阅读说明：本技术 一种农业活动区土壤层重金属的微生物修复方法 (Microbial remediation method for heavy metals in soil layer of agricultural activity area ) 是由 王雷 席北斗 刘健聪 檀文炳 李翔 王金生 闫政 徐剑锋 李彤彤 王杨杨 刘慧� 于 2020-09-11 设计创作，主要内容包括：本发明公开了一种农业活动区土壤层重金属的微生物修复方法,采用微生物修复剂对重金属污染土壤进行修复,所述微生物修复剂包括枯草芽孢杆菌和桃红荚硫菌,以及任选的巨大芽孢杆菌,特别地在重金属污染土壤中还加入零价铁负载生物炭材料,所述零价铁负载生物炭材料通过将富铁类生物质在氮气气氛下,进行程序升温热解炭化得到。该方法对污染土壤修复效果好,重金属去除效果优异。(The invention discloses a microbial remediation method for heavy metal in a soil layer of an agricultural activity area, which is characterized in that a microbial remediation agent is adopted to remediate heavy metal contaminated soil, the microbial remediation agent comprises bacillus subtilis, thiobacillus persicae and optional bacillus megatherium, particularly, a zero-valent iron loaded biochar material is also added into the heavy metal contaminated soil, and the zero-valent iron loaded biochar material is obtained by carrying out programmed heating pyrolysis carbonization on iron-rich biomass in a nitrogen atmosphere. The method has good remediation effect on the polluted soil and excellent heavy metal removal effect.)

1. A microbial remediation method for heavy metal in soil layers of agricultural activity areas adopts a microbial remediation agent to remediate heavy metal contaminated soil, wherein the microbial remediation agent comprises bacillus subtilis and rhododendron persicum, and preferably, the quantity ratio of the bacillus subtilis to the rhododendron persicum is (1-3) to (1-2).

2. The method for remediating heavy metals in soil layers of agricultural activity areas as recited in claim 1, wherein the ratio of the number of bacillus subtilis to the number of thiobacillus persicae is (2-3): 1.5.

3. The method of claim 1, wherein the microbial remediation agent further comprises Bacillus megaterium, wherein the ratio of Bacillus subtilis, Thiobacillus persimilis and Bacillus megaterium is (1-3) to (1-2): (1-2).

4. The method for microbial remediation of soil layer heavy metals of an agricultural activity area of claim 3, wherein the ratio of the number of Bacillus subtilis, Thiobacillus persicaria and Bacillus megaterium is (2-3) to 1.5:1, preferably 2:1.5: 1.

5. The microbial remediation method of soil layer heavy metals of an agricultural activity area of claim 1, wherein before remediation of heavy metal contaminated soil with the microbial remediation agent, organic fertilizer is applied, the organic fertilizer being produced by: cutting corn stalks into 2-5 cm segments, mixing animal wastes, corn stalk particles and soybean meal according to the weight ratio of 6:2:2, stacking and fermenting for 15-20 days, wherein the fermentation temperature is not more than 60 ℃.

6. The microbial remediation method of soil layer heavy metals of an agricultural activity area of claim 1, wherein a zero-valent iron-loaded biochar material is further added to the heavy metal contaminated soil, wherein the zero-valent iron-loaded biochar material is obtained by subjecting iron-rich biomass to temperature programmed pyrolysis carbonization in a nitrogen atmosphere, and the iron-rich biomass is rice straw planted in acid red soil.

7. The method for microbial remediation of soil layers of heavy metals of an agricultural active area of claim 6, wherein the programmed temperature comprises three phases:

a first temperature rise stage: heating to 300 ℃ at the heating rate of 5-10 ℃/min, and keeping the temperature for 30-40 min;

a second temperature rising stage: heating from 300 ℃ to 480 ℃ at a heating rate of 5-8 ℃/min, and keeping the temperature for 15-35 min;

a third temperature rise stage: raising the temperature from 480 ℃ to 600 ℃ and 850 ℃ at the temperature raising rate of 5-8 ℃/min, and keeping the temperature for 15-20 min.

