was added for 15 min. Excess dye was discarded, and the test tube wall was gently washed with sterile distilled water until the washing liquid was free of purple, removing unbound dye. Drying at 60°C, observing the formation of biofilm on the test tube wall under a body lens, and taking a photo record.

[0036] (4) Functional genomics analysis The functional genes of Klebsiella HDJT1 were predicted using the PICRUSt2 tool to analyze its metabolic potential and environmental adaptability. The genomic data of Klebsiella HDJT1 were imported into the KEGG database to predict its carbon, nitrogen, and sulfur metabolic pathways to reveal its carbon, nitrogen, and sulfur metabolic pathways and metabolic potential, including the biosynthetic ability of ethanol, acetic acid, and lactic acid, nitrogen fixation characteristics, dissimilatory nitrate reduction ability, and assimilatory sulfate reduction (ASR) function.

[0037] From Figure 2 It can be seen that Klebsiella HDJT1 has high activity at 30°C, and the number of viable bacteria can reach 8.15×107 CFU / mL after 24 h; however, when the temperature reaches 45°C, the activity of the strain is significantly reduced, and the number of viable bacteria after 24 h is only 1.35×107 CFU / mL. This indicates that Klebsiella HDJT1 does not have good heat resistance, and its growth and reproduction are significantly inhibited by the increase in temperature.

[0038] From Figure 3 It can be seen that the number of viable bacteria after 24 h can reach 6.37×10 7 CFU / mL at pH=7, the number of viable bacteria after 24 h can reach 5.95×10 7 CFU / mL at pH=9. However, the number of viable bacteria after 24 h is only 6.75×10 6 CFU / mL at pH=5, indicating that Klebsiella HDJT1 strain has certain alkali resistance, but poor acid resistance.

[0039] From Figure 4 (a) It can be seen that the formation of transparent circle indicates that Klebsiella HDJT1 can secrete cellulase to degrade CMC-Na. The diameter of the transparent circle is positively correlated with the cellulose degradation ability of the strain, and Klebsiella HDJT1 forms a clear transparent circle on the CMC-Na-congo red plate, indicating that it has strong cellulose degradation enzyme activity. From Figure 4(b) It can be seen that Klebsiella HDJT1 forms milky white, slimy colonies on SDY solid medium, with a colony diameter of 0.8-1.2 mm and irregular colony edges. From Figure 4 (c) It can be seen that Klebsiella HDJT1 can effectively form biofilms.

[0040] Figure 5 (a) represents the potential regulatory network of carbon metabolism. Figure 5 (b) represents a potential regulatory network for nitrogen metabolism. Figure 5 (c) shows the potential regulatory network of sulfur metabolism pathways. Predictions of the KEGG metabolic pathway using PICRUSt2 revealed the presence of carbohydrate fermentation-related genes in the Klebsiella HDJT1 genome, including alcohol dehydrogenase (ADH, EC 1.1.1.1), acetate kinase (ACK, EC 2.7.2.1), phosphate pyruvate carboxylase (PEPC, EC 4.1.1.31), and lactate dehydrogenase (LDH, EC 1.1.1.27). Simultaneously, the Klebsiella HDJT1 genome also contains a nitrogenase-encoding gene (…). nifH , nifD , nifK This indicates that it has nitrogen-fixing ability. The strain also carries a nitrate reductase-related gene (…). narG , narH , narI ) and nitrite reductase ( nirB , nirD The related genes indicate that they can reduce nitrate to ammonia, which is beneficial for intracellular amino acid synthesis.

[0041] In addition, the Klebsiella HDJT1 genome contains assimilation and dissimilation sulfate reduction (ASR and DSR) pathways, which may be accompanied by sulfur metabolism during cellulose degradation. For example, effective sulfur components such as sulfate ions in the environment are transferred into the cell and further participate in the biosynthesis of sulfur-containing amino acids such as cysteine, thereby promoting the production and excretion of cellulase. Example 5

[0042] The application and performance testing of Klebsiella HDJT1 in improving soil nutrients: (1) HDJT1 produces biohydrogen during the enzymatic hydrolysis of beet pulp.

