A Manganese-Resistant Manganese-Oxidizing Strain Klebsiella oxytoca YJ and Its Application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a manganese-tolerant and manganese-oxidizing strain Klebsiella oxytoca YJ and its application. A manganese-tolerant and manganese-oxidizing strain YJ, the taxonomic name of the manganese-tolerant and manganese-oxidizing strain YJ is Klebsiella oxytoca, and it was deposited in the Guangdong Microbial Culture Collection Center on May 21, 2024, with the deposit number GDMCC No: 64667. In the present invention, manganese-tolerant strains were screened, and a strain YJ with a manganese tolerance concentration of 200 mM was obtained. Through morphological and molecular biological identification, strain YJ was identified as Klebsiella oxytoca. Through the study of the heavy metal removal rate, it was found that the removal rate of Mn(II) in the culture solution at 500 mg / kg by strain YJ reached 92.20% on the 4th day, the removal rate of As(III) in the culture solution at 50 mg / kg reached 98.40% on the 4th day, and the removal rate of Cd(II) in the culture solution at 5 mg / kg reached 57.07% on the 5th day. It can be seen from this that strain YJ is of great significance for the treatment of heavy metal pollution such as Mn, As, and Cd.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a manganese-tolerant manganese-oxidizing strain Klebsiella oxytoca YJ and its application. Background Art

[0002] Currently in China, the cultivated land area contaminated by heavy metals such as cadmium, arsenic, and lead is estimated to be nearly 2.0×10 7 hm 2 . Soil arsenic pollution mainly comes from atmospheric deposition, sewage irrigation, and spraying of arsenic-containing pesticides. The arsenic content in the surface soil of China ranges from 2.5×10 -6 -33.5×10 -6 . The soil arsenic content shows a north-south geographical distribution, and 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 region, the southwest region, and the south China region to the northeast region, and there is a geochemical connection with the eroded materials in its upper reaches. As a metalloid element, arsenic has attracted much attention due to its high toxicity, easy enrichment, and difficult degradation characteristics, and can have harmful effects on both humans and plants. Long-term drinking of high-arsenic water can lead to chronic arsenic poisoning, as well as diseases of the skin, respiratory system, nervous system, and digestive system. Skin contact with arsenic also poses a risk of cancer. Cadmium is one of the most harmful heavy metals. Soil cadmium pollution not only reduces the yield and quality of crops, but also harms human health through the food chain enrichment effect. The "National Pollution Survey Bulletin" released by the Ministry of Ecology and Environment in 2014 shows that the exceeding standard rate of Cd in soil in China is 7.0%, ranking first among inorganic pollutants. Data shows that about 1.0×10 6 t Cd is released into the environment every year in the world; in China, the cultivated land area contaminated by Cd is about 1.0×10 7 hm 2 , and the agricultural products contaminated by Cd are as high as 1.5×10 6 t. Research shows that the heavy metal Cd content in farmland soil across the country ranges from 0.000013 - 217.23 mg / kg, with an average value of 0.39 mg / kg and a median value of 0.25 mg / kg. The Cd pollution in the country shows an uneven distribution trend. Guangdong, Hunan, and Yunnan are areas with more Cd pollution, and the Cd pollution degree in southern soil is significantly heavier than that in northern soil.

[0003] Rice is one of the main food crops in China. Due to the strong physiological tolerance and accumulation ability of rice to cadmium, the cadmium pollution situation in paddy fields is particularly serious, with a pollution area of 12,700 hm 2, the cadmium exceeding standard rate of rice in several major grain producing areas of China reached 10.3%, directly threatening food security and human health. The problem of cadmium pollution in paddy fields has become the top priority of soil heavy metal pollution and prevention. Based on the traditional mode of agricultural irrigation in China, soil arsenic pollution occurs more frequently in paddy fields, and under waterlogged conditions, the toxicity and bioavailability of arsenic in the soil will increase to varying degrees. Using chemical methods for remediation may cause secondary pollution, and more and more researchers have turned their attention to microbial bioremediation.

[0004] The inventor studied the microbial strains in the high-manganese soil environment and screened out a manganese-tolerant manganese-oxidizing strain that can oxidize Mn(II) to biological manganese oxides. Forming a composite material with biochar and adding it to the soil can significantly reduce the absorption of arsenic and cadmium by plants, which is of great significance for reducing the bioavailability of arsenic- and cadmium-polluted soils.

[0005] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0006] The purpose of the present invention is to provide a manganese-tolerant manganese-oxidizing strain Klebsiella oxytoca YJ and its application.