8. The method for microbial remediation of soil layer heavy metals of an agricultural activity area of claim 7, wherein,

in the first temperature rise stage, the temperature is raised at the temperature rise rate of 7-10 ℃/min, preferably at the temperature rise rate of 8 ℃/min, and is kept for 30-35 min, preferably 30 min after being raised to 300 ℃;

in the second temperature rise stage, the temperature is raised at the temperature rise rate of 6-8 ℃/min, preferably at the temperature rise rate of 6 ℃/min, and is kept for 20-30 min, preferably 20 min after the temperature is raised to 480 ℃;

in the third temperature raising stage, the temperature is raised at a temperature raising rate of 5-6 ℃/min, preferably at a temperature raising rate of 5 ℃/min, and is kept for 15-20 min, preferably 20 min after being raised to the temperature of 600-850 ℃.

9. The method for microbial remediation of soil layer heavy metals of an agricultural activity area of claim 7, wherein,

in the third temperature-raising stage, the temperature is raised to the temperature of 700-800 ℃, and the temperature is maintained at the temperature.

10. The microbial remediation method of claim 7 wherein the temperature is raised to a temperature of 750-800 ℃ in the third temperature raising stage, and the temperature is maintained at this temperature.

Technical Field

The invention relates to the field of environmental protection, in particular to a microbial remediation method for heavy metal contaminated farmland soil.

Background

An agricultural activity area, which is an area where agricultural production activities are performed, generally refers to an area where food crops and cash crops are mainly planted, centered on the farming industry. The most important data in the agricultural activity area is soil.

With the deterioration of the environment, such as the influence of industrial waste, dust, tail gas and the like, the soil in the agricultural activity area is increasingly polluted by heavy metals. Since heavy metals can enter all ecosystems, they have serious effects on plants, animals and humans, and in particular, heavy metals can be enriched in soil and organisms.

Due to the characteristics of heavy metal elements and the characteristics of multi-medium, multi-interface, multi-component, non-uniformity and the like of the soil environment, the soil heavy metal pollution has the following characteristics: concealment and hysteresis, irreversibility and longevity, regionality and severity, management difficulties and long periods.

The utilization and treatment of heavy metal contaminated soil are the current research focus. The method and the technology for utilizing and treating the heavy metal contaminated soil mainly take the effectiveness of plant restoration and passivation of the heavy metal in the soil by applying a passivator. In recent years, microbial remediation technology is gradually developed, and plays an increasingly important role in remediation of heavy metal contaminated soil, particularly heavy metal contaminated farmland soil.

In the research on the treatment of heavy metal sewage, researchers find that the zero-valent iron modified charcoal has a good effect of removing heavy metals and hardly causes secondary pollution.

Disclosure of Invention

The inventor researches and discovers that: for the heavy metal pollution of the soil layer in the agricultural activity area, the microorganisms without environmental load are selected for repairing, particularly the microorganisms are organically combined with the zero-valent iron modified biochar material for repairing, and particularly the carefully selected suitable microorganisms and the zero-valent iron modified biochar material with long-term stable applicability have obvious effect of removing the heavy metals.

The object of the present invention is to provide the following:

the invention provides a microbial remediation method for heavy metal in a soil layer of an agricultural activity area, which is implemented by adopting a microbial remediation agent to remediate heavy metal contaminated soil, wherein the microbial remediation agent comprises bacillus subtilis and myrothiacommune, and preferably, the quantity ratio of the bacillus subtilis to the myrothiacommune is (1-3) to (1-2).

In a second aspect, the invention provides a microbial remediation method for heavy metals in soil layers of agricultural activity areas, wherein the quantity ratio of the bacillus subtilis to the red peach pods thiobacillus is (2-3): 1.5.

In a third aspect, the invention provides a microbial remediation method for heavy metals in soil layers of agricultural activity areas, wherein the microbial remediation agent further comprises bacillus megaterium, and preferably, the number ratio of bacillus subtilis, red peach thiobacillus and bacillus megaterium is (1-3) to (1-2): (1-2).

The invention also provides a microbial remediation method for heavy metals in soil layers of agricultural activity areas, wherein the number ratio of the bacillus subtilis to the red peach thiobacillus to the bacillus megatherium is (2-3) 1.5:1, preferably 2:1.5: 1.

