LAS-DEGRADING AND/OR N-REMOVING BACTERIUM AND APPLICATION THEREOF
20210269341 · 2021-09-02
Assignee
- AGRO-ENVIRONMENTAL PROTECTION INSTITUTE, MINISTRY OF AGRICULTURE AND RURAL AFFAIRS (Tianjin, CN)
- Tianjin Agricultural University (Tianjin, CN)
Inventors
Cpc classification
International classification
Abstract
A bacterium that degrades LAS and/or removes N is provided. The bacterium is taxonomically classified as Pseudomonas sp., and was deposited in China General Microbiological Culture Collection Center on Sep. 18, 2018, with the accession number of CGMCC 16502. The bacterium can be applied in the rural domestic sewage treatment, and has the effect of removing LAS and/or N pollution.
Claims
1. A LAS-degrading and/or N-removing bacterium, taxonomically classified as Pseudomonas sp., deposited in China General Microbiological Culture Collection Center on Sep. 18, 2018, with the accession number of CGMCC 16502.
2. The bacterium according to claim 1, wherein the 16S rDNA sequence of the strain of the bacterium is SEQ ID NO: 1.
3. The bacterium according to claim 1, wherein the strain of the bacterium has the following phenotypic characteristics: when cultured at 36° C. for 24 h, the bacterial colonies are light yellow, circular and opaque, and have a wet surface and a smooth margin; and the bacterial cells are rod-shaped, 0.4˜0.8 μm×1.8˜4.7 μm in size, arranged individually or in pairs, and gram-negative.
4. The bacterium according to claim 1, wherein the strain of the bacterium has the following characteristics: (G+C) mol % is 63.4%; and the quinone components are ubiquinone Q8 and ubiquinone Q9, and their relative contents are 57.24% and 42.76%, respectively.
5. The bacterium according to claim 1, wherein the strain of the bacterium has the following characteristics: the major fatty acid components are: C.sub.16.0, Summed Feature 3 (C.sub.16:1ω7c or C.sub.16.1ω6c), and Summed Feature 8 (C.sub.18:1ω7c or C.sub.18.1ω6c), and their relative contents are 31.16%, 14.85% and 12.32%, respectively.
6. The bacterium according to claim 1, wherein the strain of the bacterium has the following characteristics: the cell wall-characteristic DAP component is meso-DPA; and the cell wall-characteristic sugar component is ribose.
7. The bacterium according to claim 1, wherein the strain of the bacterium has the following characteristics: the bacterial polar lipids comprise diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified phospholipid, an unidentified lipid and an unidentified aminophospholipid.
8. The bacterium according to claim 1, wherein the bacterium shows its LAS-degrading effect over an initial LAS concentration of 0-400 mg/L; and the bacterium shows its ammoniacal nitrogen-removing effect over an initial ammoniacal nitrogen concentration of 0-200 mg/L.
9. The bacterium according to claim 1, wherein the bacterium simultaneously degrades LAS and removes N.
10. Use of the bacterium according to claim 1 for decontamination of rural domestic sewage, wherein the rural domestic sewage has a LAS concentration of 0 to 400 mg/L and/or an ammoniacal nitrogen concentration of 0 to 200 mg/L.
11. The bacterium according to claim 8, wherein the bacterium shows its LAS-degrading effect over an initial LAS concentration of 0-200 mg/L; and the bacterium shows its ammoniacal nitrogen-removing effect over an initial ammoniacal nitrogen concentration of 0-100 mg/L.
12. A method of decontaminating rural domestic sewage, wherein the rural domestic sewage has a LAS concentration of 0 to 400 mg/L and/or an ammoniacal nitrogen concentration of 0 to 200 mg/L, said method comprising adding an effective inoculum amount of the bacterium of claim 1 to the rural domestic sewage.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0031]
[0032]
[0033]
[0034]
DETAILED DESCRIPTION OF THE INVENTION
Example 1 Isolation and Identification of the Bacterial Strain of the Present Invention
[0035] (1) Medium
[0036] {circle around (1)} Medium for nitrogen removal: 0.48 g of ammonium sulfate, 4.71 g of succinic acid and a 50 mL trace element solution were dissolved and then made to a volume of 1 L with distilled water, with the pH controlled between 7.0 and 7.2. (for a solid medium, 2% agar was added). The trace element solution: 6.5 g of K.sub.2HPO.sub.4.3H.sub.2O, 2.5 g of MgSO.sub.4.7H.sub.2O, 2.5 g of NaCl, 0.05 g of FeSO.sub.4.7H.sub.2O, and 0.04 g of MnSO.sub.4.H.sub.2O were dissolved and made to a volume of 1 L with distilled water.
