CN113862173B - Heavy metal adsorbing microbial inoculum and application thereof in soil pollution remediation - Google Patents

Abstract

The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly relates to a heavy metal adsorbing microbial inoculum and application thereof in soil pollution remediation. The microbial inoculum contains domesticated Harper spirillum huttiense HHS1; the domestication of the HHS1 of the Herbaspirillum huttiense is to perform gradient domestication in a solution containing copper ions and zinc ions, and finally obtain the HHS1 stabilized strain capable of tolerating the copper ions and the zinc ions. The microbial inoculum can be applied to stabilizing copper and zinc in the soil environment, and is beneficial to enriching and stabilizing a strain database of copper and zinc. The invention prepares the purified bacterial strain into the immobilized microbial agent, and can effectively stabilize copper and zinc in different environmental media such as water, soil and the like. Compared with single physical adsorption, chemical oxidation and other methods, the method is more efficient, energy-saving and environment-friendly. Provides an effective biological-non-biological combined repair technology for the stabilization of copper and zinc pollution.

Description

Heavy metal adsorbing microbial inoculum and application thereof in soil pollution remediation

Technical Field

The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly relates to a heavy metal adsorbing microbial inoculum and application thereof in soil pollution remediation.

Background

In order to promote the growth of livestock and poultry and improve the immunity of animals, heavy metals exceeding the absorption amount of the animals are often added into the livestock and poultry feed. A large amount of unabsorbed heavy metals are discharged along with the excrement, a large amount of livestock and poultry manure is piled on farmland soil without treatment, and once crops are planted on the soil, the growth of the crops can be damaged and injured. The excessive heavy metals threaten the normal growth of aquatic organisms and destroy microbial community structures, and can also damage human beings through food chain enrichment. Wherein, copper is found to be used in large quantities, affecting plant cell structure, photosynthesis and synthesis of plant enzymes; the zinc can be used in the piglet at a dosage of more than 2000mg/kg. However, the absorption of zinc by livestock and poultry is less than 20%.

At present, the heavy metal stabilization technology comprises four types, namely fixing/stabilizing technology, plant restoration technology and microorganism restoration technology. Wherein the engineering technology comprises two measures of deep ploughing, turning soil and changing soil; however, methods such as deep ploughing destroy the original structure of the soil, causing secondary pollution. The modes of photoelectrocatalysis degradation, advanced oxidative degradation and the like have poor specificity and high cost and are easy to generate secondary pollution. Therefore, biodegradation methods including microbial remediation techniques are attracting much attention. The microbial remediation technology has low cost, good effect, no secondary pollution and small influence on the soil microenvironment, so the application of the microbial remediation technology is a good remediation means for the site polluted by the heavy metal; however, microorganisms are often specific in repairing pollutants and only have a certain repairing effect on single heavy metals. And the microorganism is sensitive to temperature, and the low temperature environment inhibits the action of the microorganism. Therefore, the invention provides a low temperature resistant microorganism, which meets the stabilizing effect on copper and zinc at different temperatures and realizes the efficient utilization of the strain.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide a heavy metal adsorbing microbial inoculum.

The invention also aims to provide application of the microbial inoculum in soil remediation.

In order to achieve the purpose, the method is realized by the following technical scheme:

a heavy metal adsorbing microbial inoculum comprises domesticated Harper spirillum huttiense (Herbaspirillum huttiense) HHS1; the strain is Harpagophytum sp (Herbaspirillum huttiense) HHS1 which is preserved in China general microbiological culture Collection center in 2021, 07-28 days, and the address is as follows: the preservation number of No. 3 Xilu Beijing Hospital No. 1, chaoyang area, beijing, is: CGMCC No. 22975.

The domestication of the Harper spirillum HHS1 is to perform gradient domestication in a solution containing copper ions and zinc ions, and finally obtain the HHS1 stabilized strain which can tolerate the copper ions and the zinc ions.

The activated domesticated strain is mixed with the mushroom bran biochar, and then is embedded and fixed for 24-48h by using sodium alginate, so that the uniform and stable immobilized microbial agent is finally obtained.

The ratio of the mass (g) of the mushroom bran biochar to the volume (ml) of the mushroom suspension is 1:5 (w: v) -1 (w: v); the addition amount of the sodium alginate accounts for 2-3 wt% of the mass of the microbial inoculum (the mass of the microbial inoculum is the sum of the mass of the bacterial suspension, the mass of the biochar and the mass of the sodium alginate).

