Anti-radiation acinetobacter ZXP-05, heavy metal adsorbent and application of anti-radiation acinetobacter ZXP-05

Abstract

The invention relates to the field of microorganisms, in particular to anti-radiation acinetobacter ZXP-05, a heavy metal adsorbent and application of the anti-radiation acinetobacter ZXP-05 and the heavy metal adsorbent. The invention provides a strain of acinetobacter radioresistenes ZXP-05, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.32490. The invention also provides a method for preparing the acinetobacter radioresistenes ZXP-05. The anti-radiation acinetobacter ZXP-05 provided by the invention can participate in heavy metal adsorption through intracellular and extracellular combined action, and extracellular adsorption is superior to intracellular adsorption. In the specific embodiment of the invention, a plurality of studies show that the anti-radiation acinetobacter ZXP-05 has good resistance and adsorption capacity to Cd < 2 + > and Ni < 2 + >; in the heavy metal adsorption process, amino groups, hydroxyl groups, carboxyl groups, phosphate groups and the like on cells of the strain play an important role; the cells are severely recessed along with the increase of Cd < 2 + > concentration, and the adhesion is enhanced along with the increase of Ni < 2 + > concentration.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of radioresistant Acinetobacter ZXP-05, a heavy metal adsorbent and applications thereof. Background Art

[0002] Radiation-resistant microorganisms have an efficient and accurate DNA repair system and are resistant to DNA damage, making them an important resource of extreme microorganisms. Since 1956, when American scientists Anderson et al. first isolated radiation-resistant Deinococcus radiodurans R1 from canned meat products sterilized by radiation, scientists have isolated radiation-resistant microorganisms from various environments, such as 60 Micrococcus luteus was screened from soil near Co-γ rays; radiation-resistant Streptomyces (Streptomyces radiopugnans R97T) and radiation-resistant yeast fungi (Aureobasidium pullulans) were isolated from radiation-contaminated soil in Xinjiang. In addition to being resistant to ionizing radiation, most radiation-resistant microorganisms have extremely strong resistance to drought, high temperature, and high pressure; some are resistant to arsenate and arsenite; and some can decompose industrial solvents, chlorides, and other harmful substances. They represent the limits of life's survival in adverse conditions such as radiation, and have broad application prospects.

[0003] There are many acids and alkalis in industrial wastewater, which are often strong acids or alkalinities. Acinetobacter radioresistens (A. radioresistens) is a preferred strain for wastewater treatment because of its acid and alkali resistance and wide carbon source utilization. Studies have shown that A. radioresistens can degrade ammonia nitrogen in wastewater, with the advantages of strong specificity, short removal time and high removal efficiency; A. radioresistens can also degrade nicotine and produce hydrogen, and is widely used in the biodegradation of the organophosphorus pesticide methyl parathion; at the same time, it can degrade phenol, and is often used for the effective treatment of petroleum pollutants in water. In addition, A. radioresistens can be used to degrade polystyrene microplastics, herbicides and other harmful substances.

[0004] Water pollution is a global problem in the 21st century. Rapid economic development, industrial exploitation, climate change and urban expansion have caused various water pollutants, among which heavy metal pollution is the most prominent problem. Surveys show that more than 80% of China's rivers, lakes and seas are polluted by heavy metals, and local pollution is serious. Heavy metals are toxic and non-degradable. They are often combined with some substances in a specific chemical form and accumulated. It is difficult to remove them from water. Heavy metal pollution control is a hot spot and difficulty in the field of environment. In order to reduce the harm of heavy metals, environmentalists and chemists have proposed many technologies and methods for treating heavy metals in recent years. Traditional treatment methods include electrochemical method, membrane treatment method, chemical precipitation method, evaporation concentration method, ion exchange method, chemical oxidation-reduction method, activated carbon and silica gel adsorption method, etc. The above methods can achieve good treatment effects to a certain extent, but they generally have the problems of high investment, high energy consumption and difficulty in implementation. Moreover, most methods can only be used for high-concentration heavy metal wastewater, which is prone to secondary pollution. In recent years, due to the advantages of high efficiency and low cost, microbial adsorption has attracted widespread attention from scientists. Microbial adsorption is the process of removing heavy (or similar) metal ions from a medium by adsorbing the chemical composition or structural characteristics of the microorganisms themselves or their products, and then reducing the concentration of metal ions in the water through solid-liquid phase separation. Microbial adsorption has the advantages of low cost, good effect, short time, reusability, strong specificity and low risk of secondary pollution.

[0005] Environmental pollution has become an urgent concern for people. Heavy metal pollutants produced by industrial production and development seriously endanger human health and damage the ecological environment. At present, nuclear contaminated wastewater is a key issue of concern in China. Nuclear contaminated wastewater contains a large number of harmful elements, the most serious of which are radioactive pollutants produced by nuclear fission. In addition, it also contains a large number of heavy metals, such as Cd 2+ , Hg 2+ , Pb 2+ 、Ni 2+ There are radionuclides in nuclear contaminated wastewater, and ordinary microbial adsorbents are sensitive to radiation and it is difficult for them to survive for a long time under nuclear radiation. Therefore, it is very necessary to find a strain that can remove heavy metals and is resistant to radiation. Summary of the invention

[0006] The purpose of the present invention is to provide a strain of Acinetobacter radioresistance ZXP-05, a heavy metal adsorbent and its application, so as to solve the problems existing in the above-mentioned prior art. The Acinetobacter radioresistance ZXP-05 provided by the present invention can remove heavy metal ions and also has the characteristic of radiation resistance.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides a strain of Acinetobacter radioresistens ZXP-05, and the preservation number of the Acinetobacter radioresistens ZXP-05 is CGMCC No.32490.

