CN112961794B - Composite bacterium preparation for adsorbing mercury and application - Google Patents

Abstract

The invention belongs to the technical field of microbial agents, and particularly relates to a mercury-adsorbing compound bacterium preparation and application thereof. The preparation method of the compound bacteria preparation comprises the following steps: (1) Separating and purifying the mercury-contaminated soil to obtain Klebsiella and Bacillus tokyo; separating and purifying the bacillus subtilis from the microbial selenium-rich microbial inoculum to obtain bacillus subtilis; separating and purifying by using a mercury-containing culture medium; (2) Performing enrichment culture on the Klebsiella, the Bacillus tokyo and the Bacillus subtilis obtained in the step (1) and then performing enrichment culture according to the mass ratio of the enrichment solution (0.8-1.2): (0.8-1.2): (0.8-1.2) inoculating the mixture into a culture medium for culture, and obtaining a culture solution which is the composite bacterium preparation. According to the invention, the adsorption performance of the composite bacterial preparation on the effective state mercury is utilized, the migration of the mercury from soil to plants is prevented and controlled, the three microorganisms in the composite bacterial preparation act synergistically, the adsorption on the mercury is realized, and the adsorption rate has obvious advantages compared with that of a single bacterial strain.

Description

Composite bacterium preparation for adsorbing mercury and application

Technical Field

The invention belongs to the technical field of microbial agents, and particularly relates to a compound bacterium preparation for adsorbing mercury and application thereof.

Background

The serious harm caused by mercury pollution has attracted public attention of countries in the world, and the attention is increasing, and currently, 130 countries form a united nations water guarantee committee to specially regulate the emission and use of mercury. The united states environmental protection agency sets forth emission standards into the atmosphere, water and waste, and related standards for emissions in industrial processes (e.g., atmosphere protection protocols, water purification protocols, resource conservation, and recycling protocols). International collaboration council developing this chinese environment has studied and set special policies to regulate mercury emissions and use.

Aiming at the condition of continuous soil pollution aggravation in China, a soil pollution control action plan is issued in 2016, 5 and 28 days, and the situation that the soil pollution aggravation trend in China is preliminarily restrained, the soil environment quality is generally kept stable, the soil environment safety of agricultural land and construction land is basically guaranteed and the soil environment risk is basically controlled is pointed out, so that the remediation work of the mercury pollution of the soil is very urgent.

At present, various restoration technical methods aiming at mercury pollution in soil at home and abroad are available, and the restoration technical methods mainly comprise methods such as a physical chemical method, a biological restoration method, an agricultural regulation and control method and the like. The microbial remediation technology is still in the research stage of experimental research, and although mercury-resistant strains are ubiquitous in the environment on land, the number of mercury-resistant bacteria capable of serving as a biological control agent to remediate mercury-contaminated soil is extremely small. Therefore, the development of the efficient and low-cost microbial compound inoculant has important significance.

Disclosure of Invention

The invention aims to provide a composite bacterial preparation for adsorbing mercury and application thereof, which are used for solving the problems that the number of mercury-resistant bacteria used for bioremediation of mercury-contaminated soil is small and the mercury resistance is required to be improved at present.

In order to achieve the purpose, the invention provides the following technical scheme:

a composite bacteria preparation for adsorbing mercury is prepared by the following steps:

(1) Separating and purifying the mercury-polluted soil to obtain a Klebsiella N-type strain and a Bacillus tokyo K-type strain; separating and purifying the bacillus subtilis W-type strain from the microbial selenium-rich inoculant; the separation and purification are carried out by a mercury-containing culture medium;

(2) Respectively selecting a Klebsiella N-type strain, a Bacillus tokyo K-type strain and a Bacillus subtilis W-type strain obtained in the step (1) for enrichment culture to obtain three enrichment solutions with optical density (namely OD value) of 0.5-0.6, and mixing the three enrichment solutions according to the mass ratio of (0.8-1.2): (0.8-1.2): (0.8-1.2) inoculating the mixture into a culture medium for culture, and obtaining a culture solution which is the compound bacterium preparation.

The Klebsiella pneumoniae N-type strain (Klebsiella pneumoniae N-type strain) is preserved in China Center for Type Culture Collection (CCTCC) in 1 month and 15 days in 2021, and is abbreviated as CCTCC, and the preservation number is CCTCC No. M2021070;

the K-type strain of the Bacillus tokyo (Bacillus toyonensis K-type strain) is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 15 days of 2021, and the preservation number is CCTCC No. M2021072;

the Bacillus subtilis W-type strain (Bacillus subtilis W-type strain) is preserved in China Center for Type Culture Collection (CCTCC) in 2021, 1 and 15 days, and the preservation number is CCTCC No. M2021071;

the address of the above-mentioned deposit unit is: wuhan, wuhan university, zip code 430072.

Preferably, the separation and purification are repeated by adopting a plate-streaking method, and Hg in the mercury-containing culture medium is subjected to repeated operation ²⁺ The concentration increases in turn.