A preparation method of a heavy metal adsorbing microbial inoculum comprises the steps of mixing a bacterium solution obtained by activating an acclimated strain with fungus chaff biochar, and then carrying out embedding and fixing for 24-48h by utilizing sodium alginate, thus obtaining a uniform and stable immobilized microbial inoculum finally.

The method comprises the following specific steps:

1) Placing the grass snail strain HHS1 with tolerance and stabilization effects on copper and zinc in LB culture medium to culture to logarithmic phase, centrifuging to collect precipitate, and resuspending the precipitate to OD by sterile normal saline ₆₀₀ Is 0.8 to 1.2 of bacterial suspension;

2) Burning the waste mushroom sticks at 500-700 ℃ for 4-6h to form mushroom bran biochar for later use;

3) Mixing the obtained mushroom bran biochar with the mushroom suspension, adding the mushroom bran biochar and the mushroom suspension into a sodium alginate solution under the condition of stirring, and uniformly dropwise adding the mixture into CaCl through a peristaltic pump ₂ Embedding and fixing in electrolyte solution for 24-48h, discarding CaCl ₂ Electrolyte solution to prepare homogeneous and stable immobilized microbial inoculum;

wherein the proportion of the biochar in the sodium alginate is 2-5% (w: v); the proportion of the bacterial suspension in the sodium alginate is 10-20% (v: v).

The waste mushroom sticks are mushroom growth carriers of a mushroom planting base. The raw materials of the carrier mainly comprise sawdust, wheat bran and gypsum, and provide required growth elements and growth conditions for the mushrooms. When the mushrooms are mature, the nutrient components in the mushroom sticks are consumed, the mushroom sticks become waste and are stacked in the environment, and the mushroom sticks are burnt into a charcoal carrier which is placed in the soil environment to realize resource utilization. At present, the biochar material is prepared by burning corn stalks, rice husks and other raw materials. Compared with other biological materials, the fungus chaff biochar has higher nutrient content, is more favorable for improving soil fertility and realizes the recycling of wastes.

The microbial inoculum component in the step 3) comprises sodium alginate, mushroom bran biochar, mushroom suspension, ultrapure water and CaCl ₂ The electrolyte solution is an embedding fixing place.

The application of the microbial inoculum, and the application of the microbial inoculum in adsorbing heavy metals in polluted soil.

The heavy metals are the stabilization of copper and zinc.

A method for restoring heavy metals in polluted soil by using the microbial inoculum is characterized in that the microbial inoculum is added into the polluted soil, wherein the mass percentages of mushroom bran biochar and HHS1 in the soil are respectively 1% -3%;5 to 15 percent.

Compared with the prior art, the invention has the beneficial effects that:

the invention screens and separates the bacterial strain from the in-situ livestock and poultry breeding soil containing antibiotic pollution, and domesticates the bacterial strain to make the bacterial strain become a bacterial strain with stable copper ions and zinc ions, and the bacterial strain has certain tolerance and stabilization capability to heavy metal copper and zinc. The bacterium is identified as Herbaspirillum huttiense by a molecule. The strain has the function of stabilizing copper and zinc, is favorable for enriching and fixing a strain resource library of heavy metals, and provides an effective biodegradation method for the treatment of heavy metal pollution. The immobilized microorganism remediation technology is safe, effective, environment-friendly and harmless, and can not cause secondary pollution to the environment.

The method is used for obtaining the bacterial strain, and meanwhile, the waste bacterial stick is used as a carrier of the microorganism to prepare the microbial inoculum, and the carrier simultaneously stabilizes heavy metals Cu and Zn. In addition, the surface of the microbial inoculum treated by the carrier has rich functional groups and high hydrophilicity, and has the advantages of improving the water holding capacity of soil, improving the chemical properties of the soil, improving the activity of enzymes in the soil and the like. Compared with a common heavy metal adsorption strain, the strain can adsorb and stabilize two heavy metals at the same time, the microbial inoculum formed by combining the biochar and the strain greatly improves the adsorption performance of a single strain, and the content of the effective heavy metals in the soil can be rapidly reduced in a short time. The stabilization technology of the biochar can greatly reduce the content of the effective state of the heavy metal in the soil environment and improve the stabilization efficiency of the heavy metal. The microbial inoculum realizes the high-efficiency stabilization of heavy metal pollution and the resource utilization of wastes at the same time. Therefore, the biochar-microorganism immobilized fungicide prepared by the method has important significance for reducing heavy metals in soil and improving the basic environment of the soil.

