CN114480243A

CN114480243A - Microbial remediation method applied to non-foreign soil mining area

Info

Publication number: CN114480243A
Application number: CN202210116263.3A
Authority: CN
Inventors: 张春
Original assignee: Qinghai Greenway Environmental Protection Biotechnology Co ltd
Current assignee: Qinghai Greenway Environmental Protection Biotechnology Co ltd
Priority date: 2022-02-07
Filing date: 2022-02-07
Publication date: 2022-05-13

Abstract

The invention discloses a microbial remediation method applied to a non-soil-affected mining area, which mainly utilizes organic combination of various strains such as sulfate reducing bacteria, photosynthetic bacteria, azotobacter and the like to construct a healthy bottom microbial ecosystem on the surfaces of bare rocks and sandy soil, can be self-balanced and self-grown under the illumination condition, can efficiently absorb water contained in air to supply microbes by secreting extracellular polymers to grow on the surfaces of the bare rocks and the sandy soil, and realizes various remediation functions such as water retention, ecological environment improvement, plant root development promotion and the like. The produced sulphide of restoration is stable, and the solubility is very low, and environmental safety possesses the cushioning effect, through founding bottom microbial ecosystem after, heavy metal is fixed with the sulphide mineral form in the farmland, and crops can grow rapidly at heavy metal pollution farmland high efficiency, the qualified agricultural product of output. The used microorganisms are environment-friendly microorganisms, can fundamentally improve the environmental ecology and have persistence.

Description

Microbial remediation method applied to non-foreign soil mining area

Technical Field

The invention relates to the technical field of environmental remediation, in particular to a refuse slag microorganism greening remediation method for an ore area without foreign soil.

Background

Many western regions such as Qinghai and the like in China have various mineral resources, such as Qilian mountain coal belts, but the Qilian mountain coal belts have serious chromium pollution. The chromium slag belongs to heavy metal dangerous waste, whereinContaining hexavalent chromium (Cr)⁶⁺) Is easy to dissolve and unstable, has strong oxidative toxicity, and can cause damage to human body and crops. Research shows that the calcium chromate (hexavalent chromium) contained in the chromium slag also has strong carcinogenic and mutagenic properties. The chromium slag which is not harmlessly treated can seriously pollute surface water, underground water and soil, and poses great threat to the ecological environment and the safety of life, property and property of people. Therefore, many places need to implement protection and restoration projects to explore and form a new ecological management system and management capacity, build ecological parks of plateau alpine mines and form long-acting mechanisms of park operation and maintenance.

However, in high-cold high-altitude areas such as the Qinghai, the geological and climatic conditions of places such as frozen soil shallow burying and grassland wetland exist, and meanwhile, mining and excavation in deep parts of mining areas cause ground subsidence and damage to hydrological and underground water channels. In addition, excessive coal gangue and coal cinder yards cause grassland burying, the wetland disappears, and the ecology is destroyed fundamentally. The gangue and waste coal pile in coal mine contain more minerals such as pyrite, pyrrhotite, arsenopyrite, galena, chalcopyrite, sphalerite, cinnabar, realgar, orpiment and the like. According to the geochemistry, the mineral tailings are soaked in rainwater, under the action of water, oxygen and microorganisms, ionic metal is further catalytically dissolved out, heavy metal-containing wastewater is formed, and the heavy metal-containing wastewater continuously seeps out of a pile and enters an underground or surface water system. The traditional chemical restoration method has poor stability and slow effect, and the project construction needs the cooperation of resources such as electric power and the like, thereby additionally consuming energy. The real recovery of the environment can not be realized only by the surface ecological restoration of the coal gangue and slag yard, and the recovery of the prior relevant microorganism technology in a short period (within three years) is difficult to realize.

Since the autotrophic acidophilic iron-sulfur oxidizing microorganisms widely exist, the mineral leaching process mediated by the autotrophic acidophilic iron-sulfur oxidizing microorganisms occurs naturally, and the occupied area of a coal mine pile is large. The traditional chemical curing repair technology (as shown in fig. 2) mainly adopts chemical agents to perform chemical reaction with heavy metal elements, so that the heavy metal elements are not leached by rainwater or absorbed by plant roots, but the leaching of acid, iron and heavy metal mediated by autotrophic acidophilic microorganisms cannot be avoided, so that the heavy metal elements are difficult to be applied to polluted sites in which heavy metal is continuously dissolved out. In addition, the traditional vegetation restoration technology needs to cover the ground surface of the tailing pond with a large amount of extra soil and then plants, so that only the re-greening of the pile ground surface can be realized, but the problem of leaching of heavy metals mediated by microorganisms in the deep part of the pile cannot be solved. And the acid-producing leaching under rainfall of the high and steep side slope of the coal mining pit causes that the re-greening vegetation is difficult to grow after the conventional soil dressing spray seeding.