Preferably, in the step (1), hg in the mercury-containing culture medium is Hg for the Klebsiella N-type strain and the Bacillus tokyo K-type strain ²⁺ The highest concentration is 30-35 mg/L.

Preferably, for Bacillus subtilis type W strains, hg in the mercury-containing medium ²⁺ The highest concentration is 15-20 mg/L.

Preferably, in the step (2), the mass ratio of the enrichment solutions of the Klebsiella N-type strain, the Bacillus tokyo K-type strain and the Bacillus subtilis W-type strain is 1:1:1.

preferably, in step (2), the initial pH of the culture in the medium is 6 to 8.

Preferably, in the step (2), the temperature of the culture in the medium is 30 to 35 ℃.

Preferably, in the step (2), the culture time in the culture medium is 16 to 20 hours.

Preferably, in the step (2), the culture medium is subjected to shaking treatment, and the shaking rotation speed is 130-160 r/min.

The application of the compound bacterium preparation in mercury contaminated soil can adsorb effective mercury in the soil and prevent and control the migration of mercury from the soil to plants.

Compared with the closest prior art, the technical scheme provided by the invention has the following excellent effects:

(1) The Klebsiella N-type strain, the Tokyo bacillus K-type strain and the Bacillus subtilis W-type strain have good adaptability to environments with low or high mercury concentration.

(2) The three bacteria used in the invention are widely distributed in the natural environment, and have environmental ecological safety.

(3) The compound bacteria preparation can grow well under neutral and slightly acidic conditions, has strong adaptability, and can keep strong mercury adsorption capacity in slightly alkaline environments.

(4) The three microorganisms in the composite bacterial preparation provided by the invention act synergistically to realize mercury adsorption, and the adsorption efficiency has an obvious advantage compared with that of a single bacterial strain.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention. Wherein:

FIG. 1 is a comparison graph of mercury adsorption effects of single-strain and composite-strain preparations of the present invention;

FIG. 2 is a graph comparing the conversion of soil available mercury by single bacterial strain and composite bacterial preparation in the invention;

FIG. 3 is a graph of the adsorption effect of the complex bacterial preparation of the invention on mercury at different pH values;

FIG. 4 is a graph showing the adsorption effect of the complex bacterial preparation of the present invention on mercury with different concentrations;

FIG. 5 is a graph showing the mercury adsorption effect of the complex bacterial preparation of the present invention under different inoculum sizes;

FIG. 6 is a graph showing the adsorption effect of the complex bacterial preparation of the present invention on mercury under different culture times.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.

The present invention will be described in detail below with reference to the accompanying drawings in conjunction with embodiments. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

The composite bacteria preparation of the invention is mainly used for treating soil mercury pollution, and the mercury removal mechanism of mercury-resistant bacteria (such as the composite bacteria preparation of the invention) is considered to be multi-path and comprises the following steps: (1) The biological adsorption is divided into two parts of biological surface adsorption and biological accumulation, the surface adsorption mainly adopts physical adsorption, and the biological accumulation is to adsorb Hg ²⁺ Conversion to organomercury in cellular biomass; (2) The precipitation is that mercury-resistant strain converts Hg ²⁺ Solid HgCl and Hg are formed ⁰ . Bacterial transformation of Hg by multiple pathways ²⁺ Is HgSe, hgCl and Hg ⁰ . In addition, the bacteria can reduce SO ₄ ^2- Generation of H ₂ S，H ₂ S and Hg ²⁺ Directly reacting to generate HgS sediment; (3) The volatilization is to capture, transport and reduce mercury by using the mer operon in the microorganism. The protein coded by merT is involved in the transport of mercury ions from outside to inside of cells; merA-encoded mercury reductase will reduce Hg ²⁺ Reducing the mercury into metallic mercury, releasing the metallic mercury into cytoplasm and volatilizing the metallic mercury out of cells; the product of merB can break the C-Hg bond in organic mercury and degrade the organic mercury into Hg ²⁺ ，Hg ^{2 +} Then reducing the mercury into metallic mercury through the merA.

In the experimental process, no phenomenon of gas generation in the process of mercury adsorption is found, so that the mercury removal mechanism of the composite bacterial preparation (namely the mercury-resistant bacterial strain) is mainly based on bioadsorption and precipitation.

The preparation method of the compound bacteria preparation comprises the following steps:

(1) Separating and purifying the mercury-polluted soil to obtain a Klebsiella N-type strain and a Bacillus tokyo K-type strain; separating and purifying the bacillus subtilis W-type strain from the microbial selenium-rich microbial inoculum; the separation and purification are carried out by a mercury-containing culture medium.

The separation and purification are carried out by plate-scribing method, and Hg in the mercury-containing culture medium is repeatedly extracted ²⁺ The concentration increases in sequence; for Klebsiella N-type strain and Tokyo bacillus K-type strainInitial Hg in Mercury-containing Medium ²⁺ The concentration is 5mg/L and the maximum Hg ²⁺ The concentration is 30 to 35mg/L (for example, 30mg/L, 31mg/L, 32mg/L, 33mg/L, 34mg/L, 35 mg/L); for Bacillus subtilis type W strains, initial Hg in mercury-containing medium ²⁺ The concentration is 5mg/L and the highest Hg ²⁺ The concentration is 15 to 20mg/L (for example, 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20 mg/L).