The invention provides a microbial remediation method for heavy metals in soil layers of agricultural activity areas, wherein before remediation of heavy metal contaminated soil by using a microbial remediation agent, an organic fertilizer is applied, and the organic fertilizer is prepared by the following steps: cutting corn stalks into 2-5 cm segments, mixing animal wastes, corn stalk particles and soybean meal according to the weight ratio of 6:2:2, stacking and fermenting for 15-20 days, wherein the fermentation temperature is not more than 60 ℃.

The invention provides a microbial remediation method for heavy metals in soil layers of agricultural activity areas as described above, wherein a zero-valent iron-loaded biochar material is further added into the heavy metal contaminated soil, the zero-valent iron-loaded biochar material is obtained by performing temperature programming pyrolysis and carbonization on iron-rich biomass in a nitrogen atmosphere, and the iron-rich biomass is rice straw planted in acid red soil.

A seventh aspect of the invention provides a method for microbial remediation of soil layer heavy metals in an agricultural active area as described above, wherein the programmed temperature comprises three stages:

a first temperature rise stage: heating to 300 ℃ at the heating rate of 5-10 ℃/min, and keeping the temperature for 30-40 min;

a second temperature rising stage: heating from 300 ℃ to 480 ℃ at a heating rate of 5-8 ℃/min, and keeping the temperature for 15-35 min;

a third temperature rise stage: raising the temperature from 480 ℃ to 600 ℃ and 850 ℃ at the temperature raising rate of 5-8 ℃/min, and keeping the temperature for 15-20 min.

An eighth aspect of the present invention provides the method for microbial remediation of soil layer heavy metals of agricultural activity areas, as described above,

in the first temperature rise stage, the temperature is raised at the temperature rise rate of 7-10 ℃/min, preferably at the temperature rise rate of 8 ℃/min, and is kept for 30-35 min, preferably 30 min after being raised to 300 ℃;

in the second temperature rise stage, the temperature is raised at the temperature rise rate of 6-8 ℃/min, preferably at the temperature rise rate of 6 ℃/min, and is kept for 20-30 min, preferably 20 min after the temperature is raised to 480 ℃;

in the third temperature raising stage, the temperature is raised at a temperature raising rate of 5-6 ℃/min, preferably at a temperature raising rate of 5 ℃/min, and is kept for 15-20 min, preferably 20 min after being raised to the temperature of 600-850 ℃.

The ninth aspect of the present invention provides the method for remedying a heavy metal in a soil layer of an agricultural activity-area by microorganisms as described above,

in the third temperature-raising stage, the temperature is raised to the temperature of 700-800 ℃, and the temperature is maintained at the temperature.

The tenth aspect of the invention provides the microbial remediation method for the heavy metals in the soil layer of the agricultural activity area, wherein in the third temperature-raising stage, the temperature is raised to the temperature of 750-800 ℃, and the temperature is maintained at the temperature.

The microbial remediation method for the heavy metal in the soil layer of the agricultural activity area, provided by the invention, has the following advantages:

(1) the invention selects the composite microbial inoculum consisting of bacillus subtilis and rhododendron persicum, in particular to the composite microbial inoculum consisting of bacillus subtilis, rhododendron persicum and bacillus megatherium as the repairing microorganism, has obvious repairing effect on the heavy metal in the soil layer of the agricultural activity area, and has high removal rate of lead, cadmium and copper.

(2) According to the invention, the organic fertilizer is adopted to improve the soil environment, and then the compound microbial inoculum is used as a microorganism to repair, so that the heavy metal removal rate is further improved, and the soil environment is improved.

(3) The zero-valent iron load biochar material prepared from the iron-rich biomass promotes microbial remediation and is beneficial to improving the removal rate of heavy metals.

(4) The zero-valent iron loaded biochar material prepared from the iron-rich biomass has a stable long-term and lasting repairing effect because the zero-valent iron is loaded in the biochar in situ.

Detailed Description

The present invention will be described in further detail below with reference to preferred embodiments and examples. The features and advantages of the present invention will become more apparent from the description.

