A strain of Bacillus aryabhattai LW1 and its applications

Abstract

The present invention discloses the application of a strain of Phytobacter ursingii LW1 in wastewater treatment. This strain is Phytobacter ursingii LW1, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on November 4, 2021, with the deposit number GDMCC No. 62033. The Phytobacter ursingii LW1 strain has a relatively wide suitable growth temperature range and has good denitrification ability between 4°C and 40°C. It can be used for the denitrification treatment of medium- and low-temperature nitrogen-containing wastewater.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to the application of a strain of Bacillus vallismortis LW1 in biological low-temperature denitrification of wastewater. Background Art

[0002] With the continuous emergence of factories, agricultural fertilizers, and pesticides, various pollutants are discharged into the environment, resulting in various types of wastewater with complex compositions. Many factors such as the deposition of industrial waste gas nitrogen oxides, the infiltration of solid waste migration, the unreasonable discharge of wastewater, the excessive application of nitrogen fertilizers, and the reuse irrigation of sewage have caused serious nitrate pollution in water bodies.

[0003] Biological denitrification technology is a widely used and economically efficient denitrification method at present. However, the influent temperature of sewage in most regions of our country in winter is generally lower than the suitable growth temperature of nitrifying and denitrifying functional bacteria, resulting in a significant decrease in the growth and metabolic activity of microorganisms and the number of bacterial communities under low-temperature conditions in winter. Research shows that when the water temperature drops below 15°C, the nitrification effect of the system will decrease significantly, and the treatment efficiency of the traditional activated sludge process-based technology will drop greatly, and the guarantee rate of the effluent quality meeting the standards is extremely low, especially the removal of nitrogen and phosphorus is particularly difficult. In engineering, measures such as reducing the load, extending the hydraulic retention time, and heat preservation are often adopted to make the effluent quality meet the standards in winter, but these measures increase the operating cost and the treatment effect is unstable.

[0004] The advantage of Bacillus vallismortis LW1 is that it is a heterotrophic bacterium with a fast growth rate and a large biomass, and can stay in the aerobic tank for a long time. At the same time, it is cold-tolerant and can maintain a high denitrification activity under low-temperature conditions. In addition, it is resistant to organic load and can remove nitrate nitrogen and ammonia nitrogen simultaneously. Summary of the Invention

[0005] The purpose of the present invention is to provide a highly efficient denitrifying bacterium for treating low-temperature nitrogen-containing wastewater in order to solve the defects of low sewage treatment efficiency and poor treatment effect of existing biological denitrification technology under low-temperature conditions.

[0006] The invention separates a strain with low-temperature denitrification performance from activated sludge. Through morphological, physiological and biochemical and molecular biological identification, the strain belongs to Phytobacter ursingii and is named Phytobacter ursingii LW1. The 16S rRNA gene sequence obtained by sequencing the strain contains 1371 bases. The nucleotide homology comparison between the sequence registered in Genebank and the BLAST program shows that the 16S rRNA gene sequence of the strain has 99.55% homology with Phytobacter ursingii and 98.61% homology with Phytobacter diazotrophicus. The atpD gene sequence obtained by sequencing is 683 bp. The nucleotide homology comparison between the sequence registered in Genebank and the BLAST program shows that the atpD gene sequence of the strain has 99.41% homology with Phytobacter ursingii. The infB gene sequence obtained by sequencing is 763 bp. The nucleotide homology comparison between the sequence registered in Genebank and the BLAST program shows that the infB gene sequence of the strain has 98.56% homology with Phytobacter ursingii. rRNA phylogenetic tree Figure 3 shown.

[0007] The purpose of the present invention is mainly achieved through the following technical means:

[0008] A strain of Phytobacter ursingii LW1 was deposited in the Guangdong Provincial Microbial Culture Collection Center with the accession number GDMCC No: 62033. The storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City. The storage date is November 4, 2021.

[0009] Preferably, the plant bacillus of Ursinus LW1 is used in the denitrification treatment of low-medium temperature wastewater.

[0010] Preferably, the temperature of the denitrification treatment is 4-40°C.

[0011] More preferably, the temperature of the denitrification treatment is 4-15°C.

[0012] Preferably, the Bacillus ursinus LW1 can remove one or more of nitrate nitrogen and ammonia nitrogen in wastewater.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] (1) After being cultured in a basal medium with nitrate or ammonia nitrogen as the sole nitrogen source for 48 hours, the strain Phytobacter ursingii LW1 involved in the present invention can achieve a 100% removal effect on nitrate or ammonia nitrogen. In addition, this strain can tolerate relatively low temperatures and can remove nitrate or ammonia nitrogen under relatively low temperature conditions of 4 - 15°C.

[0015] (2) The Phytobacter ursingii LW1 can achieve a denitrification effect under aerobic conditions. It can be inoculated into aerobic nitrifying and denitrifying sludge to achieve simultaneous nitrification and denitrification of nitrogen-containing wastewater, and is applicable to the denitrification treatment of various nitrogen-containing wastewaters. More importantly, this bacterium can also achieve efficient nitrogen removal in a medium and low temperature environment within the range of 4 - 40°C, and thus can be used for the denitrification treatment of ammonia nitrogen-containing wastewater in low temperature seasons such as winter. Description of the Drawings

[0016] Figure 1 It is a colony morphology diagram of Phytobacter ursingii LW1.

[0017] Figure 2 It is a microscopic morphology diagram of Phytobacter ursingii LW1.

[0018] Figure 3 It is a phylogenetic tree constructed based on the 16S rRNA gene sequence of Phytobacter ursingii LW1.

[0019] Figure 4 It is a nitrate nitrogen removal curve when Phytobacter ursingii LW1 is cultured with nitrate nitrogen as the sole nitrogen source under different temperature conditions.