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

[0008] The first purpose of the present invention is to provide a manganese-tolerant manganese-oxidizing strain YJ. The taxonomic name of the manganese-tolerant manganese-oxidizing strain YJ is Klebsiella oxytoca, which was deposited at the Guangdong Microbial Culture Collection Center on May 21, 2024, with the deposit number GDMCC No: 64667.

[0009] The second purpose of the present invention is to provide a biochar-supported composite material, which is prepared from the manganese-tolerant manganese-oxidizing strain YJ.

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

[0011] The fourth purpose of the present invention is to provide the use of the manganese-tolerant manganese-oxidizing strain YJ or the biochar-supported composite material in reducing the content of As and / or Cd in crop grains.

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

[0013] (1) The present invention screens manganese-tolerant strains and obtains a strain YJ with a manganese tolerance concentration of 200 mM. Through morphological and molecular biological identification, strain YJ is Klebsiella oxytoca. Through the study of heavy metal removal rates, it is found that the removal rate of Mn(II) in the culture solution of 500 mg / kg by strain YJ reaches 92.20% on the 4th day, the removal rate of As(III) in the culture solution of 50 mg / kg reaches 98.40% on the 4th day, and the removal rate of Cd(II) in the culture solution of 5 mg / kg reaches 57.07% on the 5th day. Thus, 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-supported composite material prepared by the present invention using strain YJ can effectively reduce the accumulation of arsenic and cadmium elements in rice plants in the soil, and further reduce the arsenic and cadmium contents in rice grains.

[0015] Preservation information description

[0016] Klebsiella oxytoca YJ was preserved in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 21, 2024. The preservation address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the preservation number is GDMCC No: 64667. Description of the accompanying drawings

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

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

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

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

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

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

[0023] Figure 7 It is the electron microscope scanning diagram of the biochar-supported composite material;

[0024] Figure 8It is the control chart of arsenic content in rice grains;

[0025] Figure 9 It is the control chart of cadmium content in rice grains.

[0026] Description of main reference numerals:

[0027] Figure 8 and Figure 9 In [reference numerals] and [reference numerals], CK is the blank control group without adding the biochar-loaded composite material; BC is the treatment group adding the biochar-loaded composite material. Specific implementation manners

[0028] The technical solutions of the present invention patent will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] 1. Strain screening and identification

[0030] 1.1 Screening bacteria soil

[0031] Collect the high-manganese rice soil from Nanning, Guangxi. Take the rhizosphere soil of rice in the field by the five-point sampling method. After removing the surface sundries, take the surface soil of 5 - 20 cm, mix it evenly, put one part into a sterile centrifuge tube, place it in an ice box and bring it back to the laboratory for storage at -20 °C for the screening of manganese-oxidizing bacteria. Put one part into a self-sealing bag and bring it back to the laboratory to air-dry and then sieve it. Refer to "Methods for Agricultural Chemical Analysis of Soils" for sample preparation and determination of the manganese content and its basic physical and chemical properties in the soil.

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

[0033] Available phosphorus (mg / kg) Available potassium (mg / kg) Alkaline hydrolyzable 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 value 7.0 ± 0.2;

[0036] LB broth: Tryptone 10.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 10.0 g / L, pH value 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 value 7.0 ± 0.2.

[0038] PYCM liquid 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 value 7.0 ± 0.2.

[0039] 1.3 Strain isolation and purification

[0040] Weigh 10 g of soil sample in a sterile centrifuge tube in the laminar flow hood, add 90 mL of deionized water with glass beads that have been sterilized by high temperature and high pressure in advance, take it out after shaking in a shaking incubator at 30 °C and 180 r / min for 30 min to obtain a soil suspension.

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

[0042] Gradually dilute the PYCM bacterial liquid with sterile water to 10 -4 、10 -5 、10 -6 , take 100 μL of each and coat it on a sterile plate of PYCM solid medium. After culturing in a constant temperature incubator at 30 °C for 3 d, pick a single colony and use the streak plate method to perform 3 repeated isolations and purifications to obtain a pure culture. Inoculate the strain into sterile LB broth and culture it in a shaking incubator at 30 °C and 180 r / min until the logarithmic growth phase of the strain. Take a certain amount of the bacterial liquid and put it into a cryopreservation tube, add sterile glycerol to make the glycerol concentration between 20 - 30%, seal it and shake it well, and place the cryopreservation tube in a -80 °C refrigerator for low-temperature storage.

[0043] 1.4 Preparation of strain seed liquid

[0044] After streak plating the strain, use an inoculation loop to pick the strain into 50 mL of LB liquid medium and culture it by shaking at 28 °C and 180 r / min for 20 h, with an OD 600 value of 0.8 - 1.0.

