CN114410554A

CN114410554A - Chitin-phagocytic bacterium with aerobic arsenic methylation and volatilization functions and application thereof

Info

Publication number: CN114410554A
Application number: CN202210314513.4A
Authority: CN
Inventors: 方利平; 李芳柏; 陈冠虹
Original assignee: Institute of Eco Environmental and Soil Sciences of Guangdong Academy of Sciens
Current assignee: Institute of Eco Environmental and Soil Sciences of Guangdong Academy of Sciens
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2022-04-29
Anticipated expiration: 2042-03-29
Also published as: CN114410554B

Abstract

The invention discloses a chitin-phagocytosis strain with aerobic arsenic methylation and volatilization functions and application thereof, and belongs to the technical field of microorganisms. The invention relates to a bacterial strain named chitin phage (A)Chitinophaga filiformis) YT5, deposited at 31.12 months in 2021 at the Guangdong province collection center for microbial cultures of building 59 of Yao Dazhou 100, Pieli, Virginia, Guangdong province, with the collection numbers: GDMCC No: 62178. the chitin phagocytosis YT5 has obvious methylation effect on arsenous acid in arsenic-containing culture medium and functions of volatilizing methyl arsenic, and the main methyl arsenic product is dimethylArsenic and volatile trimethylarsenic, trimethylarsenic and dimethylarsine concentrations gradually increased with increasing incubation time, while arsenous acid concentrations decreased with increasing time. The chitin phagocytosis YT5 has important application potential in arsenic-polluted environment restoration, especially in arsenic-polluted soil restoration.

Description

Chitin-phagocytic bacterium with aerobic arsenic methylation and volatilization functions and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a chitin-phagocytosis bacterium with aerobic arsenic methylation and volatilization functions and application thereof.

Background

Arsenic is a toxic and carcinogenic substance that is ubiquitous in the environment and is an environmental problem that is commonly faced by many countries around the world. Particularly, trivalent arsenic (arsenous acid or arsenite) has higher mobility and stronger toxicity in soil and water bodies. Trivalent arsenic is easier to be absorbed by crops such as rice and the like, and the risk problems such as exceeding the standard of arsenic in grains and the like are caused. Therefore, how to effectively control arsenic pollution is the leading-edge problem of current water pollution treatment and soil remediation, and is also a national important demand. Research shows that the microorganism carrying the arsenic methylation functional gene in the soil has the state of converting inorganic arsenic into organic arsine such as methyl arsenic and the like. On one hand, the generated organic arsine is weaker in toxicity and is easier to volatilize from soil or water; in addition, organic arsine is more difficult to be absorbed by crops such as rice, and the proportion of inorganic arsenic in agricultural products such as rice can be further reduced. Thus, the methylation process of inorganic arsenic, especially trivalent arsenic, is an efficient arsenic detoxification process, helping to reduce the risk of arsenic contamination. Research shows that the growth speed of the arsenic methylation functional aerobic bacteria is high, and the arsenic methylation efficiency of the aerobic bacteria is far higher than that of the anaerobic bacteria. CN201510956127.5 identifies an aerobic arsenic methylation bacterium SM-1 of the family Cellophilicaceae, but the bacterium can only grow in a R2A culture medium and cannot grow in LB and other culture media, and has obvious limitation. In addition, the arsenic concentration of the strain is low, which is not beneficial to wide application. In view of the present extreme shortage of arsenomethylating bacteria resources, the development of novel arsenomethylating functional microbial strains is urgently needed. Therefore, the aerobic arsenic methylation and volatilization functional bacteria in the rice field soil are obtained by separation, which has important application value and significance for solving the environment of China by using the microbial technology and is also beneficial to improving the core competitiveness of the microbial treatment technology of the environment of China.

Disclosure of Invention

In order to solve the problems, the first purpose of the invention is to provide a chitin-phagocytosis strain with aerobic arsenic methylation and volatilization functions.

The second purpose of the invention is to provide the application of the chitin-phagocytosis bacteria with aerobic arsenic methylation and volatilization functions.

The purpose of the invention is realized by the following technical scheme:

a chitin-phagocytic bacterium with aerobic arsenic methylation and volatilization functions (chitin-phagocytic bacterium)Chitinophaga filiformis) YT5, deposited at 31.12 months in 2021 at the Guangdong province microbial culture Collection (GDMCC) of building 59 of Yao Dazhou 100, Pieli, Uighur, Calif., Guangzhou, Guangdong province, with the accession numbers: GDMCC No: 62178.

the application of the chitin phage YT5 is to culture the chitin phage YT5 in an environment containing inorganic trivalent arsenic to realize arsenic methylation.