The mercury-containing culture medium can be a solid flat mercury-containing culture medium or a liquid mercury-containing culture medium.

The strain used for preservation was prepared by the following method: the obtained strains are respectively inoculated in a culture medium, and the composition of the culture medium is as follows: the reagent comprises, by per liter of ultrapure water, 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 15g of agar and 0.5g of mercuric chloride. The culture temperature is 30 ℃, the culture time is 18h, and the oscillation speed of the incubator is 140r/min.

(2) Respectively selecting a Klebsiella N-type strain, a Bacillus tokyo K-type strain and a Bacillus subtilis W-type strain obtained in the step (1) for enrichment culture to obtain three enrichment solutions with OD values (namely optical densities) of 0.5-0.6, and mixing the three enrichment solutions according to the mass ratio of (0.8-1.2): (0.8-1.2): (0.8-1.2) to obtain a composite bacterial strain, inoculating the composite bacterial strain into a culture medium (such as a TSB culture medium) for culturing, wherein the initial pH of the culture is 6-8 (such as 6, 6.5, 7, 7.5 and 8), the culture temperature is 30-35 ℃ (such as 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ and 35 ℃), the culture time is 16-20 h (such as 16h, 17h, 18h, 19h and 20 h), the culture medium is subjected to oscillation treatment during culturing, the oscillation rotation speed is 130-160 r/min (such as 130r/min, 140r/min, 150r/min and 160 r/min), and the obtained culture solution is the composite bacterial preparation after the culture is finished. It is understood that, when the enrichment culture is performed, the culture medium used is a liquid medium, such as a TSB medium.

As mentioned above, the thallus used by the compound bacteria preparation of the invention is a single bacterium obtained by screening from mercury-containing contaminated soil and microorganism selenium-rich microbial inoculum, and then Hg is adopted ²⁺ Culturing single bacterium with mercury-containing culture medium with sequentially increased concentration to obtain mercury-resistant single bacterium, and culturing mercury-resistant single bacteriumAnd (4) enriching the bacteria to obtain a strain. It can be understood that the enriched strains have better mercury adsorption capacity, but the specific types of fungi of each strain need subsequent observation and identification. Therefore, for the convenience of description, the isolated strains are referred to as N-type bacteria, K-type bacteria, and W-type bacteria, respectively.

The three strains involved in the invention have the following characteristics respectively:

type N bacteria, i.e. Klebsiella pneumoniae (Klebsiella pneumoniae type N bacteria): in the form of oval rods, arranged singly, doubly or in short chains. Has capsule and no flagellum. Facultative anaerobic, capable of growing on nutrient agar. Forming raised and large mucus-like bacterial colonies on a blood agar plate, wherein the bacterial colonies are in filiform adhesion when being picked by an inoculating needle, and can be changed into smooth bacterial colonies after continuous passage. Type K bacteria, i.e., bacillus tokyo ensis type K bacteria: the single bacterial colony is round, smooth in surface, grey white, facultative anaerobic, single thallus is long rod-shaped, and spores are oval. Type W bacteria, i.e. Bacillus subtilis type W bacteria: without capsule, the perigenic flagellum can move. Oval to columnar shape, located in the center of the thallus or slightly deviated from the center, and the thallus is not expanded after sporulation. The colony surface is rough and opaque, and is dirty white or yellowish, and is facultative anaerobic when growing in a liquid culture medium.

The invention is further described with reference to specific examples.

In the following examples, the mercury contaminated soil was obtained from the soil in the red army town of ten-day-yang county of Shaanxi province, and the microbial selenium-enriched microbial inoculum was purchased from Taian Huaxin biofertilizer science and technology Limited and was a liquid preparation with a net content of 1000 mL.

In the following examples, hg is contained ²⁺ The composition of the solid plate mercury-containing culture medium with the concentration of 5mg/L is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 15g of agar and 0.5g of mercuric chloride; for other Hg ²⁺ And (3) correspondingly adjusting the content of mercuric chloride in the solid plate mercury-containing culture medium with the concentration.

In the following examples, a TSA medium was prepared from the following components in parts by mass: 15 parts of tryptone, 5 parts of plant peptone, 5 parts of sodium chloride and 15 parts of agar. When preparing the TSA culture medium, using ultrapure water as a solvent, weighing 4.6g of TSA solid powder, placing the TSA solid powder into a 250mL serum bottle, adding 100mL of ultrapure water, stirring uniformly, slightly screwing a cover, sterilizing in a 121 ℃ pressure cooker for 50min, taking out when the temperature is reduced to 45 ℃, pouring the TSA solid powder into a flat plate, and placing for 2-3 days for later use.