Disclosure of Invention

The invention aims to solve the technical problem of the prior art and provides a microbial remediation method which is more efficient in remediation process, more stable in effect, free of electric energy consumption, strong in sustainability, environment-friendly, capable of degrading and detoxifying environmental pollutants and applied to a soil-free mining area.

In order to solve the technical problems, the invention adopts the following technical scheme: a microbial remediation method applied to an alien soil-free mining area is characterized by comprising the following steps: the method comprises the following steps of (1),

(1) sampling soil in an area to be repaired;

(2) microorganism domestication and separation:

1) inoculating 10% of mixed indigenous bacteria in the slag sample to the culture medium containing Cr (VI) with a concentration of 100 mg.L^-1The enrichment medium is cultured for 7 days at 30 ℃ under aerobic and anaerobic conditions respectively;

2) transferring the obtained bacterial liquid as mother liquid to a Cr (VI) -containing concentration of 200 mg.L after 7 days^-1Respectively culturing the NB culture medium in an aerobic condition and an anaerobic condition at 30 ℃ for 7 days, and regularly detecting the reduction rate and the bacterial quantity of Cr (VI) in the culture solution in the acclimation process until the concentration of Cr (VI) in the culture medium leads the bacteria not to grow;

3) gradient 100 mg.L in Cr (VI) concentration^-1The inoculum size is 10 percent, and the mixture is placed in a constant temperature shaking box for culture, the temperature is adjusted to be 30 ℃, and the rotating speed is 150 rpm.

4) Measuring the reduction rate and the bacterial number of Cr (VI) in the solution at regular time, continuously increasing the upper limit of the concentration of Cr (VI) which can be tolerated by the solution, and continuously domesticating;

5) selecting proper dilution for the domesticated bacteria, plating on a solid enrichment medium, uniformly coating, and culturing in an incubator at 30 deg.C for 3 days;

6) observing colony morphology, and picking up a single colony on the flat plate for four-line three-area streak culture;

(3) and (3) microorganism expanding culture: picking up single colony of microorganism, inoculating to 250mL triangular flask, shaking for 7 days, transferring into two sterilized containers containing 100 mg.L^-1Culturing in a 250mL Erlenmeyer flask of NB medium of Cr (VI); after the enrichment culture is finished, performing expansion culture to a 100L plastic bottle, culturing for later use after 7 days until the concentration of the microorganisms reaches over 107cfu/mL, and preparing to add the microorganisms into a repaired pilot-plant column;

(4) repairing: adding a sample and a bacterial liquid into the soil column, wherein the lower layer of the bacterial liquid is SRB bacteria, and the upper layer is mixed functional bacteria; after the sample is added, inoculating sulfate reducing bacteria on the lower layer of the restoration group, inoculating aerobic functional bacteria on the upper layer, and adding deionized water with the same volume into the control group; after inoculation is finished, all valves are closed, and sampling is carried out for detection after functional microorganisms grow for 7 days;

(5) and (6) detecting.

Wherein, the NB culture medium comprises the following components in concentration: biometek1 at 10 g.L^-1Biometek2 is 5 g.L^-1Biometek3 is 5 g.L^-1Cr (VI) is 100 mg.L^-1Biometek4 is 20 g.L^-1300mL of distilled water, pH 9 and culture temperature of 20-35 ℃.

During restoration, 4 double-repetition soil columns are designed, wherein the soil columns are PE plastic tubes with the column length of 2m and the diameter of 30cm, two groups are restoration groups, and the other two groups are control groups; repairing sample passing by 1cm²Screening the aperture and uniformly preparing; adding 110kg of sample and 50L of bacterial liquid into each soil column of the restoration group, wherein the lower layer of the bacterial liquid is SRB bacteria, and the upper layer is mixed functional bacteria; the control group was supplemented with 110kg of sample and 50L of water.

The repaired test was carried out by sampling 50mL from different sampling ports for testing, and solid samples were taken from different sampling ports on days 15, 45 and 90 from the inoculation of the microorganism and stored at 4 ℃ for testing.

The detection items comprise detection of concentration changes of hexavalent chromium and other heavy metals in the leachate, morphological structure changes of chromium in the solid sample, functional microbial community structure and microbial total quantity changes, permeability changes, organic matter changes, stability of precipitated products and the like.

