CN106190871B - Method for treating heavy metal contaminated soil by bioleaching with composite filamentous fungi by taking straws as carbon source - Google Patents
Method for treating heavy metal contaminated soil by bioleaching with composite filamentous fungi by taking straws as carbon source Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
Abstract
The invention discloses a filamentous fungus strain NAU-12, which is classified and named as Penicillium simplicicum (Penicillium simplicicum), and is preserved in the China general microbiological culture Collection center at 6-25.2015 in the preservation number of CGMCC NO. 10990. The invention also discloses a Penicillium simplicicum NAU-12 strain (1-2 multiplied by 10) with the same volume8spore/mL) and Aspergillus niger A80 strain (1-2X 10)7spore/mL) are mixed to form the composite filamentous fungus agent. The invention also discloses a method for treating heavy metal contaminated soil by bioleaching the composite filamentous fungi which can grow rapidly in soil and have strong heavy metal resistance and can utilize the straws pretreated by alkali heat as a growth carbon source to produce various high-yield small-molecular organic acids. The method for treating the heavy metal contaminated soil has high heavy metal removal efficiency and low cost.
Description
Technical Field
The invention belongs to the technical field of environmental engineering, and relates to a method for treating heavy metal contaminated soil by bioleaching composite filamentous fungi by taking straws as a carbon source, in particular to a method for efficiently removing heavy metals in soil with low consumption by utilizing the composite filamentous fungi (penicillium simplicissimum and aspergillus niger) which has wide pH adaptation range and strong heavy metal resistance and takes agricultural waste straws as a growth carbon source to generate various small molecular organic acids.
Background
High-speed industrialization, urbanization and agricultural intensification cause heavy metal pollution of soil to be serious day by day. According to the measurement and calculation of the Ministry of agriculture, the cultivated land area polluted by heavy metals such as Pb, Cd, Cu and the like in China currently reaches 1000 million hectares, the investigation of the China's environmental monitoring general station is close to 2000 million hectares, and the amount of heavy metal polluted grain reaches 1200 million tons every year, which poses great threat to the safety of agricultural products and the health of ecological systems. Due to the specificity of soil heavy metal pollution and the complexity of regions, the treatment not only has slow effect and high cost, but also is restricted by various factors. Therefore, the treatment of the heavy metal contaminated soil is always a hotspot and a difficult point of international research and is also a problem of common interest of a plurality of disciplines in agricultural sustainable development and environmental quality improvement in China.
Bioleaching is a new method for removing heavy metals from soils that has emerged for nearly 20 years. The principle is as follows: using some extreme acidophilic bacteria (mainly one type using CO)2Bacteria that are obligate autotrophic for growing carbon sources, such as: the low pH value generated by the biological oxidation and acidification of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Thiobacillus thioparus) promotes heavy metal in an insoluble form in a soil solid phase to be dissolved in a liquid phase, and the heavy metal is removed after solid-liquid separation. The method is simple and easy to implement, has high economy, is particularly suitable for treating the composite and heavily polluted soil caused by various heavy metal elements (such as surrounding soil of mining areas and waste stacking yards), and draws high attention of scientists. Currently, the technology is in the conversion stage from laboratory research to field implementation, and small-scale projects are reported abroad.
From the existing research practice, the heavy metal bioleaching treatment of soil based on the acidophilic autotrophic bacteria as the dominant bacteria mostly takes longer time (25-30 days), the effect is unstable, and particularly, the activity of the strains is sometimes reduced under the condition of long-term continuous operation working conditions. This is because: 1) the growth rate of autotrophic bacteria is generally slow; 2) the strains are more suitable for growing in an acid environment with the pH value of 1.0-3.0, and are inoculated to soil (mostly neutral and alkaline), and a growth lag phase adapting to the pH value is needed; 3) autotrophic bacteria have certain sensitivity to organic matter, and the content of Dissolved Organic Matter (DOM) in soil often exceeds the toxic level thereof.