In the following examples, the preparation of TSB medium was as follows: adding 17.0g of tryptone casein, 3.0g of soybean casein peptone, 5.0g of sodium chloride, 2.5g of dipotassium hydrogen phosphate and 2.5g of glucose into per liter of ultrapure water, uniformly mixing, and sterilizing at 121 ℃ for 50min for later use.

Example 1

The preparation method of the composite bacteria of the embodiment comprises the following steps:

(1) Separation domestication, separation purification and enrichment culture of N-type bacteria, K-type bacteria and W-type bacteria

The N-type strain and the K-type strain are separated from mercury contaminated soil, the W-type strain is separated from a microbial selenium-rich microbial inoculum, and the specific process is as follows:

(1) respectively placing 10g of soil with low, medium and high mercury pollution levels in a 250mL triangular flask, diluting with 100mL of ultrapure water to prepare bacterial suspension, oscillating at constant temperature of 140r/min for 30min, standing, and taking 1mL of supernatant;

1mL of bacterial liquid is directly taken by the microorganism selenium-rich microbial inoculum for subsequent treatment.

(2) The obtained 3 parts of supernatant are respectively inoculated to the sterilized Hg-containing ²⁺ Streaking culture is carried out in a solid plate culture medium with the concentration of 5mg/L, and after standing culture is carried out for 24 hours at room temperature, single bacteria with good growth and different forms are picked.

(3) For the strain isolated from the soil, the above-mentioned Hg-containing strain is used ²⁺ Solid plate culture medium Hg of (2) ²⁺ The concentration was increased by 5 gradients (i.e., hg in solid plate medium) ²⁺ The concentration is increased by 5mg/L each time), the operation in the step (2) is repeated, and Hg is gradually added ²⁺ The concentration is increased to 30mg/L, the time for each domestication and separation is generally 12-30 h, and the separation time is longer as the mercury concentration is higher. Completing the separation process of the strains to obtain mercury-resistant single strains, namely an N-type strain and a K-type strain; during the test, when Hg is found ²⁺ Increase of concentration toAt 40mg/L, the bacteria in the culture medium are dropped, which shows that the N-type strain and the K-type strain are inhibited from growing in the culture medium with high mercury concentration, and the mercury-resistant concentration is less than 40mg/L. The colony status as a function of mercury concentration is shown in Table 1 below (in the table, "/" indicates that no strain could be isolated).

TABLE 1 change of colony status of N-type and K-type strains with mercury concentration

The same operation is carried out on the microorganism selenium-rich microbial inoculum, and Hg in the culture medium is added ²⁺ The concentration is increased to 15mg/L, the separation process of the strains is completed, and the mercury-resistant single strain, namely the W-type strain, is obtained. During the test, when Hg is found ²⁺ When the concentration is increased to 20mg/L, the colony in the plate is less, and the Hg is further increased ²⁺ Concentration, no strain was present in the plate. The colony status as a function of mercury concentration is shown in Table 2 below (in the table, "/" indicates that no strain could be isolated).

TABLE 2 colony status of type W strains as a function of mercury concentration

Mercury concentration (mg/L)	Growth state of bacterial colony	Incubation time (h)
			5	The flat plates are distributed densely	12～18
10	The plates are distributed more densely	18～24
			15	Uneven distribution of bacterial colonies	24～36
20	Few colonies on the plate	/
			25	No occurrence of bacterial strain	/
30	No occurrence of bacterial strain	/

(4) Inoculating the mercury-resistant single bacterium to Hg ²⁺ In a solid plate mercury-containing culture medium with the concentration of 30mg/L and 15mg/L, the plate marking method is adopted to repeat 2 to 3 times under the condition of 30 ℃ to obtain pure colonies.

(5) And (3) selecting a single bacterium from the pure colony, placing the single bacterium in 10mL of a sterilized TSB culture medium, and carrying out constant-temperature shaking culture at 30 ℃ and 140r/min for 20-24 h for enrichment to obtain strains (single bacterium), namely N-type bacteria, K-type bacteria and W-type bacteria.

(2) Observation and identification of N-type bacterium, K-type bacterium and W-type bacterium

And respectively inoculating the three separated and purified strains on a TSA culture medium, culturing at room temperature for 24h, and picking a small amount of gram-stained bacteria for observation.

The N-type bacteria are short rod-shaped, light yellow, semitransparent, convex in surface and smooth in edge, can form characteristic mucus-shaped bacterial colonies, and the long-time culture bacterial liquid has foul smell and grows in a turbid mode in a liquid culture medium, the size of the liquid culture medium is 0.23-0.39 mu m, gram-negative bacteria move.

The K-type bacteria are in a round shape, are off-white, have a convex surface and smooth edges, are in a turbid state in a liquid culture medium, have bacterial bodies sinking, grow in a turbid mode and a precipitation mode, have the size of 0.88-1.21 mu m, are gram-positive bacteria, and move.

The W-type bacteria are short rod-shaped, the bacterial colony is irregular, the surface color is dark, the bacterial colony is dirty white, turbid growth is shown in a liquid culture medium, the size is 0.52-2.02 mu m, gram-positive bacteria and movement are shown.

