CN114107059B - Application of microalgae and microbial agent and method for removing environmental heavy metals - Google Patents

Abstract

theinventionrelatestothefieldofalgaecultivationandenvironmentalpollution,inparticulartoamethodforremovingenvironmentalheavymetalsbyapplyingamicrocystisandmicrobialinoculum,whereinthemicrocystisMicranthussp(MEM-A-404)hasapreservationnumberofCCTCCNO: m20211084. The microalgae can be cultivated at the highest test temperature of low temperature of 5 ℃ and high test temperature of 45 ℃, the lowest pH value is 2.5, the highest pH value is 11, the concentration of any heavy metal ions is 20ppm to 160ppm, the microalgae strain has extremely strong heavy metal tolerance, can grow in an environment containing high heavy metal content, and meanwhile, the microalgae strain and the microbial agent prepared by the microalgae strain can efficiently remove heavy metals in water and soil.

Description

Application of microalgae and microbial agent and method for removing environmental heavy metals

Technical Field

The invention relates to the field of algae cultivation and environmental pollution, in particular to a method for removing environmental heavy metals by applying microalgae and microbial agents.

Background

Heavy metal pollution has serious toxicity to organisms. The deterioration of environmental quality can be caused by high toxicity, non-biodegradability and accumulation of heavy metals in food chains and organisms. Thus, the World Health Organization (WHO) and the United States Environmental Protection Agency (USEPA) have established strict regulations on the allowable levels of heavy metals emitted in the environment. Lead (pb2+) and copper (cu2+) are major environmental pollutants in heavy metal contamination because of their high toxicity. In addition to traditional physicochemical methods, researchers have tried various technologies for removing heavy metal pollution, and in recent years, biological methods have been mainly applied to the treatment of heavy metal pollution by microalgae. Phytoplankton, such as microalgae, adapt to the contaminated water body by adjusting their physicochemical and genetic structures. Diatom is one of the most abundant biota in the marine ecosystem. Plays an important role in the processes of removing heavy metals, detoxication and restoring aquatic ecosystems. Therefore, the screening, separation and application of the diatom with high heavy metal tolerance and removal capability have important significance for explaining the balance mechanism of the ecological system, repairing heavy metal pollution and the like.

However, the global ecological system is continuously deteriorated due to heavy metal pollution and the continuous rise of global temperature. In northern China and in most southern China, the temperature can reach 40+/-5 ℃ in summer, and the growth of microalgae is inhibited at high temperature. Meanwhile, as the industrialization degree is continuously improved, the pH value of the seawater is reduced due to the rising of the concentration of carbon dioxide, the solubility of heavy metals is further increased, and the growth of microalgae is influenced.

Although many microalgae have a certain heavy metal ion adsorption capacity, many microalgae have limited adsorption capacity for heavy metals, and the tolerance of many microalgae is poor along with the deterioration of the environment, so that the application of the microalgae in heavy metal adsorption is greatly limited. Thus, the selection of a suitable microalgae strain is critical to its use in the relevant field.

Disclosure of Invention

In view of the above, the invention provides the application of the microalgae and the microbial agent and the method for removing the environmental heavy metals. The double-headed diamond algae can grow in the environment with the temperature of 5-45 ℃, the pH concentration of 2.5-11 and the concentration of heavy metal ions of 20-160 ppm, and the algae strain has extremely strong heavy metal tolerance. Meanwhile, the algae strain and the microbial agent prepared by the algae strain can efficiently remove heavy metals in water and soil.

In order to achieve the above object, the present invention provides the following technical solutions:

theinventionprovidesamicrocystismicranthumsp(MEM-A-404),andthepreservationnumberofthemicrocystismicranthumisCCTCCNO: m20211084.

The invention also provides a microbial agent, which comprises the microalgae and acceptable auxiliary agents.

The invention also provides application of the microalgae and/or the microbial agent in heavy metal tolerance and/or removal of heavy metals in the environment.

Preferably, the environment is a body of water and/or soil.

Preferably, the heavy metal comprises one or more of Pb, cu, ag, cd and/or Cr.

The invention also provides a method for removing the environmental heavy metals, which comprises the step of treating the environmental heavy metals by the microalgae and/or the microbial agent.

Preferably, the method comprises the following steps:

s1: pretreating the environment to obtain a sample;

s2: inoculating the microalgae into the sample to obtain a culture;

s3: and (5) taking the culture, and respectively placing the culture in an illumination place and a non-illumination place for culture.

