Landfill leachate degrading bacterium S14

Abstract

The invention discloses a landfill leachate degrading bacterium S14 and belongs to the technical field of microorganisms, the landfill leachate degrading bacterium is bacillus subtilis and is preserved in the China Center for Type Culture Collection, the address of the preservation unit is Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC M 2025283. Preferably, the bacterial colony is grey white, approximately circular and opaque, and wrinkles are formed in the middle of the bacterial colony. Preferably, gram staining is positive, and thalli are slender and rod-shaped. According to the invention, the S14 strain can effectively degrade main pollutants in the landfill leachate, including COD (Chemical Oxygen Demand), ammonia nitrogen and total phosphorus. In an experiment, the removal rates of the S14 strain on ammonia nitrogen, COD (Chemical Oxygen Demand) and total phosphorus respectively reach 88.84%, 55.88% and 82.68%.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a landfill leachate-degrading bacterium S14. Background Art

[0002] As an efficient and environmentally friendly treatment technology, biological leachate treatment has attracted widespread attention and application due to its unique advantages. Therefore, biological leachate treatment has become an advanced technology widely adopted by countries around the world.

[0003] However, biological leachate treatment still faces challenges. Currently, the number of biodegrading bacteria that can be isolated and identified is relatively small, and regional variations in degrading bacteria species limit the widespread application of biodegradation technology. Furthermore, different degrading bacteria have varying requirements for temperature, pH, and nutrients, necessitating further optimization of degradation conditions to improve efficiency and stability. To this end, a leachate-degrading bacterium, S14, was proposed.

[0004] The above information disclosed in this background technology is only for enhancing understanding of the background technology of the present invention and therefore it may contain information that does not constitute the prior art that is already known to a person of ordinary skill in the art. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art, such as the limited number of degradation bacteria and insufficient optimization of degradation conditions. To this end, one object of the present invention is to provide a landfill leachate-degrading bacterium S14.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A landfill leachate-degrading bacterium S14, the leachate-degrading bacterium is Bacillus subtilis S14, which is deposited in the China Center for Type Culture Collection, the depository address of which is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit number is: CCTCC M 2025283, and the deposit date is February 24, 2025.

[0008] Preferably, the colony is off-white, approximately round, opaque, and has wrinkles in the middle.

[0009] Preferably, the Gram stain is positive and the bacteria are slender and rod-shaped.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] In the present invention, the S14 strain can effectively degrade the main pollutants in landfill leachate, including COD, ammonia nitrogen and total phosphorus. In experiments, the S14 strain achieved removal rates of 88.84%, 55.88% and 82.68% for ammonia nitrogen, COD and total phosphorus, respectively.

[0012] In the present invention, the S14 strain was isolated from the leachate of a local landfill in Xinjiang, so it can adapt to the local climate and environmental characteristics and has good local adaptability. Compared with physical and chemical treatment methods, the biological method for treating landfill leachate is low in cost, and the secondary pollution generated during the treatment process is extremely small, which meets the requirements of green environmental protection. The S14 strain can not only degrade organic pollutants, but also participate in the metabolic processes of nitrogen and phosphorus, which shows that it has multiple environmental purification functions. The S14 strain can be used for the treatment of landfill leachate, deodorization and purification of the ecological environment, and has broad application prospects.

[0013] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a bacterial colony morphology diagram of the present invention;

[0015] Figure 2 is the Gram staining image of Bacillus subtilis s14 in the present invention;

[0016] Figure 3 is a growth curve of Bacillus subtilis s14 in the present invention;

[0017] Figure 4 This is a phylogenetic tree diagram of Bacillus subtilis s1416s rDNA in the present invention;

[0018] Figure 5 The whole genome map of Bacillus subtilis s14 in the present invention;

[0019] Figure 6The figure shows the COG cluster analysis of Bacillus subtilis s14 in the present invention; wherein, A: RNA processing and modification; B: chromatin structure and dynamics; C: energy generation and conversion; D: cell cycle control of cell division and chromosome division; E: amino acid transport and metabolism; F: nucleotide transport and metabolism; G: carbohydrate transport and metabolism; H: coenzyme transport and metabolism; I: lipid transport and metabolism; J: translation, ribosome structure and biogenesis; K: transcription; L: replication, recombination and repair; M: cell wall / membrane / envelope biogenesis; N: cell activity; O: post-translational modification, protein turnover, molecular chaperone; P: transport and metabolism of inorganic ions; Q: biosynthesis, transport and catabolism of secondary metabolites; R: general function prediction; S: unknown function; T: signal transduction mechanism; U: intracellular transport, secretion and vesicle transport; V: defense mechanism; W: extracellular structure; Y: nuclear structure; Z: cytoskeleton.

