Rhodococcus pyrphila and its application in degrading nitrated cellulose

Abstract

The application discloses a rhodopirellula pyriformis strain and application thereof in degrading nitrocellulose. The rhodopirellula pyriformis strain is LZ1, and the preservation number is CCTCC NO: M 2022785. The rhodopirellula pyriformis strain can convert the oxidized nitrogen removed from the nitrocellulose into its own nutrients under the condition that the skeleton of the nitrocellulose is not damaged, realizes denitration of the nitrocellulose, and can denitrify nitrite nitrogen and nitrate nitrogen, realizes green denitrification of the nitrocellulose, and avoids water pollution in the treatment process.

Description

Technical Field

[0001] This invention belongs to the field of microbial wastewater treatment and relates to a strain of Rhodococcus pyridostigmine and its application in the degradation of nitrate cellulose. Background Technology

[0002] Propellant, as a material control factor for the effective execution of modern guided weapons, is the support and foundation of advanced guided weapon systems. Nitrocellulose is a major component of propellant, resulting in a large amount of military-grade nitrocellulose being discarded annually. Military-grade nitrocellulose is an explosive material, and any waste generated during its production is classified as K044 hazardous waste. Therefore, finding a safe and pollution-free method to degrade nitrocellulose is of great significance.

[0003] Biological treatment of nitrocellulose is a novel, pollution-free technology with advantages such as safety, low cost, and environmental friendliness, meeting the demands for ecological environmental protection amidst rapid global economic development. Early researchers used composting to denitrify nitrocellulose, a method that utilizes the combined action of thermophilic and thermostable bacteria to degrade organic matter. While the composting process is relatively simple, it is time-consuming and results in limited nitrogen reduction. Using a single bacterial strain for nitrogen reduction not only shortens the time required but also allows for easier control of denitrification, facilitating subsequent industrial-scale biological treatment of nitrocellulose. This method reduces costs, achieving an integrated economic and environmentally friendly treatment.

[0004] Chinese patent application CN202110080661.X discloses a sulfate-reducing bacterium capable of utilizing nitrates and nitrites degraded on nitrate cellulose as its own nutrients. However, this strain requires a strictly anaerobic environment, making the operation complex and costly. Chinese patent application CN202110073488.0 discloses a tall *Chaetoceros* strain, which can also denitrate cellulose. However, because *Chaetoceros* is a fungus, its long growth cycle is unsuitable for industrial-scale cellulose processing. Furthermore, both the sulfate-reducing bacteria and *Chaetoceros* exhibit low nitrogen reduction rates on nitrate cellulose and cannot fully convert nitrate nitrogen in the denitrification solution, thus failing to achieve green denitrification of nitrate cellulose. Chinese patent application CN201110006790.0 discloses a *Rhodococcus pyridococcus* strain. While this strain can effectively utilize nitrate nitrogen and ammonia nitrogen compounds in the solution and convert them to non-polluting gas N2 to the greatest extent possible, it cannot denitrate cellulose. Summary of the Invention

[0005] This invention provides a strain of Rhodococcus pyridinivorans LZ1 that can degrade nitrified cellulose. This Rhodococcus pyridinivorans can denitrify nitrified cellulose under aerobic conditions, converting nitrates and nitrites into its own nutrients. The Rhodococcus pyridinivorans described in this invention has the accession number CCTCC NO: M 2022785, the accession date was June 2, 2022, and the accession address is Wuhan University, Wuhan, China.

[0006] The present invention also provides a method for culturing the above-mentioned Rhodococcus pyridostigmine, comprising the following steps:

[0007] Rhodococcus pyridostigmine was inoculated into LB liquid medium and cultured dynamically at 37°C.

[0008] The LB liquid culture medium described in this invention is the LB liquid culture medium conventionally used in the art, with the following formula: 5 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, and natural pH.

[0009] Furthermore, the present invention also provides the application of the above-mentioned Rhodococcus pyridostigmine in the degradation of nitrate cellulose.

[0010] The specific method for the above application is as follows: Rhodococcus pyridostigmine is inoculated into wastewater containing nitrate cellulose to degrade the nitrate cellulose.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] (1) The Rhodococcus pyridostigma of the present invention can denitrify nitrate cellulose under aerobic conditions without the need for a strict anaerobic environment, effectively reducing the nitrogen content of nitrate cellulose; at the same time, Rhodococcus pyridostigma can use the nitrate nitrogen and nitrite nitrogen under the denitrification of nitrate cellulose as nutrients for its own growth and reproduction.