Drawings

FIG. 1 is a scanning electron microscope image of mushroom bran biochar provided by an embodiment of the present invention.

FIG. 2 is a morphological diagram of the fungus chaff biochar-HHS 1 microbial inoculum provided by the embodiment of the invention.

FIG. 3 is a scanning electron microscope image of the fungus chaff biochar-HHS 1 microbial inoculum provided by the embodiment of the invention.

FIG. 4 is a graph showing the effect of an immobilized microbial agent on stabilizing copper and zinc in soil in a low-temperature environment (15 ℃) according to an embodiment of the invention.

FIG. 5 is a graph showing the effect of an immobilized microbial agent on the stabilization of copper and zinc in soil at room temperature (25 ℃) in an embodiment of the present invention.

FIG. 6 is a graph showing the effect of an immobilized microbial agent on stabilizing copper and zinc in soil at room temperature (35 ℃ C.) according to an embodiment of the present invention.

FIG. 7 is a graph showing the effect of the immobilized microbial agent for degrading copper and zinc in soil at room temperature (35 ℃ C.) according to the embodiment of the present invention.

Detailed Description

The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are only for purposes of illustration and explanation and are not intended to be limiting.

Example 1 isolation and characterization of the strains:

the invention discloses a spirillum sinensis HHS1 strain which is obtained by screening and domesticating soil of a livestock and poultry breeding field polluted by heavy metal-antibiotics, and the method comprises the following specific steps:

A. screening antibiotic-resistant strains in the livestock and poultry breeding polluted soil by using terramycin:

the domestication process comprises the following steps: collecting a contaminated soil sample stacked by animal wastes produced by a livestock and poultry farm, after knowing the initial oxytetracycline concentration in the soil sample, adding the sample into an inorganic salt culture medium containing oxytetracycline for gradient domestication, gradually increasing the oxytetracycline concentration in the culture medium, sequentially and respectively culturing the oxytetracycline in a shaking table at 15 ℃ and 150r/min for 7d at each concentration for 50 mug/kg, 100 mug/kg, 200 mug/kg, 500 mug/kg, 1mg/kg and 2mg/kg in a dark mode, regularly observing the state of a bacterial liquid in the culture medium, and if the liquid in the culture medium is changed from transparent to turbid, indicating that the bacterial liquid grows well;

selecting the terramycin-tolerant mixed bacteria screened under the condition that the concentration of the gradient domesticated terramycin is 2mg/L, transferring the mixed bacteria to a solid inorganic salt culture medium containing 2mg/L terramycin, and performing light-resistant culture at 15 ℃ for 6 times; so that the terramycin degrading bacteria have growth dominance.

Inoculating the obtained mixed bacteria into an inorganic salt culture medium containing 1mg/L of oxytetracycline, culturing at 15 ℃ at 150r/min in a dark place, sampling for 0 day and 3 days, filtering with a 0.22 mu m filter membrane, and measuring the degradation effect by LC-MS; degradation experiments show that the removal efficiency of the oxytetracycline can reach 54.20 percent after 3 days. Inoculating the obtained mixed strain into liquid inorganic salt culture medium containing 2mg/L, and culturing to OD ₆₀₀ And (4) at 1.0, carrying out resuspension by using sterile normal saline to finally obtain a mixed microbial suspension which is tolerant and can degrade the terramycin.

B. Respectively inoculating the mixed microbial suspension obtained after domestication on a solid LB culture medium, a solid inorganic salt culture medium and a solid potato culture medium by using inoculating loops for streak culture; when the three culture media are used, 2mg/L of oxytetracycline is ensured to be contained; culturing at 15 deg.C under different culture media in dark place, transferring the grown microorganism every five days, repeating for six times to separate and purify the strains in each culture medium, and finally obtaining single strain;

C. and inoculating each single separated and purified strain into an inorganic salt culture medium containing 1mg/L of oxytetracycline antibiotic as a unique carbon source for degradation experiment, inspecting the degradation capability of the single strain on the oxytetracycline in the inorganic salt culture medium, and finally selecting the microorganism HHS1 with the highest antibiotic degradation effect in the single strain.

The obtained purified single strain is Herbaspirillum huttiense, is named as HHS1, and is preserved in China general microbiological culture Collection center at 28 th month 07 in 2021. The preservation number is: CGMCC No. 22975.