In order to improve the efficiency and stability of heavy metal bioleaching in soil, recently, some heterotrophic filamentous fungi which have the advantages of fast growth speed, wide pH adaptation range, strong heavy metal resistance and capability of metabolizing organic carbon to produce small-molecular organic acid (such as citric acid) have been proposed by researchers, such as: trichoderma sp, Talaromyces sp, Aspergillus niger, for heavy metal bioleaching of soil. The method can overcome the technical defect that acidophilic autotrophic bacteria bioleaches heavy metals in soil to a certain extent. The existing research on the bioleaching of soil heavy metals by filamentous fungi is mainly carried out on the basis of taking single filamentous fungi as dominant bacteria and adding a large amount of commercial carbon sources such as sucrose, glucose, starch and the like (the content of DOM carbon in the soil is not enough to maintain the growth requirement of the filamentous fungi) by external sources. However, because of the relatively single acid-producing type of a single bacterium and the limited maximum acid-producing capacity (mostly, the pH of the soil can only be reduced to about pH 3.5), it is often impossible to ensure that heavy metals of different types and forms in the soil can be removed in time and effectively after bioleaching treatment (for example, the effective removal of Pb, Cd and Ni generally requires a pH of less than 2.5). In addition, the addition of a large amount of a commercial organic carbon source has a disadvantage of excessively high treatment cost. Therefore, filamentous fungi with different acid-producing characteristics are reasonably combined, the characteristics of wide acid-producing spectrum (various organic acids) and high acid-producing efficiency of the composite bacteria are exerted, and meanwhile, agricultural wastes (such as straws) with high carbon content are tried to serve as a carbon source for microbial growth (to replace cane sugar and the like), so that the technical and economic feasibility of the heavy metal contaminated soil bioleaching treatment by the filamentous fungi can be obviously improved.
Disclosure of Invention
1. Objects of the invention
The invention aims to provide a method for removing soil heavy metals by using composite filamentous fungi (strains NAU-12 and A80) which can grow rapidly in a soil medium and have strong heavy metal resistance, can utilize agricultural waste straws as a growth carbon source and can produce a large amount of various small molecular organic acids such as citric acid, gluconic acid, oxalic acid and the like.
2. Technical scheme
A strain of filamentous fungus NAU-12 is classified and named as Penicillium simplicissimum (Penicillium simplicinum), is preserved in the China general microbiological culture Collection center (the address: Beijing, West Luo No.1, northern Cheng of the Korean district, institute of microbiology, China academy of sciences) within 25 days 6 months 2015, and has the preservation number of CGMCC NO. 10990. Penicillium simplicicum (Penicillium simplicisimum) strain NAU-12 has the following biological properties: the potato is rapidly propagated on a potato PDA plate, the potato grows for 4 days at 28 ℃, the diameter of a bacterial colony is 25mm, and a bulge and a regular radial groove are arranged in the middle of the bacterial colony; the appearance is grayish green, and the texture is velvet; a large number of conidium structures, wherein the conidium heads are spherical and have the diameter of 150-; can grow under the pH value of 3.0-8.0, and can be used for Zn2+、Cu2+、Cd2+、Pb2+Has stronger resistance, and the tolerance concentration is respectively 200mg/L, 300mg/L, 100mg/L and 1000 mg/L; the straw pretreated by alkali heat can be used as a carbon source for growth, and mainly produces gluconic acid and oxalic acid (the acid production in 12 days is respectively 56mM and 42mM), and also has a small amount of citric acid.
A filamentous fungus strain A80 is classified and named as Aspergillus niger, which is preserved in China general microbiological culture Collection center (address: Beijing city NO.1 Xilu-3 of the sunward area, Beijing, China academy of sciences microbiological research institute) at 2011, 1 month and 10 days, and the preservation number is CGMCC NO. 4533. Aspergillus niger strain A80 has the following biological properties: rapidly propagating on a potato PDA plate, growing for 4 days at 28 ℃, wherein the diameter of a bacterial colony is 60mm, the middle of the bacterial colony is provided with a bulge and regular radial furrows, the appearance is brownish black, and the texture is velvet; a large number of conidium structures, the conidium heads are spherical and have diameters300-500 μm; can grow under the pH of 2.5-7.0, and can be used for Zn2+、Cu2+、Cd2 +、Pb2+Has stronger resistance, and the tolerance concentration is respectively 300mg/L, 200mg/L, 50mg/L and 1500 mg/L; the straw pretreated by alkali heat can be used as a carbon source for growth, and the straw can mainly produce citric acid (the acid yield is 68mM in 15 days) and also has a small amount of malic acid and oxalic acid.
