Manganese-resistant manganese oxide strain Klebsiella oxytoca YJ and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a manganese-resistant manganese oxide strain Klebsiella oxytoca YJ and application of the manganese-resistant manganese oxide strain Klebsiella oxytoca YJ. The invention discloses a manganese-resistant manganese oxide strain YJ, the taxonomic name of the manganese-resistant manganese oxide strain YJ is Klebsiella oxytoca, the manganese-resistant manganese oxide strain YJ is preserved in Guangdong Microbial Culture Collection Center on May 21, 2024, and the preservation number is GDMCC No: 64667. According to the invention, manganese-resistant strains are screened to obtain a strain YJ of which the tolerance concentration to manganese is 200mM, and the strain YJ is Klebsiella oxytoca through morphological and molecular biological identification, and the strain YJ is Klebsiella oxytoca. Research on the removal rate of heavy metals shows that the removal rate of Mn (II) in a 500mg / kg culture solution reaches 92.20% at the 4th day, the removal rate of As (III) in a 50mg / kg culture solution reaches 98.40% at the 4th day, and the removal rate of Cd (II) in a 5mg / kg culture solution reaches 57.07% at the 5th day. Therefore, the strain YJ has very important significance on pollution treatment of heavy metals such as Mn, As and Cd.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a manganese-oxidizing manganese-resistant strain Klebsiella oxytocaYJ and an application thereof. Background Art

[0002] The current area of ​​cultivated land in my country contaminated by heavy metals such as cadmium, arsenic, and lead is estimated to be nearly 2.0×10 7 hm 2 Arsenic pollution in soil mainly comes from atmospheric deposition, sewage irrigation and spraying of arsenic-containing pesticides. The arsenic content in China’s surface soil is 2.5×10 -6 -33.5×10 -6 , the soil arsenic content is distributed in a north-south direction. The soil arsenic content in areas with higher altitudes is higher than that in areas with lower altitudes. It decreases from the Qinghai-Tibet Plateau, the southwest region, and the south China region to the northeast region, and there is a geochemical relationship between it and the eroded materials upstream. As a metalloid element, arsenic has attracted much attention due to its high toxicity, easy enrichment, and difficulty in degradation. It can have harmful effects on both humans and plants. Long-term drinking of high-arsenic water can cause chronic arsenic poisoning, as well as diseases of the skin, respiratory system, nervous system, and digestive system. Skin contact with arsenic also has the risk of cancer. Cadmium is one of the most serious metals poisoning among heavy metals. Soil cadmium pollution will not only reduce the yield and quality of crops, but also endanger human health through enrichment in the food chain. The "National Pollution Survey Bulletin" issued by the Ministry of Ecology and Environment in 2014 shows that the point-level excess rate of Cd in my country's soil is 7.0%, ranking first among inorganic pollutants. Data show that about 1.0×10 6 t Cd is released into the environment; in my country, the area of ​​farmland contaminated by Cd is about 1.0×10 7 hm 2 The amount of agricultural products contaminated by Cd is as high as 1.5×10 6 t. Studies have shown that the content of heavy metal Cd in farmland soils across the country ranges from 0.000013 to 217.23 mg / kg, with an average of 0.39 mg / kg and a median of 0.25 mg / kg. Cd pollution in the country shows an uneven distribution trend. Guangdong, Hunan and Yunnan are areas with more Cd pollution, and the degree of Cd pollution in the south is significantly more serious than that in the north.

[0003] Rice is one of the main food crops in my country. Due to its strong physiological tolerance and accumulation capacity to cadmium, rice fields are particularly contaminated with cadmium, with a contaminated area of ​​12,700 hm2. 2, the cadmium content of rice in several major grain-producing areas in my country exceeded the standard by 10.3%, directly threatening food security and human health. The cadmium pollution in rice fields has become the top priority for soil heavy metal pollution and prevention. Based on my country's traditional agricultural irrigation model, soil arsenic pollution occurs more in paddy fields, and under flooding conditions, the toxicity and bioavailability of arsenic in the soil will increase to varying degrees. The use of chemical methods for remediation may cause secondary pollution, and more and more researchers are turning their attention to microbial bioremediation.

[0004] The inventors studied the microbial strains in the high manganese soil environment and screened out a manganese-resistant manganese oxide strain that can oxidize Mn (II) into biological manganese oxide. They formed a composite material with biochar and added it to the soil, which can significantly reduce the absorption of arsenic and cadmium by plants. This is of great significance for reducing the biological effectiveness of arsenic and cadmium contaminated soil.