[0020] Figure 7 GO cluster analysis diagram of Bacillus subtilis s14 in the present invention;

[0021] Figure 8 is the KEGG functional classification diagram of Bacillus subtilis s14 in the present invention;

[0022] Figure 9 The diagram shows the classification of carbohydrate-related enzymes of Bacillus subtilis s14 in the present invention; wherein AA is an oxidoreductase, CE is a carbohydrate esterase, GH is a glycoside hydrolase, GT is a glycosyltransferase, and PL is a polysaccharide lyase. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] Example 1, about culture medium:

[0025] Protein decomposition medium (for culturing protein-decomposing strains): glucose 0.1 g, peptone 1 g, casein 0.5 g, agar 1.5 g, L-tyrosine 0.001 g, distilled water 100 ml, NaCl 0.5 g, CaCl2 0.1 g (Qingdao Rishui Biotechnology Co., Ltd., 250 g / bottle).

[0026] Czapek medium (for fungal culture): 1 g potassium hydrogen phosphate, 3 g sodium nitrate, 0.5 g sodium chloride, 2 g agar, 0.5 g ferrous sulfate, 1000 ml DH2O, 0.7 g MgSO4, 0.5 g H2O (Beijing Youkangjiye Biotechnology Co., Ltd., 250 g / bottle, batch number 20102223).

[0027] Starch culture medium (for cultivating starch-decomposing strains): 1 g of peptone, 0.3 g of beef extract, 2 g of soluble starch, 1.5 g of agar powder, 100 ml of distilled water, pH = 7.2-7.4.

[0028] The culture medium for bacterial suspension preparation includes the following ingredients: peptone 40.0 g, beef extract 12.0 g, NaCl 20.0 g, pH adjusted to 7.0-7.2, distilled water 4000 ml, and high pressure at 121°C for 15 min.

[0029] Experimental instruments include:

[0030] Shanghai Jinghong Experimental Equipment Co., Ltd.: MM-2 ordinary optical microscope; Jiangsu Jintan Medical Instrument Factory: SHZ-82A gas bath constant temperature shaker; Millipore Ireland BVSLGP-033RS: Millex-GP filter; Zhejiang Gongdong Medical Plastic Factory: Q / HGS 005-2004 disposable incubator; Shanghai Jinghong Experimental Equipment Co., Ltd.: GNP-9080 water-proof constant temperature incubator; Shanghai Zhiyu Analytical Instrument Manufacturing Co., Ltd.: ZHJH-C1109B clean bench; Shanghai Anting Scientific Instrument Factory: TGL-16G desktop centrifuge; Bio-Rad Corporation, USA: PCR instrument; Bio-Rad Corporation, USA: Gel-Doc 2000 gel imager; Lanzhou Lianhua Technology: COD tester; Guohua Electric Co., Ltd.: BS-IE oscillating incubator; Lanzhou Lianhua Environmental Protection Technology Co., Ltd.: 5B-6C multi-parameter water quality analyzer; Shanghai Shen'an Medical Equipment Factory: LDZX-50KB vertical pressure steam sterilizer.

[0031] Experimental methods and steps:

[0032] 1. Sample collection: Leachate from the Dongshan landfill in Urumqi was collected using a sterile method.

[0033] 2. Isolation and culture of landfill leachate-degrading bacteria: Take 1ml + 9ml DH2O of landfill leachate and dilute them in series. The dilution concentrations are 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, and 10-7, respectively. Pipette 0.1ml of the diluted sample and add it to 9cm culture medium (Gao's, Czapek, tiger red, starch, protein). Spread it evenly with an "L" stick and culture it in a 37℃ incubator for 18h-24h. Then continue to culture the obtained bacterial colony until a single colony is isolated and sealed for storage for acclimation and screening.

[0034] 3. Screening of dominant degradation strains: First, prepare sterile enhanced leachate. The collected landfill leachate is processed through natural sedimentation, centrifugation, and a bacterial filter with a pore size of 0.22 μm to obtain a sterile filtrate, which is then sealed and stored in a 4°C refrigerator for later use.