[0009] The present invention provides the use of the above-mentioned radioresistant Acinetobacter ZXP-05 in the preparation of a heavy metal adsorbent.

[0010] Preferably, the heavy metals include cadmium and nickel.

[0011] The present invention provides a heavy metal adsorbent, which comprises the above-mentioned radioresistant Acinetobacter ZXP-05.

[0012] Preferably, the heavy metals include cadmium and nickel.

[0013] Preferably, the heavy metal adsorbent further comprises auxiliary materials.

[0014] The present invention provides the use of the radioresistant Acinetobacter ZXP-05 or the heavy metal adsorbent in the purification of heavy metal polluted environments.

[0015] Preferably, the heavy metal polluted environment includes heavy metal polluted water and heavy metal polluted soil.

[0016] Preferably, the heavy metals include cadmium and nickel.

[0017] The present invention provides a method for purifying a metal-contaminated environment, comprising the step of applying the radioresistant Acinetobacter ZXP-05 or the heavy metal adsorbent in the metal-contaminated environment.

[0018] The present invention discloses the following technical effects:

[0019] The present invention provides a strain of Acinetobacter radioresistens ZXP-05, the deposit number of which is CGMCC No.32490. Radiation-resistant microorganisms are important extreme biological resources with excellent stress resistance and broad application prospects. The present invention screened 12 strains of microorganisms from irradiated hand-shredded beef, among which strain ZXP-05 was irradiated with a dose of 10 kGy. 60 After Co-γ ray irradiation, the strain ZXP-05 survived. Through morphological observation and molecular biological identification, the strain was classified and named Acinetobacter radioresistens, and was named Acinetobacter radioresistens ZXP-05. 10The value is 4.545kGy; it grows well at 30-40℃, pH 6.0-9.0, and NaCl concentration below 2%, pH 5.0 and 10.0, NaCl concentration 3-5%, PEG6000 addition amount below 20%, Cd 2+ Addition amount is below 20mg / L, Ni 2+ The addition amount is below 200 mg / L and it can grow; it is resistant to methicillin sodium, chloramphenicol and hygromycin B. The radioresistant Acinetobacter ZXP-05 provided by the present invention can participate in the adsorption of heavy metals through intracellular and extracellular effects, and the extracellular adsorption is better than the intracellular adsorption. In the specific embodiments of the present invention, multiple studies have shown that the radioresistant Acinetobacter ZXP-05 has a strong affinity for Cd 2+ and Ni 2+ It has good resistance and adsorption capacity. In the process of heavy metal adsorption, the amino, hydroxyl, carboxyl and phosphate groups on the strain cells play an important role. 2+ The depression becomes more serious as the concentration increases. 2+ The adhesion increases with increasing concentration.

[0020] Moreover, at present, there are radionuclides in nuclear contaminated wastewater, and ordinary microbial adsorbents are sensitive to radiation and are difficult to survive for a long time under nuclear radiation. However, the radioresistant Acinetobacter ZXP-05 can tolerate high-dose radiation, grow under acid-base, high-concentration salt and high-concentration heavy metal stress, and can absorb heavy metals while growing, and is expected to be used in nuclear contaminated wastewater treatment. The present invention provides a theoretical basis for its application in extreme environments and the restoration of heavy metal environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The morphological observation diagram of strain ZXP-05; A is the colony morphology of strain ZXP-05; B is the result of Gram staining; C is the result of scanning electron microscopy observation; D is the result of transmission electron microscopy observation;

[0023] Figure 2 is the phylogenetic tree of the 16S rDNA gene sequence of strain ZXP-05;

[0024] Figure 3 Anti-radiobacter radiatus ZXP-05 60 Co gamma ray capability determination results;

[0025] Figure 4 The results of the growth curve of Acinetobacter radiatus ZXP-05 under adverse conditions; A is the effect of temperature on the growth of Acinetobacter radiatus ZXP-05; B is the effect of pH on the growth of Acinetobacter radiatus ZXP-05; C is the effect of different NaCl concentrations on the growth of Acinetobacter radiatus ZXP-05; D is the effect of different PEG6000 additions on the growth of Acinetobacter radiatus ZXP-05; E is the effect of different Cd 2+ Effects of concentration stress on the growth of Acinetobacter radiatus ZXP-05; F represents different Ni 2+ Effects of concentration stress on the growth of Acinetobacter radiatus ZXP-05;

[0026] Figure 5 To study the effect of Acinetobacter radiobacter ZXP-05 on Cd 2+ The adsorption effect of 2+ Adsorption amount; B is Cd 2+ Removal rate; C is Cd 2+ Intracellular and extracellular adsorption amount; Different lowercase letters indicate significant differences among the groups (P<0.05);

[0027] Figure 6 To study the effect of Acinetobacter radiobacter ZXP-05 on Ni 2+ The adsorption effect of 2+ Adsorption amount; B is Ni 2+ Removal rate; C is Ni 2+ Intracellular and extracellular adsorption amount; Different lowercase letters indicate significant differences among the groups (P<0.05);