[0013] (2) The denitrification treatment of nitrate cellulose by Rhodococcus pyridostigma of the present invention does not produce harmful nitrates and nitrites, and does not cause secondary pollution to water quality;

[0014] (3) The pyridine-loving erythrococcus culture medium of the present invention has a simple composition, is a commonly used LB medium, the raw materials are readily available, the composition is simple, and only the nitrified cellulose needs to be sterilized before the denitrification treatment. The denitrification reaction process is simple and convenient and suitable for industrial application. Attached Figure Description

[0015] Figure 1 This is a diagram showing the colony morphology of Rhodococcus pyridostigmine on LB medium.

[0016] Figure 2 This is a microscopic image of Rhodococcus pyridostigmine;

[0017] Figure 3 Scanning electron microscope image of Rhodococcus pyridostigmine (×10000);

[0018] Figure 4 The OD value of Rhodococcus pyridostigmine colonies after co-culturing with nitrocellulose for 3-19 days;

[0019] Figure 5 The graph shows the changes in the total content of nitrite and nitrate ions in the culture medium of Rhodococcus pyridostigma after 3-19 days of co-culturing with nitrocellulose.

[0020] Figure 6 The graph shows the change in nitrogen content of nitrified cellulose after co-culturing Rhodococcus pyridostigmine with nitrified cellulose for 3 days.

[0021] Figure 7 This is a comparison of FTIR images of nitrified cellulose after co-culturing Rhodococcus pyridostigmine with nitrified cellulose for 3-19 days. Detailed Implementation

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] Example 1: Isolation, purification and identification of Rhodococcus pyridostigmine.

[0025] 1. Isolation and purification of Rhodococcus pyridostigmine

[0026] Rhodococcus pyridostigmine is a Gram-positive bacterium that was domesticated and isolated from the activated sludge of leachate treatment wastewater from Luzhou Northern Chemical Co., Ltd. in Sichuan Province. The specific steps are as follows:

[0027] Add 100 ml of LB liquid medium to a 500 ml shake flask, along with 5 g of activated sludge and 1 g / L of nitrated cellulose for enrichment culture at 25°C and a shaker speed of 200 rpm. After 7 days of culture, take 5 ml of the enriched solution and add it to 100 ml of fresh LB liquid medium, adding the same amount of nitrated cellulose. Repeat this enrichment process three times. For acclimatization culture, take 5 ml of the liquid from the third enrichment culture and add it to 100 ml of acclimatization medium, adding the same concentration of nitrated cellulose. After 7 days of culture, take 5 ml of the acclimatization medium and add it to fresh acclimatization medium, adding the same amount of nitrated cellulose. Serially dilute the final acclimatization solution and spread it on LB solid medium. Purify by streaking on single colony isolation plates. Figure 1 The obtained bacterial strain was added to LB medium, and nitrified cellulose was added as a nitrogen source for verification.

[0028] Strain characteristics: Colonies on the culture medium are red, cheese-like, with neat edges, round, opaque, smooth, moist, and easy to pick up. Transmission electron microscopy reveals that the bacteria are short rods, non-flagellated, and Gram-positive.

[0029] The LB medium formula is: yeast extract 5g / L, peptone 10g / L, NaCl 5g / L. In addition to the above components, 2% agar powder is added to the LB solid medium. The pH is natural and the solvent is deionized water.

[0030] The acclimatization culture medium formula is: glucose 15g / L, NaCl 1g / L. All the above culture media must be sterilized by high temperature and high pressure (121℃, 20min) before use.

[0031] 2.16S rDNA sequence analysis

[0032] The 16S rDNA sequence of the screened Rhodococcus pyridostigmine is 1414 bp in length, and its nucleotide sequence is shown in SEQ ID No. 1 of the sequence listing. The 16S rDNA sequence was compared with related species in the GeneBank database. The results showed that the denitrified strain and Rhodococcus pyridostigmine share 99.64% homology. Therefore, the strain of this invention is identified as Rhodococcus pyridostigmine.

[0033] Example 2: Large-scale culture of Rhodococcus pyridostigmine

[0034] (1) Preservation of glycerin tubes

[0035] Rhodococcus pyridostigmine was inoculated into LB liquid medium and activated by incubation at 37°C and 200 rpm for 2 days. The activated bacteria were then streaked onto solid LB plates and incubated at 37°C. Single colonies were taken and streaked again to test the purity of the bacteria. Single colonies were then inoculated into 10 ml of LB medium under aseptic conditions and incubated at 37°C and 200 rpm until the OD of Rhodococcus pyridostigmine reached 0.6-0.8. Under aseptic conditions, 800 μL of 30% sterile glycerol and 800 μL of bacterial suspension were added to a sterile glycerol tube and stored at -80°C. The composition of the LB liquid and solid medium was the same as in Example 1.