[0045] 1.5 Screening of manganese-tolerant strains

[0046] Add the prepared Mn(II) stock solution to the sterilized PYCM liquid medium. The Mn(II) concentration in the medium is set to 5, 10, 20, 50, 100, 150, 200, 250, 300, 400 mmol / L. And set the PYCM liquid medium with the same concentration without inoculation as the control. Inoculate the bacterial solution prepared in 1.4 into the PYCM liquid medium at an inoculation amount of 1% (v / v), and place it in a constant temperature shaking incubator at 30 °C and 180 r / min for 14 days.

[0047] Use an ultraviolet spectrophotometer to measure the OD 600 value of the culture solution as the judgment criterion: If the OD 600 of the inoculated culture solution is greater than that of the non-inoculated culture solution 600 and the difference is large, it indicates that the strain can still grow under this concentration condition. Thus, the tolerance of the manganese-oxidizing bacteria to Mn(II) is determined, and the manganese-oxidizing strain with high manganese tolerance is screened out.

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

[0049] 1.6 Observation of the morphology of strain YJ (performed with reference to the "Manual for Systematic Identification of Common Bacteria" and the "Bergey's Manual of Determinative Bacteriology")

[0050] Inoculate strain YJ onto the LB nutrient agar medium. After culturing at 28 °C for 24 h, the growth is good. The strain colonies are round, milky white and shiny, with a moist surface, a raised middle, and a neat edge, as Figure 1 shown.

[0051] 1.7 Molecular biological identification of strain YJ

[0052] Perform 16S rRNA identification on the pure culture of strain YJ: Pick about 0.5 μg of single colonies into a 1.5 mL centrifuge tube for colony PCR. Select the universal bacterial primers 27F and 1492R. The primer sequences are shown in Table 2; the PCR reaction system is shown in Table 3;

[0053] Table 2 16S rRNA primers

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

[0055] Table 3 PCR reaction system

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

[0057] The PCR program is as follows: 95 °C, 5 min; 95 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min 30 s, cycle 25 times; 72 °C, 10 min.

[0058] The raw sequences at both ends obtained were quality-controlled by Sanger sequencing to remove low-quality bases, and clean sequences were obtained. The purified PCR products were sent to Majorbio Bio-Pharm Technology Co., Ltd. for 16S rRNA sequencing. After the sequencing was completed and BLAST alignment was performed on NCBI, sequence analysis was carried out using MEGA11 software to construct a phylogenetic tree. The constructed phylogenetic tree is as Figure 2 shown.

[0059] The 16S rRNA sequence of a selected strain was amplified. The amplified result was spliced to obtain the 16S rDNA sequence of the strain. The sequence is shown in SEQ ID No. 3. After uploading the sequence to NCBI for Blast alignment, it was found that the strain YJ had a homology higher than 99% 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 Functional identification of manganese oxidation by strain YJ

[0061] The prepared Mn(II) stock solution was added to the sterilized PYCM liquid medium to make the Mn(II) concentration in the medium 1 mmol / L. The bacterial solution prepared in 1.4 was inoculated into the PYCM medium at an inoculation amount of 1% (v / v) and cultured in a constant temperature shaking incubator at 30 °C and 180 r / min. After about 7 days, when the medium became turbid and brown precipitates were produced, a 0.04% LBB (brilliant blue) color reagent solution was added to the brown bacterial solution to observe whether it turned blue to judge whether the strain had the ability of manganese oxidation. The results are shown in Figure 3 .

[0062] As Figure 3 can be seen, the bacterial solution turned blue, indicating that strain YJ has the ability of manganese oxidation and can oxidize Mn(II) to a higher valence state of manganese.

[0063] 1.9 Determination of the ability of strain YJ to remove Mn(II)

[0064] After the strain was streaked on the plate, the strain was picked up with an inoculation loop and transferred to 50 mL of LB liquid medium and cultured with shaking at 28 °C and 180 r / min for 20 h, OD 600The value is 0.8 - 1.0. Inoculate the bacterial solution into 100 mL of PYCM liquid medium with a Mn(II) concentration of 500 mg / kg at an inoculation amount of 1% (v / v), and culture it in a constant temperature shaking incubator at 28°C and 180 r / min. Sample once every 24 h. After centrifuging at 12,000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane, and then measure the manganese concentration in the supernatant by ICP-OES. Calculate the removal rate through the formula, and use the same-concentration culture solution without inoculation as the blank control. The results are as Figure 4 shown.