[0028] Figure 7 This is the infrared spectrum analysis result of heavy metal adsorption by radioresistant Acinetobacter ZXP-05;

[0029] Figure 8 Scanning electron microscope analysis of heavy metal adsorption by radioresistant Acinetobacter ZXP-05; A is 5 mg / L Cd 2+ ; B is 10mg / L Cd 2+ ; C is 15mg / L Cd 2+ ; D is 50mg / LNi 2+ ; E is 100 mg / LNi 2+ ; F is 150mg / LNi 2+ . DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] 1. Materials and methods

[0037] 1.1 Materials

[0038] (1) Sample source

[0039] Shredded beef without high temperature sterilization and vacuum packaging was purchased from a food company. Shredded beef was irradiated with 8 kGy high-energy electron beam (energy 10 MeV, power 20 kW) for screening of radiation-resistant microorganisms.

[0040] (2) Culture medium

[0041] ① Nutrient broth medium: peptone 10g, beef extract powder 3g, sodium chloride 5g, distilled water to 1000mL, sterilized at 121℃ for 15min; ② MRS broth medium: peptone 10g / L, beef powder 8g / L, yeast powder 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, soil temperature 801. 0g / L, sterilize at 115℃ for 15min for later use; ③ Malt extract medium: 130g malt extract powder, 0.1g chloramphenicol, sterilize at 121℃ for 15min for later use; ④ LB broth medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, distilled water to 1000mL, sterilize at 121℃ for 15min for later use; ⑤ Agar solid medium: add agar powder to the corresponding broth medium at a ratio of 1.5%, sterilize and set aside.

[0042] 1.2 Main Reagents

[0043] Bacterial genomic DNA extraction kit and PCR purification kit were purchased from OMEGA, USA; rTaq DNA polymerase was purchased from Takara, Japan; Biowest agarose was purchased from Invitrogen Life Sciences, Inc., USA; DL 2000 Marker was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; primer 27F / 1492R was synthesized by Chengdu Branch of Beijing Qingke Biotechnology Co., Ltd.; Gluta fixative (2.5% for electron microscopy, item number P1126) was purchased from Beijing Solebao Technology Co., Ltd.; antibiotics such as ampicillin sodium were purchased from Beijing Bio-Toda Biotechnology Co., Ltd.; other reagents were of analytical grade.

[0044] 1.3 Instruments and Equipment

[0045] The high pressure sterilizer (SX-500) was purchased from Tianmei Instrument Laboratory Equipment (Shanghai) Co., Ltd.; the electronic balance (BSA224S-CW) was purchased from Sartorius (Shanghai) Trading Co., Ltd.; the sterile homogenizer (BILON-08) was purchased from Beijing Bilang Experimental Equipment Co., Ltd.; the clean bench (SW-CJ-ICU) was purchased from Suzhou Antai Air Technology Co., Ltd.; the constant temperature shaker (ZWY-211C) was purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; the constant temperature incubator (DNP-9272) was purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.; the Eppendorf PCR instrument (5531) and the Eppendorf refrigerated centrifuge (5415R) were purchased from Beijing Siqi Biotechnology Co., Ltd.; the gel imaging system (CL1000) was purchased from Thermo Fisher Scientific (China) Co., Ltd.; the electrophoresis instrument (DYY-8C) was purchased from Beijing Liuyi Instrument Factory; the multifunctional microplate reader (SpectraMax M2) was purchased from Meigu Molecular Instrument Co., Ltd.; ICP-MS inductively coupled plasma mass spectrometer (iCAP6300) and scanning electron microscope (Zeiss Gemini 300) were provided by Hangzhou Yanqu Information Technology Co., Ltd.

[0046] 2. Methods

[0047] 2.1. Isolation and purification of strains

[0048] Weigh 25 g of irradiated hand-shredded beef under sterile conditions and place it in a sterile homogenizing bag containing 225 mL of sterile phosphate buffered saline (PBS, pH = 7.4). Homogenize for 2 min to prepare a 1:10 sample solution. Then dilute the homogenate 10-fold to 10 -6 . 100 μL of the homogenate of each gradient was applied to nutrient agar plates, MRS agar plates or malt agar plates. Nutrient agar plates and MRS agar plates were placed in a 37°C constant temperature incubator for 2-3 days; malt agar plates were placed in a 25°C constant temperature incubator for 3-5 days. According to the color, size and morphology of the colonies, different single colonies were selected and inoculated into the corresponding solid culture medium, and streaked 3-6 times to obtain single colonies. Pick a single colony, culture it in the corresponding liquid culture medium, and store it with 30% sterile glycerol and place it in a -80°C refrigerator.

[0049] 2.2. Identification of strains

[0050] (1) Morphological observation: Inoculate the strain to be tested on nutrient agar solid medium and culture at 37°C for 48 hours. Observe the growth of the colonies and record the colony morphology, color, transparency, and whether the edges are neat. At the same time, pick a single colony for Gram staining, scanning electron microscopy, and transmission electron microscopy.