[0005] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0006] The invention aims to provide a manganese-oxidation-resistant strain Klebsiella oxytoca YJ and application thereof.

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

[0008] The first object of the present invention is to provide a manganese-oxidation-resistant strain YJ, the taxonomic name of the manganese-oxidation-resistant strain YJ is Klebsiella oxytoca, and it was deposited in the Guangdong Microbiological Culture Collection Center on May 21, 2024, with a preservation number of GDMCC No: 64667.

[0009] The second object of the present invention is to provide a biochar-loaded composite material, wherein the biochar-loaded composite material is prepared from the manganese-resistant manganese-oxidizing strain YJ.

[0010] The third object of the present invention is to provide the use of the manganese-resistant manganese-oxidizing strain YJ or the biochar-loaded composite material in removing Mn, As and / or Cd from the environment.

[0011] The fourth object of the present invention is to provide the use of the manganese-resistant manganese-oxidizing strain YJ or the biochar-loaded composite material in reducing the As and / or Cd content in crop seeds.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) The present invention screens manganese-resistant strains and obtains a strain YJ with a tolerance to manganese concentration of 200mM. After morphological and molecular biological identification, strain YJ is Klebsiella oxytoca. Through the study of heavy metal removal rate, it is found that the removal rate of Mn(Ⅱ) in 500mg / kg culture solution by strain YJ reaches 92.20% on the 4th day, the removal rate of As(III) in 50mg / kg culture solution reaches 98.40% on the 4th day, and the removal rate of Cd(II) in 5mg / kg culture solution reaches 57.07% on the 5th day. It can be seen that strain YJ is of great significance for the treatment of heavy metal pollution such as Mn, As and Cd.

[0014] (2) The biochar-loaded composite material prepared by strain YJ in the present invention can effectively reduce the accumulation of arsenic and cadmium elements in the soil by rice plants, thereby reducing the arsenic and cadmium content in rice grains.

[0015] Storage Information

[0016] The strain Klebsiella oxytoca YJ was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on May 21, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the deposit number is GDMCC No: 64667. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the colony morphology of strain YJ;

[0018] Figure 2 is the phylogenetic tree of strain YJ;

[0019] Figure 3 This is the result of the identification of the manganese oxidation function of strain YJ;

[0020] Figure 4 It is the curve diagram of the removal rate change of 500 mg / kg Mn(II) in the culture medium;

[0021] Figure 5 It is the curve diagram of the removal rate change of 50 mg / kg As(III) in the culture medium;

[0022] Figure 6 It is the curve diagram of the removal rate change of 5 mg / kg Cd(II) in the culture medium;

[0023] Figure 7 This is a scanning electron microscope image of the biochar-loaded composite material;

[0024] Figure 8This is a comparison chart of arsenic content in rice grains;

[0025] Fig. 9 This is a comparison chart of cadmium content in rice grains.

[0026] Description of main reference numerals:

[0027] Figure 8 and Fig. 9 In the figure, CK is the blank control group without adding biochar-loaded composite materials; BC is the treatment group with adding biochar-loaded composite materials. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solution of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0029] 1. Strain screening and identification

[0030] 1.1 Screening soil for bacteria

[0031] High-manganese rice soil was collected from Nanning, Guangxi. The five-point sampling method was used to collect rice rhizosphere soil in the field. After removing the surface debris, 5-20 cm of the surface soil was collected. After mixing evenly, one portion was placed in a sterile centrifuge tube, placed in an ice box and brought back to the laboratory and stored at -20°C for the screening of manganese oxidizing bacteria. One portion was placed in a ziplock bag and brought back to the laboratory for air drying and sieving. The sample was prepared and the manganese content and basic physical and chemical properties in the soil were determined with reference to the "Soil Agricultural Chemical Analysis Method".

[0032] Table 1 Soil physical and chemical properties and selenium content

[0033] Available phosphorus (mg / kg) Available potassium (mg / kg) Alkaline nitrogen (mg / kg) Organic matter (g / kg) Total manganese (mg / kg) pH 23.96 192.98 71.4 25.28 3034.09 6.46

[0034] 1.2 Culture medium

[0035] LB nutrient agar: tryptone 10.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 10.0 g / L, agar powder 15 g / L, pH 7.0 ± 0.2;

[0036] LB broth: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, pH 7.0 ± 0.2;

[0037] PYCM solid medium: peptone 0.8 g / L, yeast extract powder 0.2 g / L, dipotassium hydrogen phosphate 0.1 g / L, magnesium sulfate 0.2 g / L, sodium nitrate 0.2 g / L, calcium chloride 0.1 g / L, ammonium chloride 0.1 g / L, ammonium carbonate 0.1 g / L, ammonium ferric citrate 1.0 g / L, agar 20.0 g / L, pH 7.0±0.2.