Further, the methylation is dimethylation and/or trimethylation.

Further, the inorganic trivalent arsenic is arsenous acid and/or arsenite.

Further, the concentration range of the inorganic trivalent arsenic in the environment is 0-100 mu mol/L; preferably 0 to 50 mu mol/L; more preferably 0 to 12. mu. mol/L; most preferably 0 to 6. mu. mol/L.

Further, the environment includes, but is not limited to, culture medium, soil and water.

Further, the culture medium is any one of an R2A culture medium, an LB culture medium and a TSB culture medium.

Further, the formula of the R2A culture medium is as follows: 0.3-0.4 g/L tryptone, 0.4-0.6 g/L acid hydrolyzed casein, 0.4-0.6 g/L yeast extract powder, 0.4-0.6 g/L soluble starch, 0.2-0.4 g/L dipotassium hydrogen phosphate, 0.05-0.15 g/L magnesium sulfate, 0.2-0.4 g/L sodium pyruvate, 0.2-0.3 g/L peptone, 0.4-0.6 g/L glucose, the balance of water, and the pH value of 7.2 +/-0.2; preferably 0.35g/L of tryptone, 0.5g/L of acid hydrolyzed casein, 0.5g/L of yeast extract powder, 0.5g/L of soluble starch, 0.3g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate, 0.3g/L of sodium pyruvate, 0.25g/L of peptone, 0.5g/L of glucose and the balance of water, and the pH value is 7.2 +/-0.2.

The LB culture medium comprises the following components in percentage by weight: 9-11 g/L peptone, 4-6 g/L yeast powder, 9-11 g/L NaCl and the balance of water; preferably: 10g/L peptone, 5g/L yeast powder, 10g/L NaCl and the balance of water.

The TSB culture medium comprises the following components in percentage by weight: 16-18 g/L of tryptone, 2-4 g/L of soybean papain digest, 4-6 g/L of sodium chloride, 2-3 g/L of potassium dihydrogen phosphate, 2-3 g/L of glucose and the balance of water, wherein the pH value is 7.3 +/-0.2; preferably: 17g/L tryptone, 3g/L soybean papain digest, 5g/L sodium chloride, 2.5g/L potassium dihydrogen phosphate, 2.5g/L glucose, and the balance of water, wherein the pH value is 7.3 +/-0.2.

Further, when the environment is a culture medium, the inoculum size of the chitin-phagocytizing bacteria YT5 is determined by the bacterial cell density OD₆₀₀And (4) =0.01 meter.

Further, the culture conditions comprise that the temperature is 28-32 ℃, the rotating speed is 150-250 rpm, and the time is 60-80 h; preferably, the temperature is 30 ℃, the rotation speed is 200rpm, and the time is 72 h.

A microbial preparation with aerobic arsenic methylation and volatilization functions comprises a culture medium and the chitin phage YT 5.

The preparation method of the microbial preparation with aerobic arsenic methylation and volatilization functions comprises the steps of inoculating the chitin phage YT5 into a culture medium, and performing shake culture to obtain the microbial preparation with aerobic arsenic methylation and volatilization functions.

The shake culture conditions comprise that the temperature is 28-32 ℃, the rotating speed is 150-250 rpm, and the time is 8-14 h; preferably, the temperature is 30 ℃, the rotating speed is 200rpm, and the time is 10-12 h.

The microbial preparation with the aerobic arsenic methylation and volatilization functions is applied to the arsenic-polluted environment remediation.

Further, the environment includes, but is not limited to, a body of water and soil.

Further, the specific operation of the application is as follows: and (3) placing the microbial preparation with the aerobic arsenic methylation and volatilization functions in an environment to be repaired for culture.

Compared with the prior art, the invention has the following advantages and effects:

the chitin phagocytosis bacterium YT5 provided by the invention has obvious methylation effect on inorganic trivalent arsenic and the ability of volatilizing methyl arsenic, and the main methyl arsenic products are trimethyl arsenic and dimethyl arsenic, wherein the proportion of trimethyl arsenic with lower toxicity is taken as the main component. Therefore, the chitin phage YT5 can obviously reduce the content of virulent inorganic arsenic, generate organic arsine with lower toxicity and volatile arsenic, and has important application potential in arsenic-polluted environment treatment and restoration, especially in arsenic-polluted soil treatment and restoration.