[0051] (2) Molecular biological identification: DNA of bacteria was extracted from overnight cultured bacterial solution according to the kit instructions. The extracted DNA sample was used as PCR template, and universal primers 27F (5'-AGTTTGATCMTGGCTCAG-3', SEQ ID No.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID No.2) were used for PCR amplification of 16S rDNA. The amplification system was: 10×Taq Buffer 5μL, Taq DNA polymerase 0.5μL, dNTP Mixture 4μL, upper and lower primers 2μL each, DNA template 1μL, ddH2O 35.5μL. The amplification program was: 94℃ pre-denaturation for 5min, cycle stage 94℃ denaturation for 30s, 57℃ annealing for 40s, 72℃ extension for 90s, 35 cycles, and then 72℃ extension for 10min. The amplified PCR products were detected by 1% agarose gel electrophoresis. After clear bands were detected, they were sent to the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed by online BLAST comparison at the National Center of Biotechnology Information (NCBI). The strain sequences with high homology were selected, and the strain phylogenetic tree was constructed using MEGA5.0 software according to the Neighbor-Joining method.

[0052] 2.3. Determination of the radiation resistance of strains

[0053] The strain was cultured in 100 mL LB broth at 37°C and 160 rpm for 16 h, and then centrifuged at 4°C and 6000 rpm for 5 min to collect the cells. The cells were washed twice with sterile PBS buffer and then mixed with sterile PBS buffer to a concentration of 10 7 -10 8 CFU / mL. The mixed bacterial solution was evenly loaded into 9 sterilized centrifuge tubes. 60 Co-γ-ray irradiation. The irradiation dose was set to 0.5, 1.0, 1.5, 2, 4, 6, 8, and 10 kGy, with the unirradiated (0 kGy) sample as the control. The actual absorbed dose of the sample was measured using a silver dichromate dosimeter, which was 0.50, 1.20, 1.60, 2.30, 3.70, 6.20, 7.90, and 9.70 kGy. The number of surviving microorganisms was determined after irradiation. Three replicates were performed for each dose level.

[0054] 2.4. Determination of growth curve of strains under adverse conditions

[0055] LB broth was used as the basic medium, and 1% of the overnight culture solution was inoculated. The culture was shaken at 160 rpm at 35°C (except for the study on the effect of temperature on growth). Samples were taken every 1.5 h or 3 h and the OD was measured using a multifunctional microplate reader. 600 The growth curve was drawn based on the absorbance value.

[0056] Different culture temperatures (20, 25, 30, 35, 40, 45°C) were set to explore the effect of temperature on the growth of the strain. The growth of the strain in an acid-base environment was studied by adjusting the pH of the culture medium (pH 1-12). The NaCl concentration of the culture medium was adjusted (0.0%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%) to simulate a high-salt environment and explore the salt tolerance of the strain. 20% and 30% polyethylene glycol 6000 (PEG6000) were added to the LB broth culture medium to simulate drought and evaluate the drought resistance of the strain. Different concentrations of heavy metals (Cd 2+ The concentration is 5, 10, 15, 20, 25 mg / L, Ni 2+ The concentrations were 50, 100, 150, 200, and 250 mg / L) to simulate heavy metal stress and check the strain's ability to resist heavy metal stress.

[0057] 2.5. Determination of strain resistance to antibiotics

[0058] Prepare a certain concentration of stock solution of antibiotics such as ampicillin sodium, carbenicillin sodium, and methicillin sodium. The specific preparation method is shown in Table 1. The prepared solution is filtered through a 0.45μm membrane filter and stored in a -20℃ refrigerator. After high-temperature sterilization, the LB agar medium is cooled to about 45℃, and a certain amount of antibiotic stock solution is added and mixed evenly. After the LB agar medium solidifies, take 100μL of overnight cultured bacterial solution and spread it on the plate, culture it overnight at 37℃, and check the growth of the strain. Set two concentrations for each antibiotic. The specific concentrations are shown in Table 1.

[0059] Table 1 Antibiotic preparation method and antibacterial concentration

[0060]

[0061] Note: Hygromycin B is purchased as a liquid at a concentration of 50 mg / mL.

[0062] 2.6. Effect of strains on heavy metal adsorption

[0063] Add a certain amount of Cd to 100 mL of LB broth 2+ and Ni 2+ , medium Cd 2+ The concentration is 5, 10, 15 mg / L, Ni 2+The concentrations were 10, 30, 50, 100, and 150 mg / L. 1 mL of overnight culture was inoculated, and cultured at 37°C and 160 rpm for 1, 2, 4, 8, 12, 24, 48, and 72 h. 5 mL of the mixture was taken and centrifuged at 6000 rpm for 5 min. The supernatant was filtered through a 0.45 μm membrane filter and the heavy metal concentration was determined by ICP-MS inductively coupled plasma mass spectrometry. The heavy metal adsorption and heavy metal removal rates were calculated according to the following formulas.

[0064] The calculation formula for heavy metal adsorption is: Q = C0-C t ;

[0065] The calculation formula of heavy metal removal rate is: RE = (C0-C t ) / C0×100;

[0066] In the formula, Q (mg / L) is the adsorption amount, RE (%) is the removal rate, C0 (mg / L) is the initial concentration of heavy metals, and C t (mg / L) is the heavy metal concentration at time t.