[0038] PYCM liquid culture medium: peptone 0.8 g / L, yeast extract powder 0.2 g / L, dipotassium hydrogen phosphate 0.1 g / L, magnesium sulfate 0.2 g / L, sodium nitrate 0.2 g / L, calcium chloride 0.1 g / L, ammonium chloride 0.1 g / L, ammonium carbonate 0.1 g / L, ammonium ferric citrate 1.0 g / L, pH 7.0±0.2.

[0039] 1.3 Strain isolation and purification

[0040] In a clean bench, weigh 10 g of soil sample from a sterile centrifuge tube, add it to 90 mL of deionized water with glass beads that has been sterilized in advance at high temperature and high pressure, and shake it in an oscillation box at 30°C and 180 r / min for 30 min, then take it out to obtain a soil suspension.

[0041] Take 10mL of soil suspension and add 90mL of sterilized LB broth, and culture it in a shaker at 30℃ and 180r / min for 72h as the initial inoculum. Then transfer the mixed bacterial liquid to PYCM liquid medium with a Mn(Ⅱ) concentration of 1mmol / L, and continue to culture under the above conditions for 3d.

[0042] The PYCM bacterial solution was diluted to 10 -4 , 10 -5 , 10 -6 , 100 μL of each was spread on a sterile plate of PYCM solid medium, and after culturing in a 30°C constant temperature incubator for 3 days, a single colony was picked and separated and purified three times by the plate streak method to obtain a pure culture. The strain was inoculated into sterile LB broth, and cultured in an oscillating box at 30°C and 180r / min until the growth logarithmic phase of the strain. A certain amount of bacterial solution was placed in a cryopreservation tube, and sterile glycerol was added to make the glycerol concentration between 20-30%. After sealing, it was shaken thoroughly and the cryopreservation tube was placed in a -80°C refrigerator for low temperature storage.

[0043] 1.4 Preparation of strain seed solution

[0044] After streaking the strain plate, pick the strain with an inoculation loop and add it to 50 mL LB liquid medium. Cultivate it at 28°C and 180 rpm for 20 h. 600 The value is 0.8-1.0.

[0045] 1.5 Screening of manganese-resistant strains

[0046] The prepared Mn(Ⅱ) mother solution was added to the sterilized PYCM liquid culture medium. The Mn(Ⅱ) concentration in the culture medium was set to 5, 10, 20, 50, 100, 150, 200, 250, 300, and 400 mmol / L. A PYCM liquid culture medium with the same concentration but without bacteria was set as a control. The bacterial solution prepared in 1.4 was inoculated with 1% (v / v) into the PYCM liquid culture medium and cultured in a constant temperature shaking incubator at 30°C and 180 r / min for 14 days.

[0047] Use UV spectrophotometer to measure the OD of culture medium 600 Value as the judgment standard: OD of the culture medium of the inoculation 600 Greater than the OD of the uninoculated culture medium 600 If the difference is large, it means that the strain can still grow under this concentration condition. This is used to determine the tolerance of manganese oxidizing bacteria to Mn(Ⅱ) and screen out manganese oxidizing strains with high manganese tolerance.

[0048] As a result, a strain with a tolerance to manganese concentration of 200mM was screened and marked as strain YJ.

[0049] 1.6 Observation of strain YJ morphology (refer to the Manual of Identification of Common Bacteria and the Bergey Manual of Bacteria Identification)

[0050] Strain YJ was inoculated on LB nutrient agar medium and cultured at 28°C for 24 hours. The growth was good. The colonies of the strain were round, milky white and shiny, with a moist surface, a protrusion in the middle, and neat edges. Figure 1 shown.

[0051] 1.7 Molecular biological identification of strain YJ

[0052] The pure culture of strain YJ was subjected to 16S rRNA identification: about 0.5 μg of a single colony was picked and placed in a 1.5 mL centrifuge tube for colony PCR, and bacterial universal primers 27F and 1492R were selected. The primer sequences are shown in Table 2; the PCR reaction system is shown in Table 3;

[0053] Table 2 16S rRNA primers

[0054] Universal primer name Gene sequence Sequence Listing Number 27F 5'-AGAGTTTGATCCTGGCTCAG-3' SEQ ID No.1 1492R 5'-TACGGYTACCTTGTTACGACTT-3' SEQ ID No.2

[0055] Table 3 PCR reaction system

[0056] Reagents Addition amount Template DNA 0.5μL 27F (10uM) 1μL 1492R (10uM) 1μL 10×Ex Taq buffer 2μL 5u Ex Taq 0.2μL 2.5mM dNTP Mix 1.6μL <![CDATA[ddH2O]]> 13.7μL

[0057] The PCR program was: 95°C, 5 min; 95°C 30 s, 56°C 30 s, 72°C 1 min 30 s, 25 cycles; 72°C, 10 min.