[0037] {circle around (2)} LAS-degrading selective medium: 0.48 g of ammonium sulfate, 4.71 g of succinic acid, 0.10 g of LAS, and a 50 mL trace element solution. The trace element solution: 6.5 g of K.sub.2HPO.sub.4.3H.sub.2O, 2.5 g of MgSO.sub.4.7H.sub.2O, 2.5 g of NaCl, 0.05 g of FeSO.sub.4.7H.sub.2O, and 0.04 g of MnSO.sub.4.H.sub.2O, which were dissolved and made to a volume of 1 L with distilled water.
[0038] (2) Isolation and screening of nitrogen removing strains
[0039] 10 mL of sewage from the septic tank in Ankang, Shaanxi was taken with a sterile pipette, placed in a 250 ml Erlenmeyer flask (containing 100 mL sterile medium for nitrogen removal), and cultured at 30° C. on a shaker at 160 rpm. Each passage was cultured for three days. The presence of nitrobacteria was detected by using a diphenylamine reagent. The bacterial liquid was cultured until the presence of nitrobacteria was detected in the medium.
[0040] The culture liquid containing the nitrobacteria was plated by limiting dilution, and cultured in an incubator at 37° C. for 48 hours. Afterwards single colonies were picked, and inoculated by the streak plate method for multiple times. When no contaminating bacteria were observed under a microscope, the purification of the strain was completed.
[0041] The purified strain was subjected to a screening experiment for ammoniacal nitrogen removal. The strains were individually inoculated in the medium for nitrogen removal, and cultured in an incubator at 37° C. for 48 hours. The ammoniacal nitrogen removal was determined, and the strains with the ammoniacal nitrogen removal higher than 85% were selected as the nitrogen-removing strains.
[0042] (3) Screening for nitrogen-removing and LAS-degrading strains
[0043] The selected nitrogen-removing strains were inoculated into the liquid LAS-degrading selective medium in an inoculation amount of 10%, and cultured on a shaker for 48 hours, and the degradation of LAS was measured. A strain showing a degradation higher than 80% is a LAS-degrading and/or N-removing bacterium.
[0044] (4) A LAS-degrading and/or N-removing bacterium was identified by screening.
[0045] Furthermore, the bacterial strain was assayed and has the following characteristics: (G+C)mol % is 63.4%; the quinone components are ubiquinone Q8 and ubiquinone Q9, and their relative contents are 57.24% and 42.76%, respectively; the major fatty acid components (relative content>10%) are: C.sub.16:0, Summed Feature 3 (C.sub.16:1ω7c or C.sub.16:1ω6c), and Summed Feature 8 (C.sub.18:1ω7c or C.sub.18:1ω6c), and their relative contents are 31.16%, 14.85% and 12.32%, respectively; the cell wall-characteristic DAP component is meso-DPA; and the cell wall-characteristic sugar component is ribose; the polar lipid of this strain comprises diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified phospholipid, an unidentified lipid, and an unidentified aminophospholipid; and the hybridization homology of the bacterial strain with the model strain Pseudomonas guangdongensis CCTCC AB2012022.sup.T is 35.4%.
[0046] (5) 16S rDNA sequence analysis and phylogenetic analysis The 16S rDNA gene sequence was 1498 bp in length as confirmed by PCR amplification and sequencing of the 16S rDNA (see SEQ ID NO: 1). Through the alignment by submission to Genbank, it was found that the LAS-degrading and/or N-removing bacterium according to the present invention can have a homology as high as 97.88% with Pseudomonas linyingensis LYBRD3-7.sup.T. The 16S rDNA sequence genetic tree of the strain and some related species was generated with the software MEGA 5.0 by the neighbor-joining method, and the similarity was repeatedly calculated for 1,000 times. In
[0047] (6) The physiological and biochemical results of the LAS-degrading and/or N-removing bacterium according to the present invention are shown in Table 2 below.