[0036] (2) Expand cultivation

[0037] The bacterial cells preserved in the glycerol tubes from step (1) were inoculated into LB liquid medium and cultured at 37°C and 200 rpm until the medium turned red. The composition of the LB liquid medium was the same as in Example 1.

[0038] Example 3: Comparison of nitrogen reduction effects of nitrified cellulose by Rhodococcus pyridinivorans on different number of days.

[0039] The Rhodococcus pyridostigmine bacterial suspension from Example 2 was inoculated into 100 ml of LB medium and cultured. 5 g / L of nitrated cellulose was added to the LB medium. After the nitrated cellulose was thoroughly moistened by gentle shaking, it was sterilized in an autoclave (121°C, 20 min). The co-culture conditions of nitrated cellulose and Rhodococcus pyridostigmine were 37°C and 200 rpm.

[0040] After the transformation reaction proceeded for 2-19 days, the OD value of the resulting liquid was determined. The specific testing method was as follows: 5 ml of the reaction liquid was taken, filtered through filter paper, and the absorbance of the filtered liquid was measured at 600 nm using a UV spectrophotometer. The results are shown below. Figure 4 As shown, the blank control is pure water. From Figure 4 It can be seen that the growth of the strains in the liquid after the reaction is characterized by a phenomenon where the OD of the strains first decreases, then grows, and then decreases again as the reaction time increases. This indicates that after the initial strains adapt to the growth environment, they begin to grow using nitrocellulose. As nutrients are continuously consumed, the number of strains shows a downward trend.

[0041] After the conversion reaction proceeded for 2-19 days, the contents of nitrite nitrogen and nitrate nitrogen in the solution were determined. The specific test methods were as follows: the method for detecting nitrate nitrogen concentration was ultraviolet spectrophotometry (HJ / T 346-2007); the method for detecting nitrite nitrogen concentration was N-(1-naphthyl)-ethylenediamine spectrophotometry (GB 7493-84). Figure 5 The graph shows the changes in the total content of nitrite and nitrate ions in the culture medium after co-culturing Rhodococcus pyridostigma with nitrified cellulose for 3-19 days. It was found that when the reaction time reached 14 days, the nitrite and nitrate nitrogen in the solution continuously increased, and when the reaction time reached 19 days, the nitrite and nitrate nitrogen in the solution decreased sharply. It can be seen that Rhodococcus pyridostigma can use nitrified cellulose as a nutrient for growth and reproduction, and the bacteria can denitrify nitrite and nitrate nitrogen, thereby avoiding water pollution in the treatment process.

[0042] The cultured substrate, nitrocellulose, was re-extracted. The extraction method was as follows: First, the precipitate was collected by centrifugation at 8000 rpm and washed once with pure water; it was poured into a glass dish and dried in a 60℃ oven; acetone solution was added to the precipitate at a ratio of 40 ml acetone per 1 g of precipitate, and the mixture was thoroughly dissolved in a 45℃ water bath for 3-4 hours, then centrifuged to collect the supernatant; a mixture of water and ethanol at a volume ratio of 2:1 was added dropwise to the supernatant until no more nitrocellulose precipitated; the precipitate was collected by centrifugation, and the centrifuge tube was leveled with 1M NaCl solution and washed three times; after completion, the precipitate was washed three times with pure water by vacuum filtration, dried, and the nitrogen content was analyzed using an Elemantar Vario EL Cube elemental analyzer. The results are as follows. Figure 6 As shown, the nitrogen content of the original nitrated cellulose decreased after being treated with Rhodococcus pyridostigma for 3 days, indicating that Rhodococcus pyridostigma can indeed denitrate nitrated cellulose.

[0043] Example 4: Comparison of FTIR results of nitrate cellulose after treatment with Rhodococcus pyridostigma for different number of days

[0044] Fourier transform infrared spectroscopy was performed to compare nitrified cellulose treated with Rhodococcus pyridostigma for different numbers of days. The 1638 cm⁻¹ sample was among the samples treated with Rhodococcus pyridostigma. -1 The vibration is -NO2 asymmetric stretching vibration at 1256 cm. -1 The vibrations at these locations are -NO2 symmetric stretching vibrations, with values ​​of 836, 746, and 679 cm. -1 For O-NO2 tensile and torsional vibrations, 1156, 1058 cm -1 The vibrations at the points represent symmetric and asymmetric vibrations caused by oxygen bridges and ring groups in nitrate cellulose. It was found that the major groups of nitrate cellulose did not disappear, nor were any new groups generated, indicating that, under certain time conditions, co-culturing nitrate cellulose with Rhodococcus pyridostigma will not destroy the structure of nitrate cellulose. See details... Figure 7 .