[0058] Sanger sequencing was used to perform quality control on the raw sequences at both ends to remove low-quality bases and obtain clean sequences. The purified PCR products were sent to Meiji Biopharmaceutical Technology Co., Ltd. for 16S rRNA sequencing. After sequencing, BLAST comparison was performed at NCBI, and sequence analysis was performed using MEGA11 software to construct a phylogenetic tree. The constructed phylogenetic tree is shown in the figure. Figure 2 shown.

[0059] The 16S rRNA sequence of a strain screened out was amplified, and the amplified results were spliced ​​to obtain the 16S rDNA sequence of the strain, as shown in SEQ ID No. 3. The sequence was uploaded to NCBI for Blast comparison, and it was found that strain YJ had more than 99% homology with multiple strains of Klebsiella oxytoca sp. According to sequence homology analysis, YJ was determined to be Klebsiella oxytoca, and was named Klebsiella oxytoca YJ.

[0060] 1.8 Identification of the manganese oxidation function of strain YJ

[0061] Add the prepared Mn(Ⅱ) mother solution to the sterilized PYCM liquid medium to make the Mn(Ⅱ) concentration of the medium 1mmol / L, add the bacterial solution prepared in 1.4 to the PYCM medium at a 1% (v / v) inoculation rate, and culture in a constant temperature shaking incubator at 30℃ and 180r / min. After about 7 days, when the medium becomes turbid and produces brown precipitate, add 0.04% LBB (Brilliant Blue) colorimetric solution to the brown bacterial solution to observe whether it turns blue, so as to determine whether the strain has the ability to oxidize manganese. The results are shown in Figure 3 .

[0062] Depend on Figure 3 It can be seen that the bacterial solution turned blue, indicating that strain YJ has the ability to oxidize manganese and can oxidize Mn (II) into high-valent manganese.

[0063] 1.9 Determination of Mn(Ⅱ) removal ability of strain YJ

[0064] After streaking the strain plate, pick the strain with an inoculation loop and add it to 50 mL LB liquid medium. Cultivate it at 28°C and 180 rpm for 20 h. 600The value is 0.8-1.0. The bacterial solution was inoculated with 1% (v / v) in 100mL PYCM liquid culture medium with a Mn(II) concentration of 500mg / kg, and cultured in a constant temperature shaking incubator at 28°C and 180r / min. Samples were taken every 24h, and the supernatant was filtered through a 0.22μm filter membrane after centrifugation in a 12000r / min centrifuge for 10min. The manganese concentration of the supernatant was then determined by ICP-OES. The removal rate was calculated using the formula, and the culture solution with the same concentration without bacteria was used as a blank control. The results are as follows: Figure 4 shown.

[0065] Removal efficiency = (initial solution manganese concentration - treated solution manganese solubility) / initial solution manganese concentration × 100%

[0066] Depend on Figure 4 It can be seen that with the increase of time, the removal rate of Mn(Ⅱ) in 500 mg / kg culture medium by strain YJ gradually increased, reaching 92.20% on the 4th day.

[0067] 1.10 Determination of As(III) removal capacity of strain YJ

[0068] After streaking the strain plate, pick the strain with an inoculation loop and add it to 50 mL LB liquid medium. Cultivate it at 28°C and 180 rpm for 20 h. 600 The value is 0.8-1.0. The bacterial solution was inoculated with 1% (v / v) in 100mL PYCM liquid culture medium with an As(III) concentration of 50mg / kg, and cultured in a constant temperature shaking incubator at 28℃ and 180r / min. Samples were taken every 24h, and the supernatant was filtered through a 0.22μm filter membrane after centrifugation in a 12000r / min centrifuge for 10min. The arsenic concentration of the supernatant was then determined by ICP-OES. The removal rate was calculated using the formula, and the culture solution with the same concentration without bacteria was used as a blank control. The results are shown in Figure 5 .