Preferably, the concentration of heavy metal ions in the sample in S2 is 20 to 160ppm.

Preferably, the pH concentration of the sample in S2 is 2.5 to 11.

Preferably, the temperature of the sample in S2 is 5 to 45 ℃.

The invention provides an application of microalgae and a microbial agent and a method for removing environmental heavy metals, wherein the preservation number of the microalgae (Micracinium sp) is CCTCC NO: m20211084.

The algae strain can grow in an environment with the temperature of 5-45 ℃, the pH concentration of 2.5-11 and the concentration of heavy metal ions of 20-160 ppm, has extremely strong heavy metal tolerance, and can grow in an environment with high heavy metal content. Meanwhile, the algae strain and the microbial agent prepared by the algae strain can efficiently remove heavy metals in water and soil.

Description of biological preservation

Micraciniumsp.mem-a-404,25thdayofthepreservationdate2021,andcctccno: m20211084, accession number: china center for type culture Collection, with the addresses: chinese university of Wuhan and Wuhan.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a nucleotide sequence of an 18S rRNA gene of an alga strain Micracinium sp;

FIG. 2 shows the cell morphology of the algal strain Micracinium sp magnified 100-fold under a microscope;

FIG. 3 shows PCR amplification products of 18S rRNA gene of Micracinium sp strain;

lane M is DL2000 Marker, lane 1 is 18SrRNA gene amplification product, lane 2 is 18SrRNA gene amplification product;

FIG. 4 shows a phylogenetic tree of the 18S rRNA gene sequence of the algal strain Micracinium sp;

FIG.5showstheexposuregrowthofMEM-A-404at20ppm,40ppmand80ppmof(a)lead(Pb)and(b)copper(Cu);

FIG.6showsthegrowthbehaviorofMEM-A-404at120ppmand160ppm(a)lead(Pb)and(b)copper(Cu)exposure;

FIG.7showsthatMEM-A-404canbegrownatdifferenttemperaturelevels;

FIG.8showsthatMEM-A-404canbegrownatdifferentpHlevels;

FIG.9showsanoptimizedslopeofaresponsesurfacemethodquadraticmodelfordeterminingMEM-A-404enhancedlead(Pb)metalremovalconditions;

wherein (a) and (b) are the desired optimized slopes selected to enhance Pb removal;

FIG.10showstheresistanceofMEM-A-404todifferenttemperatures,pHvaluesanddifferentlead(Pb)concentrations;

FIG. 11 shows a 3D surface map of a response surface approach;

wherein: (a) A 3D surface plot of the response surface method showing the interaction of pH and lead (Pb) to Pb metal removal efficiency;

(b) The 3D surface plot of the response surface method shows the interaction of temperature and lead (Pb) to Pb metal removal efficiency.

Detailed Description

The invention discloses application of microalgae and microbial agents and a method for removing environmental heavy metals, and the technical parameters can be properly improved by those skilled in the art by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention. The preparation of the culture medium, the separation and identification of algae strains and the reagents used in tolerance verification can be obtained by the market or related scientific research units.

Example 1 preparation of F/2 algal Medium

(1) Preparation of solution I

Dissolving 35g/L sea salt in distilled water (dH) ₂ After O), filtering by 0.45 mu m pore diameter filter paper to obtain filtrate;

mixing the filtrate with 1.21g/L of tries (formula C ₄ H ₁₁ NO ₃ ) Mixing, adjusting pH to 7.6, and autoclaving at 121deg.C for 20min.

(2) Preparation of solution II

CuSO is performed ₄ ·5H ₂ O (final concentration of 19.6 g/L), na ₂ MoO ₄ ·2H ₂ O (final concentration of 12.6 g/L), znSO ₄ ·7H ₂ O (final concentration of 44 g/L), coCl.6H ₂ O (final concentration 10.92 g/L) MnCl ₂ ·4H ₂ O (final concentration of 360 g/L) and sterilizing at 121deg.C for 20min.

(3) Preparation of solution III

5mL NaNO ₃ (final concentration is 200 g/L), 5mL NaH ₂ PO4·H ₂ O (final concentration is 13.3 g/L) and 1mL of solution II are mixed, and the mixture is heated and sterilized at 121 ℃ for 15min and then mixed with the solution I to obtain the F/2 algae culture medium.

Example 2 isolation and identification of algal strains

(1) Collecting soil and water sample

Soil and water samples were collected from the copper mine of eastern city, hainan province, china.