The invention further determines an optimal culture substrate for regulating arsenic methylation by the chitinase YT 5.

Drawings

FIG. 1 is a colony morphology of arsenic methylation functional chitinase YT5 in R2A medium.

FIG. 2 is a graph representing the morphology of cells in the logarithmic phase of the arsenic methylation functional chitinase YT 5; wherein A is an SEM image and B is a TEM image.

FIG. 3 is a phylogenetic tree diagram of the 16S rRNA gene of the arsenic methylation functional chitinase YT 5.

FIG. 4 is a graph showing the growth of the arsenic methylation functional chitin phage YT5 in the culture medium containing arsenic R2A, LB and TSB.

FIG. 5 is a graph showing the results of analysis of the efficiency of arsenic methylation of the arsenic methylation function of chitinase YT5 in the culture medium containing arsenic R2A, LB and TSB.

FIG. 6 is a graph showing the results of analysis of the effect of As methylation of the arsenic methylation function Chitosan phagemid YT5 after culturing in TSB medium containing different initial arsenous acids for 72 hours.

FIG. 7 is a graph showing the result of analysis of the arsenic volatilization accumulation of the arsenic methylation functional chitin phage YT5 in the culture medium containing arsenic R2A, LB and TSB for 72 hours.

FIG. 8 is a graph showing the results of analysis of inorganic arsenic conversion in a soil system by chitin phagocytosis YT5 with arsenic methylation function.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

Example 1: separation and identification of arsenic methylation functional chitin phagocytosis YT5

Samples were collected from arsenic contaminated paddy field soil in certain areas of Hunan province. Weighing 100g of a soil sample which is naturally dried and sieved by a 10-mesh sieve into a 250ml conical flask, adding 100ml of sterilized ultrapure water, sealing the flask mouth with a breathable film, and standing and culturing for 15 days at room temperature to activate soil microorganisms. Using sterilized ultrapure water to carry out gradient dilution on the soil suspension, wherein the concentration gradient is 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6And 10^-7Respectively taking 100 mul of the solution with the concentration of 10^-4、10^-5、10^-6And 10^-7The diluted solution of (3) was applied to ST10 containing 20. mu.M arsenous acid^-1Solid medium plate. ST10^-1The formula of the solid culture medium is as follows: tryptone 0.5g, yeast extract 0.05g, glucose 0.5g, constant volume to 1000ml, high temperature and high pressure sterilization at 121 ℃ for 20 min. After the 12mM arsenous acid stock solution was sterilized by filtration through a 0.22 μ M pore filter, 1.7mL of the stock solution was added to 1000mL of the medium to give a final arsenous acid concentration of 20 μ M. The coated plate was placed upside down in an incubator at 30 ℃ for 72 hours, and a single colony with a distinct morphological feature was picked and inoculated into 100mlR2A medium containing 10. mu.M arsenous acid. The formula of the R2A liquid medium is as follows: 0.35g of tryptone, 0.5g of acid hydrolyzed casein, 0.5g of yeast extract powder, 0.5g of soluble starch, 0.3g of dipotassium phosphate, 0.1g of magnesium sulfate, 0.3g of sodium pyruvate, 0.25g of peptone, 0.5g of glucose and pH 7.2, adding water to a constant volume of 1000ml, and sterilizing at 121 ℃ for 20min under high temperature and high pressure. After single colony inoculation culture is carried out for 72h, the content of the methyl arsenic, the dimethyl arsenic, the trimethyl arsenic, the arsenous acid and the arsenic acid in the culture solution is detected by a high performance liquid chromatography-inductively coupled plasma mass spectrometer (HPLC-ICP-MS, PerkinElm), the bacterial solution generated by the methyl arsenic is selected for flat plate streak purification, and a bacterial strain with the arsenic methylation function is obtained after three times of purification.

Morphological feature analysis: the strain is gram-negative bacteria, has no endospore and no motility. As shown in figure 1, the bacterial strain grows aerobically on R2A at 30 ℃ for 3 days, the bacterial colony is round, convex, smooth in surface, neat in edge, golden in color, and the diameter of the bacterial colony is 0.5-1.0 mm. Scanning electron microscopy (SEM, A in figure 2) shows that the strain is rod-shaped as a whole, and the ends of the strain are blunt and round; transmission electron microscopy (TEM, B in FIG. 2) showed that strain YT5 was oval, sporulated, pilus-free, electron dense in the nuclear region, and cells were approximately 1.5 μm. + -. 0.4 μm in average length and 0.4 μm. + -. 0.1 μm in width.