[0067] 2.7. Determination of extracellular adsorption and intracellular accumulation of heavy metals by strains

[0068] The adsorption of heavy metals by microorganisms is a complex process. According to the difference in the distribution of adsorbed ions in microbial cells, adsorption is divided into intracellular adsorption, extracellular adsorption and surface adsorption. The strain containing heavy metals was cultured at 37°C and 160rpm for 72 hours, and centrifuged at 4°C and 6000rpm for 5 minutes to collect the bacteria. The bacteria were washed twice with sterile PBS buffer and resuspended with 5mM sterile EDTA. The suspension was shaken at 200rpm for 30 minutes, and then centrifuged at 6000rpm for 5 minutes to separate the supernatant and bacteria. The heavy metal concentration in the supernatant was determined by ICP-MS inductively coupled plasma mass spectrometry, and the bacteria were dried and the dry weight was determined. The dry bacteria were then digested, the bioaccumulation of heavy metals in the cells was determined, and the amount of heavy metal adsorption in the cells was calculated by the following formula.

[0069] The calculation formula for the extracellular heavy metal adsorption is: Q e =C e ×V e / m / 1000;

[0070] The calculation formula for the amount of heavy metal adsorption in cells is: Q i =C i ×V i / m / 1000;

[0071] In the formula, Q e is the extracellular heavy metal adsorption amount (mg / g), Q iis the intracellular heavy metal adsorption amount (mg / g), C e (mg / L) is the heavy metal concentration in the EDTA eluent, V e (mL) is the amount of EDTA used; C i (mg / L) is the heavy metal concentration after bacterial digestion, V i (mL) is the volume of the sample after digestion, and m(g) is the dry weight of the bacteria.

[0072] 2.8. Study on the adsorption characteristics of strains for heavy metals

[0073] The strains were exposed to different concentrations of heavy metals (Cd 2+ The concentration is 5, 10, 15 mg / L, Ni 2+ The cells were grown in LB broth with concentrations of 50, 100, and 150 mg / L, and the cells without heavy metals were used as controls for scanning electron microscopy (SEM) and infrared spectroscopy (FTIP) analysis. The bacterial solution was cultured at 37°C and 160 rpm for 72 hours, centrifuged at 4°C and 6000 rpm for 5 minutes, and the collected cells were washed 3 times with sterile PBS buffer at 4°C. After centrifugation, some cells were fixed with 2.5% glutaraldehyde solution at 4°C for 24 hours, and then dehydrated with different concentrations of ethanol (20% to anhydrous ethanol), each dehydration for 20 minutes, and critical point drying was performed. The dehydrated cell samples were sprayed with gold, and the changes in cell morphology before and after heavy metal bioaccumulation were determined by SEM. The remaining cells were freeze-dried, ground into powder, and the changes in cell functional groups were analyzed by FTIR spectroscopy.

[0074] 3. Results and analysis

[0075] 3.1. Identification of strains

[0076] (1) Morphological observation

[0077] After initial screening, 12 strains of bacteria were obtained and these 12 strains were subjected to 10 kGy dose 60 After Co-γ-ray irradiation and culturing on the corresponding agar medium, only one strain grew on the plate, and this strain was named ZXP-05. Strain ZXP-05 was used as the research object for subsequent research.

[0078] The strain ZXP-05 was streaked on a nutrient agar plate and cultured at 37°C for 24 h. The colony morphology was observed and found to be round, milky white, with a raised center, and neat, smooth, moist, and easy to pick up ( Figure 1 A in the figure). Gram staining was red, indicating that strain ZXP-05 was a Gram-negative bacterium ( Figure 1B in the figure). Under scanning electron microscopy, the cells of strain ZXP-05 are oval, exist in monomeric or dimeric forms, have no spores, and are approximately (0.85-0.95) μm × (0.65-0.81) μm in size ( Figure 1 C in the figure. Under transmission electron microscopy, the cells contain highly electron-dense areas and pili outside the cells ( Figure 1 D).

[0079] (2) 16S rDNA PCR amplification and sequence analysis of strain ZXP-05

[0080] The PCR amplification product was sent to the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing. After sequencing, a DNA fragment of 1417 bp was obtained. The online BLAST results showed that the 16S rDNA PCR sequence of strain ZXP-05 was most closely related to Acinetobacter sp. (Accession No. HM352326.1), with a similarity of 99.72%, followed by Acinetobacter radioresistens (Accession No. JN669117.1), with a similarity of 99.65%. The strain sequences with high homology were downloaded from GenBank, and the strain phylogenetic tree was constructed using MEGA5.0 software according to the Neighbor-Joining method. The results showed that strain ZXP-05 and Acinetobacter radioresistens were clustered into the same branch ( Figure 2 ); Combined with morphological characteristics, strain ZXP-05 was determined to be Acinetobacter radioresistens. The present invention named strain ZXP-05 as Acinetobacter radioresistens ZXP-05, and sent it to the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration for preservation on November 4, 2024. The classification of the strain was named Acinetobacter radioresistens, the preservation number was CGMCC No.32490, and the preservation address was the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0081] 3.2. Anti-radiation Acinetobacter ZXP-05 60 Co gamma ray radiation capability determination

[0082] D 10The value refers to the radiation dose required to reduce the total number of microorganisms to 10% of their initial number. It reflects the resistance of microorganisms to radiation. The larger the value, the stronger the radiation resistance. 60 After Co gamma ray irradiation, the relationship between the number of surviving bacteria and the irradiation dose is shown in Figure 3 The relationship between irradiation dose and microbial changes was analyzed by linear regression, and the equation y = -0.24205x + 8.79191 was obtained, R 2 = 0.98219, the D of Acinetobacter radioresistance ZXP-05 was calculated by Eq. 10 The value is 4.545kGy.