Inoculating N-type bacteria, K-type bacteria and W-type bacteria in a TSB culture medium, culturing at 30 ℃ and 140r/min for 20h to obtain fresh bacteria liquid, and extracting genome DNA.

16S rDNA of the strain was amplified using primer 1 (sequence: 5. Sequencing the PCR product, and the result is: the N-type bacterium has a nucleotide sequence shown as SEQ ID No.1 in a sequence table; the K-type bacterium has a nucleotide sequence shown as SEQ ID No.2 in a sequence table; the W-type bacterium has a nucleotide sequence shown as SEQ ID No.3 in a sequence table.

The sequenced sequences were aligned in NCBI (https:// blast.ncbi.nlm.nih.gov/blast.cgi. The results of partial DNA comparison of N-type bacteria, K-type bacteria and W-type bacteria are shown in tables 3, 4 and 5, respectively.

TABLE 3 partial DNA alignment results of N-type bacteria

TABLE 4 partial DNA alignment results of type K bacteria

TABLE 5 partial DNA alignment results of type W bacteria

The 16S rDNA gene fragment obtained by sequencing the N-type strain has the sequence length of 1436bp, and has 99.44% similarity with Klebsiella pneumoniae (Klebsiella pneumoniae) with the closest genetic relationship.

The sequence length of a 16S rDNA gene fragment obtained by sequencing the K-type strain is 1429bp, and the 16S rDNA gene fragment has 99.93% similarity with the genetic relationship of Bacillus toyonensis (Bacillus Tokyo).

The sequence length of a 16S rDNA gene fragment obtained by sequencing the W-type strain is 1442bp, and the W-type strain has 99.79 percent of similarity with the genetic relationship of Bacillus subtilis.

(3) Culturing to obtain compound bacteria preparation

Respectively selecting an obtained strain of N-type bacteria, K-type bacteria and W-type bacteria to perform enrichment culture in a TSB culture medium to obtain three enrichment solutions with OD values of 0.5-0.6, and mixing the three enrichment solutions according to a mass ratio of 1:1:1, obtaining a composite strain, inoculating 5mL of the composite strain into a TSB culture medium for culturing, wherein the initial pH of the culture is 7.02, the culture temperature is 30 ℃, the culture time is 18h, the culture medium is subjected to oscillation treatment during the culture, the oscillation speed is 140r/min, and when the OD value is between 0.5 and 0.6, the obtained composite bacterial liquid after the culture is finished is the composite bacterial preparation of the embodiment.

Example 2

The preparation method of the complex bacteria preparation of this embodiment is different from that of embodiment 1 in step (3), and other steps are the same as those in embodiment 1 and are not described again. The step (3) is specifically as follows:

respectively selecting an obtained strain of N-type bacteria, K-type bacteria and W-type bacteria to perform enrichment culture in a TSB culture medium to obtain three enrichment solutions with OD values of 0.5-0.6, and mixing the three enrichment solutions according to a mass ratio of 0.8:1.0:1.2, obtaining a composite strain, inoculating 5mL of the composite strain into a TSB culture medium for culturing, wherein the initial pH of the culture is 7.08, the culture temperature is 30 ℃, the culture time is 18h, the culture medium is subjected to oscillation treatment during the culture, the oscillation speed is 140r/min, and when the OD value is between 0.5 and 0.6, the culture is finished, and the obtained composite bacterial liquid is the composite bacterial preparation of the embodiment.

Example 3

The preparation method of the complex bacteria preparation of this embodiment is different from that of embodiment 1 in step (3), and other steps are the same as those in embodiment 1 and are not described again. The step (3) is specifically as follows:

respectively selecting an obtained strain of N-type bacteria, K-type bacteria and W-type bacteria to perform enrichment culture in a TSB culture medium to obtain three enrichment solutions with OD values of 0.5-0.6, and mixing the three enrichment solutions according to a mass ratio of 1.2:1.0:0.8, obtaining a composite strain, inoculating 5mL of the composite strain into a TSB culture medium for culture, wherein the initial pH of the culture is 7.05, the culture temperature is 30 ℃, the culture time is 18h, the culture medium is subjected to oscillation treatment during culture, the oscillation speed is 140r/min, and when the OD value is between 0.5 and 0.6, the culture is finished, and the obtained composite bacterial liquid is the composite bacterial preparation of the embodiment.

Examples of the experiments

In the following experimental examples, the influence of factors such as the type of bacteria, the concentration of mercury in soil, time, pH, temperature, inoculum size, and coexisting heavy metals on the effect of the composite bacterial preparation on the adsorption of mercury was analyzed through experiments.

In the following experimental examples:

the single bacterium refers to K-type bacterium, N-type bacterium and W-type bacterium obtained by separation and purification;

the complex bacterium preparation (NKW complex bacterium) was the complex bacterium preparation in example 1;

the KN complex bacteria, KW complex bacteria, and NW complex bacteria were complex bacteria preparations composed of two kinds of bacteria obtained by the same method as in example.