The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

In the invention, the composite microbial agent consisting of the bacillus subtilis and the rhododendron persicum is used as the microbial repairing agent, so that the heavy metal contaminated soil of a farmland can be remarkably repaired, and the soil environment is hardly influenced or damaged.

In a preferred embodiment, the ratio of the number of Bacillus subtilis to the number of Thiobacillus persimilis is (1-3): (1-2), preferably (2-3):1.5, and more preferably 2: 1.5. Under the condition, the soil stabilizer has ideal effect of removing heavy metals in soil, and particularly has high removal rate of lead, cadmium and copper.

In a preferred embodiment, the microbial remediation agent used in the present invention further comprises Bacillus megaterium, which facilitates the removal of heavy metals. Preferably, the number ratio of the bacillus subtilis to the rhododendron persicum to the bacillus megaterium is (1-3) to (1-2): (1-2) more preferably, the ratio of the number of Bacillus subtilis, Thiobacillus persimilis and Bacillus megaterium is (2-3) 1.5:1, most preferably 2:1.5:1, and the comprehensive remediation effect on the heavy metals in the soil is excellent.

In the present invention, Bacillus subtilis, Thiobacillus persimilis and Bacillus megaterium are not particularly limited, and commercially available bacteria may be used or the bacteria may be cultured by themselves.

The Bacillus subtilis is enlarged and cultured to 1 x 10⁸CFU/ml, enlarged culture of Thiobacillus persimilis to 1 × 10⁸CFU/ml, scale-up culture of Bacillus megaterium to 1X 10⁸CFU/ml, then according to the volume ratio (1-3):(1-2): (1-2), preferably 2:1.5:1, most preferably 2:1.5:1, to obtain the complex microbial inoculum.

In the case of the soil in the agricultural activity area contaminated with heavy metals, the inoculation amount of the microorganism as the complex microbial inoculum is 30 to 50ml/kg of soil, preferably 50ml/kg of soil.

As an alternative embodiment, the bacillus subtilis, the rhododendron persicum and the bacillus megaterium can be respectively and independently inoculated into the heavy metal contaminated soil.

If the inoculation amount is too low, the heavy metal removal effect is poor, and if the inoculation amount is too high, adverse effects may be brought to the soil environment.

As the heavy metal contaminated farmland soil to be remediated, the depth of inoculation of the microorganism is preferably 10 to 40cm, preferably 20 cm.

In the invention, before the microbial repairing agent is used for repairing the heavy metal contaminated soil, the organic fertilizer is applied to the soil to be repaired, so that the repairing effect can be promoted, and the comprehensive performance of the soil can be improved.

In a preferred embodiment, the organic fertilizer is prepared by: cutting corn stalks into 2-5 cm segments, mixing animal wastes, corn stalk particles and soybean meal according to the weight ratio of 6:2:2, stacking and fermenting for 15-20 days, wherein the fermentation temperature is not more than 60 ℃. And if the temperature exceeds 60 ℃, turning the pile.

The inventor also finds that the zero-valent iron loaded biochar material can remarkably improve the repairing effect compared with the method that only the composite microbial inoculum microorganism is used as the repairing material.

In a preferred embodiment, the zero-valent iron-loaded biochar material is obtained by performing temperature programmed pyrolysis carbonization on iron-rich biomass in a nitrogen atmosphere, wherein the iron-rich biomass is rice straws planted in acid red soil.

In the acid red soil, the iron ion content is high, the iron ion is absorbed by rice and transmitted to various organ tissues such as roots, stems, leaves and the like, the iron ion/ferrous ion is enriched in crop plants after long-term accumulation of the growth cycle, and the zero-valent iron is loaded in the charcoal in situ through in-situ reaction in pyrolysis and carbonization.

In a preferred embodiment, the temperature programming comprises the following three phases:

the first temperature raising stage is carried out at a temperature raising rate of 5 to 10 ℃/min, preferably at a temperature raising rate of 7 to 10 ℃/min, preferably at a temperature raising rate of 8 ℃/min, to 300 ℃, and is maintained at this temperature for 30 to 40 minutes, preferably 30 to 35 minutes, more preferably 30 minutes.