[0083] D of microorganisms in different media 10 The values ​​are different. For example, the D values ​​of Bacillus flexus screened from the Chinese patent medicine Jinsuo Pills in physiological saline and filter paper are different. 10 The values ​​are 6.75 and 7.18 kGy respectively. The radiation resistance of microorganisms in different states is also different. The D 10 The value is significantly higher than that of vegetative bacteria. 10 The average value is 2.48 kGy, and the vegetative cell D 10 The average value is 0.762 kGy. Deinococcus radiodurans is the most radiation-resistant microorganism found in the world. Studies have found that the D 10 The value is as high as 13kGy. The radioresistant Acinetobacter ZXP-05 screened by the present invention can be 60 Co survives gamma rays, D 10 The value is 4.545 kGy, indicating that it has strong resistance to gamma rays.

[0084] 3.3. Growth curve determination of Acinetobacter radioresistance ZXP-05 under adverse conditions

[0085] (1) Growth curve of radioresistant Acinetobacter ZXP-05 at different temperatures

[0086] LB broth culture is the most suitable culture medium for Acinetobacter radioresistens, and subsequent studies will use LB broth as the basic culture medium. The culture temperature was set at 20, 25, 30, 35, 40, and 45°C to explore the effect of temperature on the growth of Acinetobacter radioresistens. The results are as follows Figure 4 The results showed that the optimal growth temperature of radioresistant Acinetobacter ZXP-05 was 30-40℃, with rapid growth in the early stage and reaching equilibrium after 16.5h of growth. The OD 600The value is 0.95; lowering the temperature slows down the growth of radioresistant Acinetobacter ZXP-05. The lower the temperature, the slower the growth and the longer it takes to reach the equilibrium period. At 20-25℃, the OD 600 The value is about 0.92; however, when the temperature is raised to 45°C, the growth of radioresistant Acinetobacter ZXP-05 will be severely inhibited.

[0087] (2) Growth curve of radioresistant Acinetobacter ZXP-05 at different pH

[0088] LB broth medium is used as the basic medium, and the pH of the medium is adjusted to 1.0-12.0 with hydrochloric acid and sodium hydroxide. Culture at 35°C with shaking, pH 4.0-11.0. The growth curve is as follows Figure 4 The results show that the optimal growth pH of radioresistant Acinetobacter ZXP-05 is 7.0, and it reaches the equilibrium phase after 16.5h of growth. The OD at the equilibrium phase is 600 The value was 0.95; it grew well at pH 6.0 and 8.0, with fast growth in the early stage; at pH 5.0 and 9.0, the growth of Acinetobacter radioresistance ZXP-05 was slightly inhibited in the early stage; at pH 10.0, the growth of Acinetobacter radioresistance ZXP-05 was severely inhibited in the early stage, and it began to grow after 9 hours of adaptation. Under weak acid and weak base, the growth of Acinetobacter radioresistance ZXP-05 was inhibited in the early stage, and when it reached the equilibrium period, the OD 600 The values ​​were not much different. When the OD value reached the equilibrium period under the condition of pH 5.0-10.0, 600 The value is 0.90~0.95. However, when the pH is lower than 5.0 or higher than 10.0, the growth of radioresistant Acinetobacter ZXP-05 can be inhibited.

[0089] (3) Growth curve of radioresistant Acinetobacter ZXP-05 at different salt concentrations

[0090] LB broth was used as the basic medium, and NaCl was used to adjust the salt concentration. The growth curves at different NaCl concentrations are shown in Figure 4 The results show that the radioresistance Acinetobacter ZXP-05 grows well at salt concentrations below 1%. When the salt concentration is 2%, the growth of the radioresistance Acinetobacter ZXP-05 is slightly inhibited in the early stage. When the OD reaches the equilibrium period 600 The value can still reach 0.95; as the salt concentration increases, the inhibition effect increases, and the OD 600 When the salt concentration was 6%, the growth of Acinetobacter radioresistance ZXP-05 was severely inhibited and it started to grow after 12 hours of adaptation. Meanwhile, the growth condition was poor. When the growth equilibrium state was reached, OD 600 The value is only 0.2.

[0091] (4) Growth curve of radioresistant Acinetobacter ZXP-05 in arid environment

[0092] In this example, PGE6000 was added to LB broth to simulate a drought environment and explore the drought resistance of radioresistant Acinetobacter ZXP-05. The growth curve is shown in Figure 4 The results showed that when the addition amount of PEG6000 was 20%, the growth of radioresistant Acinetobacter ZXP-05 was slightly inhibited, and it began to grow after 6 hours of adaptation and reached the equilibrium period after 18 hours of growth. The OD at the equilibrium period was 600 The value is 0.55; when the concentration of PRG6000 is increased to 30%, the growth of radioresistant Acinetobacter ZXP-05 is severely inhibited and almost does not grow.