The filamentous fungus strain NAU-12 disclosed by the invention is applied to treatment of heavy metal contaminated soil.
A composite filamentous fungus preparation is prepared from the fungus powder containing 1-2X 108spore/mL Penicillium simplicicum NAU-12 microbial inoculum and containing 1-2 × 107spore/mL Aspergillus niger A80 microbial inoculum is prepared by mixing the same volumes of the microbial inoculum.
The preparation method of the composite filamentous fungus agent comprises the following steps: selecting NAU-12 thallus, inoculating into liquid growth culture medium, and shake culturing at 25-35 deg.C for 5-7 days; selecting A80 thallus, inoculating into liquid growth culture medium, and shake culturing at 25-35 deg.C for 4-6 days; after the culture is finished, mixing the two cultures according to the volume ratio of 1:1 to obtain the composite microbial inoculum.
The application of the composite filamentous fungus agent in treating heavy metal contaminated soil is disclosed.
The invention relates to a method for treating heavy metal contaminated soil by bioleaching of composite filamentous fungi by taking straws as a carbon source, which comprises the following steps:
step (1), preparing a composite filamentous bacterium agent for inoculation: selecting NAU-12 thallus, inoculating into liquid growth culture medium, and shake culturing at 25-35 deg.C for 5-7 days; selecting A80 thallus, inoculating into liquid growth culture medium, and shake culturing at 25-35 deg.C for 4-6 days; after the culture is finished, mixing the two cultures according to the volume ratio of 1:1 to obtain a compound filamentous fungus agent;
step (2), preparation of carbon source: crushing the straws to 1-1.5cm, and soaking in 0.5-1.5% NaOH solution at 80-100 ℃ for 1-2h for alkali-heat pretreatment;
step (3), treating heavy metal contaminated soil: diluting heavy metal soil with water to prepare soil slurry with the slurry concentration of 1-5%, putting the soil slurry into a reactor, adding 40-80g/L of straws or cane sugar subjected to alkali-heat pretreatment as a carbon source, adding 1-3g/L of yeast extract, inoculating a composite filamentous fungus agent according to 5-10% of the volume of the treated slurry, stirring the mixture at the temperature of 25-35 ℃ and the aerobic rotation speed of 120-150r/min for 15-20 days, and pumping out the treated soil slurry;
step (4), solid-liquid separation: carrying out soil-water solid-liquid separation on the soil slurry treated in the step (3) to obtain a soil slurry precipitate and a clear liquid containing heavy metal ions;
step (5), neutralizing and precipitating: adding alkali into the clear liquid containing heavy metal to precipitate heavy metal ions; and neutralizing the soil mud sediment by using quicklime.
In the step (1), the liquid growth medium is: NaNO3 1.5g/L、K2HPO4 0.5g/L，KCl 0.025g/L，MgSO40.025g/L, 1.5g/L of yeast extract, 35g/L of sucrose, natural pH and sterilization.
The rotation speed of the shaking culture is 120-150 r/min.
In the step (2), the straws are air-dried wheat or rice straws, and the total solid content of the straws is more than 90%.
In the step (3), straws subjected to alkali heat pretreatment are preferably used as a carbon source. The addition amount of the straws subjected to the alkali heat pretreatment is preferably 50-70g/L, and the addition amount of the yeast extract is preferably 1-2 g/L. In the invention, the addition amount is calculated by the mass of the straw or the yeast extract and the volume ratio of the soil slurry, and the volume of the soil slurry is equal to the volume of water.
In the step (5), the alkaline substance is magnesium oxide, quicklime or sodium hydroxide.
The heavy metal comprises Pb, Zn, Cd, Cu, Ni, Mn and the like.