And a second temperature raising stage for raising the temperature at a temperature raising rate of 5 to 8 ℃/min, preferably at a temperature raising rate of 6 to 8 ℃/min, more preferably at a temperature raising rate of 6 ℃/min, from 300 ℃ to 480 ℃, and holding the temperature for 15 to 35 minutes, preferably for 20 to 30 minutes, more preferably for 20 minutes.

In the third temperature raising stage, the temperature is raised at a temperature raising rate of 5-8 ℃/min, preferably at a temperature raising rate of 5-6 ℃/min, more preferably at a temperature raising rate of 5 ℃/min, from 480 ℃ to 600-.

In the invention, the microorganism and the zero-valent iron-loaded biochar are respectively inoculated and mixed into the heavy metal contaminated soil to be repaired, or the microorganism and the zero-valent iron-loaded biochar are mixed uniformly and then mixed into the soil. However, the microorganism and the zero-valent iron-loaded biochar are preferably inoculated and mixed into the heavy metal contaminated soil to be repaired respectively, and practice suggests that the effect is better.

Examples

The present invention is further described below by way of specific examples, which are merely exemplary and do not limit the scope of the present invention in any way.

Example 1

Example 1

And planting the rice in the red soil for 100 days. After 100 days, the rice is pulled out, the paddy is removed, and the straw is remained. Washing straws with deionized water, soaking in 0.01M dilute hydrochloric acid for 1 hr, dividing the straws into root and straw parts, respectively drying at 70.0 deg.C, and pulverizing into 1mm granules. The root particles are placed in a tubular muffle furnace, nitrogen is introduced, and the pyrolysis carbonization reaction is carried out by the following temperature programming: heating to 300 ℃ at the heating rate of 8 ℃/min, and carrying out heat preservation reaction for 30 min; heating from 300 ℃ to 480 ℃ at the heating rate of 6 ℃/min, and carrying out heat preservation reaction for 20 minutes; heating from 480 ℃ to 750 ℃ at the heating rate of 5 ℃/min, and carrying out heat preservation reaction for 20 minutes to obtain the zero-valent iron-loaded biochar material.

Drying the pollution-free black soil taken from the test field, rolling, naturally drying, sterilizing and sieving by a 100-mesh sieve. Adding PbCl₂、CdCl₃、CuSO₄The water solution comprises 300mg/kg of lead (calculated by Pb), 50mg/kg of cadmium (calculated by Cd) and 100mg/kg of copper (calculated by Cu), is uniformly stirred, aged and placed for a week, naturally air-dried, crushed, sieved by a 100-mesh sieve and simulates a polluted soil sample.

Will be 1 × 10⁸CFU/ml Bacillus subtilis and 1X 10⁸And mixing the CFU/ml of the thiomyces persicae at a volume ratio of 2:1.5 to obtain the composite microbial inoculum.

Inoculating the composite microbial inoculum into a polluted soil sample according to the amount of 50ml/kg soil, then mixing a zero-valent iron-loaded biochar material into the soil according to the amount of 200mg/kg soil, placing the mixture into a phytotron for 60 days, and supplementing water by deionized water every other day to keep the soil humidity.

After 60 days, the contents of Pb, Cd and Cu were 40.6mg/kg, 0.28mg/kg and 44.9mg/kg, respectively.

Example 2

The rice is planted in the red soil for 100 days, and 100mg/L citric acid aqueous solution is sprayed every week. After 100 days, the rice is pulled out, the paddy is removed, and the straw is remained. Washing straws with deionized water, soaking in 0.01M dilute hydrochloric acid for 1 hr, dividing the straws into root and straw parts, respectively drying at 70.0 deg.C, and pulverizing into 1mm granules. The root particles are placed in a tubular muffle furnace, nitrogen is introduced, and the pyrolysis carbonization reaction is carried out by the following temperature programming: heating to 300 ℃ at the heating rate of 10 ℃/min, and carrying out heat preservation reaction for 30 min; heating from 300 ℃ to 480 ℃ at the heating rate of 6 ℃/min, and carrying out heat preservation reaction for 20 minutes; heating from 480 ℃ to 750 ℃ at the heating rate of 5 ℃/min, and carrying out heat preservation reaction for 20 minutes to obtain the zero-valent iron-loaded biochar material.