TABLE-US-00001 TABLE 2 Physiological and Biochemical Characteristics: Ala-Phe-Pro − adonitol − L-pyrrolidonyl − L-Arabitol − arylamidase arylamidase D-cellobiose − β-galactosidase − H.sub.2S Production − β-N-acetylamino- − glucosidase Glutamyl − D-glucose − γ-glutamyl- − fermentation/glucose − arylamidase transferase pNA β-Glucosidase − D-maltose + D-mannitol − D-mannose − β-xylosidase − β-Alanine − L-proline + lipase − arylamidase pNA arylamidase palatinose − tyrosine + urease − D-sorbitol − arylamidase sucrose − D-tagatose − D-trehalose − Citrate (sodium) − malonate − 5-keto-D-Gluconate − L-lactate − α-glucosidase − alkalinisation succinate − β-N-Acetyl- − α-galactosidase − phosphatase − alkalinisation galactosaminidase Glycine − ornithine − lysine − L-Histidine − arylaminase decarboxylase decarboxylase assimilation coumaric acid − β-glucuronidase − O/129 + Glu-Gly-Arg- − resistance arylamidase L-malate + ELLMAN − L-lactate − assimilation assimilation Symbol Description: “+”, positive; “−”, negative.
Example 2 LAS-Degrading Test
[0048] The activated LAS-degrading and/or N-removing bacterium of the present invention was respectively inoculated into the media having LAS concentrations of 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L by a dilution coating method, with an inoculum amount of 1%. One blank control group and 3 parallel test groups were set up. After culturing under shaking for 48 hours, the degradation of LAS was determined. The results are shown in Table 3 below.
TABLE-US-00002 TABLE 3 Degradation of the strains of the present invention at different initial concentrations of LAS Initial concentration 0 50 100 200 400 (mg/L) Removal of LAS — 87 82 78 50 (%)
[0049] It can be seen from the results in Table 3 that the LAS-degrading and/or N-removing bacterium of the present invention can degrade LAS, and can exert this LAS-degrading function at an initial LAS concentration from 0 to 400 mg/L. In particular, the degradation of LAS was higher at the initial LAS concentration from 0 to 200 mg/L.
Example 3 Nitrogen Removing Effect Experiment
[0050] The LAS-degrading and/or N-removing bacterial strain of the present invention was inoculated into 100 mL media for nitrogen removal at different nitrogen levels, with initial ammoniacal nitrogen concentrations of 50 mg/L (Treatment 1), 100 mg/L (Treatment 2) and 200 mg/L (Treatment 3), respectively. A medium not inoculated with the strain was used as a blank control. The media were cultured in an incubator at 37° C. under shaking at 160 rpm for 30 h. The culture solution was centrifuged at 10,000 rpm for 6 minutes, and filtered through a 0.45 μm filter. The contents of ammoniacal nitrogen and nitrate nitrogen in the culture solution were measured. The detailed measurement results are shown in
[0051] It can be seen from
Example 4 Degradation Experiment on Rural Domestic Sewage
[0052] (1) In the Tanjiawan area of Shiyan, Hubei Province (China), the domestic sewage of the rural septic tank was subjected to grid filtration and sedimentation, and 20 L of the supernatant was added to an integrated sewage treatment apparatus. Test results: the pH of wastewater was 7.2, the ammoniacal nitrogen concentration was 60 mg/L, CODcr was 300 mg/L, and LAS was 5 mg/L.
[0053] (2) The liquid containing the LAS-degrading and/or N-removing bacterium of the present invention was directly added to the integrated sewage treatment apparatus containing the 20 L of rural domestic sewage, in an inoculum amount of 10% by volume. The culturing temperature was 30° C. The pH of the target water did not need to be controlled during the sewage decontamination process, and aeration was performed for 1 hour every day. Results: after 2 days of treatment, the removal of ammoniacal nitrogen from the water can reach 88%, the removal of CODcr can reach 60%, and the degradation of LAS was 97%.
[0054] The above examples are merely illustration of the present invention and are not intended to limit the scope of protection of the present invention. Any designs identical or similar to the present invention are within the scope of protection of the present invention.