(2) Pretreatment of

The stones, cobbles and straw were removed and washed and filtered to give samples which were placed at 4 ℃ for use.

(3) Cultivation of algae

10mL of sample was taken and mixed with 100mL of F/2 algal culture in a 250mL Erlenmeyer flask, and 2mL of PbNO was added separately ₃ And 2mL of CuSO4.5H ₂ O, lead (Pb) ²⁺ ) And copper (Cu) ²⁺ ) The final concentration of ions was 20ppm;

the flask was placed at 25℃and 100. Mu. Mol m ^-2 s ^-1 Culturing for 16h under illumination, and culturing for 8h at 25deg.C without illumination to obtain culture;

preparing heavy metal ion solution: is configured into a heavy metal ion mother solution with the concentration of 1000 ppm.

(4) Purification of algae

80. Mu.L of the culture was inoculated into F/2 algal medium and after several days of spreading the algae suspension on the F/2 algal medium, approximately uniform mixed microalgae and fungal contamination was observed. And selecting microalgae strains with high growth speed, and purifying by repeated diffusion and a streak plate method.

(5) Activation of algae

Inoculating the purified algae into F/2 algae liquid culture medium, shake culturing, detecting OD every 24 hr ₇₅₀ And (5) obtaining an alga culture.

(6) PCR amplification of 18S rRNA Gene

Taking 1mL of algae culture under a sterile environment, and centrifuging at 8000rpm for 5min to obtain algae cells; cleaning cells with sterile deionized water (dH 2O) to remove residual salts; extracting algae cell DNA; the algae cell DNA is used as a template, and is optimized, and PCR amplification (a PCR reaction system is shown in table 1, and a PCR reaction program is shown in table 2) is carried out on the 18S rRNA gene.

TABLE 1 PCR reaction System

TABLE 2 PCR reaction procedure

(7) Data processing

The algae belongs to the genus of the microcystis through 18s rRNA gene sequencing identification. The microalgae (micracinium sp) has been deposited at the China center for type culture Collection, university of Wuhan, hubei province, china, 1/9/2021, with a deposit number: cctccc NO: m20211084; the gene sequence table is shown in figure 1;

the cell morphology of the microalgae is shown in figure 2, the cell morphology is green sphere, and the cell diameter is 15 mu m plus or minus 2 mu m under normal conditions;

the gene length of the microalgae is 994bp, and the RNA gel electrophoresis diagram is shown in figure 3;

the phylogenetic development of this microalgae was analyzed by Mega-X software, as shown in fig. 4.

EXAMPLE 3 tolerance of algal strains to Low concentration heavy metals

Respectively dispose Pb ²⁺ And Cu ²⁺ 100mL of F/2 medium at concentrations of 20ppm, 40ppm and 80ppm were added to the tube bioreactor (diameter 3.3cm, height 60 cm), respectively. The strain isolated in example 2 was inoculated into each tube bioreactor at an initial inoculation concentration OD ₇₅₀ 0.30, temperature 25 ℃, photoperiod 16 (light): 8 (dark) illumination intensity 100. Mu. Mol m ² s ^-1 . toexaminethesurvivalandgrowthrateofMEM-A-404,ODwasmeasuredevery2days ₇₅₀ For a total of 12 days. theresultsshowthatMEM-A-404isfoundinthesethreePb's ²⁺ Grows well at concentration, however, on exposure to Cu ²⁺ In the lower part, the growth is relatively slow. As shown in fig. 5.

EXAMPLE 4 tolerance of algal strains to high concentrations of heavy metals

According to the screening result of example 3, 80, 120 and 160ppm Pb was further supplemented in the F/2 medium prepared ²⁺ And Cu ²⁺ Then inoculated into the culture medium of example 3In a tubular bioreactor under identical conditions. At OD ₇₅₀ Pb at concentrations of 120ppm and 160ppm in the measurement of (3) ²⁺ thegrowthofMEM-A-404wasrestarted,withCuatalowerconcentrationthanthesame ²⁺ . incontrast,MEM-A-404grewslowlyataconcentrationof160ppm,butwasstillpossible,asshowninFIG.6.

EXAMPLE 5 tolerance of algal strains to different temperatures, pH

theresultsindicatethatMEM-A-404canbegrownatdifferenttemperaturesandpHlevels,asshowninFIGS.7and8.