3. Advantageous effects
The filamentous fungus strain Penicillium simplicicum SimplicissimuNAU-12 and Aspergillus niger A80 of the present invention are both non-pathogenic and safe to humans and animals. The method utilizes agricultural waste straws pretreated by alkali heat as a growth carbon source and utilizes the composite filamentous fungi which can produce various micromolecular organic acids to bioleach and treat the heavy metal contaminated soil, thereby overcoming the defects of low efficiency of removing heavy metal by using a single strain and high cost of adding commercial carbon source in the prior art. The treatment technology is carried out at normal temperature and normal pressure, and has the advantages of less reaction equipment, low energy consumption and easy practical application after large scale production.
Drawings
FIG. 1 shows the colony morphology of Penicillium simplicicum NAU-12 on PDA plates.
FIG. 2 shows the dynamic of small organic acid production in pure culture in Penicillium simplicissimum NAU-12.
FIG. 3 shows the colony morphology of A.niger A80 on PDA plates.
FIG. 4 shows the dynamic of production of small organic acids in Aspergillus niger A80 during pure culture.
Biological material preservation information
NAU-12, classified and named Penicillium simplicinum (Penicillium simplicinum), was deposited in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 25/6/2015 with the deposition address: no. 3 of Xilu No.1 of Beijing, Chaoyang, the institute of microbiology of Chinese academy of sciences, the preservation number is CGMCC NO. 10990.
A80, classified and named Aspergillus niger, deposited in China general microbiological culture Collection center (CGMCC) at 1 month and 10 days 2011, with the deposition address: the collection number of the microorganism is CGMCC NO.4533, No. 3 of Xilu No.1 of Beijing, Chaoyang, Beijing, and institute of microbiology of Chinese academy of sciences.
Detailed Description
The technical solution of the present invention is further illustrated by the following examples.
Example 1 isolation and cultivation of Penicillium simplisimum NAU-12 and Aspergillus niger A80:
weighing 10g of soil collected from a certain copper mine of Anhui cuprum, pouring the soil into a triangular flask filled with a certain volume of sterile water, performing shaking culture at 28 ℃ and 120r/min for 1d, sucking 2mL of bacterial suspension from the triangular flask, and coating the bacterial suspension on a solid growth medium (NaNO)3：1.5g/L，K2HPO4：0.5g/L、KCl：0.025g/L，MgSO4: 0.025g/L, yeast extract: 1.5g/L, sucrose: 35g/L, agar: 20g/L), and culturing at 28 ℃ for 4 days. Selecting a suspected strain with developed hyphae, and streaking the suspected strain in cresol containing 3 percent of bromineOn a solid growth culture medium of green (acid production indicator), after complete color development, selecting a colony with a large yellow transparent ring around the colony, and carrying out streaking separation for 2 times to obtain two purified strains which are respectively named as NAU-12 and A80. Referring to the handbook of fungal identification and the book of Chinese funguses, strains NAU-12 and A80 were preliminarily identified as Penicillium and Aspergillus, respectively. Extracting DNAs of NAU-12 and A80 by adopting a fungus DNA kit, amplifying 18S rDNA-ITS sequences by PCR, and finally identifying NAU-12 and A80 as penicillium simplicissimum and aspergillus niger by rDNA-ITS gene sequencing and BLAST homology comparison with NCBI database.
Liquid growth culture medium is adopted to carry out amplification culture on the strains NAU-12 and A80 respectively:
selecting Penicillium notatum NAU-12 thallus from the inclined plane, inoculating into liquid growth culture medium, performing shake culture at 25-35 deg.C for 5-7 days at 120-8spores/mL NAU-12 microbial inoculum. The acid production type and acid production amount of the strain NAU-12 during the amplification culture process are detected by high performance liquid chromatography, as shown in FIG. 2, it is found that the strain NAU-12 mainly produces gluconic acid and oxalic acid (the acid production amount in 12 days is 56mM and 42mM respectively), and also has a small amount of citric acid (the yield in 12 days is 8 mM).