The invention can utilize high-efficiency iron reducing bacteria to convert Fe³⁺Reduction to Fe²⁺Reducing the environmental potential of the system to make the environmental potential lower than 700mV, so that the pyrite can be basically insoluble; on the basis, the sulfate radicals are reduced to negative divalent sulfur ions by utilizing the efficient sulfate reducing bacteria, metal sulfide precipitates are formed by in situ and free metal ions, and the sulfide-state heavy metal is relatively stable in the environment, has small solubility product and can realize environmental safety.

The microbial remediation cost is low, and no secondary pollution is caused. And the microorganism can survive in the repair environment for a long time, and the repair effect is stable and sustainable. Microbial remediation itself is a self-regulating process, with H being produced by the biological sulfate reduction process₂If no heavy metal is coprecipitated with the S in a local environment, the S inhibits the harmful microorganism per se (product inhibition) and inhibits the sulfate reduction process; generation of H₂If S has heavy metal coprecipitated with it in local environment, H is obtained₂S is reduced (product inhibition is reduced), and the process of restarting the sulfate reduction process has no secondary pollution. The sulfide generated by the restoration is stable, the solubility is very low, the environment is safe, and the buffer effect is achieved.

In the aspect of damaged ecological greening and restoration, a healthy bottom layer microbial ecosystem can be constructed on the surfaces of bare rocks and sandy soil mainly by using organic combinations of various strains such as sulfate reducing bacteria, photosynthetic bacteria, azotobacter and the like, the self-balanced bottom layer ecosystem can be self-balanced and self-grow under the condition of illumination, and can efficiently absorb water contained in air to supply microbes by secreting extracellular polymers to grow on the surfaces of the bare rocks and the sandy soil, so that various restoration functions such as water retention, ecological environment improvement, plant root development promotion and the like are realized. After the bottom layer microbial ecosystem is constructed by the technology, heavy metals are fixed in the farmland in the form of sulfide minerals, crops can grow rapidly in the farmland polluted by the heavy metals in a high-efficiency mode, and finally qualified agricultural products are produced. The used microorganism is an environment-friendly microorganism, so the method is an environment-friendly restoration technology, can fundamentally improve the environmental ecology, and has durability.

Drawings

FIG. 1 is a schematic diagram of a microorganism in-situ mineralization repair mechanism of a coal mine gangue pile;

FIG. 2 is a schematic diagram of a mechanism for dissolving out heavy metals from a coal mine waste pile;

FIG. 3 is a graph showing the tolerance of a microorganism for removing chromium to hexavalent chromium;

FIG. 4 is a map of separation, purification and microscopic examination of functional microorganisms for removing chromium, wherein a, c, e, g represent morphological changes of microorganisms and chromium salts in different stages in a culture dish, and b, d, f, h correspond to the above gram stain, in order to observe the coexisting change characteristics of heavy metal chromium and microorganisms under a microscope, namely, observe the management and control of the microorganisms on the heavy metal chromium and the change of trivalent chromium and hexavalent chromium under the conditions of the microorganisms;

FIG. 5 is a growth curve of a chromium-removing functional microorganism;

FIG. 6 shows the upper Cr layer of the column pilot scale⁶⁺A concentration variation trend graph;

FIG. 7 shows the pilot Cr upper layer of the pillar⁶⁺A removal rate change map;

FIG. 8 shows a layer Cr of a pilot scale pillar⁶⁺A concentration variation trend graph;

FIG. 9 shows a layer Cr of a pilot scale pillar⁶⁺A removal rate change map;

FIG. 10 shows the pilot bottom Cr of the pillar⁶⁺A concentration variation trend graph;

FIG. 11 shows the pilot Cr layer of the pillar⁶⁺A removal rate change map;

FIG. 12 is a block diagram comparing repair data of a repair group and a control group in a repair process.

Detailed Description

The invention is further illustrated by the following specific embodiments in conjunction with the accompanying drawings:

the microbial remediation method applied to the non-loam mining area is carried out according to the following steps,

(1) sampling soil in an area to be repaired;

(2) microorganism domestication and separation:

1) inoculating 10% of mixed indigenous bacteria in the slag sample to the culture medium containing Cr (VI) with concentration of 100 mg.L^-1The enrichment medium is cultured for 7 days at 30 ℃ under aerobic and anaerobic conditions respectively;

2) transferring the obtained bacterial liquid as mother liquid to a Cr (VI) -containing concentration of 200 mg.L after 7 days^-1Respectively culturing the NB culture medium in aerobic and anaerobic conditions at 30 ℃ for 7 days, and regularly detecting the reduction rate and the bacterial number of Cr (VI) in the culture solution in the acclimation process until the concentration of Cr (VI) in the culture medium leads the bacteria not to grow;

6) observing colony morphology, and picking up single colony four-line three-zone streaking culture on a plate, as shown in figures 3 and 4;

(3) and (3) microorganism expanding culture: picking up single colony of microorganism, inoculating to 250mL triangular flask, shaking for 7 days, transferring into two sterilized containers containing 100 mg.L^-1Culturing in a 250mL Erlenmeyer flask of NB medium of Cr (VI); after the enrichment culture is finished, performing expansion culture to a 100L plastic bottle, culturing for later use after 7 days until the concentration of the microorganisms reaches over 107cfu/mL, and preparing to add the microorganisms into a repaired pilot-plant column; as shown in fig. 5.