Example 6 efficiency of removal of heavy metals by algal strains

Based on the experimental results, the orthogonal response surface method of software "Design Expert" was used to determine that the algal strain isolated in example 2 removed Pb from F/2 medium under different experimental conditions ²⁺ And Cu ²⁺ Capability of ions. The experiment was performed at 5 pH values, including 2, 3, 6.5, 10 and 12.5;5 different temperatures including 5 ℃, 10 ℃, 20 ℃, 35 ℃ and 50 ℃; pb of 4 different concentrations ²⁺ And Cu ²⁺ Including 50ppm, 100ppm, 150ppm and 200ppm, when OD ₇₅₀ The metal removal efficiency was assessed for 4 different time intervals of 3, 6, 9 and 12 days after inoculation of the algal strain for 0.25.

The results are shown in Table 3 and FIGS. 9 and 10. Pb was observed at a pH of between 6 and 10 and a temperature in the range of between 10℃and 35 DEG C ²⁺ And Cu ²⁺ Wherein the time has a significant effect on the metal removal efficiency of the algal strain.

As shown in FIG. 11, temperature and pH are plotted against Pb in the F/2 medium ²⁺ And Cu ²⁺ Has a significant effect on the removal of (C). A 3-dimensional (3D) surface plot of Response Surface Method (RSM) is an orthogonal statistical design showing the interaction effect of pH and metal ions (a) and temperature and metal ions (b) on the efficiency of the Pb removal by the microalgae. Figure a shows that maximum Pb removal can be achieved in the pH range between pH 6 and pH 8. Similarly, panel b shows that maximum Pb removal can be achieved at any temperature below 20 ℃ and above 30 ℃. However, at temperatures above 40 ℃, the growth rate of microalgae can decrease. Therefore, to the maximum extentPb is removed, and the preferred pH and temperature may be 8 and 35 ℃.

TABLE 3 response surface method for removing lead (Pb) ions (RSM) quadratic model analysis of variance (ANOVA)

The F value of the Quadratic model was 15.15, indicating that the model was significant. P values less than 0.0500 indicate significant model values.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

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<120> application of microalgae and microbial agent and method for removing environmental heavy metals

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ctaaacccct taacgaggat caattggagg gcaagtctgg tgccagcagc cgcggtaatt 120

ccagctccaa tagcgtatat ttaagttgct gcagttaaaa agctcgtagt tggatttcgg 180

gtggggcctg ccggtccgcc gtttcggtgt gcactggcag ggcccacctt gttgccgggg 240

acgagctcct gggcttcact gtccgggact cggagtcggc gctgttactt tgagtaaatt 300

agagtgttca aagcaggcct acgctctgaa tacattagca tggaataaca cgataggact 360

ctggcctatc ctgttggtct gtaggaccgg agtaatgatt aagagggaca gtcgggggca 420

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gcatttgcca aggatgtttt cattaatcaa gaacgaaagt tgggggctcg aagacgatta 540

gataccgtcc tagtctcaac ataaacgatg ccgactaggg atcggcggat gtttcttcga 600

tgactccgcc ggcaccttat gagaaatcaa agtttttggg ttccgggggg agtatggtcg 660

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ataacaggtc tgtgatgccc ttagatgttt ctgg 994

Claims (8)

1. Microalgae (Sphaerophyta)Micractinium sp) The method is characterized in that the preservation number is CCTCC NO: m20211084.

2. A microbial agent comprising the microalgae of claim 1 and an acceptable adjuvant.

3. Use of the microalgae according to claim 1 and/or the microbial agent according to claim 2 for heavy metal tolerance and/or removal of heavy metals in an environment;

the environment is water and/or soil;

the heavy metals are as follows: pb.

4. A method for removing heavy metals in the environment, which is characterized by comprising the step of taking the microalgae according to claim 1 and/or the microbial agent according to claim 2 to treat the heavy metals in the environment;

the heavy metals are as follows: pb.

5. The method according to claim 4, comprising the steps of:

s1: pretreating the environment to obtain a sample;

s2: inoculating the microalgae and/or the microbial agent into the sample to obtain a culture;

s3: and (5) taking the culture, and respectively placing the culture in an illumination place and a non-illumination place for culture.

6. The method of claim 5, wherein the concentration of heavy metal ions in the sample in S2 is 20 to 160ppm.

7. The method of claim 6, wherein the sample in S2 has a pH concentration of 2.5 to 11.

8. The method of claim 7, wherein the sample is incubated at a temperature of 5-45 ℃ in S2.