CN109420675B - Soil remediation composition and application and soil bioremediation method - Google Patents
Soil remediation composition and application and soil bioremediation method Download PDFInfo
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Abstract
The invention relates to the field of environmental protection, in particular to a soil remediation composition, application and a soil bioremediation method. The soil remediation composition contains a water-retaining agent, a soil conditioner, a nutrient substance and a mixed microbial inoculum. The application is the application of the soil remediation composition in soil remediation. The method comprises remediating soil using a soil remediation composition as described above. The microbial agent capable of degrading petroleum hydrocarbon is introduced into the soil, and components (such as soil quality regulator, nutrient substances, water-retaining agent and the like) having an improvement effect on soil quality are introduced to promote the microbial agent to propagate in the soil and promote pollutants in the soil to be degraded, so that a good soil remediation effect is obtained. The soil remediation composition disclosed by the invention cannot cause secondary soil pollution, cannot damage the soil environment, and is low in cost and simple to operate.
Description
Technical Field
The invention relates to the field of environmental protection, in particular to a soil remediation composition, an application and a soil bioremediation method.
Background
In the processes of petroleum exploration, exploitation, transportation, extraction and the like, due to misoperation or accident leakage, part of petroleum is scattered into soil, so that the petroleum hydrocarbon pollution of the soil is caused. The petroleum pollutants discharged into the environment by the ground crude oil, accident leakage and other ways all over the world every year are about 8 multiplied by 106t, petroleum hydrocarbon contamination has become a focus of world concern. In the 80 s of the 20 th century, the United states presented a serious problem of petroleum hydrocarbon soil pollution, about half of 45 ten thousand of brown land had petroleum hydrocarbon pollution, and the same Canada had petroleum hydrocarbon pollution of 60% of the land, and many regions and plants in the United kingdomThere is also severe oil pollution, and relevant repair engineering plans have been made in germany, the netherlands, the uk and other countries to strengthen the repair and treatment of the petroleum hydrocarbon polluted soil.
Since 1978 in China ascending ten oil producing countries in the world, more and more oil and gas reservoirs and oil and gas fields are explored and developed, oil enterprises are increased, about 70 ten thousand tons of crude oil are exploited every year, one tenth of the crude oil enters the soil environment, and the newly increased polluted soil in China reaches about 10 ten thousand tons every year. The situation of petroleum hydrocarbon pollution of soil is severe, and the sustainable development process of China is hindered.
After the petroleum hydrocarbon pollutants enter the soil, the soil structure and the composition of organic matters are changed, the petroleum hydrocarbon is easy to adhere to soil particles, soil pores are blocked, the water permeability and the air permeability of the soil are influenced, nutrient elements are limited from entering the soil solution from the soil particles, and the soil fertility is reduced; and the large amount of organic carbon contained in petroleum hydrocarbons can cause serious imbalance in the carbon, nitrogen and phosphorus ratios of the soil. In addition, the metabolic intermediate products of petroleum hydrocarbon in soil have some characteristic functional groups, and can absorb and complex heavy metal ions, thereby influencing the migration and transformation of heavy metals in the soil/plant system. Meanwhile, some hydrocarbon components which cannot be adsorbed by the soil can permeate into underground water to cause pollution of the underground water, and potential harm is brought to human domestic water.
Soil in nature has a certain self-cleaning capacity for petroleum hydrocarbon pollution, but when the petroleum hydrocarbon entering the soil exceeds the self-cleaning capacity of the environment, the petroleum hydrocarbon is accumulated and polluted in the soil. The existing methods for restoring petroleum hydrocarbon polluted soil mainly comprise a physical restoration method, a chemical restoration method, a biological restoration method, a combination of multiple restoration techniques and the like.
Microorganisms are important members of the soil ecosystem and play a critical role in the material circulation and energy flow of soil and biospheres. Many microorganisms in nature can take petroleum hydrocarbon as a sole carbon source and energy source, degrade petroleum hydrocarbon pollutants in the environment through metabolism, and finally convert the petroleum pollutants into harmless carbon dioxide and water. Under aerobic conditions:
petroleum hydrocarbon + microorganism + O2Ying (nutrient supplement)Nutrient substance → CO + H2O + by-product + microbial cell biomass.