(5) and (6) detecting.

During restoration, 4 double-repetition soil columns are designed, wherein the soil columns are PE plastic tubes with the column length of 2m and the diameter of 30cm, two groups are restoration groups, and the other two groups are control groups; repairing sample passing by 1cm²The aperture is sieved and the preparation is uniform; adding 110kg of sample and 50L of bacterial liquid into each soil column of the restoration group, wherein the lower layer of the bacterial liquid is SRB bacteria, and the upper layer is mixed functional bacteria; the control group was supplemented with 110kg of sample and 50L of water.

Analysis of results

1. Soil column pilot test upper layer results and analysis

Soil column pilot test upper layer Cr⁶⁺Concentration change meter

	Bioremediation-1	Control-1
			Background sample	528.6	528.6
10 days	463.05	477.18
			20 days	423.7	435.75
30 days	399.11	348.11

According to the column pilot test upper layer Cr⁶⁺The concentration change table and the trend chart are shown in FIG. 6, and it can be seen that 180.49 and 129.49, Cr, were reduced in the control group and the repaired group, respectively, after the 30-day test⁶⁺The concentration is in a descending trend. The decrease range of the control group is higher than that of the repair group, because the water added into the control group is easy to have certain leaching effect on the system while the culture medium is supplemented every week, and the free chromium is migrated to the lower layer of the system; the culture medium supplemented by the restoration group has a certain concentration of microorganisms which have a certain blocking effect on the leaching process of the system, so that the concentration of upper chromium ions in the restoration group is slightly higher than that in the control group due to the adsorption and reduction effects of the microorganisms.

Referring to fig. 7, as the repair time increases, the removal rate of free chromium in the upper layer of the system tends to increase, wherein the removal rate of free chromium in the control group and the repair group reaches 34.14% and 24.49% respectively within 30 days, the microorganism concentration in the repair group increases continuously, and the removal rate of the control group is higher than that of the repair group.

2. Column pilot test layer results and analysis

Middle layer Cr of column pilot scale⁶⁺Table of variation of concentration

	Bioremediation-2	Control-2
			Background sample	1057.2	1057.2
10 days	952.64	977.31
			20 days	925.05	956.76
30 days	803.47	950.95

Layer Cr from column pilot scale⁶⁺The concentration change table and the trend chart are shown in FIG. 8, and it can be seen that the control group and the repair group respectively reduced Cr by 106.25 and 253.53 after the 30-day test⁶⁺The concentration is in a descending trend. Repair group Cr⁶⁺The concentration decreased more than the control group.

In the middle system, the microbial repair effect is more remarkable compared with the control group, and the microbial concentration also increases along with the repair time. Although the leaching of the solution in the middle system caused a slight decrease in the free chromium concentration (increase in removal rate) in the control compared to the initial period, the change in chromium removal rate was small in the samples at 10, 20 and 30 days in the process, indicating that there was a state of balance in the fluidity of the chromium in the middle layer in the control; the repair group in the middle layer system increases with the increase of time due to the removal of chromium by adding microorganisms, the removal rate of free chromium reaches 26.02% in 30 days, the result is probably equal to that of a flowing layer of the middle layer, the concentration of the free chromium is in the tolerance range of the microorganisms, the migration of the free chromium in the middle layer is effectively limited through the adsorption and reduction of the microorganisms, and the effect of solidification and precipitation is realized. As shown in fig. 9.

3. Column pilot run bottom results and analysis

Column pilot test bottom layer Cr⁶⁺Concentration change meter

	Bioremediation-3	Control-3
			Background sample	2643	2643
10 days	2381.60	2613.58
			20 days	2312.625	2563.96
30 days	2008.68	2566.73

According to column pilot test bottom Cr⁶⁺The concentration change table and the trend chart, as shown in FIG. 10, can be seen that the control group and the repair group were reduced by 76.27 and 634.32, Cr, respectively, after the 30-day test⁶⁺The concentration is in a descending trend. The free chromium concentration of the control group is not changed greatly, and the Cr of the repair group⁶⁺The concentration decreased more than the control group.

