CN116064248A - Aspergillus flavus and application thereof in lead-polluted soil remediation - Google Patents

Abstract

The invention discloses aspergillus flavus (Aspergillus tamarii) and application thereof in lead-polluted soil restoration. The strain has been deposited in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms at 5.13 of 2022, and has a deposit number of: CGMCC No.40173. Aspergillus flavus (Aspergillus tamarii) separated and screened by the method for separating and screening various heavy metals, especially Pb ²⁺ The ion has good tolerance, and Pb in the soil can be removed ²⁺ Ion mineralization into solid lead-containing mineral, pb within 90-120 d ²⁺ The solidifying rate of the ions reaches 72.0 to 75.7 percent, and can be used for the microbial passivation restoration of lead-polluted soil.

Description

Aspergillus flavus and application thereof in lead-polluted soil remediation

Technical Field

The invention belongs to the technical field of environmental microorganisms and ecological restoration, and particularly relates to aspergillus flavus (Aspergillus ustamarili) and application thereof in lead-polluted soil restoration.

Background

In recent years, with the rapid development of economy and the acceleration of industrialized urban process in China, the problem of heavy metal pollution of soil is becoming serious. According to the national soil pollution Condition issued by the Environment protection part and the national resource part in 2014 in a combined wayThe gazette shows that the cultivated land polluted by heavy metals such As Cd, ni, cu, as, hg, pb and the like occupies 19.4 percent, wherein the cultivated land polluted by heavy metals such As Cd, as, pb and the like has the area of 2 multiplied by 10 ⁵ km ² About 1/5 of the total area of the cultivated land. When the crops are cultivated on the polluted farmland, heavy metals are transferred from soil to the crops and are enriched in the crops, so that the growth and development of the crops are affected, and the crops can enter the human body through a food chain, thereby threatening the physical health of the human body. Therefore, how to prevent and treat the heavy metal pollution of the soil or to adopt effective restoration measures to the soil polluted by the heavy metal has important significance for achieving sustainable and healthy development of agriculture for the well-remolded soil ecology.

The phenomenon of lead pollution in soil is common, and the source of the phenomenon is natural, mainly the sedimentation of volcanic eruption ash; secondly, lead pollution caused by human activities is also a cause of large lead pollution amount and frequent occurrence of soil, such as development and smelting of lead-containing minerals; use of lead acid batteries in the battery industry; the production and use of products such as fuel oil, combustion exhaust gas of fuel coal, lead-containing paint, coating, pigment, medicine and the like. Lead in these sources of pollution can enter the soil through a variety of channels, causing lead contamination of the soil and a series of ecological problems that result. Therefore, how to repair lead-contaminated soil has attracted considerable attention; at present, lead-polluted soil remediation technology mainly comprises physical remediation, chemical remediation and biological remediation. The physical repair is mainly to adopt a method of soil dressing or soil replacement, namely, the contaminated soil is covered with non-contaminated soil or the contaminated soil is partially or completely excavated and replaced with the non-contaminated soil, but the method has larger engineering quantity and high investment; the chemical restoration mostly adopts the phenomenon that a passivating agent is added into soil to change the occurrence form of heavy metals, so that the bioavailability of lead is reduced, but the repeated use of the passivating agent can damage the soil property, so that nutrition loss and even secondary pollution can be caused. Bioremediation mostly adopts a phytoremediation method, namely a method for removing heavy metals in soil through mechanisms such as absorption, aggregation, degradation and fixation of the heavy metals by plants, but the phytoremediation action process is slower.

In contrast, the microbial remediation method has a plurality of advantages, and as the microbial individuals are small, the propagation is quick, the adaptability is strong, and part of microorganisms have strong tolerance to heavy metals, the microbial remediation method can be used for the remediation of soil heavy metal pollution. However, the currently reported microorganism has little report on microbial transformation and mineralization of heavy metals on the heavy metal pollution restoration mechanism of the soil, and particularly, the lead ions in the soil are mineralized through aspergillus flavus (Aspergillus tamarii), so that passivation restoration of the lead-polluted soil is not reported.