Physiological and biochemical characteristic analysis: the strain can grow at 37 ℃ and cannot grow under anaerobic conditions. A catalase test (catalase test) is negative, indicating that the strain does not have the capacity of catalyzing the decomposition of hydrogen peroxide into molecular oxygen so as to detoxify. The oxidase was positive, indicating that the strain had cytochrome oxidase which oxidized cytochrome C to oxidized cytochrome C which oxidized phenylenediamine to develop color. The strain can hydrolyze esculin to generate glucose and esculin. The gelatin liquefaction is positive, which indicates that the strain has gelatinase and has the function of liquefying gelatin. The positive experiment of 2-nitrophenyl-beta-D-galactopyranoside (ONPG) shows that the strain has beta-galactosidase activity, decomposes ONPG to generate yellow o-nitrophenol and can ferment lactose. Indole production is negative, i.e. the strain is not able to break down tryptophan in peptone, is not able to produce indigo substrate (indole); nor the ability to produce acid from glucose. Arginine double hydrolase and urease negatives, indicating that the strain has no arginine deacylase and citrullinase activity and no urea hydrolysis activity. The nitrate is reduced to be positive, and the bacterial strain has nitrate reductase and can reduce the nitrate into nitrite. As for the sugar-utilizing ability, the strain can utilize glucose, mannose, maltose, sucrose, arabinose and rhamnose, and also has the ability to utilize salicin and melibiose, but cannot utilize ribose, serine, mannitol and citrate.

16S rRNA gene sequence analysis: using bacterial DNA extraction kit (DNeasy Blood)&Tissue Kit, Qiagen, Valencia, CA) genomic DNA of the strain was extracted, and the obtained DNA was used as a template with primer 27F (AGAGTTTGATCMTGGCTC)AG) and 1492R (GGTTACCTTGTTACGACTT) were used to amplify the 16S rRNA gene, and the PCR products were sent to Shanghai Biometrics Ltd for Sanger sequencing. The length of the 16S rRNA gene fragment sequence of the strain is 1354bp, and the specific sequence is shown in SEQ.ID NO. 1. Nucleotide homology comparison is carried out on the registered sequences in a Genbank database by using Blast, and the result shows that the aerobic arsenic methylation functional strain YT5 is combined with the registered sequencesChitinophaga filiformisThe homology of the gene reaches 99.48 percent (great name)Flexibacter filiformis). 16S rRNA gene sequence of the strain andChitinophagabelongs to other strains, multiple alignment is carried out in MEGA software by MUSCLE algorithm, gaps before and after the alignment are deleted, and phylogenetic trees are constructed by an adjacent method. As can be seen from the topology of the tree in FIG. 3, this strain is similar to the model strainChitinophaga filiformis IFO 15056^TThe homology is highest.

Preliminarily judging the strain to be chitin phagocytic bacteria by combining morphological characteristics, physiological and biochemical characteristics and 16S rRNA gene sequence analysis resultsChitinophagaThe strain is named as chitin phage (A)Chitinophaga filiformis) YT5, deposited at 31.12.31.2021 at the Guangdong province microbial culture Collection (GDMCC) of building 59 of Yao 100, Pieli Zhongluo, Zyguyu, Guangdong province, with the deposit numbers: GDMCC No: 62178.

16S rRNA gene sequence (seq. id No. 1):