For the bottom layer system belonging to the layer enriched with free chromium, the layer can better reflect the repairing effect of microorganisms, and the detection result shows that the removal rate of the free chromium in the control group is lower and is almost in the error range, and the result is related to the fact that the bottom layer belongs to the free chromium accumulation in the leaching process. The removal rate of free chromium in the repair group gradually increases along with the change of time, the removal rate of free chromium in the bottom layer reaches 18.29 percent in 30 days, the result is far lower than the removal rate of chromium in the middle layer system, and the concentration of microorganisms is also lower than that of the middle layer system. This result may be associated with a certain pressure on the microorganisms due to the high concentration of free chromium in the bottom layer, and the reduction of the concentration of microorganisms in the bottom layer indicates that the activity of the microorganisms with high concentration of free chromium is limited, and even a great deal of death of the microorganisms may occur due to the toxic nature of chromium. One month of repair experiments show that the microorganism repair functional bacteria have obvious solidification and reduction effects on chromium from the change of the concentration and the removal rate of hexavalent chromium at the bottom layer of the upper layer and the middle layer. The microbial activity as a whole shows an upward trend, but the upward trend gradually becomes slower with climate change.

Through the repair test, obvious test data comparison occurs between the repair group and the control group, and through analysis in fig. 12, the removal rate of free chromium is as follows: upper > middle > bottom; respectively, the upper layer is 31.14 percent, the middle layer is 26.02 percent and the bottom layer is 18.29 percent. The microbial activity is as follows: the upper layer, the middle layer and the bottom layer are shown, and the detection results show that the microorganism has good effect of repairing free chromium.

The results of the pilot-scale tests are consistent with the variation trend of laboratory research, and the removal of soluble chromium by microorganisms in the field pilot-scale tests has certain deviation compared with the laboratory results, so that the phenomenon is caused by a plurality of reasons, such as physicochemical properties, temperature, oxygen demand, pH, microorganism concentration, nutrition consumption and the like of the sample.

The removal rate of the control group comes from the fact that the leaching action causes the free chromium to migrate to the lower layer of the system; the removal rate of the restoration group has the solidification effect of microorganisms besides the migration of chromium from the bacterial liquid leaching process.

The present invention has been described in detail, and it should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Claims

1. A microbial remediation method applied to an alien soil-free mining area is characterized by comprising the following steps: the method comprises the following steps of (1),

(1) sampling soil in an area to be repaired;

(2) microorganism domestication and separation:

2) transferring the obtained bacterial liquid as mother liquid to a Cr (VI) -containing concentration of 200 mg.L after 7 days^-1Respectively culturing the NB culture medium in aerobic and anaerobic conditions at 30 ℃ for 7 days, and periodically detecting the reduction rate and the bacterial quantity of Cr (VI) in the culture solution in the acclimation process until the concentration of Cr (VI) in the culture medium leads the bacteria not to grow;

(5) and (6) detecting.

2. The microbial remediation method of claim 1 applied to an earthen-free mine site, wherein: the NB medium had the following components and concentrations: biometek1 at 10 g.L^-1Biometek2 is 5 g.L^-1Biometek3 is 5 g.L^-1Cr (VI) is 100 mg.L^-1Biometek4 is 20 g.L^-1300mL of distilled water, pH 9 and culture temperature of 20-35 ℃.

3. The microbial remediation method of claim 1 applied to an earthen-free mine site, wherein: during repair, firstly, designing a total of 4 double-repeated soil columns which are PE plastic pipes with the column length of 2m and the diameter of 30cm, wherein two groups are repair groups, and the other two groups are control groups; repairing sample passing by 1cm²Sieving the pore diameter and preparing the mixture evenly(ii) a Adding 110kg of sample and 50L of bacterial liquid into each soil column of the restoration group, wherein the lower layer of the bacterial liquid is SRB bacteria, and the upper layer is mixed functional bacteria; the control group was supplemented with 110kg of sample and 50L of water.

4. The microbial remediation method of claim 1 applied to an earthen-free mine site, wherein: the repaired test was carried out by sampling 50mL from different sampling ports for testing, and solid samples were taken from different sampling ports on days 15, 45 and 90 from the inoculation of the microorganism and stored at 4 ℃ for testing.

5. The microbial remediation method of claim 1 applied to an earthen-free mine site, wherein: the detection items comprise the detection of the concentration change of hexavalent chromium and other heavy metals in the leachate, the morphological structure change of chromium in the solid sample, the change of the functional microbial community structure and the total microbial quantity, the change of the permeability, the change of organic matters and the stability of precipitated products.