[0069] Removal efficiency = (initial solution arsenic concentration - treated solution arsenic solubility) / initial solution arsenic concentration × 100%

[0070] Depend on Figure 5 It can be seen that with the increase of time, the removal rate of strain YJ for As(III) in 50 mg / kg culture medium gradually increased, reaching 98.40% on the 4th day.

[0071] 1.11 Determination of Cd(II) removal capacity of strain YJ

[0072] After streaking the strain plate, pick the strain with an inoculation loop and add it to 50 mL LB liquid medium. Cultivate it at 28°C and 180 rpm for 20 h. 600The value is 0.8-1.0. The bacterial solution was inoculated with 1% (v / v) in 100mL PYCM liquid culture medium with a Cd(II) concentration of 5mg / kg, and cultured in a constant temperature shaking incubator at 28℃ and 180r / min. Samples were taken every 24h, and the supernatant was filtered through a 0.22μm filter membrane after centrifugation in a 12000r / min centrifuge for 10min. The cadmium concentration of the supernatant was then determined by ICP-OES. The removal rate was calculated using the formula, and the culture solution with the same concentration without bacteria was used as a blank control. The results are shown in Figure 6 .

[0073] Removal efficiency = (initial solution cadmium concentration - treated solution cadmium solubility) / initial solution cadmium concentration × 100%

[0074] Depend on Figure 6 It can be seen that with the increase of time, the removal rate of Cd(II) in 5 mg / kg culture medium by strain YJ gradually increased, reaching 57.07% on the 5th day.

[0075] 2. Biochar-loaded composites

[0076] 2.1 Preparation of biochar-loaded composites

[0077] Prepare 100 mL LB liquid culture medium, sterilize and cool it, then inoculate the strain YJ bacterial solution into the LB liquid culture medium at a rate of 1% (v / v), and add sterilized biochar at a rate of 1% (m / v), and culture it in a constant temperature shaking incubator at 30°C and 180 r / min for 7 days.

[0078] The culture was poured into a sterile centrifuge tube and centrifuged at 12000 rpm for 10 min using a high-speed centrifuge, and the supernatant was discarded; after washing with sterile water, the culture was centrifuged at 12000 rpm for 10 min using a high-speed centrifuge, and the supernatant was discarded. The operation was repeated 3 times. The precipitate after centrifugation was freeze-dried, and the morphology of the biochar-loaded manganese-resistant manganese-oxidizing strain YJ composite material was observed using a scanning electron microscope (SEM). The results of the electron microscope scanning are shown in Figure 7 .

[0079] 2.2 Potted plant experiment

[0080] 2.2.1 A rice pot experiment was conducted using soil with an arsenic content of 50.89 mg / kg as planting soil. 1% (m / m) biochar-loaded composite material was added to the potted plants. The blank control group CK was set without biochar-loaded composite material. The rice plants were harvested after they matured, and the sample processing and arsenic content of rice grains were determined according to the "Determination of Total Arsenic and Inorganic Arsenic in Food" (GB 5009.11-2014). The results are shown in Figure 8 .

[0081] Depend on Figure 8 It can be seen that compared with the blank control CK, the addition of biochar-loaded composite materials can effectively reduce the As content in rice grains.

[0082] 2.2.2 A rice pot experiment was conducted using soil with a cadmium content of 0.97 mg / kg as planting soil. 1% (m / m) biochar-loaded composite material was added to the potted plants. The blank control group CK was set without biochar-loaded composite material. The rice plants were harvested after they matured, and the sample processing and cadmium content of rice grains were determined according to the National Food Safety Standard for the Determination of Cadmium in Food (GB 5009.15-2014). The results are shown in Fig. 9 .

[0083] Depend on Figure 8 It can be seen that compared with the blank control CK, the addition of biochar-loaded composite materials can effectively reduce the Se content in rice grains.

[0084] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A manganese oxide resistant strain YJ, characterized in that: The taxonomic name of the manganese-resistant manganese oxide strain YJ is Klebsiella oxytoca, which was deposited in the Guangdong Microbiological Culture Collection Center on May 21, 2024, with the deposit number GDMCC No: 64667.

2. A biochar-loaded composite material, characterized in that: The biochar-loaded composite material is prepared from the manganese-resistant manganese-oxidizing strain YJ described in claim 1.

3. Use of the manganese-resistant manganese-oxidizing strain YJ according to claim 1 or the biochar-loaded composite material according to claim 2 in removing Mn, As and / or Cd from the environment.

4. Use of the manganese-resistant manganese-oxidizing strain YJ according to claim 1 or the biochar-loaded composite material according to claim 2 in reducing the As and / or Cd content in crop seeds.

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