Drying the pollution-free black soil taken from the test field, rolling, naturally drying, sterilizing and sieving by a 100-mesh sieve. Adding PbCl₂、CdCl₃、CuSO₄The water solution comprises 250mg/kg of lead (calculated by Pb), 70mg/kg of cadmium (calculated by Cd) and 100mg/kg of copper (calculated by Cu), is uniformly stirred, aged and placed for a week, naturally air-dried, crushed, sieved by a 100-mesh sieve and simulates a polluted soil sample.

Will be 1 × 10⁸CFU/ml Bacillus subtilis, 1X 10⁸CFU/ml of Thiobacillus persimilis and 1X 10⁸And mixing the CFU/ml bacillus megaterium according to the volume ratio of 2:1.5:1 to obtain the composite microbial inoculum.

Inoculating the composite microbial inoculum into a polluted soil sample according to the amount of 50ml/kg soil, then mixing a zero-valent iron-loaded biochar material into the soil according to the amount of 200mg/kg soil, placing the mixture into a phytotron for 60 days, and supplementing water by deionized water every other day to keep the soil humidity.

After 60 days, the contents of Pb, Cd and Cu were 34.7mg/kg, 0.26mg/kg and 40.5mg/kg, respectively.

Example 3

Drying the pollution-free black soil taken from the test field, rolling, naturally drying, and sieving with a 100-mesh sieve. Adding PbCl₂、CdCl₃、CuSO₄The water solution comprises 500mg/kg of lead (calculated by Pb), 500mg/kg of cadmium (calculated by Cd) and 100mg/kg of copper (calculated by Cu), is uniformly stirred, aged and placed for a week, naturally air-dried, crushed, sieved by a 100-mesh sieve and simulated to be a polluted soil sample.

Cutting corn straws into 2 cm fragments, mixing the cattle manure, the corn straw particles and the bean pulp according to the weight ratio of 6:2:2, and stacking and fermenting for 18 days at the fermentation temperature of not more than 60 ℃ to obtain the biological organic fertilizer. It was applied to a simulated contaminated soil sample in an amount of 5 wt%, mixed well, and placed in a climatic incubator for 3 days.

Will be 1 × 10⁸CFU/ml Bacillus subtilis, 1X 10⁸CFU/ml of Thiobacillus persimilis and 1X 10⁸CFU/ml Bacillus megaterium in volume ratio2:1.5:1 to obtain the composite microbial inoculum.

The rice is planted in the red soil for 100 days, and 300mg/L citric acid aqueous solution is sprayed every week. After 100 days, the rice is pulled out, the paddy is removed, and the straw is remained. Washing straws with deionized water, soaking in 0.01M dilute hydrochloric acid for 1 hr, dividing the straws into root and straw parts, respectively drying at 70.0 deg.C, and pulverizing into 1mm granules. The root particles are placed in a tubular muffle furnace, nitrogen is introduced, and the pyrolysis carbonization reaction is carried out by the following temperature programming: heating to 300 ℃ at the heating rate of 8 ℃/min, and carrying out heat preservation reaction for 30 min; heating from 300 ℃ to 480 ℃ at the heating rate of 6 ℃/min, and carrying out heat preservation reaction for 20 minutes; heating from 480 ℃ to 750 ℃ at the heating rate of 5 ℃/min, and carrying out heat preservation reaction for 20 minutes to obtain the zero-valent iron-loaded biochar material.

The complex microbial inoculum is inoculated in the amount of 50ml/kg soil, and then the zero-valent iron-loaded biochar material is blended into the soil in the amount of 200mg/kg soil. Then placing the soil in an artificial climate incubator for 60 days, and supplementing water by deionized water every other day to keep the soil humidity.

After 60 days, the contents of Pb, Cd and Cu were 26.4mg/kg, 0.23mg/kg and 36.3mg/kg, respectively.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention.