Pseudomonas aeruginosa with monomethylamine degradability and application thereof

Abstract

The present invention discloses a strain of Pseudomonas aeruginosa with monomethylamine degradability and the application thereof. This strain, named Pseudomonas aeruginosa GDUTAN1, was deposited on May 24, 2017 in the China Center for Type Culture Collection in Wuhan University, Wuhan City, Hubei Province with a deposit number of CCTCC NO.: M 2017283. This Pseudomonas aeruginosa GDUTAN1 was Gram-negative and rod-like, and round, green and opaque in the colony morphology, having a diameter of 1-2 mm. The Pseudomonas aeruginosa GDUTAN1 of the present invention can be applied to environmental remediation, degrading monomethylamine in the environment at a high degradation efficiency. When it degrades monomethylamine for 96 h at a substrate concentration of 50-140 mg/L, the degradation efficiency can reach more than 99%.

Claims

1. A method of degrading monomethylamine, comprising: adjusting the pH of a substance containing monomethylamine to 5-9; adding to the substance an isolated or purified strain of Pseudomonas aeruginosa named GDUTAN1, the strain deposited on May 24, 2017 in the China Center for Type Culture Collection in Wuhan University, Wuhan City, Hubei Province with a deposit number of CCTCC NO: M 2017283, in an inoculating amount of 0.5-2.5 mL; and reacting the substance and the strain of Pseudomonas aeruginosa at 20° C. to 40° C. to thereby degrade monomethylamine in the substance.

2. The method according to claim 1, wherein the substance comprises, water or soil.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the morphology of Pseudomonas aeruginosa GDUTAN1 of the present invention under an electron microscope.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(2) The content of the present invention is further illustrated by the following specific examples, but these examples should not be construed as limiting the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise specified. Unless otherwise indicated, the reagents, methods, and devices employed in the present invention are routine in the art.

Example 1

(3) A strain of Pseudomonas aeruginosa with monomethylamine degradability, named Pseudomonas aeruginosa GDUTAN1, was deposited on May 24, 2017 in the China Center for Type Culture Collection in Wuhan University (No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province) with a deposit number of CCTCC NO: M 2017283.

(4) The Pseudomonas aeruginosa GDUTAN1 of the present example was isolated and screened from leachate of a landfill in Guangzhou City, Guangdong Province. The separation and purification methods were as follows: The acclimation medium used was an inorganic salt medium (g/L) (each 1000 mL of the inorganic salt medium contains: K.sub.2HPO.sub.4.3H.sub.2O 1.2 g, KH.sub.2PO.sub.4 1.2 g, NH.sub.4Cl 0.4 g, MgSO.sub.4.7H.sub.2O 0.2 g, and FeSO.sub.4.7H.sub.2O 0.01 g; each 1 mL of the trace element solution contains: CaCl.sub.2.2H.sub.2O 0.2 g, MnSO.sub.4.4H.sub.2O 0.2 g, CuSO.sub.4.2H.sub.2O 0.01 g, ZnSO.sub.4.7H.sub.2O 0.2 g, CoCl.sub.2.6H.sub.2O 0.09 g, Na.sub.2MoO.sub.4.2H.sub.2O 0.12 g, and H.sub.3BO.sub.3 0.006 g). First, 1 mL of the landfill leachate was taken and diluted 100 times, inoculated into a nutrient broth, and aerobically cultured at 37° C. for 1 day in a shaker at a rotational speed of 150 rpm. 1 mL of the enriched bacterial solution was taken and inoculated in a nutrient solution containing monomethylamine, and aerobically cultured at 37° C. for 5 days in a shaker at a rotational speed of 150 rpm, and then moved to the next concentration in an inoculating amount of 10%, with the substrate acclimation gradients respectively at 10, 20, 50 and 100 mg/L. After acclimation, the acclimation solution was applied to a solid agar plate with monomethylamine as the sole carbon source (the solid medium containing monomethylamine was obtained by adding 18 g of agar and 4 mg of monomethylamine to per liter of the above inorganic salt medium), and cultured at 35° C. for 3 days; a single colony was selected and placed in a beef extract peptone medium (beef extract 3.0 g/L, peptone 10.0 g/L, NaCl 5.0 g/L, pH 7.4-7.6), and enriched and cultivated; the degradation efficiency of monomethylamine was determined, and the strain with the highest degradation efficiency was selected for purification.