Microorganisms capable of degrading petroleum hydrocarbons are widely distributed, but are mainly present in polluted areas, because natural microbial community structures or populations are not suitable for petroleum-polluted environments, and after the environments are polluted by petroleum, suitable persons live, so that populations suitable for petroleum pollution are developed, and the growth of other microbial populations is inhibited or eliminated. Microbial remediation generally refers to remediation of contaminated soil by accelerating the decomposition of organic pollutants using indigenous microorganisms in the soil or supplementing domesticated highly-efficient degrading microorganisms to the contaminated environment under optimized environmental conditions, and can be divided into in-situ bioremediation (a method of treating petroleum hydrocarbon-contaminated soil in situ) and ex-situ bioremediation (a method of digging out petroleum hydrocarbon-contaminated soil, treating the petroleum hydrocarbon-contaminated soil outside a contaminated site or transporting the petroleum hydrocarbon-contaminated soil to a special remediation site).
Compared with physical and chemical methods, the microbial remediation method has the advantages of low cost, good treatment effect, no secondary pollution, wide application range, no damage to soil environment and the like, and is suitable for being widely applied to the treatment of petroleum hydrocarbon polluted soil. China starts late in the aspects of contaminated site soil remediation technology, engineering application, equipment research and development and the like, and is still in a low-level loitering stage at present.
In the aspect of petroleum hydrocarbon polluted soil remediation technology, research work is mainly carried out in laboratory or pilot-scale, and on-site petroleum hydrocarbon polluted remediation cases are rare. In the aspect of petroleum hydrocarbon polluted soil remediation equipment, the petroleum hydrocarbon polluted soil remediation equipment mostly stays in the pilot-scale stage of development of scientific research equipment or experimental prototype, and the industrial application of the remediation equipment is very little. Therefore, the method screens out the bacterial strains with high-efficiency degradation effect on the petroleum hydrocarbon pollutants, and has very important research value on bioremediation of the petroleum hydrocarbon polluted soil by matching with a proper promotion technology.
Disclosure of Invention
The invention aims to overcome the defect of poor soil remediation effect on petroleum hydrocarbon polluted soil by using a bioremediation method in the prior art, and provides a soil remediation composition and a soil bioremediation method.
In order to achieve the above objects, the present invention provides, in one aspect, a soil remediation composition comprising a water-retaining agent, a soil conditioner, a nutrient, and a mixed microbial agent.
Preferably, the mixed bacterial agent is selected from at least two of Stenotrophomonas (Stenotrophomonas), Dietzia (Dietzia), Micrococcus luteus (Micrococcus luteus), Acinetobacter (Acinetobacter), and Microbacterium aromaticum (Microbacterium esteroraticum).
Another aspect of the present invention provides the use of a soil remediation composition as described above for the remediation of soil, in particular petroleum hydrocarbon contaminated soil.
In a further aspect of the invention there is provided a method of bioremediation of soil comprising remediating soil using the soil remediation composition of the invention.
In the soil bioremediation method of the invention, the microbial agent capable of degrading pollutants (such as petroleum hydrocarbon) in the soil is introduced into the soil, and the components (such as soil quality regulator, nutrient substance, water retention agent and the like) having the effect of improving the soil quality are introduced to promote the microbial agent to propagate in the soil and promote the pollutants in the soil to be degraded, thereby obtaining better soil remediation effect.
In addition, the soil remediation composition provided by the invention is used for soil remediation, secondary pollution is avoided, the soil environment is not damaged, the cost is low, and the operation is simple. The soil remediation composition is particularly suitable for remediation of soil polluted by petroleum hydrocarbon, and is particularly suitable for remediation of volatile and semi-volatile soil polluted by petroleum hydrocarbon.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In one aspect, the invention provides a soil remediation composition comprising a water retention agent, a soil conditioner, a nutrient, and a mixed microbial inoculum.