Disclosure of Invention

The invention aims to provide aspergillus flavus and application thereof in lead-polluted soil restoration.

The aspergillus flavus provided by the invention is aspergillus flavus (Aspergillus tamarii) BDH33 and CGMCC No.40173.

The aspergillus flavus (Aspergillus tamarii) BDH33 is preserved in China general microbiological culture collection center (CGMCC) of China for 5 months and 13 days, and the preservation number is CGMCC No.40173.

The aspergillus flavus (Aspergillus tamarii) BDH33 has the following characteristics:

growing for 7d on a PSA culture medium at the temperature of 28 ℃, wherein the diameter of a colony reaches 54mm; the colony is velvet-shaped, the central part is flocculent, the color is golden, and the peripheral part is earthy yellow; the conidium is yellow brown, and has a shape from spherical to ellipsoidal, and can produce purple black sclerotium.

The invention has the beneficial effects that:

aspergillus flavus (Aspergillus tamarii) BDH33 vs. Cu in the present invention ²⁺ 、Pb ²⁺ 、Cr ³⁺ Has good tolerance and can grow and reproduce under the heavy metal concentration of 0-5 mmol/L.

Aspergillus (Aspergillus tamarii) BDH33 against Pb in the present invention ²⁺ Has excellent tolerance at 5mmol/LPb ²⁺ Culturing for 7d under the ion concentration, wherein the colony diameter reaches 45mm, and the colony diameter can reach 51mm after culturing for 28 d;at 10mmol/L Pb ²⁺ The colony diameter of the strain reaches 25mm after 7d of culture under the ion concentration, and the colony diameter can reach 48mm after 28d of culture.

The aspergillus flavus (Aspergillus tamarii) BDH33 provided by the invention can be used for preparing Pb in soil ²⁺ Mineralizing into Pb-containing minerals, for Pb ²⁺ Plays a passivation role.

The aspergillus flavus (Aspergillus tamarii) BDH33 provided by the invention contains Pb ²⁺ The lead solidification rate reaches 75.7 percent after the lead is treated in soil with ion concentration of 20mg/g for 3 months.

Drawings

FIG. 1 shows Aspergillus swift BDH33 at different Pb ²⁺ Growth patterns of 28d culture on ion concentration medium.

Figure 2 is an X-ray crystal diffraction (XRD) measurement chart of minerals in soil.

Fig. 3 is a soil electron scanning microscope (SEM) and energy spectrum (EDS). A is the original soil of a control group; b and C are BDH33 treated lead-containing soil; A. the boxes in B and C are the energy spectrum analysis regions.

FIG. 4 shows the effect of Aspergillus flavus BDH33 on Pb in soil ²⁺ Mineralization rate of (2).

Detailed Description

The media used in the examples are as follows:

high salt Bosch's Medium (g/L): sodium nitrate 2, monopotassium phosphate 1, magnesium sulfate heptahydrate 0.5, potassium chloride 0.5, ferrous sulfate 0.01, sodium chloride 60, sucrose 30 and agar 15, and the pH is regulated to 7.0-7.2.

PSA medium (g/L): potato (peeled) 200, sucrose 20, agar 20.

Maltose liquid Medium (g/L): malt extract 20, glucose 20, peptone 6, natural pH.

Heavy metal mother liquor: cu of 0.5mol/L is prepared respectively ²⁺ 、Pb ²⁺ 、Cr ³⁺ 、Cd ²⁺ Mother liquor.

Example 1: isolation, screening and identification of fungal strains

1. Isolation of fungal strains

Collecting 5-10cm sample of sediment at the mouth of North Daihe river, weighing 10g sediment, and adding into the containerMixing in triangular flask with 90ml seawater, standing for 2 hr, and serial diluting the upper layer suspension 10 times to 10 ^-4 Respectively dripping 200 μl of each gradient in the middle of a sterile culture dish, cooling the melted high-salt culture medium to about 50deg.C, pouring 20ml of culture medium into the culture dish, rapidly rotating the culture dish to uniformly mix the bacterial suspension with the culture medium, solidifying the culture medium, and culturing in a constant temperature incubator at 28deg.C for 7d; after the colony grows out, different single colonies are picked up and transferred onto a PSA culture medium, and are cultured for 7 days under the same condition, so that pure culture strains are obtained; the colonies of the pure culture strains were scraped off and placed in 20% glycerol for storage in a-80℃refrigerator.