[0043] All batches of fermentation used 250 mL serum bottles, with a working volume of 220 mL for anaerobic fermentation under nitrogen aeration. Before aeration and culture medium sterilization, different concentrations of pulverized, desaccharified beet pulp (passed through a 40-mesh sieve) were added to the serum bottles as a carbon source. After inoculation with Klebsiella HDJT1 (10%, v / v), fermentation was carried out in a shaking incubator (30℃, 125 rpm) for 48 h. Gas samples were collected every 6 or 12 hours by transferring the gas from the top space of the bottle into a gas sampling bag. The hydrogen-producing fermentation medium formulation was: potassium dihydrogen phosphate (KH2PO4) 1.5 g / L; dipotassium hydrogen phosphate (K2HPO4) 3.93 g / L; magnesium chloride (MgCl2) 1.0 g / L; anhydrous calcium chloride (CaCl2) 0.1 g / L; ammonium sulfate ((NH4)2SO4) 1.3 g / L; yeast extract 4 g / L; cysteine ​​0.5 g / L; resazurin 1 mg / L. The hydrogen produced during fermentation was measured using a CCCC Gold Source GC-7920 gas chromatographic analyzer. The gas chromatographic conditions were: TCD detector, high-purity argon as carrier gas, and three repetitions at each time point.

[0044] from Figure 6 It can be seen that when the substrate addition is 10g beet pulp per liter of fermentation broth, the hydrogen production per unit time is the largest and the hydrogen production effect is long-lasting. Within 48 hours, the hydrogen production per liter of fermentation broth reaches as high as 1170.3 mmol, indicating that Klebsiella HDJT1 has the function of enzymatically hydrolyzing beet pulp to produce biohydrogen. (2) HDJT1 converts organic nitrogen to inorganic nitrogen during the enzymatic hydrolysis of beet pulp.

[0045] One gram of desaccharified beet pulp was buried in 100 grams of sterilized soil with a moisture content of 60%. 10 mL of OD was added to each treatment group. 600 =1 (to ensure the bacterial concentration is 10) 8 A bacterial suspension (CFU / mL) was prepared, and an equal volume of deionized water was added to the control group. After 7 days of degradation, the available nitrogen content in the soil surrounding the beet residue was measured in both the control and bacterial treatment groups, with six replicates. The determination of available nitrogen content in the soil was carried out in accordance with Sichuan Provincial Standard DB51 / T 1975-2014.

[0046] from Figure 7 It can be seen that the content of available nitrogen in the soil of the bacterial treatment group increased by 28.6% compared with the control. (3) HDJT1 reduces the effective sulfur content of sulfides while degrading cellulose.

[0047] In each 9.5 mL of tenth inorganic salt medium (containing 5.25 mg / L of effective sulfur concentration) with 1 g / L sodium carboxymethyl cellulose or salicylic acid as the sole carbon source, 0.5 mL of 0.1 M sodium sulfide standard solution was added to make the initial sulfur ion concentration reach 5 mol / L. The inoculation amount of the inoculation treatment group was 100 μL of OD600=1 (to ensure the bacterial concentration of 10 8 CFU / mL), and the same volume of deionized water was added to the control group. The sulfide and effective sulfur concentrations were measured every 12 h, and each time point was repeated 3 times. The sulfide was determined by the methylene blue spectrophotometry of the national standard GB16489-1996, and the effective sulfur was determined by the barium sulfate turbidimetry shown in the agricultural industry standard NY / T 1121.14-2023.

[0048] From Figure 8 It can be seen that Klebsiella HDJT1 has a very high efficiency of reducing sulfide regardless of using sodium carboxymethyl cellulose and salicylic acid as the sole carbon source. When using sodium carboxymethyl cellulose as the sole carbon source, the reduction of sulfide is accompanied by an increase in the content of effective sulfur. When salicylic acid is used as the sole carbon source, the effective sulfur concentration shows a trend of first increasing and then decreasing, which may be due to the functions of assimilating sulfate and participating in the biosynthesis of sulfur-containing amino acids (such as cysteine and methionine). Example 6

[0049] The application of Klebsiella HDJT1 in degrading sugar beet residues includes the following steps: The precipitated bacterial cells after enzyme production fermentation were mixed with the supernatant crude enzyme solution, and adjusted to OD 600 =1 (to ensure the bacterial concentration of 10 8 CFU / mL), 10 mL of which was uniformly sprayed on 100 g of sterilized soil as the treatment group soil. The control group soil was directly sprayed with 10 mL of supernatant crude enzyme solution uniformly on 100 g of sterilized soil. A certain amount of 1.0 g of dried and sugar-removed sugar beet residues was accurately weighed and placed in a 6 cm × 8 cm small nylon bag. Each 1 nylon bag containing dried and sugar-removed sugar beet residues was completely immersed in every 100 g of treatment group soil or control group soil, and each treatment group had 6 replicates. After sealing, it was placed in a constant temperature condition of 30℃ for 7 d of degradation.