According to the invention, the soil is soil in the meaning known in the art, being a mixture of minerals, organic matter, living organisms and water and air. Preferably, the minerals comprise 90-95% or more of the solid phase fraction (dry weight of the soil) and the organic matter comprises about 1-10% by weight, with the soil constituents being predominantly minerals. Soil organic matter is an organic compound present in various forms in soil. In addition to this, there is a soil solution, which is a generic term for soil moisture and dissolved and suspended substances contained therein. Soil solutions are the vehicles by which plants and microorganisms absorb nutrients from the soil and are also the primary pathways by which contaminants migrate through the soil.
The soil remediation composition does not contain components which can destroy the soil environment, so that secondary pollution can not be caused while soil remediation is carried out.
According to the invention, the mixed bacterial agent can be a bacterial strain which can be used for degrading petroleum hydrocarbon in the prior art, but the inventor of the invention finds that the mixed bacterial agent obtained by randomly combining single petroleum hydrocarbon degrading bacteria can not necessarily improve the degradation rate of the petroleum hydrocarbon and can also degrade the degradation effect of the soil petroleum hydrocarbon in the research process. Further, Stenotrophomonas (Stenotrophomonas), Dietzia (Dietzia), Micrococcus luteus (Micrococcus luteus), Acinetobacter (Acinetobacter), and Microbacterium odoratum (Microbacterium odoratum) have good degradation activity on crude oil, normal paraffin, branched paraffin, aromatic hydrocarbon and other components, and the symbiotic and synergistic effects of the Stenotrophomonas, the Dietzia (Dietzia), the Micrococcus (Micrococcus luteus), the Acinetobacter and the Microbacterium odoratum are obvious. Therefore, based on the process or mode difference of different microorganisms on petroleum hydrocarbon degradation, the single petroleum hydrocarbon degradation bacteria are mixed, a petroleum hydrocarbon degradation microbial community can be constructed in soil, and the microbial communities can play an obvious symbiotic and synergistic role, so that the petroleum hydrocarbon degradation efficiency can be obviously improved.
Wherein Stenotrophomonas (Stenotrophomonas) belongs to strict non-fermentation aerobic gram-negative bacillus and has low nutritional requirement. The inventor of the invention finds that stenotrophomonas has unique biochemical activity and metabolic characteristics and can degrade benzene, toluene, ethylbenzene and aniline.
Among them, the bacterium Dietzia (Dietzia) is a strain having the ability to degrade crude oil and long-chain hydrocarbons, and has the ability to degrade petroleum hydrocarbons in a high-salt environment. In addition, in the process of research, the inventor of the invention also finds that the bacteria can generate surface active substances in the process of degrading petroleum hydrocarbon, and the surface active substances are beneficial to the direct contact utilization of the petroleum hydrocarbon by microorganisms.
Among them, Micrococcus luteus (Micrococcus luteus) belongs to the obligate aerobic gram-positive bacteria, and the requirement for survival conditions is loose. The inventor of the invention finds that the bacterium has better degradation effect on normal alkane, isoparaffin and cycloparaffin in petroleum components in the process of research.
Among them, Acinetobacter (Acinetobacter) is a non-fermentative bacterium which degrades saturated hydrocarbon through a terminal oxidation pathway under aerobic conditions, and has strong growth ability and rapid propagation.
Among them, the Microbacterium estericum (Microbacterium estericum) is a kind of bacteria which is widely distributed in soil, has many varieties and is rarely studied. In the process of research, the inventor of the invention finds that the bacteria of the genus Microbacterium estericum have strong aerobic property and strong organic matter degradation capability.