2. Screening for heavy metal resistant fungi

Firstly preparing a PSA culture medium, sterilizing by high-pressure steam, and respectively adding filtered and sterilized heavy metal mother liquor to prepare a single heavy metal Cu-containing liquid ²⁺ 、Pb ²⁺ 、Cr ³⁺ And Cd ²⁺ PSA medium of (C) to final concentrations of Cu ²⁺ 5mmol/L、Pb ²⁺ 5mmol/L、Cr ³⁺ 5mmol/L and Cd ²⁺ 2mmol/L, then pour plate for use.

Screening heavy metal fungi. Taking 1.5ml of centrifuge tubes, respectively adding 100 mu l of sterile water, then picking a proper amount of spores of pure culture strains, adding the spores into the centrifuge tubes, and uniformly mixing to prepare bacterial suspension; then 1 mul of fungus suspension liquid is respectively dropped in the center of a PSA plate containing heavy metal, the plate is placed in an incubator at 28 ℃ in an inverted mode for 7d, the degree of fungus heavy metal resistance is judged by measuring the colony diameter, and fungus strains with good heavy metal resistance are screened. Through the screening, a strain which can resist Cu respectively is obtained ²⁺ 、Pb ²⁺ And Cr (V) ³⁺ The results of strain BDH33 of (C) are shown in Table 1.

TABLE 1 growth diameter of strain BDH33 on media containing different heavy metals

Note that: CK is a medium which does not contain heavy metals; "-" indicates no growth.

3. Fungus strain identification

3.1 morphological identification.

BDH33 is inoculated on a PSA culture medium and cultured for 7 days at 28 ℃, and the colony diameter can reach 54mm; the colony is velvet-shaped, the central part is flocculent, the color is golden, and the peripheral part is earthy yellow; the conidium is yellow brown, and has a shape from spherical to ellipsoidal, and can produce purple black sclerotium.

3.2 molecular biology identification.

The genome of the fungus BDH33 was PCR amplified using fungal ITS primers ITS1:5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. And (3) carrying out agarose gel electrophoresis and gel cutting recovery purification on the PCR amplification product, and then carrying out ITS sequence sequencing, wherein the sequence is shown as a sequence 1 in a sequence table. Blast comparison analysis is carried out on the sequence in NCBI database, and the result shows that the ITS sequence of the strain has the highest similarity with aspergillus swift (Aspergillus tamarii) SKF8, which reaches 99.84%; this strain was designated as Aspergillus oryzae (Aspergillus tamarii) BDH33.

Aspergillus swift (Aspergillus tamarii) BDH33 was deposited in the China general microbiological culture Collection center, CGMCC, with the address of No. 1, 3, and the deposit number of No.40173.

Example 2: aspergillus swift BDH33 against Pb ²⁺ Ion tolerance determination

Preparing Pb-containing powder ²⁺ PSA culture medium with ion concentration of 5mmol/L, 10mmol/L and 15mmol/L respectively, pouring the culture medium into a flat plate, and then respectively taking 1 mu L of fungus suspension of aspergillus flavus BDH33 to be inoculated with different Pb ²⁺ The ion concentration of the PSA plate center, placed in 28 degrees C incubator culture, and in 2d, 5d, 7d, 14d, 21d and 28d observation and record of its growth.

The results are shown in Table 2 when Pb ²⁺ At an ion concentration of 5mmol/L, the inoculated strain 2d starts to grow, and at the subsequent time of 5-14 d, the growth speed is faster, and at the time of 21-28 d, the colony diameter is 51mm, and basically no longer grows. When Pb ²⁺ Ion(s)At a concentration of 10mmol/L, growth starts at 5d after inoculation, the strain grows faster in the following 7-21 d, and the colony diameter reaches 48mm at 28 d. But when Pb ²⁺ When the ion concentration was increased to 15mmol/L, the growth thereof was not seen even by the culture to the 28 th d, whereby it was confirmed that A.fumigatus BDH33 was against Pb ²⁺ The upper limit concentration of the ion tolerance is 10-15 mmol/L.