Selecting Aspergillus niger A80 thallus from the inclined plane, inoculating into liquid growth culture medium, and performing shaking culture at 25-35 deg.C for 4-6 days at 120-7spores/mL A80 inoculum. The acid production type and acid production amount of the strain A80 in the process of the expanded culture are detected by high performance liquid chromatography, as shown in FIG. 4, the strain A80 is mainly used for producing citric acid (the acid production amount in 15 days is 68mM), and is combined with a small amount of malic acid and oxalic acid (the acid production amount in 15 days is 3mM and 7mM respectively).
The liquid growth culture medium comprises: NaNO3 1.5g/L，K2HPO4 0.5g/L，KCl 0.025g/L，MgSO40.025g/L, 1.5g/L of yeast extract, 35g/L of cane sugar, natural pH and sterilization.
Example 2 treatment of soil contaminated with Cu, a heavy Metal alone
The method for treating Cu-polluted soil by bioleaching with composite filamentous fungi comprises the following steps:
step (1), preparation of microbial inoculum: according to the method of example 1Respectively obtaining the density of the bacteria of 108spore/mL NAU-12 and 107Mixing spore/mL A80 microbial inoculum according to the volume ratio of 1:1 to obtain a composite microbial inoculum for inoculation;
step (2), preparing a wheat straw carbon source: mechanically pulverizing air-dried wheat straw (total solid content 92.5%) to 1cm, soaking in 1.5% NaOH solution at 80 deg.C for 2 hr for alkali-heat pretreatment;
step (3), soil Cu biological removal: the soil sample is taken from the soil in a certain copper ore area of the cuprum of Anhui, is yellow red soil, and has the pH value of 6.22. Through detection, the soil sample is mainly polluted by single Cu, and the Cu content reaches 1098 mg/kg.
2g of Cu-contaminated soil is taken, 200mL of water is added for dilution, the prepared soil slurry is put into a triangular flask, 10g of wheat straw (used as a carbon source) subjected to alkali heat pretreatment and 0.3g of yeast extract are added, and then 12mL of composite microbial inoculum is inoculated.
In addition, four control treatment groups were set: a. no inoculation of any microbial inoculum; b. only 12mL of the penicillium notatum NAU-12 microbial inoculum is inoculated; c. only 12mL of Aspergillus niger A80 microbial inoculum is inoculated; d. 12mL of the composite filamentous fungus agent was inoculated, but 10g of sucrose was used as a carbon source instead of wheat straw.
Subsequently, the different treatments were placed in a rotary shaker at 30 ℃ and 150r/min and aerobically incubated for 15 days. And monitoring the pH value of the soil slurry at the 15 th day, reducing the pH value of the soil slurry inoculated with the composite filamentous fungi agent by taking wheat straws as a carbon source to 2.46, reducing the pH value of the soil slurry inoculated with the composite filamentous fungi agent by taking cane sugar as a carbon source to about 2.42, and reducing the pH value of the soil slurry singly inoculated with the penicillium simplicissimum NAU-12 microbial agent or the aspergillus niger A80 microbial agent to 3.52.
Step (4), pouring out the treated soil slurry, centrifuging for 15min at 3000r/min, and filtering by using filter paper to obtain the slurry containing Cu2+The filtrate and soil mud precipitate.
Step (5) of adding Cu2+NaOH is added into the filtrate to adjust the pH value to 8.0 so as to ensure that Cu is added2+Conversion to Cu (OH)2Precipitating and discharging wastewater; adding quicklime into the soil slurry sediment to adjust the pH to 6.0, drying the sample, and adopting HCl-HNO3-HClO4And (3) digesting, inductively coupling and detecting the content of the residual Cu in the soil sample by plasma emission spectroscopy (see table 1). According toAnd calculating the Cu removal rate according to the difference of the Cu contents of the soil before and after treatment. Through calculation, the Cu removal rates of two groups of treatments (using wheat straws or sucrose as a carbon source) inoculated with the composite microbial inoculum are similar (82-83%), both can reach the national soil environment quality standard (GB15618-1995), and are obviously higher than the Cu removal rate of the treatment inoculated with single NAU-12 or A80.