According to the invention, the size distribution or the size combination of the solid particles in the soil is referred to as the mechanical composition of the soil, also called the soil texture. Soil can be classified according to its mechanical composition. The texture of soil is one of the basic factors that affect the fertility of soil, the tiltability and the pollutant capacity. The inventor researches and discovers that the soil quality regulator can regulate the particle size of solid particles of soil, enables the particle size of the soil to be more uniform, has higher oxygen content, can break soil hardening, loosen the soil, improve the air permeability of the soil, reduce the volume weight of the soil, promote the activity of soil microorganisms, strengthen the contact of the microorganisms and pollutants and enhance the soil fertilizer-water permeability. Therefore, the soil property regulator of the present invention may be a material having the above function in the prior art, and preferably, the soil property regulator may be selected from one or more of sawdust, straw and zeolite powder.
According to the invention, the water-retaining agent is also called super absorbent polymer, can absorb deionized water which is several hundred times to thousands times of the self weight and several tens times to more than one hundred times of saline water, forms gel substance after absorbing water and expanding, and can slowly release water under dry condition. The inventor researches and discovers that the water-retaining agent can remarkably improve the water content, the volume expansion rate, the liquid phase percentage, the total porosity and the capillary pore percentage of soil, improve the soil structure, relieve the ground temperature change, promote the activity of microorganisms and improve the soil fertility so as to stimulate and maintain the growth of the microorganisms by using the water-retaining agent properly. Therefore, the water retaining agent of the present invention may be a material having the above function in the prior art, and preferably, the water retaining agent is selected from one or more of acrylic acid polymer, acrylamide polymer and starch-acrylic acid copolymer.
In the present invention, the nutrient may be a soil nutrient containing nitrogen and phosphorus. Preferably, the nutrient is urea and/or corn steep liquor.
The substances (the water-retaining agent, the soil conditioner, the nutrient substances and the mixed microbial inoculum) are added into the petroleum hydrocarbon polluted soil in proper compositions, and the components are organically combined to form a whole so as to play a role of synergistic compatibility, so that the petroleum hydrocarbon polluted soil can be effectively repaired.
According to the present invention, the content of each component in the soil remediation composition may be varied within a wide range, and preferably, the content of the soil conditioner in the soil remediation composition is 1500 parts by weight, more preferably 300-1250 parts by weight (e.g., 300 parts by weight, 500 parts by weight, 700 parts by weight, 300 parts by weight, or the like) based on 100 parts by weight of the water retaining agent,900 parts by weight, 1100 parts by weight, 1250 parts by weight, and any value within the range of any two of these point values. ) (ii) a The nutrient is contained in an amount of 10 to 500 parts by weight, more preferably 10 to 360 parts by weight (e.g., 10 parts by weight, 20 parts by weight, 30 parts by weight, 40 parts by weight, 50 parts by weight, 80 parts by weight, 120 parts by weight, 150 parts by weight, 180 parts by weight, 200 parts by weight, 250 parts by weight, 280 parts by weight, 300 parts by weight, 330 parts by weight, 360 parts by weight, and any value within a range of any two of these points); the content of the mixed microbial inoculum is 107-1012CFU, preferably 108-1011CFU, e.g. (10)8CFU、109CFU、1010CFU、1011CFU, and any value within a range of any two of these point values).
Wherein, CFU is a colony forming unit, which is defined as that a certain amount of diluted bacterial liquid is poured or coated to make the single cells of the microorganisms in the bacterial liquid disperse on the culture medium plate one by one, and after the bacterial liquid is cultured, each living cell forms a colony.
In a second aspect, the present invention provides the use of a soil remediation composition as described above for remediation of soil.
As described above, the soil remediation compositions of the present invention are particularly useful for remediation of soil contaminated with petroleum hydrocarbons.
Among these, petroleum hydrocarbons refer to hydrocarbon-containing mixtures found in crude oil. Petroleum hydrocarbon is one of the organic pollutants widely existing in the current environment, including gasoline, kerosene, diesel oil, lubricating oil, paraffin, asphalt and the like, and is a mixture of various hydrocarbons (normal paraffin, branched paraffin, cyclane and aromatic hydrocarbon) and small amount of other organic matters, such as sulfide, nitride, naphthenic acid and the like.