TABLE 2 growth dynamics and p. Pb of Aspergillus swift BDH33 ²⁺ Ion tolerance assay

Note that: the data in the table are colony diameter (mm); "-" indicates no growth.

It was also found from the above observation that, with Pb ²⁺ As shown in FIG. 1, the biomass of colonies grown on the plate by A.fumigatus BDH33 gradually decreased, although A.fumigatus BDH33 was grown at 5mmol/L and 10mmol/L Pb ²⁺ The diameter difference is not obvious when 28d is cultivated on the culture medium, but the biomass difference is obvious, and the ratio of the biomass to Pb is 10mmol/L ²⁺ Biomass on the medium was about 5mmol/L Pb ²⁺ One fifth of the biomass on the medium; but at 15mmol/L Pb ²⁺ On the medium, no growth of Aspergillus flavus BDH33 was seen, and the biomass was 0.

Example 3: aspergillus swift BDH33 against Pb ²⁺ Mineralization and repair effects of ions

1. Pb-containing ²⁺ Fungus repair experiment of soil

Preparation of experimental soil. Taking a plurality of soil, sieving with a round hole sieve with the aperture of 1mm, reserving soil with the particle size of less than 1mm, and filling the soil into 250ml conical flasks with 100g of soil per flask.

Pb ²⁺ And (3) preparing a solution. 1.5ml of 0.5mol/L Pb was extracted ²⁺ Adding mother liquor into 73.5ml maltose liquid culture medium to obtain Pb ²⁺ A maltose solution with an ion final concentration of 10 mmol/L.

Pb-containing ²⁺ Fungus repair experiments of soil. Will contain Pb ²⁺ 75ml of ionic maltose solution was added to the above-mentioned 100g of soilShaking, mixing, sterilizing in high pressure steam sterilizing pot at 121deg.C for 30min, inoculating 100 μl of Aspergillus oryzae BDH33 spore suspension, shaking triangular flask, and mixing thoroughly to obtain Pb-containing powder without fungi ²⁺ Soil was used as a control, cultures were performed at room temperature, shake flasks once every 10d interval, and culture was terminated for 120 d.

2. Determination of minerals in soil

X-ray crystal diffraction (XRD) analysis. After the experiment is finished, about 5-10 g of soil is taken from a triangular flask, placed in a culture dish, naturally air-dried at room temperature, and an air-dried soil sample is ground into powder by an agate mortar and then subjected to XRD test. The test conditions were: 30kv,10ma,2θ scan angle=10° to 70 °, scan speed=6°/s. The obtained X-ray crystal diffraction pattern was subjected to phase search and analysis by using JADE 6 software.

XRD measurement of minerals in soil is shown in figure 2, and the results show that the mineral composition of the soil is mainly composed of anorthite, quartz, calcite, calcium oxalate and other minerals, whether the soil is a control group soil (containing lead and not inoculated with BDH 33) or an experimental group soil (containing lead and inoculated with BDH 33). However, the XRD patterns of the soil inoculated with the aspergillus flavus BDH33 are obviously different between 27-29 degrees of 2 theta angles, and it is presumed that the lead-containing soil forms a new lead-containing mineral under the action of the aspergillus flavus BDH 33; but due to Pb added in the soil ²⁺ The amount of newly formed lead-containing minerals was thus very small compared to the amount of other minerals in the soil, so that the diffraction peaks of the mineral crystals thereof were weak, and it was difficult to search and distinguish what minerals they were, and therefore it was analyzed by the following analysis means.

3. Observation and analysis of minerals in soil

Electron scanning microscopy (SEM) and energy spectroscopy (EDS) observations and analyses. And (3) performing electron microscope sample preparation on the original soil of the control group and the soil of the experimental group, and spraying Pt on the sample to enhance the conductivity of the sample in order to obtain a clear electronic image. The sample is placed in an electron scanning microscope, the lead-containing mineral is observed under the condition of back scattering by adopting an accelerating voltage of 15kV, and the elements in the mineral are qualitatively and semi-quantitatively analyzed by an X-ray energy spectrum analyzer (EDS).