After DNA extraction, PCR amplification, sequence sequencing and sequence comparison of the obtained strain, the 16s rDNA sequence of the strain HHS1 has homology of more than 99.93 percent with the Herbaspirillum huttiense, and the strain is identified as the Herbaspirillum huttiense with the nearest genetic relationship, which is called as the HHS1 for short.

Screening each medium component:

inorganic salt medium (g/L): ((NH) ₄ ) ₂ SO ₄ Is 1, KH ₂ PO ₄ Is 1, K ₂ HPO ₄ 1, naCl 0.5, feCl ₃ 6H ₂ O is 0.05 and CaCl ₂ 0.02, 10 for anhydrous glucose, pH = 7.0)

Basal medium (g/L): 5 of yeast powder, 10 of peptone and 10 of NaCl; the medium was sterilized at 121 ℃ for 20min before use.

Potato culture medium: boiling 200g of fresh potato blocks in a heating furnace for 20min, filtering to remove potato blocks, adding 20g of glucose into the supernatant, and diluting to 1L.

Inorganic salt solid medium (g/L): ((NH) ₄ ) ₂ SO ₄ Is 1, KH ₂ PO ₄ Is 1, K ₂ HPO ₄ 1, naCl 0.5, feCl ₃ 6H ₂ O is 0.05 and CaCl ₂ 0.02, 10 for anhydrous glucose, pH = 7.0), 15-20g of agar powder was boiled with heating.

LB medium (g/L): 5 parts of yeast powder, 10 parts of peptone and 10 parts of NaCl; 15-20g of agar powder is heated and boiled.

Potato solid medium: boiling 200g of fresh potato blocks in a heating furnace for 20min, filtering to remove potato blocks, adding 20g of glucose into the supernatant, and diluting to 1L.15-20g of agar powder is heated and boiled.

The medium was sterilized at 115 deg.C (containing glucose)/121 deg.C (containing no glucose) for 30min before use.

The 16s rDNA sequence was:

TGGCTCAGATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGCAGCATAGGA GCTTGCTCCTGATGGCGAGTGGCGAACGGGTGAGTAATATATCGGAACGTGCCCTAGAGTGGG GGATAACTAGTCGAAAGACTAGCTAATACCGCATACGATCTACGGATGAAAGTGGGGGATCGCA AGACCTCATGCTCCTGGAGCGGCCGATATCTGATTAGCTAGTTGGTGGGGTAAAAGCCTACCAA GGCAACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCAATG CCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTTGTCAGGGAAGAAACGGTAGTAG CGAATAACTATTACTAATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCC GCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGT TGTGTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAATTGCATTTGAGACTGCACGGCTA GAGTGTGTCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGATATGTGGAGGAA TACCGATGGCGAAGGCAGCCCCCTGGGATAACACTGACGCTCATGCACGAAAGCGTGGGGAGC AAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCTACTAGTTGTCGGGTCTTAAT TGACTTGGTAACGCAGCTAACGCGTGAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAA ACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACG CGAAAAACCTTACCTACCCTTGACATGGATGGAATCCCGAAGAGATTTGGGAGTGCTCGAAAG AGAACCATCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACGAAAGGGCACTCTAATGAGACTGCCGGT GACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCAC ACGTCATACAATGGTACATACAGAGGGCCGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAGT GTATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTTGGAATCGCTAGTAATCGCG GATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAG CGGGTTTTACCAGAAGTGGGTAGCCTAACCGCAAGGAGGGCGCTCACCACGGTAGGATTCGTG ACTGGGGTGAAGTCGTA

example 2

The obtained HHS1 strain is transferred to a new liquid inorganic salt culture medium, and 10mg/L Cu is added to the inorganic salt culture medium ²⁺ Solution and 10mg/L of Zn ²⁺ The solution is used as the initial concentration of domestication and is cultured in a shaking table at 15 ℃ and 150r/min for 7 days in a dark place, and then Cu in the inorganic salt culture medium is gradually increased ²⁺ And Zn ²⁺ Concentration of (C), cu ²⁺ And Zn ²⁺ The concentration is 10mg/L, 20mg/L, 30mg/L, 40mg/L and 50mg/L in sequence, the concentration is increased once and is cultured in a shaking table for 7 days at 15 ℃ and 150r/min in a dark place, the state of the bacterial liquid in the culture medium is observed regularly, and the liquid in the culture medium turns turbid from transparent, so that the bacterial liquid grows well; the domesticated HHS1 is transferred once every three days, and is repeated for six times, and Cu in the culture medium is transferred every time ²⁺ And Zn ²⁺ Culturing at 50mg/L at 15 deg.C and 150r/min in shaking table under dark condition for 3d; finally obtaining the alloy with Cu ²⁺ And Zn ²⁺ The HHS 1-tolerant strain of (1); centrifuging the obtained HHS1 strain tolerant to heavy metals for 10min at 8000rpm of a centrifuge, thoroughly separating from inorganic salt culture medium, and resuspending with sterile physiological saline to make the OD of the bacterial suspension ₆₀₀ And the concentration is 1.0, and the mixture is stored for standby.