The "adsorption effect on mercury" referred to in the following experimental examples is the mercury removal effect in terms of the change in the mercury content of the mercury-containing culture solution, and is hereinafter expressed as "mercury removal rate".

In the following experimental examples, three parallel samples were prepared for each set, a blank set was set, and Hg was measured by a two-pass atomic spectrophotometer ²⁺ The initial concentration and the final concentration, and calculating the mercury removal rate, thereby detecting the mercury of the single strain and the compound strainThe calculation method is shown in the following formula (1):

mercury removal rate = [ (C) ₀ -C ₁ -C ₂ )/C ₀ ]X 100% formula (1)

In the formula: c ₀ Is Hg in solution ²⁺ The initial concentration of (a);

C ₁ for Hg in the solution after the culture is finished ²⁺ (ii) a final concentration of (d);

C ₂ is a blank group of Hg ²⁺ The natural volatilization amount of (2).

Experimental example 1 comparison of adsorption effects of single-bacterium and composite-bacterium preparations on mercury

5mL of the single bacterium, 5mL of the complex bacterium preparation (abbreviated as NKW complex bacterium) of example 1, 5mL of the KN complex bacterium, 5mL of the KW complex bacterium, and 5mL of the NW complex bacterium were inoculated into Hg-containing bacteria ²⁺ The culture medium with the concentration of 10mg/L is subjected to shaking culture for 72 hours at the temperature of 30 ℃ and the speed of 140r/min. Using the above Hg ²⁺ The mercury removal rate was calculated by the concentration measurement method of (1) and the result is shown in FIG. 1. The adsorption results are shown in FIG. 1.

The results of fig. 1 show that: after 72h of culture, the mercury removal rates of the W-type bacteria, the N-type bacteria, the K-type bacteria, the KN, the KW, the NW and the NKW compound bacteria reach 30.1%, 32.2%, 32.3%, 50.1, 55.1, 60.6% and 80.2% respectively, namely the mercury removal rates of the NKW compound bacteria are that the NKW compound bacteria > NW > KN > KW > K-type bacteria > N-type bacteria > W-type bacteria from large to small, the mercury removal rate of the NKW compound bacteria is obviously superior to that of single bacteria, and the mercury removal rate of the KNW compound bacteria is obviously superior to that of the compound bacteria consisting of the two bacteria as can be seen in the figure.

Experimental example 2 conversion of soil available mercury by single and composite bacterium preparations

5mL of single bacterium and 5mL of the complex bacterium preparation of example 1 were taken, respectively, and 6mL of ultrapure water was added to prepare a bacterial suspension, which was uniformly sprayed on the surface of 150g of a soil sample in three layers, and the soil sample was wetted with ultrapure water, and cultured at room temperature while making a sterility control, and the results are shown in FIG. 2.

The results of fig. 2 show that: after the culture for 7d, the content of the available mercury in the soil is in a decreasing trend; the mercury removal rate of the composite bacteria on the soil available mercury can reach 80%.

In the experimental example, "effective mercury" refers to the existence form of mercury which is easily absorbed by plants and has great harm to crops.

Experimental example 3 adsorption Effect of the Complex bacterium preparation on Mercury at different pH values

5mL of the composite bacteria preparation is inoculated in Hg ²⁺ Adjusting pH with HCl and NaOH in TSB culture medium with concentration of 10mg/L, and culturing at constant temperature of 30 deg.C and 140r/min under shaking for 24h.

Hg determination using a two-pass atomic spectrophotometer ²⁺ The mercury removal rate was calculated from the initial and final concentrations of (a) and (b), thereby measuring the mercury adsorption effect of the complex strain under different pH conditions, and the results are shown in fig. 3.

The results of fig. 3 show that: the neutral alkaline condition is more beneficial to mercury adsorption than the acidic condition, and the mercury removal rate is about 10-20% in a culture medium with pH = 4-5. The maximum mercury removal rate of 71.84% is reached around pH =6.45, and the mercury removal rate is slightly reduced to between 60 and 65% at pH 7 and 9.

Experimental example 4 adsorption Effect of Compound bacterium preparation on Mercury of different concentrations

5mL of the composite bacteria preparation is inoculated to Hg ²⁺ The TSB medium (9) is subjected to constant temperature shaking culture at 30 ℃ and 140r/min for 4d by adjusting the pH to 6.45.

Hg determination using a two-pass atomic spectrophotometer ²⁺ The mercury removal rate was calculated from the initial and final concentrations of (a) to (b), thereby measuring the ability of the composite strain to adsorb mercury, and the results are shown in fig. 4.

The results of fig. 4 show that: the strain grows with Hg ²⁺ The increase in the content of (a) is suppressed and the mercury removing ability thereof is also lower. When Hg is contained ²⁺ At 10mg/L, the mercury removal rate is highest and reaches 79.5 percent along with Hg ²⁺ Gradually decrease the mercury removal rate in Hg ²⁺ 53.5-58.4% at 15-20 mg/L in Hg ²⁺ The mercury removal rate is reduced to 50% when the concentration is 25 mg/L. When the maximum tolerance of the strain to mercury is exceeded, the cell growth is seriously inhibited, and the mercury removal rate is obviously reduced by about 20.3%.