(5) Determination of degradation efficiency: Sampling periodically during the biodegradation of monomethylamine, and determining the degradation efficiency spectrophotometrically. Degradation efficiency=(initial concentration−final concentration)/initial concentration.

(6) Spectrophotometric determination of monomethylamine concentration: Taking a certain amount of the monomethylamine degradation solution into a 10 mL colorimetric tube, diluting to 2.0 mL with an absorption solution (0.01 M HCl), and respectively adding 4.0 mL of buffer (obtained by dissolving 4.08 g of potassium dihydrogen phosphate and 1.6 g of borax in 80 mL of distilled water, adding 6.35 mL of 5.0 M NaOH solution, and diluting to 100 mL with water) and 0.4 mL of diazonium salt solution (obtained by adding 1.0 mL of sodium nitrite solution to 10 mL of p-nitrophenylamine hydrochloride solution and mixing), shaking well, letting stand for 40 min, adding 1.0 mL of 5 M NaOH solution, mixing, letting stand for 20 min, and performing colorimetric quantification at 510 nm.

(7) The purified colonies were identified, with the results as follows:

(8) (1) Morphological Characteristics of the Bacteria:

(9) a. By using the conventional physiological and biochemical identification methods of bacteria and electron microscopy, it was revealed that the Pseudomonas aeruginosa screened out was Gram-negative with cell staining; under the electron microscope, the bacterium was rod-like with a single flagellum, and had a size of (0.5 to 0.9)×(0.5 to 1.5) μm, as shown in FIG. 1;

(10) b. morphological characteristics of the colonies: after 24 h of culture in an LB solid medium, the colony appeared to be neatly edged, round, green and opaque, having a diameter of 1-2 mm; and

(11) c. the main physiological and biochemical characteristics of Pseudomonas aeruginosa are shown in Table 2:

(12) TABLE-US-00002 TABLE 2 Physiological and biochemical characteristics of Pseudomonas aeruginosa Items Test results Arabinose − Xylose − Glucose + Mannitol − Citrate utilization + DNA hydrolysis − V-P test − Nitrate (reduction) − Starch hydrolysis − Gelatin liquefaction + Anaerobic growth + 2% NaCl growth + 5% NaCl growth + pH = 5.5 growth + pH = 9.0 growth + Gram staining − 50° C. growth − 15° C. growth +

(13) The above results indicate that the selected bacteria of the present invention had physiological and biochemical characteristics very similar to those of Pseudomonas aeruginosa.

(14) (2) Extracting Bacterial Genomic DNA and Using Bacterial 16S rDNA Universal Primers:

(15) TABLE-US-00003 Upstream primer: F27  (5′-AGTTTGATCMTGGCTCAG-3′) Downstream primer: R1492 (5′-GGTTACCTTGTTACGACTT-3′)

(16) The entire 16S rDNA gene was amplified, with the sequencing results as shown in SEQ ID NO: 1.

(17) By aligning the 16S rRNA gene sequence of 1331 bp in length as shown in SEQ ID NO: 1 with the gene sequence registered in the Genbank, it was found that the strain had 100% homology with Pseudomonas aeruginosa DSM 5007.

(18) Based on the above physiological and biochemical characteristics and 16S rRNA gene sequencing results, the selected strain of the present invention should belong to a new variant of Bacillus, and was named Pseudomonas aeruginosa GDUTAN1.