[0093] (5) Radioresistant Acinetobacter ZXP-05 in Cd 2+ Growth curve under stress

[0094] Cd 2+ The toxicity is strong, and it is difficult for general microorganisms to survive in high concentrations. 2+ Growth curves under stress, such as Figure 4 The results show that the radioresistant Acinetobacter ZXP-05 can tolerate 20 mg / L Cd 2+ When Cd 2+ When the concentration was 5 mg / L, it had no inhibitory effect on the growth of Acinetobacter radiodurans ZXP-05, and even promoted it, reaching the equilibrium period after 15 hours of growth. 2+ When the concentration was 10 mg / L, the plants started to grow after 24 hours of adaptation. 2+ When the concentration was 15 mg / L, the plants started to grow after 30 hours of adaptation. 2+ Under these conditions, the growth reached equilibrium after 36 hours; increasing Cd 2+ When the concentration was 20 mg / L, the plants started to grow after 39 hours of adaptation and reached the equilibrium period after 60 hours of growth. 2+ It will delay the growth of radioresistant Acinetobacter ZXP-05, but when reaching the equilibrium period, each concentration of Cd 2+ OD 600 The value is close to 1.00, which is larger than that without heavy metals. It is speculated that this is because the stress of heavy metals activates the physiological response of Acinetobacter radioresistance ZXP-05, enabling it to grow better. 2+ When the concentration was 25 mg / L, the radioresistant Acinetobacter ZXP-05 stopped growing, which suggested that it was sensitive to Cd 2+ The MIC of this drug is 25 mg / L.

[0095] (6) Radioresistant Acinetobacter ZXP-05 in Ni 2+ Growth curve under stress

[0096] Radioresistant Acinetobacter ZXP-05 in Ni 2+ Growth curves under stress, such as Figure 4 As shown in F. The results showed that the radioresistant Acinetobacter ZXP-05 could 2+ Able to survive under stress, and can tolerate up to 200 mg / L Ni 2+ When Ni 2+ When the concentration was 50 mg / L, there was almost no inhibition on the growth of Acinetobacter radiodurans ZXP-05. When the equilibrium period was reached, OD 600 The value is close to 1.00; when Ni is increased 2+ When the concentration was 100 mg / L, the cells started to grow after 24 hours of adaptation. When the equilibrium period was reached, OD 600 The value is about 0.90, which is slightly suppressed; increasing Ni 2+ When the concentration was 150 mg / L, the cells started to grow after 33 hours of adaptation, and the OD at the equilibrium period was 600 The value is about 0.90, which is slightly suppressed; increasing Ni 2+ When the concentration was 200 mg / L, the cells started to grow after 39 hours of adaptation. When the equilibrium period was reached, OD 600 is about 0.83, which is slightly suppressed. 2+ When the concentration was 250 mg / L, the radioresistant Acinetobacter ZXP-05 stopped growing, which suggested that it was sensitive to Ni. 2+ The MIC of this drug is 250 mg / L.

[0097] 3.4. Antibiotic resistance of Acinetobacter radioresistance ZXP-05

[0098] Acinetobacter baumanii has good antibiotic resistance and is a major pollutant in hospitals. Acinetobacter radioresistens and Acinetobacter baumanii are both Acinetobacter genus, and it is speculated that Acinetobacter radioresistens ZXP-05 is resistant to antibiotics. Different types of antibiotics were used with different concentrations to explore the resistance of Acinetobacter radioresistens ZXP-05 to antibiotics. The results are shown in Table 2. Acinetobacter radioresistens ZXP-05 has low resistance to antibiotics. Among the 12 antibiotics tested, it is only resistant to methicillin sodium, chloramphenicol and hygromycin B.

[0099] Table 2 Antibiotic resistance of Acinetobacter radioresistance ZXP-05

[0100] antibiotic Concentration (μg / mL) Whether to grow Concentration (μg / mL) Whether to grow Ampicillin Sodium(Amp) 25 - 50 - Carbenicillin Sodium(Car) 25 - 50 - Methicillin Sodium(Met) 20 + 37.5 + Kanamycin Sulfate (Kan) 10 - 50 - Chloramphenicol (Chl) 12.5 + 25 + Streptomycin (Str) 10 - 50 - Nalidixic acid (Nal) 7.5 - 15 - Tetracycline (Tet) 10 - 50 - Hygromycin B (HygB) 10 + 100 + Rifampicin (Rif) 25 - 50 - Spectinomycin hydrochloride (Spe) 100 - 350 - Ofloxacin (Ofl) 10 - 20 -

[0101] Note: - indicates no growth, + indicates growth.

[0102] 3.5. Adsorption effect of radioresistant Acinetobacter ZXP-05 on heavy metals

[0103] (1) Heavy metal Cd 2+ Adsorption

[0104] The effects of Acinetobacter radioresistance ZXP-05 on Cd at different initial concentrations 2+ The adsorption amount is shown in Figure 5 A in, removal rate see Figure 5 B in the figure, the intracellular and extracellular adsorption amounts are shown in Figure 5 In C. Figure 5 As shown in A, different Cd 2+ At the same time, the toxicity of Acinetobacter radioresistance ZXP-05 to Cd 2+ The adsorption amount of Cd increased; the adsorption amount of Cd 2+ The adsorption amount showed irregular changes at different concentrations. It is speculated that this is due to the different concentrations of Cd 2+ The growth of radioresistant Acinetobacter ZXP-05 was different under different conditions ( Figure 4 E). At 72h, along with Cd 2+ As the concentration increases, the adsorption amount increases; but as the Cd 2+ As the concentration increases, the removal rate decreases ( Figure 5 B). Further analysis of the radioresistant Acinetobacter ZXP-05 against Cd 2+ The results showed that with the increase of Cd 2+ With the increase of Cd concentration, both the extracellular adsorption and intracellular adsorption increased significantly (P<0.05). 2+ Whatever the initial concentration, the extracellular adsorption amount is much greater than the intracellular adsorption amount ( Figure 5 C).