[0050] Performance determination: (1) Corrected weight loss rate determination: After the degradation of sugar beet residues was completed and dried, the weight of the remaining residues in each nylon bag was weighed, and the corrected weight loss rate was calculated to evaluate the effect of bacterial addition on the degradation of sugar beet residues. The corrected weight loss rate calculation formula is:

[0051] The average weight loss rate of the control group with only crude enzyme solution was 40.82%, and the average weight loss rate of the group with bacterial treatment was 48.49%. The corrected weight loss rate was 12.97%, indicating that the addition of 10 7 CFU·g -1 The addition of bacterial bodies can continue to promote the degradation of sugar beet residues based on the crude enzyme solution, increasing the degradation efficiency of sugar beet residues by 12.97%. This may be related to the specific metabolic pathways of the strain.

[0052] (2) Determination of cellulose content in the remaining residues after 7 days of sugar beet residue degradation: After degradation, the sugar beet residues were dried and crushed, then passed through a 30-mesh sieve to ensure uniform particle size. Accurately weigh 0.10 g of the sugar beet residue sample and place it in a dry test tube. Add 5 mL of a mixture of acetic acid and nitric acid (volume ratio 1:1) to the test tube.

[0053] Cover the spherical glass plug and place the test tube in a boiling water bath for 25 minutes, stirring constantly to ensure complete decomposition of the sample. After cooling to room temperature, centrifuge and discard the supernatant, retaining the precipitate. Rinse the precipitate with distilled water three times, centrifuging after each rinse to ensure complete removal of residual acid. Add 10 mL of 10% sulfuric acid and 10 mL of 0.1 mol / L potassium dichromate solution to the treated precipitate and shake well. Place the test tube in a boiling water bath again for 10 minutes to promote the oxidative decomposition of cellulose.

[0054] After heating, pour the contents of the test tube into a triangular flask and rinse the test tube with distilled water three times to ensure that all reactants are transferred to the triangular flask. After the solution cools, add 5 mL of 20% potassium iodide (KI) solution to the triangular flask and shake well.

[0055] Add 1 mL of 0.5% starch solution to make the solution blue. Titrate with 0.2 mol / L anhydrous sodium thiosulfate (Na2S2O3) solution until the blue color disappears, and record the titration volume V x (mL). Prepare a blank sample by adding 10 mL of 10% sulfuric acid and 10 mL of 0.1 mol / L potassium dichromate solution without adding the sugar beet residue sample, and record the blank titration volume V0 (mL). The cellulose content calculation formula for the remaining residues after sugar beet residue degradation is:

[0056] According to the calculation, the cellulose content in the remaining residues of the control group with only crude enzyme solution was 21.73%, while the cellulose content in the treatment group with the addition of HDJT1 bacterial bodies was 16.12%, indicating that the addition of Klebsiella HDJT1 not only accelerated the degradation of sugar beet residues, but also made the degradation of cellulose more complete.

[0057] (3) Cellulase activity test: (3.1) Determination of endo-type β-glucanase (CMCase) enzyme activity Prepare 1% sodium carboxymethyl cellulose solution as substrate, take 1 mL of 1% methyl cellulose sodium solution, add 0.5 mL of citric acid buffer with pH = 4.5, add 0.5 mL of enzyme solution, and put the mixture into a 50°C water bath for reaction for 30 min. After the reaction is completed, add 1.5 mL of DNS reagent, shake the tubes well. Put the test tubes in a boiling water bath for 5 min, then take out the test tubes and cool them to room temperature. Use distilled water to dilute the solution in the test tube to 20 mL, heat and dissolve, and mix well. The blank group: do not perform 50°C water bath, directly add DNS reagent to inactivate the enzyme activity, and the other steps are the same as the test group. Use a spectrophotometer to measure the OD 540 value of the solution.

[0058] (3.2) Determination of filter paper enzyme activity (FPA) Take 50 mg (about 1x60 cm) of enzyme-free filter paper (DEPC water de-enzyme treated) as substrate, add 1.5 mL of citric acid buffer with pH = 4.5; add 0.5 mL of enzyme solution to the substrate, and put the mixture into a 50°C water bath for reaction for 60 min. After the reaction is completed, follow the CMC enzyme treatment method to perform the same treatment steps. The blank group: do not perform 50°C water bath, directly add DNS reagent to inactivate the enzyme activity, and the other steps are the same as the test group. Use a spectrophotometer to measure the OD 540 value of the solution; calculate the enzyme activity according to the enzyme activity unit of 1 μg of glucose generated per minute.