[0065] Removal efficiency = (Initial solution manganese concentration - Treated solution manganese concentration) / Initial solution manganese concentration × 100%

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

[0067] 1.10 Determination of the removal ability of strain YJ to As(III)

[0068] After streaking the strain on a plate, pick the strain with an inoculation loop into 50 mL of LB liquid medium and culture it with shaking at 28°C and 180 r / min for 20 h. OD 600 value is 0.8 - 1.0. Inoculate the bacterial solution into 100 mL of PYCM liquid medium with an As(III) concentration of 50 mg / kg at an inoculation amount of 1% (v / v), and culture it in a constant temperature shaking incubator at 28°C and 180 r / min. Sample once every 24 h. After centrifuging at 12,000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane, and then measure the arsenic concentration in the supernatant by ICP-OES. Calculate the removal rate through the formula, and use the same-concentration culture solution without inoculation as the blank control. The results are shown in Figure 5 .

[0069] Removal efficiency = (Initial solution arsenic concentration - Treated solution arsenic concentration) / Initial solution arsenic concentration × 100%

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

[0071] 1.11 Determination of the removal ability of strain YJ to Cd(II)

[0072] After streaking the strain on a plate, pick the strain with an inoculation loop into 50 mL of LB liquid medium and culture it with shaking at 28°C and 180 r / min for 20 h. OD 600The value is 0.8 - 1.0. The bacterial solution was inoculated into 100 mL of PYCM liquid medium with a Cd(II) concentration of 5 mg / kg at an inoculation amount of 1% (v / v), and cultured in a constant temperature shaking incubator at 28 °C and 180 r / min. Samples were taken every 24 h. After centrifugation at 12,000 r / min for 10 min, the supernatant was taken and filtered through a 0.22 μm filter membrane, and then the cadmium concentration in the supernatant was measured by ICP-OES. The removal rate was calculated by the formula, and the same-concentration culture solution without inoculation was used as a blank control. The results are shown in Figure 6 .

[0073] Removal efficiency = (Initial cadmium concentration in the solution - Cadmium concentration in the treated solution) / Initial cadmium concentration in the solution × 100%

[0074] It can be seen from Figure 6 that as time increases, the removal rate of Cd(II) in the 5 mg / kg culture solution by strain YJ gradually increases and reaches 57.07% on the 5th day.

[0075] 2. Biochar-supported composite materials

[0076] 2.1 Preparation of biochar-supported composite materials

[0077] 100 mL of LB liquid medium was prepared, sterilized and cooled. The bacterial solution of strain YJ was inoculated into the LB liquid medium at an inoculation amount of 1% (v / v), and at the same time, sterilized biochar was added at an addition amount of 1% (m / v), and cultured 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 12,000 rpm for 10 min, and the supernatant was poured off; after washing with sterile water, it was centrifuged at 12,000 rpm for 10 min, and the supernatant was poured off, and the operation was repeated 3 times. The centrifuged precipitate was freeze-dried, and the morphology of the biochar-supported manganese-resistant manganese-oxidizing strain YJ composite material was observed by scanning electron microscopy (SEM). The SEM results are shown in Figure 7 .

[0079] 2.2 Pot experiment

[0080] 2.2.1 A rice pot experiment was carried out using soil with an arsenic content of 50.89 mg / kg as the planting soil. 1% (m / m) of the biochar-supported composite material was added to the pot, and the one without the biochar-supported composite material was set as the blank control group CK. After the rice matured, its plants were harvested, and the sample was processed and the arsenic content in the rice grains was measured with reference to "Determination of total arsenic and inorganic arsenic in foods" (GB 5009.11-2014). The results are shown in Figure 8 .

[0081] It can be seen fromFigure 8 It can be seen that compared with the blank control CK, adding the biochar-supported composite material can effectively reduce the As content in rice grains.

[0082] 2.2.2 Using soil with a cadmium content of 0.97 mg / kg as the planting soil for a rice pot experiment, adding 1% (m / m) of the biochar-supported composite material to the pots, and setting the group without the biochar-supported composite material as the blank control group CK. After the rice matured, the plants were harvested, and the sample treatment and the determination of the cadmium content in rice grains were carried out with reference to the "National Food Safety Standard Determination of Cadmium in Foods" (GB 5009.15-2014). The results are shown in Figure 9 .

[0083] From Figure 8 It can be seen that compared with the blank control CK, adding the biochar-supported composite material can effectively reduce the Se content in rice grains.

[0084] The foregoing description of specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the present invention to the precise forms disclosed, and obviously, many changes and variations are possible in light of 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 applications, so that those skilled in the art can implement and utilize various different exemplary embodiments of the present invention, as well as various different selections and changes. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

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

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

3. Use of the manganese-tolerant manganese-oxidizing strain YJ described in claim 1 or the biochar-supported composite material described in claim 2 for removing Mn, As, and / or Cd from the environment.

4. Use of the manganese-tolerant manganese-oxidizing strain YJ described in claim 1 or the biochar-supported composite material described in claim 2 for reducing the content of As and / or Cd in crop grains.

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