Experimental example 5 adsorption Effect of the composite bacteria preparation on mercury under different inoculum sizes

Preparation of Hg-containing ²⁺ Adding different inoculum sizes of composite bacteria preparation into 10mg/L culture solution, and culturing at 30 deg.C and 140r/min under constant temperature shaking for 24h.

Hg determination using a two-pass atomic spectrophotometer ²⁺ The mercury removal rate was calculated from the initial and final concentrations of (a) and (b), thereby examining the mercury removal effect of the composite strain inoculum amount, and the results are shown in fig. 5.

The results of fig. 5 show that: the amount of inoculation has a certain influence on the mercury removal effect. Along with the increase of the inoculation amount, the mercury removal rate shows a trend of increasing first and then decreasing, when the inoculation amount is 1-5 mL, the mercury removal rate is gradually increased and is 41.1-58.6%, and when the inoculation amount is 7mL, the mercury removal rate is obviously decreased and is about 11.5%. This is probably due to the fact that the inoculum size is too large, the nutrient content is limited, the strain does not reach the best growth activity, and the mercury removal rate is reduced.

Experimental example 6 adsorption Effect of the Complex microbial preparation on Mercury at different incubation times

5mL of the composite bacterial preparation is inoculated in Hg ²⁺ Adjusting pH to 6.5 with HCl in TSB culture medium with concentration of 10mg/L, and culturing at 30 deg.C and 140r/min.

Hg determination using a two-pass atomic spectrophotometer ²⁺ The mercury removal rate was calculated from the initial and final concentrations of (a) to (b), and thus the mercury adsorption effect of the composite strain at different incubation times was examined, and the results are shown in fig. 6.

The results of fig. 6 show that: along with the change of time, the mercury removal rate is gradually increased, the mercury removal rate range of the NKW composite bacteria is 33.12-88.51%, wherein after the NKW composite bacteria are cultured for 4 days, the mercury removal rate of the strain reaches the maximum value of 88.51%, and after the NKW composite bacteria are cultured for 5 days, the mercury removal rate is not obviously changed, which shows that the composite bacteria have stronger mercury removal capability in a growth period and a stabilization period.

The experimental results show that the composite bacteria preparation provided by the invention can adsorb mercury, the adsorption rate (namely mercury removal rate) can reach more than 80%, and the growth environment has strong adaptability, so that the composite bacteria preparation has important application value in the aspect of mercury pollution treatment.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Xian Zhongdi environmental science and technology Limited, xian science and technology university

<120> composite bacterium preparation for efficiently adsorbing mercury and application