[0105] (2) Heavy metal Ni 2+ The removal effect

[0106] The effects of Acinetobacter radiodurans ZXP-05 on Ni at different initial concentrations 2+ The adsorption amount is shown in Figure 6 A in, removal rate see Figure 6 B in the figure, the intracellular and extracellular adsorption amounts are shown in Figure 6 In C. Figure 6 As shown in A, the radioresistant Acinetobacter ZXP-05 has a significant effect on Ni 2+ The adsorption amount increased; different Ni 2+The adsorption amount changes irregularly at different concentrations. It is assumed that this is due to the 2+ Under different conditions, the growth of radioresistant Acinetobacter ZXP-05 was different ( Figure 4 F in). At 72h, the adsorption amount increased with the increase of Ni 2+ The concentration increases first, reaches the maximum at 50 mg / L, and then decreases; the removal rate increases with the increase of Ni 2+ The increase in concentration decreased ( Figure 6 B) Analysis of intracellular adsorption and extracellular adsorption, the results are as follows Figure 6 As shown in C, the extracellular adsorption increased significantly with the increase of heavy metal concentration (P<0.05), and the intracellular adsorption amount first increased with the increase of heavy metal concentration, reached the maximum at 50 mg / L, and then decreased.

[0107] 3.6 Study on the adsorption characteristics of strains for heavy metals

[0108] (1) Infrared spectroscopy analysis

[0109] FTIR spectra of radioresistant Acinetobacter ZXP-05 before and after heavy metal adsorption Figure 7 In microorganisms, 3438cm -1 The stretching vibration peaks of -OH and -NH of polysaccharides and proteins are at Cd 2+ The effect of -1 , in Ni 2+ Under the action of -1 At the same time, under heavy metal stress, the peak intensity of this wave is significantly reduced. 2928cm -1 The peak at 1644 cm is formed by the symmetric and antisymmetric stretching vibration of -CH2- and -CH3 of proteins and lipids. -1 The C=O stretching vibration peak of protein amide I band, 1545 cm -1 The NH bending vibration peak and CN stretching vibration peak of protein amide II are at 1403 cm -1 The absorption peak of peptide side chain -COOH is 1077cm -1 The peaks of these groups showed some changes under heavy metal stress, indicating that the surface of Acinetobacter radioresistance ZXP-05 cells contained amino, hydroxyl, carboxyl and other active groups, which participated in the binding of heavy metal Cd through complexation, chelation and coordination. 2+ and Ni 2+ adsorption.

[0110] (2) Scanning electron microscopy analysis

[0111] Observation of radioresistant Acinetobacter ZXP-05 in different concentrations of Cd by scanning electron microscopy2+ and Ni 2+ Cell morphological changes after stress. When the radioresistant Acinetobacter ZXP-05 was not subjected to heavy metal stress, the cells were plump and dispersed, and the pili on the surface were dispersed ( Figure 1 C in ). At 5mg / LCd 2+ Under stress, the cells are still plump and dispersed, but a membrane appears on the cell surface and pili adhere to the surface ( Figure 8 A); at 10mg / LCd 2+ Under stress, cells become slightly concave, filaments appear on the surface, and cells stick together and aggregate ( Figure 8 B); at 15mg / LCd 2+ Under stress, cells are severely sunken, surface filaments increase, and there are abundant cross-links between cells ( Figure 8 C). At 50 mg / L Ni 2+ Under stress, the cell shape did not change significantly, and the cell wall did not rupture, but the cell pili adhesion ( Figure 8 D); at 100 mg / LNi 2+ Under stress, cells develop filaments and cells adhere to each other ( Figure 8 E); at 150mg / LNi 2+ Under stress, cell filaments increase and cell adhesion increases ( Figure 8 F). In general, cells grow with Cd 2 + As the concentration increases, the depression becomes more serious. 2+ As the concentration increases, the number of surface filaments increases and cell adhesion strengthens.

[0112] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Acinetobacter radioresistens ZXP-05, characterized in that: The deposit number of the radioresistant Acinetobacter ZXP-05 is CGMCC No.32490.

2. Use of the radioresistant Acinetobacter ZXP-05 according to claim 1 in the preparation of heavy metal adsorbents.

3. The use according to claim 2, characterized in that: The heavy metals include cadmium and nickel.

4. A heavy metal adsorbent, characterized in that: The heavy metal adsorbent includes the radioresistant Acinetobacter ZXP-05 described in claim 1.

5. The heavy metal adsorbent according to claim 4, characterized in that: The heavy metals include cadmium and nickel.

6. The heavy metal adsorbent according to claim 4, characterized in that: The heavy metal adsorbent also includes auxiliary materials.

7. Use of the radioresistant Acinetobacter ZXP-05 described in claim 1 or the heavy metal adsorbent described in any one of claims 4 to 6 in the purification of heavy metal polluted environments.

8. The use according to claim 7, characterized in that: The heavy metal polluted environment includes heavy metal polluted water and heavy metal polluted soil.

9. The use according to claim 7, characterized in that: The heavy metals include cadmium and nickel.

10. A method for purifying a metal-contaminated environment, characterized in that: The method comprises the step of applying the radioresistant Acinetobacter ZXP-05 described in claim 1 or the heavy metal adsorbent described in any one of claims 4 to 6 in a metal-contaminated environment.

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