In a third aspect, the present invention provides a method for bioremediation of soil, the method comprising: soil remediation is carried out using a soil remediation composition as described above.
The inventor of the present invention needs to explain here that when the soil remediation composition as above is contacted with soil to remediate the soil, the components of the soil remediation composition may be added to the soil separately or may be added to the soil together in a ratio. The soil remediation composition used is considered to remediate soil, as is the form in which the above components are added to the soil.
According to the present invention, the water retaining agent may be used in an amount of 0.01 to 1 part by weight, preferably 0.1 to 0.3 part by weight, specifically, for example, 0.1, 0.15, 0.2, 0.25, 0.3 part by weight and any value within a range of any two of these points, based on 100 parts by weight of the soil to be remediated.
According to the present invention, the soil property-adjusting agent may be used in an amount of 0.1 to 5 parts by weight, preferably 0.5 to 2.5 parts by weight, specifically, for example, 0.5, 0.8, 1.1, 1.4, 1.7, 2.0, 2.3, 2.5 parts by weight and any value within a range of any two of these points, based on 100 parts by weight of the soil to be restored.
According to the invention, the chemical composition and the nutritional type of microbial cells determine the nutritional requirements of the cells, the petroleum degrading bacteria are mainly chemoheterotrophic bacteria, and when the soil polluted by petroleum hydrocarbon is subjected to microbial treatment, the petroleum hydrocarbon provides a large amount of available carbon substrates for the microbes in the system, but the petroleum hydrocarbon provides mainly carbon and hydrogen elements, and the lack of other elements can make the microbes difficult to degrade the petroleum hydrocarbon in the soil. The inventor researches and discovers that the nutrient substances such as nitrogen and phosphorus are supplemented, so that the ratio of C \ N \ P in the soil is close to the ratio of elements in the microorganism, and the mass propagation of the microorganism can be effectively promoted. Due to ammoniacal Nitrogen (NH)4 +the-N) nutrient substances can be converted into ammonia gas under the weak alkaline condition and diffused into the air, so that the loss of the nitrogen nutrient substances is caused. The phosphorus nutrient substances are relatively stable in the soil and are not easy to lose. Therefore, the influence of the addition amount of the nitrogen nutrient substances on the degradation effect is mainly considered. In the method of the present invention, preferably, the nutrient is used in an amount such that the carbon-nitrogen mass ratio C: n is (5-15):1, and specifically, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 and any two of these ratios may be usedAny other ratio within the range of ratios formed. Wherein the content of C is calculated according to 90 percent of the total mass of petroleum hydrocarbon in the soil. When the nutrient is urea and/or corn steep liquor, the content of N is 46.67% of the total mass of urea or 3.5% of the total weight of corn steep liquor.
According to the present invention, preferably, the microbial agent may be used in an amount of 10 parts by weight based on 100 parts by weight of soil to be remediated9-1011The CFU may be, for example, 1 × 109、5×109、1×1010、5×1010、1×1011、5×1011CFU and any value within the range of any two of these point values.
According to the invention, the soil water content is preferably maintained between 15 and 25% during the remediation process.
According to the invention, the soil is preferably a petroleum hydrocarbon contaminated soil, in particular a petroleum hydrocarbon micro-contaminated soil and a volatile, semi-volatile petroleum hydrocarbon contaminated soil.
Examples
The present invention will be described in detail below by way of examples.
1. Materials used in the following examples and comparative examples
Polyacrylic acid was purchased from Heizhou Chunjiang chemical Co., Ltd under the trade designation SL-324;
the polyacrylamide is purchased from Shenyang Anruixiang chemical hardware Limited company, and the product number is polyacrylamide-03;
starch-acrylic acid polymers were purchased from Tuozhi Macro technologies, Inc.;
zeolite powder was purchased from Cibotai mineral processing plant, Lingshou county;
urea was purchased from denxin macrochemical ltd;
the corn steep liquor is purchased from Shandong Hongxing corn development Co Ltd;
acinetobacter is purchased from China general microbiological culture Collection center with the preservation number of CGMCC1.10395T；
The Dietzia is purchased from China general microbiological culture Collection center with the collection number CGMCC1.6332T；
Micrococcus luteus is purchased from China general microbiological culture Collection center with the preservation number of CGMCC 1.10554;
the ester fragrance microbacterium is purchased from China general microbiological culture collection center with the preservation number of CGMCC 1.10674;
stenotrophomonas is purchased from China general microbiological culture collection center with the preservation number of CGMCC 1.6393;
n-hexadecane was purchased from Aladdin under the trade designation H103406.