Example 3

And (3) a soil remediation test of stabilizing copper in soil by using the immobilized microbial agent in a low-temperature environment (15 ℃): preparation of the microbial inoculum:

1. the HHS1 heavy metal tolerant strain domesticated in the example 2 is activated by a liquid culture medium according to a conventional mode, the bacterial suspension is inoculated into an LB culture medium after activation, the content of the bacterial suspension accounts for 2wt% of the LB culture medium, the bacterial suspension is cultured at the constant temperature of 15 ℃ for 48h to a logarithmic phase, and the culture solution is collected and resuspended to obtain bacterial liquid. Specifically, HHS1 strain is inoculated in LB culture medium, after the culture is carried out to logarithmic phase, bacteria are washed down by sterile physiological saline to prepare OD ₆₀₀ The bacterial suspension was 1.0.

2. Drying and grinding the waste mushroom sticks after mushroom planting, putting the waste mushroom sticks into a muffle furnace, and burning the waste mushroom sticks for 4 hours at 500 ℃ to form mushroom residue biochar; then the formed mushroom bran biochar is cleaned and dried by tap water, sieved by a 100-mesh sieve, and then is sterilized by an autoclave at 121 ℃,sterilizing under 1.5Mpa, collecting, and standing by, wherein the biochar has porous structure and specific surface area up to 105m ² The scanning electron micrograph is shown in figure 1; adding 30g of sterilized fungus chaff biochar into 150mL of HHS1 bacterial suspension obtained in the step 1, simultaneously adding 30g of sodium alginate into 1350mL of sterile water to prepare a sodium alginate solution, then pouring the mixed solution of the fungus chaff biochar and the microorganisms into the sodium alginate solution, continuously stirring the mixed system, dropwise adding the mixed system into 4 percent CaCl by using a latex tube and a peristaltic pump ₂ Embedding and fixing in electrolyte solution. The immobilization time is 24h, and finally the uniform and stable immobilized microbial agent is obtained. At the moment, the biochar in the system accounts for 2 percent of the mass of the microbial inoculum; the microorganism accounts for 10 percent of the mass of the microbial inoculum; sodium alginate accounts for 2% of the mass of the microbial inoculum. The form of the immobilized microbial preparation is shown in FIG. 2. The scanning electron microscope of the immobilized microbial inoculum is shown in figure 3.

As can be seen in fig. 2 and 3: the immobilized microbial inoculum is uniform black pellets, and the surface is smooth and full; the scanning electron microscope can clearly see that the uniform microorganisms are wrapped in the mixed system of the sodium alginate and the biochar, the strains are in a uniform rod-shaped form, and no mixed bacteria exist.

The soil to be tested is the polluted soil of a certain livestock and poultry breeding field in autonomous county of Hsiuyen rock Mandarin of Liaoning province, the texture of the soil is loam, the pH value is 7.05, the effective state contents of copper and zinc in the soil are 124mg/kg and 242mg/kg respectively, and the soil is sieved by a 1mm sieve; 100g of soil was inoculated with the microbial inoculum prepared in example 3, and the inoculum was cultured for one month at a constant temperature of 15 ℃. The heavy metal contaminated soil remediation test is provided with two treatments:

treatment 1 control treatment: control treatment 1 original soil (without added bacteria) for 0 days; control treatment 2 original soil (without added bacteria) was allowed to stand for 30 days;

and (2) treating with microbial inoculum (wherein the content of the fungus chaff biochar accounts for 2wt% of the soil, and the microbial inoculum accounts for 10wt% of the soil).