[0035] Subsequently, the dominant degrading bacteria were domesticated by gradually increasing the leachate concentration (to improve tolerance and degradation capacity to high-concentration leachate). A certain amount of environmental samples containing bacterial species were suspended in a sterilized 250 mL Erlenmeyer flask containing glass beads and fully shaken for about 30 minutes to disperse the microbial cells and form a uniform bacterial suspension. 10 mL of the bacterial suspension was taken and added to a conical flask containing 100 mL of enrichment liquid culture medium. The shaker was shaken and cultured at a speed of 180 r / min. The concentration (volume fraction) of the initial enrichment culture medium leachate was 10%. After one acclimation cycle, 10 mL of the culture solution was transferred to fresh liquid culture medium with leachate concentrations (volume fractions) of 20%, 40%, 60%, 80% and 100%. After 6 acclimation cycles, under sterile conditions, a small amount of culture solution was dipped with an inoculating loop, and partitions were drawn on the plate culture medium. The inoculated plate was inverted and cultured in a 35°C incubator for 3-5 days. Then, typical colonies of the dominant bacteria were selected and partitioned and streaked on the plate of the separation culture medium for purification to obtain single colonies. The purified single strain was then inoculated onto the slant culture medium and stored at 4°C for later use.

[0036] The leachate-degrading bacteria is Bacillus subtilis, which is deposited in the China Center for Type Culture Collection. The address of the depository is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number: CCTCCM 2025283 and the deposit date: February 24, 2025.

[0037] Colony morphology: The colony is grayish white, nearly round, opaque, with wrinkles in the middle. Figure 1 shown.

[0038] Bacterial morphology: Gram staining is positive, the bacteria are slender and rod-shaped, as shown in the following Figure 2 shown.

[0039] Example 2, regarding growth curve:

[0040] A single colony of s14 was inoculated into LB medium and cultured at 35°C. Within 24 hours, 100 μl of samples were taken at 0, 1.5, 3, 4, 6, 8, 10, 12, 14, 16, 20, and 22 hours, and measured with a microplate reader (three samples in parallel). The curve drawn with the OD value as the vertical axis and the culture time as the horizontal axis is the growth curve, which reflects four periods: adaptation period (0-3 hours), logarithmic period (3-10 hours), stable period (10-12 hours), and decline period (13-24 hours). Figure 3 shown.

[0041] Example 3, Determination of the Degradation Efficiency of S14:

[0042] In the triangular flask containing landfill leachate, 10 8 , 10 9 , 10 10 The concentration of s14 bacterial suspension was cultured at 37°C, and the leachate without bacteria was cultured under the same conditions as the control. The culture was shaken at 180 r / min. The COD, ammonia nitrogen and total phosphorus values were measured after 120 h, and the degradation rate was calculated, see Table 1.

[0043] Degradation rate calculation method:

[0044] COD degradation rate = (COD value of original solution - COD value of blank degradation - COD value after degradation) / COD value of original solution * 100%

[0045] Ammonia nitrogen degradation rate = (ammonia nitrogen measured value of original solution - ammonia nitrogen measured value of blank degradation - ammonia nitrogen measured value after degradation) / ammonia nitrogen measured value of original solution * 100%

[0046] Total phosphorus degradation rate = (total phosphorus determination value of original solution - total phosphorus determination value of blank degradation - total phosphorus determination value after degradation) / total phosphorus determination value of original solution * 100%

[0047] concentration Ammonia nitrogen COD Total phosphorus <![CDATA[10 8 ]]> 82.68％ 87.20％ <![CDATA[10 9 ]]> 86.71％ 54.95％ <![CDATA[10 10 ]]> 88.84％ 55.88％

[0048] Table 1 Removal rate of ammonia nitrogen, COD and total phosphorus by s14 (%)

[0049] Example 3, identification of strain s14:

[0050] The 16S rDNA sequence of s14 was compared with the 16S rDNA sequences of other related species, and a phylogenetic tree was constructed, as shown in the attached figure. Figure 4As shown in the sequence comparison results, the strain with the highest homology is GCA_000227465. The homology is as high as 98.6%, corresponding to the strain Bacillus subtilis_TU-B-10, which was identified as Bacillus subtilis and named Bacillus subtilis s14. Figure 4 shown.

[0051] Example 4: Gene sequencing analysis:

[0052] The whole genome sequence of Bacillus subtilis s14 was assembled after second-generation and third-generation sequencing. The total length is 4302644 bp, with a GC content of 43.58%, encoding 4228 genes. The coding sequence accounts for 86.42% of the whole genome sequence, with an average length of 364.77 bp. Among them, there are 27 rDNAs and 86 tRNAs in the non-coding RNA. Figure 5 shown.