CAGGTAGCAATACTGGGTGGCGACCGGCAAACGGGTGCGGAACACGTACGTAACCTTCCTTTAAGTGGGAGATAGCCCGAAGAAATTCGGATTAATACCCCATAAGATCATGGAGTGGCATCACTCAGTGATTAAAGAATTTCGCTTAAAGATGGGCGTGCGGCTGATTAGGTAGTTGGTGAGGTAACGGCTCACCAAGCCAACGATCAGTAACTGGCGTGAGAGCGCGACCAGTCACACGGGCACTGAGACACGGGCCCGACTCCTACGGGAGGCAGCAGTAAGGAATATTGGTCAATGGACGGAAGTCTGAACCAGCCATGCCGCGTGAAGGATGAAGGTCCTCTGGATTGTAAACTTCTTTTATATGGGAAGAAACCACTTTTTtCTAAAGGTGTtGACgGTACCATAGGAATAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTATCCGGATTCACTGGGTTTAAAGGGTGCGTAGGCGGATTAGTAAGTCCGTGGTGAAATCTCCGAGCTTAACTCGGAAACTGCCGTGGATACTATTAATCTTGAATGCTGTGGAGGTTAGCGGAATATGTCATGTAGCGGTGAAATGCATAGATATGACATAGAACACCAATTGCGAAGGCAGCTGGCTACACAGAGATTGACGCTGAGGCACGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATTACTCGACATTTGCGATATACAGTAAGTGTCTGAGCGAAAGCATTAAGTAATCCACCTGGGAAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGTCCGCACAAGCGGTGGAGCATGTGgTTTAATTCGATGATACGCGAGGAACCTTACCTgGGCTAGAATGCTGGGGGACTGGATCTGAAAGGGTCCTTTGTAGCAATACACCGCCAGTAAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTTAGTTGCCAACAGGTCAAGCTGGGAACTCTAAAGAAACTGCCGTCGTAAGACGCGAGGAAGGAGGGGATGATGTCAAGTCATCATGGCCTTTATGCCCAGGGCTACACACGTGCTACAATGGTAGGAACAAAGGGCTGCTACCTGGTAACAGGATGCTAATCTCAAAAATCCTATCTCAGTTCGAATTGAGGGCTGCAACTCGCCCTCATGAAGCTGGAATCGCTAGTAATCGTATATCAGCAATGATACGGTGAATACGTTCCCGGACCTTGTACACACCGCCCGTCAAGCCATGAAAGCCGGGGGGACCTGAAG 。

example 2: growth curve and arsenic methylation efficiency of arsenic methylation functional chitin phagocytosis YT5 in culture medium containing arsenic R2A, LB and TSB

And carrying out amplification culture and arsenic methylation efficiency verification on YT5 by using three liquid culture media of R2A, LB and TSB. The method comprises the following specific steps: 500ml of sterilized R2A liquid culture medium is prepared, and the single colony in the streak plate is inoculated into the culture medium for shake culture (30 ℃, 200 rpm) for 10-12 hours, the OD600 is 0.4-0.5, and is in the middle logarithmic phase. The strain liquid after the enlargement culture is centrifuged for 10min at 5000rpm for collecting the strain. 0.55ml of YT5 bacterial suspension (OD 600= 1.8) was inoculated into 100ml of sterilized R2A, LB and TSB liquid medium containing 12. mu.M arsenous acid, respectively. The LB liquid culture medium formula is: 10g/L peptone, 5g/L yeast powder, 10g/L NaCl and the balance of water; the TSB liquid culture medium formula is as follows: tryptone 17.0 g/L, soybean papain digest 3.0 g/L, sodium chloride 5.0 g/L, potassium dihydrogen phosphate 2.5g/L, glucose 2.5g/L, and water in balance, wherein the pH value is 7.3 +/-0.2. The OD600 of the initial bacteria for reaction is 0.01, the culture solution is cultured for 72 hours under the conditions of 30 ℃ and 200rpm constant temperature shaking, 200 mu L of the bacteria solution is taken to be positioned in an enzyme label plate for measuring the OD600 respectively at each sampling time point of culture, 1ml of the bacteria solution sample is centrifuged for 2min at 12000rpm, and after the supernatant is filtered by a 0.22 mu m filter membrane, the HPLC-ICP-MS is used for arsenic morphological analysis and quantification.

As shown in fig. 4, the growth curve of the strain YT5 in the arsenic-containing medium shows that the strain YT5 can grow in three media and present different growth characteristics; log phase lags in LB, YT5 growth accelerated after 24 hours of culture in TSB and exceeded R2A at 40 hours. The stationary phase was entered at 24, 58 and 40 hours in R2A, LB and TSB, respectively, and the biomass in LB and TSB was 3.5-4.4 times greater than in R2A, indicating that LB and TSB are the best media for the expanded culture of strain YT 5.

As shown in FIG. 5, the strain YT5 showed significant methylation of 12. mu.M arsenous acid in the medium containing arsenic R2A, LB and TSB. In the three media, the concentrations of trimethylarsenic and dimethylarsine gradually increased with increasing culture time, while the concentration of inorganic arsenous acid decreased with increasing culture time. The methyl arsenic was most efficient in TSB at 48 hours of culture, followed by LB and R2A. After the reaction is finished for 72 hours, the conversion ratio of arsenous acid to methyl arsenic in the culture media of R2A, LB and TSB is respectively 13%, 38% and 53%, which proves that the strain YT5 can well convert arsenous acid to methyl arsenic under the conditions of three culture media. The main products of methyl arsenic are trimethyl arsenic and dimethyl arsenic, and the proportions of trimethyl arsenic, which are less toxic, are 40%, 44% and 52% in R2A, LB and TSB, respectively.