Three times of treatment are set for each treatment, and soil samples are taken respectively at 0 day and 30 days to analyze the content of the Cu and Zn heavy metals in the soil in effective states. The water content of the soil is ensured to be 20% in the whole experimental process. The experimental results show that: the HHS1 strain can tolerate heavy metal copper and has good adsorption and stabilization effects on copper and zinc. The effective states of heavy metals copper and zinc are respectively reduced by 71.4 percent and 59.73 percent after the bactericide is applied for 30 days. The results are shown in FIG. 4.

Example 4:

soil remediation test for stabilizing copper in soil by immobilized microbial agent in normal temperature environment (25℃)

The difference from the example 3 lies in that the temperature for culturing the strain is different when the microbial agent is prepared by solidification, and specifically comprises the following steps: HHS1 is activated, inoculated in solid LB culture medium, placed in a constant temperature incubator at 25 ℃ for culture to logarithmic phase, and then washed by sterile physiological saline to prepare OD ₆₀₀ A bacterial suspension of 1.0 was used as the inoculum. And mixing the sterilized mushroom bran biochar, HHS1 bacterial liquid and a sodium alginate solution to prepare the mushroom bran biochar-spirillum microbial inoculum.

The soil to be tested is the polluted soil of a certain livestock and poultry breeding field in autonomous county of Hsiuyan Man nationality in Liaoning province, the texture of the soil is loam, the pH value is 7.05, the effective copper and zinc content of the soil is respectively 134mg/kg and 284mg/kg, and the soil is sieved by a 1mm sieve; 100g of soil is taken to inoculate the microbial inoculum and is cultured for one month in a constant temperature environment of 25 ℃. The heavy metal contaminated soil remediation test is provided with two treatments:

treatment 1, original soil (without bacteria) is treated for 0 day in a control way 1; control treatment 2 original soil (without added bacteria) was allowed to stand for 30 days;

and 2, microbial inoculum treatment (wherein the biochar content accounts for 2wt% of the soil, and the microbial inoculum content accounts for 10wt% of the soil).

Three times of treatment are set in parallel, and soil samples are taken respectively at 0 day and 30 days to analyze the content of the heavy metal effective state of Cu in the soil. The water content of the soil is ensured to be 20% in the whole experimental process. The experimental results show that: the HHS1 strain can tolerate heavy metal copper and has good stabilizing effect on copper. The effective state of the heavy metal copper is reduced by 55.56 percent and 76.63 percent after the microbial inoculum is applied for 30 days. The results are shown in FIG. 5.

Example 5:

soil remediation test for stabilizing copper in soil by immobilized microbial agent in normal temperature environment (35℃)

The difference from example 3 is that curingThe temperature of strain culture is different when the microbial agent is prepared, and the method specifically comprises the following steps: HHS1 is activated, inoculated in solid LB culture medium, cultured in a constant temperature incubator at 35 ℃ to logarithmic phase, and washed with sterile physiological saline to prepare OD ₆₀₀ A bacterial suspension of 1.0 was used as the inoculum. And mixing the sterilized mushroom bran biochar with the HHS1 bacterial liquid and a sodium alginate solution to prepare the mushroom bran biochar-glusula bacterial agent.

The soil to be tested is polluted soil from a certain livestock and poultry breeding field in autonomous county of Hsiuyan Man nationality in Liaoning province, the texture of the soil is loam, the pH value is 7.05, the effective state contents of copper and zinc in the soil are respectively 122mg/kg and 232mg/kg, and the soil is sieved by a 1mm sieve; 100g of soil is inoculated with a microbial agent and cultured for one month at the constant temperature of 35 ℃. The heavy metal contaminated soil remediation test is provided with two treatments:

treatment 1, original soil (without bacteria) is treated for 0 day in a control way 1; control treatment 2 original soil (without addition of bacteria) was allowed to stand for 30 days;

and 2, microbial inoculum treatment (wherein the biochar content accounts for 2wt% of the soil, and the microbial inoculum content accounts for 10wt% of the soil).

Three times of treatment are set for each treatment, and soil samples are taken respectively at 0 day and 30 days to analyze the content of the heavy metal effective state of Cu and Zn in the soil. The water content of the soil is ensured to be 20% in the whole experimental process. The experimental results show that: the HHS1 strain can tolerate heavy metals of copper and zinc and has good stabilizing effect on copper. The effective state of the heavy metal copper is reduced by 87.33 percent and 73.03 percent after the microbial inoculum is applied for 30 days. The results are shown in FIG. 6.

According to results of examples 3-5, the microbial inoculum has good copper-zinc adsorption effect on contaminated soil at different temperatures. Wherein, the effect is optimal under the normal temperature condition (35 ℃), the effect is good under the low temperature environment, and the low temperature resistant effect of the microorganism is obvious.