[0045] In summary, the Rhodococcus pyridostigma of the present invention can perform denitrification and denitrification reactions on nitrate cellulose. Through the co-reaction of the strain and the substrate, the strain completes the denitrification treatment of nitrate cellulose. Furthermore, Rhodococcus pyridostigma can use the nitrates and nitrites generated during the denitrification of nitrate cellulose, which are polluting to the environment, as nutrients for its own growth and reproduction. The entire process is economical and green, which is conducive to the industrial processing of nitrate cellulose.

[0046] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention. sequence list <110> Nanjing University of Science and Technology <120> Rhodococcus pyridostigmine and its application in the degradation of nitrate cellulose <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1414 <212> DNA <213> Rhodococcus pyridinivorans <400> 1 tcatgcgtca tgcatgcagt cgacgatgag cccagcttgc tgggtggatt agtggcgaac 60 gggtgagtaa cacgtgggtg atctgccctg cactctggga taagcctggg aaactgggtc 120 taataccgga tatgacctcg ggatgcatgt tctggggtgg aaagtttttc ggtgcaggat 180 gagcccgcgg cctatcagct tgttggtggg gtaatggcct accaaggcga cgacgggtag 240 ccggcctgag agggcgaccg gccacactgg gactgagaca cggcccagac tcctacggga 300 ggcagcagtg gggaatattg cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg 360 gatgacggcc ttcgggttgt aaacctcttt cacccatgac gaagcgcaag tgacggtagt 420 gggagaagaa gcaccggcca actacgtgcc agcagccgcg gtaatacgta gggtgcgagc 480 gttgtccgga attactgggc gtaaagagct cgtaggcggt ttgtcgcgtc gtctgtgaaa 540 tcccgcagct caactgcggg cttgcaggcg atacgggcag actcgagtac tgcaggggag 600 actggaattc ctggtgtagc ggtgaaatgc gcagatatca ggaggaacac cggtggcgaa 660 ggcgggtctc tgggcagtaa ctgacgctga ggagcgaaag cgtgggtagc gaacaggatt 720 agataccctg gtagtccacg ccgtaaacgg tgggcgctag gtgtgggttt ccttccacgg 780 gatccgtgcc gtagccaacg cattaagcgc cccgcctggg gagtacggcc gcaaggctaa 840 aactcaaagg aattgacggg ggcccgcaca agcggcggag catgtggatt aattcgatgc 900 aacgcgaaga accttacctg ggtttgacat gtaccggacg actgcagaga tgtggtttcc 960 cttgtggccg gtagacaggt ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg 1020 ttaagtcccg caacgagcgc aacccttgtc ctgtgttgcc agcacgtgat ggtggggact 1080 cgcaggagac tgccggggtc aactcggagg aaggtgggga cgacgtcaag tcatcatgcc 1140 ccttatgtcc agggcttcac acatgctaca atggtcggta cagagggctg cgataccgtg 1200 aggtggagcg aatcccttaa agccgatctc agttcggatc ggggtctgca actcgacccc 1260 gtgaagtcgg agtcgctagt aatcgcagat cagcaacgct gcggtgaata cgttcccggg 1320 ccttgtacac accgcccgtc acgtcatgaa agtcggtaac acccgaagcc ggtggcctaa 1380 cccctgtgga agcagccgag aataggataa attc 1414

Claims

1. Pyridine-loving Rhodococcus ( Rhodococcuspyridinivorans ), characterized in that, It is Rhodococcus pyridostigmine LZ1, with accession number CCTCC NO: M 2022785.

2. The method for culturing Rhodococcus pyridostigmine according to claim 1, characterized in that, Includes the following steps: Rhodococcus pyridostigmine was inoculated into LB liquid medium and cultured dynamically at 37°C.

3. The cultivation method according to claim 2, characterized in that, The LB liquid culture medium is formulated as follows: 5 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, pH natural.

4. The application of Rhodococcus pyridostigmine according to claim 1 in the degradation of nitrate cellulose.

5. The application according to claim 4, characterized in that, The specific method is as follows: Inoculate Rhodococcus pyridostigmine into wastewater containing nitrate cellulose to degrade the nitrate cellulose.

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