The soil is dry soil sterilized at 250 ℃ for 24 h.
2. Evaluation of bioremediation effect of soil petroleum hydrocarbon pollution
Enriching a microbial inoculum in an inorganic salt culture medium with target pollutants as a unique carbon source; centrifugally separating to obtain enriched microbial inoculum; thirdly, the microbial inoculum, the water-retaining agent, the soil conditioner and the nutrient substance are put into the soil together; adding target pollutants into the soil; and fifthly, periodically detecting the content of the petroleum hydrocarbon in the soil and the total number of soil bacteria, and calculating the degradation effect of the petroleum hydrocarbon.
The specific operation steps are as follows:
(1) adding 1.5mL of bacterial liquid into a triangular flask filled with 150mL of inorganic salt culture medium, adding the target pollutant, and oscillating at 150r/min for 7 days to obtain enriched bacterial liquid.
(2) And performing 8000r/min centrifugal solid-liquid separation on the obtained enrichment culture solution, discarding the upper layer culture medium solution, adding 100mL of inorganic salt culture medium, fully oscillating, performing 8000r/min centrifugal solid-liquid separation again, discarding the upper layer solution, and reserving the bottom layer solid for later use.
(3) Adding the water-retaining agent, the soil conditioner, the nutrient substances and the bottom solid into the soil, fully stirring, and simultaneously adding deionized water to maintain the water content of the soil within a certain range.
(4) Adding the target pollutant into the soil, and uniformly stirring.
(5) Soil petroleum hydrocarbon content and soil bacterial count were measured weekly.
(6) Supplying water every two days, keeping the water content of the soil within a certain range, and turning the soil 3 times every week.
Inorganic salt culture medium: NH (NH)4Cl 0.67g, Na2CO3Is 1.06g, MgSO4·7H2O is 0.1g and CaCl20.1g, K2HPO4·3H2O is 1.5g, KH2PO40.5g of FeCl3.6H2O is 0.1g, deionized water is 1L, and the pH value is 7.0-7.5.
3. Determination of petroleum hydrocarbon content in soil
Drying a fresh soil sample by adopting anhydrous sodium sulfate, taking carbon tetrachloride as an extracting solution, oscillating and extracting petroleum substances in the soil sample, and determining the concentration of the petroleum substances. The content of petroleum is 2930cm-1 (CH) from wave number2The absorbance at the band of 2960cm-1 (stretching vibration of C-H bond in CH3 group) and 3030cm-1 (stretching vibration of C-H bond in aromatic ring) was calculated.
The specific determination steps are as follows:
(1) and removing impurities such as gravel, plant roots and the like from the collected soil sample, and fully and uniformly mixing.
(2) Accurately weighing 10.000g of soil sample, adding 10g of anhydrous sodium sulfate, mixing well, standing for 30min, solidifying and crushing.
(3) And transferring the dried soil sample into a 50ml centrifuge tube, adding 25ml carbon tetrachloride, fully mixing, placing in an ultrasonic oscillator, and oscillating for 10 min.
(4) And centrifuging the vibrated centrifugal tube 8000r/min for solid-liquid separation, and collecting the upper layer carbon tetrachloride.
(5) And adding 25ml of carbon tetrachloride into the residual soil solids at the bottom layer of the centrifuge tube again, fully mixing, placing in an ultrasonic oscillator, and oscillating for 10 min.