<130> M1CNCN201607_IV20-0329

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1436

<212> DNA

<213> Klebsiella pneumoniae

<400> 1

cggcaggcct acacatgcaa gtcgagcggt agcacagaga gcttgctctc gggtgacgag 60

cggcggacgg gtgagtaatg tctgggaaac tgcctgatgg agggggataa ctactggaaa 120

cggtagctaa taccgcataa cgtcgcaaga ccaaagtggg ggaccttcgg gcctcatgcc 180

atcagatgtg cccagatggg attagctggt aggtggggta acggctcacc taggcgacga 240

tccctagctg gtctgagagg atgaccagcc acactggaac tgagacacgg tccagactcc 300

tacgggaggc agcagtgggg aatattgcac aatgggcgca agcctgatgc agccatgccg 360

cgtgtgtgaa gaaggccttc gggttgtaaa gcactttcag cggggaggaa ggcggtgagg 420

ttaataacct cgtcgattga cgttacccgc agaagaagca ccggctaact ccgtgccagc 480

agccgcggta atacggaggg tgcaagcgtt aatcggaatt actgggcgta aagcgcacgc 540

aggcggtctg tcaagtcgga tgtgaaatcc ccgggctcaa cctgggaact gcattcgaaa 600

ctggcaggct agagtcttgt agaggggggt agaattccag gtgtagcggt gaaatgcgta 660

gagatctgga ggaataccgg tggcgaaggc ggccccctgg acaaagactg acgctcagct 720

gcgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgctg taaacgatgt 780

cgatttggag gttgtgccct tgaggcgtgg cttccggagc taacgcgtta aatcgaccgc 840

ctggggagta cggccgcaag gttaaaactc aaatgaattg acgggggccc gcacaagcgg 900

tggagcatgt ggtttaattc gatgcaacgc gaagaacctt acctggtctt gacatccaca 960

gaactttcca gagatggatt ggtgccttcg ggaactgtga gacaggtgct gcatggctgt 1020

cgtcagctcg tgttgtgaaa tgttgggtta agtcccgcaa cgagcgcaac ccttatcctt 1080

tgttgccagc ggttaggccg ggaactcaaa ggagactgcc agtgataaac tggaggaagg 1140

tggggatgac gtcaagtcat catggccctt acgaccaggg ctacacacgt gctacaatgg 1200

catatacaaa gagaagcgac ctcgcgagag caagcggacc tcataaagta tgtcgtagtc 1260

cggattggag tctgcaactc gactccatga agtcggaatc gctagtaatc gtagatcaga 1320

atgctacggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagtgg 1380

gttgcaaaag aagtaggtag cttaaccttc gggagggcgc ttaccacttt ggatca 1436

<210> 2

<211> 1429

<212> DNA

<213> Bacillus toyonensis

<400> 2

gtcgagcgaa tggattgaga gcttgctctc aagaagttag cggcggacgg gtgagtaaca 60

cgtgggtaac ctgcccataa gactgggata actccgggaa accggggcta ataccggata 120

acattttgaa ctgcatggtt cgaaattgaa aggcggcttc ggctgtcact tatggatgga 180

cccgcgtcgc attagctagt tggtgaggta acggctcacc aaggcaacga tgcgtagccg 240

acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc 300

agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat 360

gaaggctttc gggtcgtaaa actctgttgt tagggaagaa caagtgctag ttgaataagc 420

tggcaccttg acggtaccta accagaaagc cacggctaac tacgtgccag cagccgcggt 480

aatacgtagg tggcaagcgt tatccggaat tattgggcgt aaagcgcgcg caggtggttt 540

cttaagtctg atgtgaaagc ccacggctca accgtggagg gtcattggaa actgggagac 600

ttgagtgcag aagaggaaag tggaattcca tgtgtagcgg tgaaatgcgt agagatatgg 660

aggaacacca gtggcgaagg cgactttctg gtctgtaact gacactgagg cgcgaaagcg 720

tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaagt 780

gttagagggt ttccgccctt tagtgctgaa gttaacgcat taagcactcc gcctggggag 840

tacggccgca aggctgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 900

gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgaaaaccc 960

tagagatagg gcttctcctt cgggagcaga gtgacaggtg gtgcatggtt gtcgtcagct 1020

cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1080

tcattaagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1140

cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gacggtacaa 1200

agagctgcaa gaccgcgagg tggagctaat ctcataaaac cgttctcagt tcggattgta 1260

ggctgcaact cgcctacatg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1320

tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1380

gaagtcggtg gggtaacctt tatggagcca gccgcctaag gtggacaga 1429

<210> 3

<211> 1442

<212> DNA

<213> Bacillus subtilis

<400> 3

tgcctataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga 60

cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg 120

ctaataccgg atggttgttt gaaccgcatg gttcaaacat aaaaggtggc ttcggctacc 180

acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa 240

cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac 300

tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg 360

ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtac 420

cgttcgaata gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc 480

cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc 540

tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg 600

gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg 660

cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg 720

aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780

atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac 840

tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca 900

agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 960

cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg 1020

gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080

atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg 1140

aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca 1200

atggacagaa caaagggcag cgaacccgcg aggttaagcc aatcccacaa atctgttctc 1260

agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat 1320

cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga 1380

gtttgtaaca cccgaagtcg gtgaggtaac cttttaggag ccagccgccg aaggtggaca 1440

ga 1442

Claims (7)

1. The preparation method of the composite bacterial preparation for adsorbing mercury is characterized by comprising the following steps of:

respectively picking a strain of Klebsiella (A), (B), (C) and (C)Klebsiella pneumoniae) N-type strain, bacillus Tokyo: (Bacillus toyonensis) K-type strain, bacillus subtilis (B) ((B))Bacillus subtilis) Carrying out enrichment culture on the W-type strain to obtain three enrichment solutions with optical densities of 0.5-0.6, wherein the three enrichment solutions are mixed according to the mass ratio of (0.8-1.2): (0.8-1.2): (0.8-1.2) inoculating the mixture into a culture medium for culture, wherein the obtained culture solution is the compound bacterium preparation;

the Klebsiella N-type strain is preserved in China center for type culture Collection with a preservation number of CCTCC No. M2021070;

the K-type strain of the Bacillus tokyo is preserved in China center for type culture Collection with the preservation number of CCTCC No. M2021072;

the bacillus subtilis W-type strain is preserved in China center for type culture Collection with the preservation number of CCTCC No. M2021071.

2. The compound bacteria preparation of claim 1, wherein the mass ratio of the enriched liquid of the Klebsiella N-type strain, the Bacillus tokyo K-type strain and the Bacillus subtilis W-type strain is 1:1:1.

3. the complex bacterial preparation according to claim 1, wherein the initial pH of the culture in said medium is 6 to 8.

4. The complex bacterial preparation according to claim 1, wherein the temperature for culturing in said medium is 30-35 ℃.

5. The complex bacterial preparation according to claim 1, wherein the culture time in the medium is 16 to 20 hours.

6. The complex bacterium preparation according to claim 1, wherein the culture medium is subjected to shaking treatment at a rotation speed of 130 to 160r/min.

7. Use of the complex bacterial preparation according to any one of claims 1 to 6 in the treatment of mercury contaminated soil.

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