Example 3: comparison of efficiency of converting As-methylated functional Chitosan YT5 to arsenous acid of different concentrations in TSB Medium

In reference example 2, 0.55ml of YT5 bacterial suspension (OD 600= 1.8) was inoculated into 100ml of sterilized TSB liquid medium containing 3, 6, 12, 24, and 48 μ M arsenous acid, respectively. The strain YT5 has initial strain OD600 of 0.01, the culture solution is cultured for 72 hours under the conditions of 30 ℃ and 200rpm constant temperature shaking, points are respectively taken on each treated sample, 1ml of the strain solution sample is centrifuged for 2min at 12000rpm, and after supernatant is filtered by a 0.22 mu m filter membrane, the arsenic morphological analysis and the quantification are carried out by HPLC-ICP-MS.

As shown in FIG. 6, the strain YT5 has good arsenic methylation conversion efficiency on different gradients of arsenous acid in a TSB culture medium; the methylation conversion rate of arsenous acid is 100%, 98.2%, 52.1%, 24.3% and 10.5% in 72 hours; the production amount of methyl arsenic reaches 3.05, 6.1, 6.3, 5.83 and 5.1 mu M respectively; the result shows that the transformation amount of the strain is not obviously reduced along with the increase of the initial concentration of the arsenous acid on the premise of the same strain amount, and the strain YT5 is proved to have better tolerance to the arsenous acid.

Example 4: arsenic volatilization efficiency of arsenic methylation functional chitin phagocytosis YT5 in culture medium containing arsenic R2A, LB and TSB

120ml of R2A, LB and TSB medium containing 12. mu.M of inorganic arsenous acid were prepared in 500ml ground gas-collecting glass bottles, respectively. The volatile arsenic collecting device comprises 500ml ground glass bottle, two ventilating ports and a catcher, wherein one ventilating port is used for blowing and ventilating, the other ventilating port is used for wrapping a 1ml gun head by tinfoil paper, quartz wool is arranged at two ends of the gun head, and the filler in the middle is 1% AgNO₃Soaking 35-60 mesh high-purity silica gel particles overnight to play a role in exhausting gas and oxidizing and capturing volatile arsenic. The OD600 of the initial bacteria liquid is 0.01, the culture is carried out on a shaking table at 30 ℃ and 200rmp, and the catching heads are recovered after the culture is carried out for 72 hours. In order to collect volatile arsenic from the entire glass bottle and from the culture, air was blown from the air inlet for 20 minutes each time, the glass bottle was shaken intermittently, and the trap was replaced with a new one after completion. Taking out and recovering silica gel particles in the trapping gun head at 2ml of 0.5 (V/V) HNO₃And refluxing, digestion and elution are carried out for 2 hours at the medium temperature of 90 ℃, and the form and the content of arsenic are measured by HPLC-ICP-MS. As the volatile arsenic detected in the experiment is only trimethyl arsenic and contains AgNO₃The soaked silica gel particles are capable of oxidizing trimethylarsenic to trimethylarsenic oxide. Thus, the arsenic content in the eluate represents the trimethylarsenic content.

Strain YT5 produced volatile arsenic after 72 hours of culture in R2A, LB and TSB. As shown in fig. 7. The volatile methyl arsenic product is low-toxicity trimethyl arsenic, and no other volatile arsenic is detected. Volatile methyl arsenic in R2A, LB and TSB media was 0.014 μ g, 0.16 μ g and 0.07 μ g (as arsenic), respectively. The volatile methyl arsenic is mainly trimethyl arsenic, and the volatilization rates of arsenic in 72 hours are respectively 0.01% (R2A), 0.2% (LB) and 0.1% (TSB) of the total arsenic proportion of the three culture media. The results show that the strain YT5 has better volatilization capacity on arsenic.

Example 5: arsenic methylation function chitin phagocytosis YT5 effect of repairing arsenic in arsenic-contaminated soil

Collecting soil (the soil pH = 6.4 and the total arsenic = 45.6 mg/kg) of arsenic-polluted paddy field in the Hunan area, naturally drying the soil and sieving the soil with a 10-mesh sieve to obtain a uniform soil sample. Wherein, a part of soil sample is taken and sterilized by Co-gamma irradiation to obtain the sterilized soil sample. Respectively weighing 4g of original soil sample and 4g of sterilized soil sample in a 50ml conical flask, and respectively adding 40ml of sterilized ultrapure water; 0.55ml of YT5 bacterial liquid (OD 600= 1.8) was taken from the YT5 bacterial liquid obtained in example 2, and added to the original soil and the sterilized soil of the conical flask, and the corresponding control group (i.e., the sample without YT5 bacterial liquid) was set, and after the conical flask was placed at room temperature for standing and culturing for 14 days, 1ml of bacterial liquid sample was taken, and a predetermined amount of phosphoric acid extract was added, followed by shaking extraction, centrifugation at 12000rpm for 2min, and filtration of the supernatant on a 0.22 μm filter, followed by morphological analysis and quantification of arsenic by HPLC-ICP-MS.