(6) And centrifuging and carrying out solid-liquid separation on the vibrated centrifugal tube 8000r/min, collecting upper carbon tetrachloride, mixing the upper carbon tetrachloride with the upper carbon tetrachloride collected before, and waiting for detection.
(7) The water content w of the soil samples was determined according to the relevant provisions of GB 7172w。
The content ω (mg/kg) of petroleum in the soil was calculated according to the following formula.
In the formula:
omega is the content of petroleum in soil, mg/kg;
ρ1the concentration of the sample petroleum, mg/L, is obtained by checking on a standard curve;
ρ0the petroleum concentration of the blank sample searched on the standard curve is mg/L;
d is the dilution multiple of the extracting solution;
v, the volume of the extracting solution is 50 ml;
ms-sample size, g;
ww-water content of soil,%.
The measuring range of the method is 50-500mg/kg, and if the petroleum hydrocarbon content of the soil to be measured exceeds the range, the petroleum hydrocarbon content is measured after dilution.
And (3) performing the measurement on the soil sample before restoration, and after restoration is started, performing the measurement on the soil sample every week to count the content of the petroleum hydrocarbon, wherein the unit of the content of the petroleum hydrocarbon is mg/kg.
4. Determination of total number of soil bacteria
The determination method comprises the following steps: plating colony counting method
The specific determination steps are as follows:
weighing 10g of soil sample, placing the soil sample into a triangular flask containing 90mL of sterile deionized water, placing the triangular flask on a shaking table, oscillating the triangular flask at the room temperature for 4 hours at the speed of 160 times/min, fully mixing the soil sample with water, fully dispersing microbial cells in the soil, and separating the microbial cells from the soil. This is 10-1Sucking 1mL of the soil suspension into 9mL of sterile water containing 8.5g/L NaCl, sucking with a sterile pipette for 3 times, and mixing to obtain 10-2And (3) soil suspension. And so on to form 10-3、10-4、10-5、10-6、10-7、10-8Soil suspensions of various dilutions. Selecting proper dilution and determining fineness of soil suspension by combining plate counting methodAnd (4) total number of bacteria.
The total number of bacteria in the soil, x (CFU/g), was calculated according to the following formula.
In the formula:
x is the total number of soil bacteria, CFU/g;
e-total number of bacteria in soil suspension, CFU/ml;
v-volume of soil suspension, ml;
m is the sample size, g;
w-soil moisture content,%.
Meat extract peptone medium: 10g of NaCl, 10g of peptone, 5g of beef extract, 18g of agar and 1L of deionized water, and the pH value is 7.2-7.4.
The soil sample before remediation was subjected to the above measurement, and after remediation was started according to the method of examples or experimental examples, the soil sample was subjected to the above measurement every week, and the total number of soil sample bacteria (unit is 10) was counted6CFU/g)。
The following test methods were used in the following examples and comparative examples. The soil used for the test was dry soil sterilized at 250 ℃ for 24 h. And (4) centrifugally separating the bacterial liquid, adding the solid part into deionized water, fully mixing, and adding into soil.
Example 1
Weighing 6g of polyacrylic acid, and dissolving in 200g of deionized water to obtain an aqueous solution 1; weighing 0.96g of urea, and dissolving the urea in 100g of deionized water to obtain an aqueous solution 2; mixing stenotrophomonas and Microbacterium estericum (1:1) in 300g of deionized water to obtain an aqueous solution 3; weighing 45g of sawdust, mixing the sawdust with 2400g of soil, adding the aqueous solutions 1, 2 and 3 into the soil, and uniformly mixing, wherein the dosage of the stenotrophomonas and the microbacterium fragrans is such that the dosage of the mixed microbial inoculum in the soil is shown in table 2. Then 16g of n-hexadecane was added to the uniformly mixed soil and uniformly mixed. Supplying water every two days, keeping the water content of the soil at 15-25 wt%, and turning the soil 3 times every week. The petroleum hydrocarbon content of the soil and the total number of soil bacteria were measured every week, and the results are shown in tables 1 and 2, respectively.