As a result, see fig. 3, the lead-containing minerals tend to appear bright in the back-scattered electron image. As can be seen from the graph, the original soil of the control group is dark, and the energy spectrum result shows that elements in the soil mainly comprise C, O, si, al and the like; in the back scattering electronic image of the lead-containing soil treated by BDH33, soil minerals with different brightness degrees appear, and the energy spectrum element analysis is respectively carried out on the soil minerals with different brightness, so that the result shows that the elements in the dark soil minerals mainly have C, O, si, al elements and the like, which are similar to the original soil of a control group; while the elements in the bright soil minerals mainly contain C, O, pb and the like, wherein the relative mass percent of Pb elements is 86.32 percent, and the Pb in the soil is seen to be Pb after BDH33 treatment ²⁺ The mineral particles which have been mineralized to be lead-rich are presumed to be organic lead-containing minerals based on their elemental composition and the characteristics of the species.

4. Pb in soil ²⁺ Mineralization rate of (2)

The soil is sampled respectively after being treated for 30d, 60d, 90d and 120d, a proper amount of soil is taken and put into a blast drying box for drying, 1g of soil is taken and put into a 15ml centrifuge tube, 12ml of deionized water is added, the mixture is fully and uniformly vortex, supernatant fluid (10.8 ml) is collected centrifugally, and Pb in the supernatant fluid is measured by adopting an ICP-MS inductively coupled plasma mass spectrometry ²⁺ The concentration of ions and then the Pb in the soil is calculated ²⁺ Mineralization rate of (2). Mineralization rate (%) = (C ₀ -C _t )/C ₀ *100%, C in ₀ Is Pb in soil ²⁺ Is present in the composition (in mg/g); c (C) _t Pb in soil after fungus treatment ²⁺ Content (mg/g).

The results are shown in FIG. 4, pb-containing ²⁺ After the soil is treated by aspergillus flavus BDH33 for 30d, 60d, 90d and 120d, free Pb in the soil ²⁺ The contents of (3) are 1.85, 1.03, 0.68 and 0.59mg/g respectively, and Pb in soil ²⁺ The mineralization rates were 23.9%, 57.6%, 72.0% and 75.7%, respectively, compared to the initial content of 2.43 mg/g. As can be seen, aspergillus flavus BDH33 is a strain which is tolerant to high concentrations of lead and which is capable of converting Pb ²⁺ Strains mineralized into solid lead-containing minerals have wide prospects in passivation and restoration application of lead-contaminated soil.

The foregoing is illustrative of the present invention and is not to be construed as limiting the scope of the present application; it will be apparent to those skilled in the art that any modifications, variations, or equivalents may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (7)

1. An aspergillus flavus is characterized in that the aspergillus flavus is named as aspergillus flavus (Aspergillus tamarii) BDH33, and the preservation number of the aspergillus flavus in the China general microbiological culture collection center of the China Committee for culture Collection of microorganisms is CGMCC No.40173.

2. A microbial preparation comprising the aspergillus flavus (Aspergillus tamarii) BDH33 as claimed in claim 1 as an active ingredient.

3. Use of aspergillus flavus (Aspergillus tamarii) BDH33 according to claim 1 for remediation of heavy metal contaminated soil.

4. Use according to claim 3, characterized in that the heavy metals comprise: cu (Cu) ²⁺ 、Pb ²⁺ 、Cr ³⁺ The method comprises the steps of carrying out a first treatment on the surface of the More preferably, the heavy metal is Pb ²⁺ 。

5. Use according to claim 3, characterized in that said heavy metal contaminated soil Pb ²⁺ Soil with ion range of 0-2.43 mg/g.

6. Use according to claim 3, characterized in that the minerals in the heavy metal contaminated soil comprise quartz, anorthite, calcite, calcium oxalate and the like.

7. The method of mineralizing Pb by Aspergillus (Aspergillus tamarii) BDH33 ²⁺ Application in ions.

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