As shown in fig. 8, with respect to the methyl arsenic content (1.5 mg/kg) of the soil control group and the methyl arsenic content (0.1 mg/kg) of the sterilized soil control group, the total amount of methyl arsenic in the phosphate extraction state in the arsenic-contaminated soil inoculated with YT5 strain reaches 15.5mg/kg, which is significantly higher than the methylation rates of arsenic in the soil control group and the sterilized soil control group, and it is proved that YT5 strain can effectively improve the methylation rate of the arsenic-contaminated soil; meanwhile, the rate of the arsenomethyl group directly inoculated into the sterilized soil is similar to that (16.9 mg/kg), and the addition of YT5 strain and the effect thereof are less influenced by soil indigenous microorganisms.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> institute for ecological environment and soil of academy of sciences of Guangdong province

<120> chitin phage strain with aerobic arsenic methylation and volatilization functions and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1354

<212> DNA

<213> Chitinophaga chitin bacteria (Chitinophaga filiformis)

<400> 1

caggtagcaa tactgggtgg cgaccggcaa acgggtgcgg aacacgtacg taaccttcct 60

ttaagtggga gatagcccga agaaattcgg attaataccc cataagatca tggagtggca 120

tcactcagtg attaaagaat ttcgcttaaa gatgggcgtg cggctgatta ggtagttggt 180

gaggtaacgg ctcaccaagc caacgatcag taactggcgt gagagcgcga ccagtcacac 240

gggcactgag acacgggccc gactcctacg ggaggcagca gtaaggaata ttggtcaatg 300

gacggaagtc tgaaccagcc atgccgcgtg aaggatgaag gtcctctgga ttgtaaactt 360

cttttatatg ggaagaaacc acttttttct aaaggtgttg acggtaccat aggaataagc 420

accggctaac tccgtgccag cagccgcggt aatacggagg gtgcaagcgt tatccggatt 480

cactgggttt aaagggtgcg taggcggatt agtaagtccg tggtgaaatc tccgagctta 540

actcggaaac tgccgtggat actattaatc ttgaatgctg tggaggttag cggaatatgt 600

catgtagcgg tgaaatgcat agatatgaca tagaacacca attgcgaagg cagctggcta 660

cacagagatt gacgctgagg cacgaaagcg tggggatcaa acaggattag ataccctggt 720

agtccacgcc ctaaacgatg attactcgac atttgcgata tacagtaagt gtctgagcga 780

aagcattaag taatccacct gggaagtacg accgcaaggt tgaaactcaa aggaattgac 840

gggggtccgc acaagcggtg gagcatgtgg tttaattcga tgatacgcga ggaaccttac 900

ctgggctaga atgctggggg actggatctg aaagggtcct ttgtagcaat acaccgccag 960

taaggtgctg catggctgtc gtcagctcgt gccgtgaggt gttgggttaa gtcccgcaac 1020

gagcgcaacc cctatcttta gttgccaaca ggtcaagctg ggaactctaa agaaactgcc 1080

gtcgtaagac gcgaggaagg aggggatgat gtcaagtcat catggccttt atgcccaggg 1140

ctacacacgt gctacaatgg taggaacaaa gggctgctac ctggtaacag gatgctaatc 1200

tcaaaaatcc tatctcagtt cgaattgagg gctgcaactc gccctcatga agctggaatc 1260

gctagtaatc gtatatcagc aatgatacgg tgaatacgtt cccggacctt gtacacaccg 1320

cccgtcaagc catgaaagcc ggggggacct gaag 1354

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agagtttgat cmtggctcag 20

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggttaccttg ttacgactt 19

Claims

1. A chitin-phagocytic bacterium with aerobic arsenic methylation and volatilization functions is characterized in that: named chitin-phagocytizing bacteria (Chitinophaga filiformis) YT5, deposited at 31.12 months in 2021 at the Guangdong province collection center for microbial cultures of building 59 of Yao Dazhou 100, Pieli, Virginia, Guangdong province, with the collection numbers: GDMCC No: 62178.