TABLE 1 variation of Cu content in soil before and after different treatments
Example 3 treatment of soil contaminated with Complex heavy Metal Pb-Zn-Cd
The method for treating the soil polluted by the composite heavy metal Pb-Zn-Cd by bioleaching the composite filamentous fungi comprises the following steps:
step (1), preparation of microbial inoculum: the same as example 2;
step (2), preparing a rice straw carbon source: mechanically pulverizing air-dried rice straw (total solid content is 90.2%) to 1cm, soaking in 1% NaOH solution at 100 deg.C for 1.5 hr for alkali-heat pretreatment;
step (3), biologically removing Zn, Pb and Cd in soil: the soil sample is collected from the polluted soil around a lead and zinc mine plant in Dexing, Jiangxi, is the quaternary red soil, and has the pH value of 5.7. Through detection, the Zn, Pb and Cd contents in the soil sample are 1520mg/kg, 570mg/kg and 28mg/kg respectively, and exceed the national soil environment quality standard (GB 15618-1995).
250g of Pb-Zn-Cd combined contaminated soil is taken, 10L of water is added for dilution, the prepared soil slurry is put into a reactor, 700g of rice straw (used as a carbon source) and 15g of yeast extract are added, and then 1L of the compound microbial inoculum is inoculated. Referring to example 2, four control groups were set: a. inoculating only 1L of penicillium simplicissimum NAU-12 microbial inoculum, inoculating only 1L of Aspergillus niger A80 microbial inoculum, and inoculating 1L of composite filamentous microbial inoculum but using 700g of sucrose to replace wheat straw as a carbon source.
Subsequently, each set of treated reactors was treated with aerobic stirring at 30 ℃ and 150r/min for 20 d. And monitoring the pH value of the soil slurry at the 20 th day, reducing the pH value of the soil slurry inoculated with the composite filamentous bacterium agent by taking wheat straws as a carbon source to about 2.4, reducing the pH value of the soil slurry inoculated with the composite filamentous bacterium agent by taking cane sugar as a carbon source to about 2.5, and reducing the pH value of the soil slurry singly inoculated with the penicillium simplicissimum NAU-12 bacterium agent or the aspergillus niger A80 bacterium agent to about 3.5.
Step (4), pouring out the treated soil slurry, centrifuging for 15min at 3000r/min, and filtering to obtain the Zn-containing soil slurry2+、Pb2+And Cd2+The filtrate and the heavy metal-removed soil slurry precipitate.
Step (5) of adding Zn2+、Pb2+And Cd2+Adding MgO into the filtrate to adjust the pH value to 7.5, converting heavy metal into hydroxide precipitate, and discharging wastewater; adding quicklime into the soil mud precipitate to adjust pH to 6.0, drying the sample, and adding HCl-HNO3-HClO4And (3) digesting, and measuring the content of residual Zn, Pb and Cd in the soil sample by inductively coupled plasma emission spectroscopy (see table 2). And calculating the removal rate of the heavy metal according to the difference of the heavy metal contents in the soil sample before and after treatment. Through calculation, the removal rates of Zn, Pb and Cd in two groups of treatments (taking rice straws or sucrose as a carbon source) inoculated with the composite microbial inoculum are not greatly different and are respectively 83-85%, 40-45% and 93-95%, the removal rates can reach the national soil environment quality standard (GB15618-1995), and are obviously higher than the removal rate of Cu treated by inoculating single NAU-12 or A80.
TABLE 2 variation of Zn, Pb and Cd content in soil before and after different treatments
Claims (9)
1. Filamentous fungus strain NAU-12, classified and named as Penicillium simplicissimum (A)Penicillium simplicissimum) And is preserved in the China general microbiological culture Collection center at 25.6.2015, with the preservation number of CGMCC NO. 10990.
2. The use of the filamentous fungal strain NAU-12 of claim 1 in the treatment of heavy metal contaminated soil, said heavy metal being at least one of Pb, Zn, Cd, Cu.