Example 6:

soil remediation test for stabilizing copper and zinc in soil by immobilized microbial agent in normal temperature environment (35℃)

The difference from the embodiment 3 lies in that the biochar and the microbial inoculum account for different percentages of the whole microbial inoculum system when the microbial inoculum is prepared by solidification, and specifically the following steps are carried out: after HHS1 activationInoculating to solid LB culture medium, culturing in constant-temperature incubator at 35 deg.C to logarithmic phase, and washing with sterile physiological saline to obtain OD ₆₀₀ A bacterial suspension of 1.0 was used as the inoculum. And mixing the sterilized mushroom bran biochar with the HHS1 bacterial liquid and a sodium alginate solution to prepare the mushroom bran biochar-glusula bacterial agent.

The soil to be tested is taken from the polluted soil of a certain livestock and poultry breeding field in Shenyang city of Liaoning province, the texture of the soil is loam, the pH value is 7.05, the effective state content of copper and zinc in the soil is respectively 122mg/kg and 232mg/kg, and the soil is sieved by a 1mm sieve; 100g of soil is inoculated with a microbial agent and cultured for one month at the constant temperature of 35 ℃. The heavy metal contaminated soil remediation test is provided with two treatments:

treatment 1 control treatment: control treatment 1 original soil (without addition of bacteria) for 0 days; control treatment 2 original soil (without added bacteria) was allowed to stand for 30 days;

and 2, treating with microbial inoculum (wherein the content of the biochar accounts for 2wt% of the soil, and the microbial inoculum size accounts for 5wt% of the soil).

Three times of treatment are set for each treatment, and soil samples are taken respectively at 0 day and 30 days to analyze the content of the heavy metal effective state of Cu and Zn in the soil. The water content of the soil is ensured to be 20% in the whole experimental process. The experimental results show that: the HHS1 strain can tolerate heavy metals of copper and zinc and has a good stabilizing effect on copper. The effective state of the heavy metal copper is reduced by 82.35 percent and 71.35 percent after the microbial inoculum is applied for 30 days. The results are shown in FIG. 7. The result shows that the stabilization effect for changing the biochar in the microbial inoculum and the heavy metal in the bacterial suspension is good.

In conclusion, the immobilized microbial agent provided by the invention is a biochar microbial compound microbial agent which has the advantages of high microbial density, strong antitoxicity, strong adsorption capacity, strong tolerance to target antibiotics and good degradation effect. The free single bacterium screened by the invention can rapidly grow to the logarithmic phase within a few hours, has strong adaptability, can rapidly stabilize the effective heavy metal in the soil and reduce the content of the terramycin in the soil. The biodegradation method has the advantages of low toxicity, high speed, no secondary pollution and high popularization value.

Sequence listing

<110> Shenyang application ecological research institute of Chinese academy of sciences

<120> heavy metal adsorbing microbial inoculum and application thereof in soil pollution remediation

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1471

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tggctcagat tgaacgctgg cggcatgcct tacacatgca agtcgaacgg cagcatagga 60