(19) The Pseudomonas aeruginosa was deposited on May 24, 2017 in the China Center for Type Culture Collection (CCTCC) in Wuhan University (No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province) with a deposit number of CCTCC NO: M 2017283.

Example 2

(20) This example is the application of Pseudomonas aeruginosa GDUTAN1 in environmental remediation, which can degrade monomethylamine in the environment. The environment includes the atmosphere, water or soil.

(21) The method for verifying the monomethylamine degradability of the selected Pseudomonas aeruginosa GDUTAN1 of the present invention is as follows:

(22) Inoculating the Pseudomonas aeruginosa GDUTAN1 strain in the slant preservation into an LB enrichment culture solution, activating the bacteria at 35° C. for 24 h in a shaker at 150 rpm, and centrifuging the bacterial solution, and then collecting the bacteria. Resuspending with 5 mL of an inorganic salt solution (each 100 mL of the inorganic salt solution contains the following components: K.sub.2HPO.sub.4.3H.sub.2O 0.12 g, KH.sub.2PO.sub.4 0.12 g, NH.sub.4Cl 0.04 g, MgSO.sub.4.7H.sub.2O 0.02 g, FeSO.sub.4.7H.sub.2O 0.001 g, CaCl.sub.2.2H.sub.2O 0.02 g, MnSO.sub.4.4H.sub.2O 0.02 g, CuSO.sub.4.2H.sub.2O 0.001 g, ZnSO.sub.4.7H.sub.2O 0.02 g, CoCl.sub.2.6H.sub.2O 0.009 g, Na.sub.2MoO.sub.4.2H.sub.2O 0.012 g, H.sub.3BO.sub.3 0.0006 g, and double distilled water 100 mL), and inoculating 2.0 mL of the bacterial solution into 100 mL of an inorganic salt solution containing 50 mg/L of monomethylamine, with the pH of the inorganic salt at 6; reacting at 35° C. for 96 h in a shaker at 200 rpm, sampling periodically and determining the degradation efficiency spectrophotometrically. The degradation efficiency, measured in the same way as in Example 1, was determined to be 99.2%.

Example 3

(23) Inoculating the Pseudomonas aeruginosa GDUTAN1 strain in the slant preservation into an LB enrichment culture solution, activating the bacteria at 35° C. for 24 h in a shaker at 150 rpm, and centrifuging the bacterial solution, and then collecting the bacteria. Resuspending with 5 mL of an inorganic salt solution, and inoculating 2.0 mL of the bacterial solution into 100 mL of an inorganic salt solution containing 50 mg/L of monomethylamine (the same as in Example 2), with the pH of the inorganic salt at 8; reacting at 35° C. for 96 h in a shaker at 100 rpm, sampling periodically and determining the degradation efficiency spectrophotometrically. The degradation efficiency, measured in the same way as in Example 1, was determined to be 41.5%.

Example 4

(24) Inoculating the Pseudomonas aeruginosa GDUTAN1 strain in the slant preservation into an LB enrichment culture solution, activating the bacteria at 35° C. for 24 h in a shaker at 150 rpm, and centrifuging the bacterial solution, and then collecting the bacteria. Resuspending with 5 mL of an inorganic salt solution, and inoculating 1.5 mL of the bacterial solution into 100 mL of an inorganic salt solution containing 80 mg/L of monomethylamine (the same as in Example 2), with the pH of the inorganic salt at 7; reacting at 20° C. for 96 h in a shaker at 150 rpm, sampling periodically and determining the degradation efficiency spectrophotometrically. The degradation efficiency, measured in the same way as in Example 1, was determined to be 98.9%.