3. A composite filamentous fungus preparation is characterized by that it contains 1-2X 10 of fungus8spore/mL Penicillium simplicicum NAU-12 microbial inoculum and containing 1-2 × 107spore/mL Aspergillus niger A80 microbial inoculum is formed by equal-volume mixing; wherein, Penicillium simplicissimum NAU-12 is classified and named as Penicillium simplicissimum (A)Penicillium simplicissimum) The strain is preserved in the China general microbiological culture Collection center at 25.6.2015, with the preservation number of CGMCC NO. 10990; aspergillus niger A80, classified and named as Aspergillus niger (A)Aspergillus niger) And is preserved in China general microbiological culture Collection center (CGMCC) at 2011, 1 month and 10 days, with the preservation number of CGMCC NO. 4533.
4. The method for preparing the composite filamentous fungal agent as claimed in claim 3, characterized by comprising the steps of: selecting NAU-12 thallus, inoculating into liquid growth culture medium, and shake culturing at 25-35 deg.C for 5-7 days; selecting A80 thallus, inoculating into liquid growth culture medium, and shake culturing at 25-35 deg.C for 4-6 days; after the culture is finished, mixing the two cultures according to the volume ratio of 1:1 to obtain the composite microbial inoculum.
5. The method for preparing the composite filamentous fungal agent as claimed in claim 4, wherein the liquid growth medium is: NaNO3 1.5 g/L、K2HPO4 0.5 g/L，KCl 0.025 g/L，MgSO40.025g/L, 1.5g/L of yeast extract, 35g/L of sucrose, natural pH and sterilization.
6. The use of the composite filamentous fungal agent as claimed in claim 3 in the treatment of heavy metal contaminated soil, wherein the heavy metal is at least one of Pb, Zn, Cd and Cu.
7. A method for treating heavy metal contaminated soil by bioleaching is characterized by comprising the following steps:
step (1), preparing a composite filamentous fungus agent for inoculation: selecting NAU-12 thallus with preservation number of CGMCC NO.10990, inoculating into liquid growth culture medium, and shake culturing at 25-35 deg.C for 5-7 days; selecting A80 thallus with preservation number of CGMCC NO.4533, inoculating into liquid growth culture medium, and performing shake culture at 25-35 deg.C for 4-6 days; after the culture is finished, mixing the two cultures according to the volume ratio of 1:1 to obtain a compound filamentous fungus agent;
step (2), preparation of carbon source: crushing the straws to 1-1.5cm, and soaking in 0.5-1.5% NaOH solution at 80-100 ℃ for 1-2h for alkali-heat pretreatment;
step (3), treating heavy metal contaminated soil: diluting heavy metal soil with water to prepare soil slurry with the slurry concentration of 1-5%, putting the soil slurry into a reactor, adding 40-80g/L of straws or cane sugar subjected to alkali-heat pretreatment as a carbon source, adding 1-3g/L of yeast extract, inoculating a composite filamentous fungus agent according to 5-10% of the volume of the treated soil slurry, stirring the mixture at the temperature of 25-35 ℃ and the rotation speed of 120-150r/min for 15-20 days, and pumping out the treated soil slurry; wherein the heavy metal is at least one of Pb, Zn, Cd and Cu;
step (4), solid-liquid separation: carrying out soil-water solid-liquid separation on the soil slurry treated in the step (3) to obtain a soil slurry precipitate and a clear liquid containing heavy metal ions;
step (5), neutralizing and precipitating: adding alkali into the clear liquid containing heavy metal to precipitate heavy metal ions; and neutralizing the soil mud sediment by using quicklime.
8. The method for bioleaching heavy metal contaminated soil according to claim 7, wherein in the step (1), the liquid growth mediums are all: NaNO3 1.5 g/L、K2HPO4 0.5 g/L，KCl 0.025 g/L，MgSO40.025g/L, 1.5g/L of yeast extract, 35g/L of sucrose, natural pH and sterilization;
the rotation speed of the shaking culture is 120-150 r/min.
9. The method for bioleaching heavy metal contaminated soil according to claim 7, wherein in the step (2), the straws are air-dried wheat or rice straws, and the total solid content of the straws is more than 90%;
in the step (3), the adding amount of the straw subjected to alkali-heat pretreatment is 50-70g/L, and the adding amount of the yeast extract is 1-2 g/L;
in the step (5), the alkali is magnesium oxide, quicklime or sodium hydroxide.
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