[0020] Figure 5 It is an ammonia nitrogen removal curve when Phytobacter ursingii LW1 is cultured with ammonia nitrogen as the sole nitrogen source under different temperature conditions. Detailed Embodiments

[0021] The present invention will be further described in detail below with reference to specific embodiments, but the implementation manners of the present invention are not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0022] The low-temperature denitrifying bacteria provided in the present invention: Phytobacter ursingii LW1, is preserved in the Guangdong Provincial Microbial Culture Collection Center, and its preservation number is: GDMCC No: 62033. The preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is November 4, 2021.

[0023] Example 1 Isolation and performance identification of strains

[0024] 1. Screening and separation of low-temperature denitrifying bacteria

[0025] The activated sludge was added to the inorganic salt culture medium with nitrate as the only nitrogen source, and the culture was acclimated and cultured for many times at 10°C. The obtained bacterial solution was gradient diluted and spread on the plate, and then the single colony was streaked and purified to obtain a pure single colony. The selected single colony was inoculated into the culture medium with nitrate as the only nitrogen source, and the concentration changes of nitrate, nitrite and ammonia nitrogen in the culture medium were measured regularly, so as to screen out the most efficient low-temperature denitrification bacteria, namely strain LW1.

[0026] 2. Identification of strains

[0027] 2.1 Morphology and culture characteristics

[0028] The colonies were cultured on LB plates at 30°C for 2 days. The colonies were round, convex, smooth, with neat edges and off-white color. Figure 1 .

[0029] 2.2 Microscopic morphological characteristics

[0030] This bacterium is a Gram-negative bacillus. Figure 2 .

[0031] 2.3 Physiological and biochemical characteristics

[0032]

[0033]

[0034] (“+” indicates a positive reaction, “-” indicates a negative reaction)

[0035] Example 2 Molecular Biology Identification Results

[0036] The 16S rRNA gene sequence obtained by sequencing the low-temperature denitrifying strain is 1371 bp. The nucleotide homology of this strain's 16S rRNA gene sequence was compared with the registered sequences in Genebank using the Blast program. The homology of this strain's 16S rRNA gene sequence with Phytobacter ursingii reached 99.55%, and with Phytobacter diazotrophicus reached 98.61%. The atpD gene sequence obtained by sequencing is 683 bp. The nucleotide homology of this strain's atpD gene sequence was compared with the registered sequences in Genebank using the Blast program. The homology of this strain's atpD gene sequence with Phytobacter ursingii reached 99.41%. The infB gene sequence obtained by sequencing is 763 bp. The nucleotide homology of this strain's infB gene sequence was compared with the registered sequences in Genebank using the Blast program. The homology of this strain's infB gene sequence with Phytobacter ursingii reached 98.56%. Based on the 16S rRNA gene sequence, a phylogenetic tree was constructed by the neighbor-joining method, as shown in Figure 3 Based on the above identification results, this strain was named Phytobacter ursingii LW1 and was preserved in the Guangdong Provincial Microbial Culture Collection Center on November 4, 2021, with the preservation number GDMCC No: 62033, and the preservation address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0037] Example 3 Denitrification effect of Phytobacter ursingii LW1

[0038] The bacterial liquid expanded and cultured in a liquid agar medium (tryptone 10, yeast extract 5, sodium chloride 10, unit g / L) was transferred into 150 mL of an inorganic salt medium containing 100 mg / L NO 3 - -N (K 2 HPO 4 (0.5), KH 2 PO 4 (1), MgSO 4 (0.246), CaCl 2 (0.0111), FeSO 4 (0.005), glucose (2), unit (g / L)). Six temperatures of 4°C, 10°C, 15°C, 20°C, 30°C, and 40°C were set, and the culture was carried out at 120 r / min for 48 h. The degradation rate of nitrate nitrogen at 10°C, 15°C, 20°C, and 30°C was 96 - 100%, the removal rate at 40°C was 88%, and the removal effect at 4°C reached 58%. The removal curves of nitrate nitrogen under different temperature conditions are shown inFigure 4 This bacterium degrades nitrate into clean nitrogen, and there is no secondary pollution during the removal process, that is, no release of N 2 O.

[0039] Removal effect of ammonia nitrogen by Phytobacter ursingii LW1 in Example 4

[0040] The bacterial liquid expanded and cultured in a liquid agar medium (tryptone 10, yeast extract 5, sodium chloride 10, unit g / L) was transferred into 150 mL of an inorganic salt medium containing 50 mg / L NH 4 + -N (K 2 HPO 4 (0.5), KH 2 PO 4 (1), MgSO 4 (0.246), CaCl 2 (0.0111), FeSO 4 (0.005), glucose (1), unit (g / L)). Six temperatures of 4°C, 10°C, 15°C, 20°C, 30°C, and 40°C were set, and the culture was carried out for 48 h at 120 r / min. The degradation rate of ammonia nitrogen was 100% at 10°C, 15°C, 20°C, and 30°C, the removal rate was 90% at 40°C, and the removal effect reached 62% at 4°C. The removal curves of ammonia nitrogen under different temperature conditions are shown in Figure 5 .

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A strain of Phytobacter ursingii LW1, characterized in that, it is deposited in the Guangdong Microbial Culture Collection Center, with the deposit number GDMCC No: 62033, the deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, and the deposit date is November 4, 2021.

2. The application of the Phytobacter ursingii LW1 according to claim 1 in the denitrification treatment of low- and medium-temperature wastewater, wherein the temperature of the denitrification treatment is 4 - 40 °C; the denitrification treatment is to remove one or both of nitrate nitrogen and ammonia nitrogen in the wastewater.

3. According to the application described in claim 2, characterized in that, the temperature of the denitrification treatment is 4 - 15 °C.

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