Example 5

(25) Inoculating the Pseudomonas aeruginosa GDUTAN1 strain in the slant preservation into an LB enrichment culture solution, activating the bacteria at 35° C. for 24 h in a shaker at 150 rpm, and centrifuging the bacterial solution, and then collecting the bacteria. Resuspending with 5 mL of an inorganic salt solution, and inoculating 2.0 mL of the bacterial solution into 100 mL of an inorganic salt solution containing 110 mg/L of monomethylamine (the same as in Example 2), with the pH of the inorganic salt at 6; reacting at 25° C. for 96 h in a shaker at 200 rpm, sampling periodically and determining the degradation efficiency spectrophotometrically. The degradation efficiency, measured in the same way as in Example 1, was determined to be 99.9%.

Example 6

(26) Inoculating the Pseudomonas aeruginosa GDUTAN1 strain in the slant preservation into an LB enrichment culture solution, activating the bacteria at 35° C. for 24 h in a shaker at 150 rpm, and centrifuging the bacterial solution, and then collecting the bacteria. Resuspending with 5 mL of an inorganic salt solution, and inoculating 1.5 mL of the bacterial solution into 100 mL of an inorganic salt solution containing 140 mg/L of monomethylamine (the same as in Example 2), with the pH of the inorganic salt at 7; reacting at 30° C. for 96 h in a shaker at 150 rpm, sampling periodically and determining the degradation efficiency spectrophotometrically. The degradation efficiency, measured in the same way as in Example 1, was determined to be 99.0%.

(27) The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited thereto, and any other alterations, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should all be equivalent replacements and included in the scope of protection of the present invention.

(28) TABLE-US-00004 Sequence Listing 110 Guangdong University of Technology 120 Pseudomonas aeruginosa with monomethylamine degradability and  application thereof 130 OP180087 160    1 170 SIPOSequenceListing 1.0 210    1 211 1331 212 DNA 213 Pseudomonas aeruginosa 400    1 attcagcggc ggacgggtga gtaatgccta ggaatctgcc tggtagtggg ggataacgtc      60 cggaaacggg cgctaatacc gcatacgtcc tgagggagaa agtgggggat cttcggacct    120 cacgctatca gatgagccta ggtcggatta gctagttggt ggggtaaagg cctaccaagg    180 cgacgatccg taactggtct gagaggatga tcagtcacac tggaactgag acacggtcca    240 gactcctacg ggaggcagca gtggggaata ttggacaatg ggcgaaagcc tgatccagcc    300 atgccgcgtg tgtgaagaag gtcttcggat tgtaaagcac tttaagttgg gaggaagggc    360 agtaagttaa taccttgctg ttttgacgtt accaacagaa taagcaccgg ctaacttcgt  420 gccagcagcc gcggtaatac gaagggtgca agcgttaatc ggaattactg ggcgtaaagc  480 gcgcgtaggt ggttcagcaa gttggatgtg aaatccccgg gctcaacctg ggaactgcat  540 ccaaaactac tgagctagag tacggtagag ggtggtggaa tttcctgtgt agcggtgaaa  600 tgcgtagata taggaaggaa caccagtggc gaaggcgacc acctggactg atactgacac  660 tgaggtgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa  720 cgatgtcgac tagccgttgg gatccttgag atcttagtgg cgcagctaac gcgataagtc  780 gaccgcctgg ggagtacggc cgcaaggtta aaactcaaat gaattgacgg gggcccgcac  840 aagcggtgga gcatgtggtt taattegaag caacgcgaag aaccttacct ggccttgaca  900 tgctgagaac tttccagaga tggattggtg ccttcgggaa ctcagacaca ggtgctgcat  960 ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgtaacgag cgcaaccctt 1020 gtccttagtt accagcacct cgggtgggca ctctaaggag actgccggtg acaaaccgga 1080 ggaaggtggg gatgacgtca agtcatcatg gcccttacgg ccagggctac acacgtgcta 1140 caatggtcgg tacaaagggt tgccaagccg cgaggtggag ctaatcccat aaaaccgatc 1200 gtagtccgga tcgcagtctg caactcgact gcgtgaagtc ggaatcgcta gtaatcgtga 1260 atcagaatgt cacggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg 1320 gagtgggttg c  1331