2. the use of chitinophaga YT5 of claim 1, wherein: the chitin phagocytosis bacterium YT5 is cultured in an environment containing inorganic trivalent arsenic to realize arsenic methylation.

3. The use of chitinophaga YT5 according to claim 2, wherein:

the methylation is dimethylation and/or trimethylation;

the inorganic trivalent arsenic is arsenous acid and/or arsenite.

4. The use of chitinophaga YT5 according to claim 2 or 3, wherein:

the environment is a culture medium, soil or a water body;

the concentration range of the inorganic trivalent arsenic in the environment is 0-100 mu mol/L.

5. The use of chitinophaga YT5 according to claim 4, wherein:

the culture medium is any one of an R2A culture medium, an LB culture medium and a TSB culture medium;

the formula of the R2A culture medium is as follows: 0.3-0.4 g/L tryptone, 0.4-0.6 g/L acid hydrolyzed casein, 0.4-0.6 g/L yeast extract powder, 0.4-0.6 g/L soluble starch, 0.2-0.4 g/L dipotassium hydrogen phosphate, 0.05-0.15 g/L magnesium sulfate, 0.2-0.4 g/L sodium pyruvate, 0.2-0.3 g/L peptone, 0.4-0.6 g/L glucose, the balance of water, and the pH value of 7.2 +/-0.2;

the LB culture medium comprises the following components in percentage by weight: 9-11 g/L peptone, 4-6 g/L yeast powder, 9-11 g/L NaCl and the balance of water;

the TSB culture medium comprises the following components in percentage by weight: 16-18 g/L of tryptone, 2-4 g/L of soybean papain digest, 4-6 g/L of sodium chloride, 2-3 g/L of potassium dihydrogen phosphate, 2-3 g/L of glucose and the balance of water, wherein the pH value is 7.3 +/-0.2;

the culture conditions are that the temperature is 28-32 ℃, the rotating speed is 150-250 rpm, and the time is 60-80 h.

6. The use of chitinophaga YT5 according to claim 5, wherein:

the concentration range of the inorganic trivalent arsenic in the environment is 0-50 mu mol/L;

the formula of the R2A culture medium is as follows: 0.35g/L of tryptone, 0.5g/L of acid hydrolyzed casein, 0.5g/L of yeast extract powder, 0.5g/L of soluble starch, 0.3g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate, 0.3g/L of sodium pyruvate, 0.25g/L of peptone, 0.5g/L of glucose and the balance of water, wherein the pH value is 7.2 +/-0.2;

the LB culture medium comprises the following components in percentage by weight: 10g/L peptone, 5g/L yeast powder, 10g/L NaCl and the balance of water;

the TSB culture medium comprises the following components in percentage by weight: 17g/L tryptone, 3g/L soybean papain digest, 5g/L sodium chloride, 2.5g/L potassium dihydrogen phosphate, 2.5g/L glucose and the balance of water, wherein the pH value is 7.3 +/-0.2;

the inoculation amount of the chitin-phagocytizing bacteria YT5 is determined according to the bacterial cell density OD₆₀₀=0.01 meter;

the culture conditions are 30 ℃, 200rpm of rotation speed and 72h of time.

7. A microbial preparation with aerobic arsenic methylation and volatilization functions is characterized in that: comprises a culture medium and the chitinophaga YT5 of claim 1;

the culture medium is any one of the R2A culture medium, LB culture medium and TSB culture medium of claim 5 or 6.

8. The method for preparing a microbial preparation having aerobic arsenic methylation and volatilization function as claimed in claim 7, wherein: inoculating the chitin-phagocytizing bacteria YT5 in a culture medium, and performing shake culture to obtain the microbial preparation with aerobic arsenic methylation and volatilization functions.

9. The method for preparing a microbial preparation with aerobic arsenic methylation and volatilization functions as claimed in claim 8, wherein the aerobic arsenic methylation step comprises the following steps:

the conditions of shaking culture are that the temperature is 28-32 ℃, the rotating speed is 150-250 rpm, and the time is 8-14 h.

10. The use of the microbial preparation with aerobic arsenic methylation and volatilization function as claimed in claim 8, wherein the microbial preparation is used for arsenic-polluted environment remediation, and the microbial preparation comprises the following components:

and (3) placing the microbial preparation with the aerobic arsenic methylation and volatilization functions in an environment to be repaired for culture.