[0059] The enzyme activity calculation formula is as follows:

[0060] (3.3) Determination of exo-type β-glucanase activity Add 1% microcrystalline cellulose substrate solution to the test tube, add 0.5 mL of buffer, add 0.5 mL of enzyme solution to the test tube, put the mixture into a 50°C water bath for reaction for 30 min, and after the reaction is completed, follow the CMC enzyme treatment method to perform the same treatment steps. The blank group: do not perform 50°C water bath, directly add DNS reagent to inactivate the enzyme activity, and the other steps are the same as the test group. Use a spectrophotometer to measure the OD 540 value of the solution; calculate the enzyme activity according to the enzyme activity unit of 1 μg of glucose generated per minute.

[0061] (3.4) Determination of β-glucosidase activity Take 10 mL of 1% salicylic acid solution as the substrate, add 0.5 mL of buffer; add 0.5 mL of enzyme solution to the substrate, and put the mixture into a 50°C water bath for 30 min; after the reaction is completed, the same treatment steps are performed according to the CMC enzyme treatment method. Blank group: do not perform 50°C water bath, directly add DNS reagent to inactivate the enzyme activity, and the other steps are the same as the test group. The OD value of the solution is measured by a spectrophotometer; and the enzyme activity is calculated according to the enzyme activity unit of 1 μg of glucose generated per minute. 540

[0062]

[0063] As can be seen from Table 4, the endo-type β-glucanase activity of the crude enzyme solution of Klebsiella HDJT1 reaches 39.85 U / mL, the filter paper enzyme activity is 20.87 U / mL, the exo-type β-glucanase activity is 63.29 U / mL, and the β-glucosidase activity reaches 8.87 U / mL.

[0064] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification of the above embodiments according to the technical essence of the present application, which does not depart from the technical solutions of the present application, still belongs to the protection scope of the technical solutions of the present application.​

Claims

1. Klebsiella HDJT1 was deposited at the China General Microbiological Culture Collection Center on December 24, 2024, with accession number CGMCC NO.33184.

2. The Klebsiella HDJT1 according to claim 1, characterized in that, The optimal culture temperature for Klebsiella HDJT1 is 30±1℃, and the optimal culture pH is 7.0±0.

5.

3. The use of Klebsiella HDJT1 according to any one of claims 1-2 in cellulase production.

4. The use of Klebsiella HDJT1 according to any one of claims 1-2 in the efficient degradation of beet pulp.

5. The use of Klebsiella HDJT1 according to any one of claims 1-2 in improving soil nutrients.

6. The use according to claim 4, characterized in that, Includes the following steps: S1. Crush or cook the sugar-reduced beet pulp; S2. Klebsiella HDJT1 strain was inoculated into SDY medium for activation culture to restore its activity and proliferate. The bacterial concentration was adjusted to obtain activated Klebsiella HDJT1 bacterial solution. S3. Klebsiella HDJT1 activated bacterial solution was inoculated into an inorganic salt liquid culture medium containing beet pulp and fermented at 30℃ and pH 7.0, with stirring during the fermentation process.

7. The use according to claim 6, characterized in that, In step S2, the concentration of the activated Klebsiella HDJT1 bacterial solution is OD. 600 =1.

0.

8. The use according to claim 6, characterized in that, In step S3, the activated Klebsiella HDJT1 bacterial solution is 2% of the volume of an inorganic salt liquid culture medium containing beet pulp.

9. The use according to claim 6, characterized in that, The fermentation medium containing beet pulp in step S3 is an inorganic salt liquid medium with the following formula: 1.0 g / L dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O), 1.0 g / L sodium chloride (NaCl), 1.0 g / L ammonium chloride (NH4Cl), 0.01 g / L ferric chloride hexahydrate (FeCl3·6H2O), 0.4 g magnesium sulfate heptahydrate (MgSO4·7H2O), and 0.075 g anhydrous calcium chloride (CaCl2).

10. The use according to claim 5, characterized in that, The improvement of soil nutrients mainly includes increasing the content of available nitrogen and available sulfur in the soil.

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