gcttgctcct gatggcgagt ggcgaacggg tgagtaatat atcggaacgt gccctagagt 120

gggggataac tagtcgaaag actagctaat accgcatacg atctacggat gaaagtgggg 180

gatcgcaaga cctcatgctc ctggagcggc cgatatctga ttagctagtt ggtggggtaa 240

aagcctacca aggcaacgat cagtagctgg tctgagagga cgaccagcca cactgggact 300

gagacacggc ccagactcct acgggaggca gcagtgggga attttggaca atgggggcaa 360

ccctgatcca gcaatgccgc gtgagtgaag aaggccttcg ggttgtaaag ctcttttgtc 420

agggaagaaa cggtagtagc gaataactat tactaatgac ggtacctgaa gaataagcac 480

cggctaacta cgtgccagca gccgcggtaa tacgtagggt gcaagcgtta atcggaatta 540

ctgggcgtaa agcgtgcgca ggcggttgtg taagtcagat gtgaaatccc cgggctcaac 600

ctgggaattg catttgagac tgcacggcta gagtgtgtca gaggggggta gaattccacg 660

tgtagcagtg aaatgcgtag atatgtggag gaataccgat ggcgaaggca gccccctggg 720

ataacactga cgctcatgca cgaaagcgtg gggagcaaac aggattagat accctggtag 780

tccacgccct aaacgatgtc tactagttgt cgggtcttaa ttgacttggt aacgcagcta 840

acgcgtgaag tagaccgcct ggggagtacg gtcgcaagat taaaactcaa aggaattgac 900

ggggacccgc acaagcggtg gatgatgtgg attaattcga tgcaacgcga aaaaccttac 960

ctacccttga catggatgga atcccgaaga gatttgggag tgctcgaaag agaaccatca 1020

cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080

cgagcgcaac ccttgtcatt agttgctacg aaagggcact ctaatgagac tgccggtgac 1140

aaaccggagg aaggtgggga tgacgtcaag tcctcatggc ccttatgggt agggcttcac 1200

acgtcataca atggtacata cagagggccg ccaacccgcg agggggagct aatcccagaa 1260

agtgtatcgt agtccggatt ggagtctgca actcgactcc atgaagttgg aatcgctagt 1320

aatcgcggat cagcatgtcg cggtgaatac gttcccgggt cttgtacaca ccgcccgtca 1380

caccatggga gcgggtttta ccagaagtgg gtagcctaac cgcaaggagg gcgctcacca 1440

cggtaggatt cgtgactggg gtgaagtcgt a 1471

Claims (7)

1. The heavy metal adsorbing microbial inoculum is characterized in that: the microbial inoculum contains domesticated Harper snail bacteria (A. Merrill.)Herbaspirillumhuttiense) HHS1; the Harte grass Spirobacteria (A)Herbaspirillumhuttiense) HHS1 was deposited at the china general microbiological culture collection center on 28 th month 07/2021, address: xilu No. 1 Hospital No. 3, beijing, chaoyang, with the deposit number: c GMCC No. 22975;

the domestication of the Harper spirillum HHS1 is to perform gradient domestication in a solution containing copper ions and zinc ions, and finally obtain the HHS1 domesticated strain tolerant to the copper ions and the zinc ions.

2. The heavy metal adsorbing microbial inoculum according to claim 1, which is characterized in that: and mixing the activated domesticated strain with the fungus chaff biochar, and embedding and fixing by using sodium alginate for 24-48h to finally prepare the uniform and stable immobilized microbial agent.

3. The heavy metal adsorbing bacterial agent as set forth in claim 2, wherein: the mass ratio of the mushroom bran biochar to the volume ratio of the mushroom suspension is 1:5 (w: v) -1 (w: v); the addition amount of the sodium alginate accounts for 2-3 wt% of the mass of the microbial inoculum.

4. The preparation method of the heavy metal adsorbing microbial inoculum according to claim 1, which is characterized by comprising the following steps: and mixing the activated domesticated strain with the fungus chaff biochar, and embedding and fixing by using sodium alginate for 24-48h to finally prepare the uniform and stable immobilized microbial agent.

5. The preparation method of the heavy metal adsorbing microbial inoculum according to claim 4, which is characterized in that:

1) Placing Harpagophytum procumbens strain HHS1 with tolerance and stabilization effects on copper and zinc in LB culture medium, culturing to logarithmic growth phase, centrifuging, collecting precipitate, and resuspending the precipitate to OD with sterile physiological saline ₆₀₀ Is 0.8 to 1.2 of bacterial suspension;

2) Burning the waste mushroom sticks at 500-700 ℃ for 4-6h to form mushroom residue biochar for later use;

3) Mixing the obtained mushroom bran biochar with the mushroom suspension, adding the mushroom bran biochar and the mushroom suspension into a sodium alginate solution under the condition of stirring, and uniformly dropwise adding the mixture into CaCl through a peristaltic pump ₂ Embedding and fixing in electrolyte solution for 24-48h, discarding CaCl ₂ Electrolyte solution to prepare homogeneous and stable immobilized microbial inoculum;

wherein the proportion of the biochar in the sodium alginate is 2-5% (w: v); the proportion of the bacterial suspension in the sodium alginate is 10-20% (v: v).

6. The use of the microbial inoculum according to claim 1, wherein: the application of the microbial inoculum in adsorbing heavy metals in polluted soil; the heavy metals are copper and zinc.

7. A method for remediating heavy metals in contaminated soil by using the microbial inoculum according to claim 2, which is characterized by comprising the following steps: adding the microbial inoculum into polluted soil, wherein the mass percentages of the fungus chaff biochar and the HHS1 in the soil are respectively 1-3%; 5% -15%; the heavy metals are copper and zinc.

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