[0053] Example 5: Annotation of COG database:

[0054] By comparing the homologous genes of Bacillus subtilis s14 to the COG database, we found four major functional protein categories, namely information storage and processing, cellular processes, signal transduction, metabolism, and proteins of unknown function. There are 2,860 proteins with COG functional annotations, of which metabolism-related proteins account for the largest number, with 1,266, or 44.3% of the total annotated genes. These proteins are mainly involved in the transport and metabolism of amino acids and carbohydrates. In addition, there are 11.4% of proteins with unknown functions in the genome that cannot be functionally annotated, as shown in the attached figure. Figure 6 shown.

[0055] Example 6, for GO database annotation:

[0056] The gene ID encoding the Bacillus subtilis s14 protein was mapped to the GO term to obtain GO annotation information. The GO functional classification of the bacterium was mainly distributed in biological pathways, cellular components, and molecular functions. It was further divided into about 41 subcategories, with a total of 1,166 functional genes annotated. Among them, the cellular process and metabolic process genes of the biological pathways accounted for the largest percentages, at 32% and 27.5%, respectively; the protein complex part, cell membrane part, and organelle part of the cellular components accounted for the largest percentages, at 20.3%, 57.6%, and 14.4%, respectively; the catalytic activity, adhesion, and transport activity genes of the molecular functions accounted for the largest percentages, at 55.8%, 24.8%, and 12.6%, respectively. Figure 7 shown.

[0057] Example 7, for KEGG database annotation:

[0058] The KEGG functional annotation of the full genome of Bacillus subtilis s14 revealed a total of 1263 genes, of which 1261 genes were related to metabolic pathways, mainly carbohydrate metabolism (256), amino acid metabolism (206), cofactor and vitamin metabolism (145), and others (206). Figure 8 shown.

[0059] Example 8: Annotation of the unique function database:

[0060] As attached Figure 9 As shown, metabolic relevance screening using specific functional databases, combined with KEGG metabolic pathway analysis, revealed that the Bacillus subtilis s14 genome contains the largest number of GH-class glycoside hydrolase genes, as noted in the CAZy database. Furthermore, the genome also contains genes encoding glycosyltransferases, polysaccharide lyases, carbohydrate esterases, and a series of carbohydrate-binding domains. These enzymes and domains contribute to the efficient utilization and metabolism of carbohydrates by participating in carbohydrate binding, degradation, and transport.

[0061] Example 9: Analysis of genes related to landfill leachate biodegradation:

[0062] KEGG metabolic pathway analysis revealed that 18 genes involved in nitrogen metabolism (ko00910) were identified in the Bacillus subtilis s14 genome, mainly genes involved in nitrification and denitrification denitrification pathways and ammonia assimilation pathways.

[0063]

[0064] Table 2s14 Enzymes and genes related to ammonia nitrogen metabolism

[0065] As shown in Table 2, during gene annotation, genes encoding nitrate reductase (Nar) and nitrite reductase (Nir) in the ammonia nitrogen denitrification pathway, and nitrite oxidoreductase (NXR) in the ammonia nitrogen nitrification pathway were found in the Bacillus subtilis s14 genome. In addition, genes encoding key enzymes involved in ammonia assimilation were found, including genes encoding glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase.

[0066] As shown in Table 3, the genes gltA, encoding citrate synthase, and PhoD, encoding alkaline phosphatase, were found in the Bacillus subtilis s14 genome, indicating that the bacterium can hydrolyze organic phosphorus using both acidolysis and enzymatic hydrolysis. Furthermore, the bacterium can absorb phosphorus through specific phosphorus transporters on the plasma membrane. These include inorganic phosphate-specific transporters synthesized by the genes encoding pstS, pstC, pstA, and pstB, and phosphate transporters synthesized by the genes encoding phnE, phnD, and phnC.

[0067]

[0068] Table 3s14 Enzymes and genes related to phosphorus metabolism

[0069] Parts not described in the present invention are the same as those in the prior art or can be implemented using the prior art. Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A landfill leachate-degrading bacterium S14, characterized in that: The leachate-degrading bacteria was Bacillus subtilis, which was deposited in the China Center for Type Culture Collection with the deposit number: CCTCC M2025283.

2. The landfill leachate-degrading bacteria S14 according to claim 1, characterized in that: The colony is off-white, approximately round, opaque, and has wrinkles in the middle.

3. The landfill leachate-degrading bacteria S14 according to claim 1, characterized in that: Gram staining is positive, and the bacteria are slender and rod-shaped.

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