Example 2
Weighing 3.6g of polyacrylamide and dissolving in 200g of deionized water to obtain an aqueous solution 1, weighing 12.86g of corn steep liquor and dissolving in 100g of deionized water to obtain an aqueous solution 2, weighing 45g of straws and mixing in 2400g of soil, adding the aqueous solutions 1, 2 and 3 into the soil, and uniformly mixing, wherein the dosage of the dietzia and the microbacterium estericum leads the dosage of the mixed microbial inoculum in the soil to be shown in table 2. And (3) adding 16g of n-hexadecane into the uniformly mixed soil, and uniformly mixing. Supplying water every two days, keeping the water content of the soil at 15-25 wt%, and turning the soil 3 times every week. The petroleum hydrocarbon content of the soil and the total number of soil bacteria were measured every week, and the results are shown in tables 1 and 2, respectively.
Example 3
Weighing 8.4g of starch-acrylic acid polymer and dissolving in 200g of deionized water to obtain an aqueous solution 1, weighing 0.96g of urea and dissolving in 100g of deionized water to obtain an aqueous solution 2, weighing 45g of zeolite powder and mixing in 2400g of soil, adding the aqueous solutions 1, 2 and 3 into the soil and mixing uniformly, wherein the dosage of micrococcus luteus and Microbacterium estericum is that the amount of the mixed microbial inoculum in the soil is shown in table 2. And (3) adding 16g of n-hexadecane into the uniformly mixed soil, and uniformly mixing. Supplying water every two days, keeping the water content of the soil at 15-25 wt%, and turning the soil 3 times every week. The petroleum hydrocarbon content of the soil and the total number of soil bacteria were measured every week, and the results are shown in tables 1 and 2, respectively.
Example 4
Weighing 6g of polyacrylic acid to be dissolved in 200g of deionized water to obtain an aqueous solution 1, weighing 0.96g of urea to be dissolved in 100g of deionized water to obtain an aqueous solution 2, weighing acinetobacter and micro-bacterium carlsbergii (1:10) to be mixed in 300g of deionized water to obtain an aqueous solution 3, weighing 18g of sawdust to be mixed in 2400g of soil, adding the aqueous solutions 1, 2 and 3 to the soil, and uniformly mixing, wherein the using amounts of the acinetobacter and the micro-bacterium carlsbergii enable the amount of the mixed fungicide in the soil to be shown in table 2. And (3) adding 16g of n-hexadecane into the uniformly mixed soil, and uniformly mixing. Supplying water every two days, keeping the water content of the soil at 15-25 wt%, and turning the soil 3 times every week. The petroleum hydrocarbon content of the soil and the total number of soil bacteria were measured every week, and the results are shown in tables 1 and 2, respectively.
Example 5
Weighing 6g of polyacrylamide, dissolving the polyacrylamide in 200g of deionized water to obtain an aqueous solution 1, weighing 12.86g of corn steep liquor, dissolving the corn steep liquor in 100g of deionized water to obtain an aqueous solution 2, mixing the dietzia and the acinetobacter (10:1) in 300g of deionized water to obtain an aqueous solution 3, weighing 72g of straws, mixing the straws in 2400g of soil, adding the aqueous solutions 1, 2 and 3 into the soil, and uniformly mixing, wherein the dosage of the dietzia and the acinetobacter leads the amount of the mixed microbial inoculum in the soil to be shown in table 2. And (3) adding 16g of n-hexadecane into the uniformly mixed soil, and uniformly mixing. Supplying water every two days, keeping the water content of the soil at 15-25 wt%, and turning the soil 3 times every week. The petroleum hydrocarbon content of the soil and the total number of soil bacteria were measured every week, and the results are shown in tables 1 and 2, respectively.
Example 6
Weighing 6g of polyacrylic acid to be dissolved in 200g of deionized water to obtain an aqueous solution 1, weighing 1.92g of urea to be dissolved in 100g of deionized water to obtain an aqueous solution 2, weighing 45g of zeolite powder to be mixed in 2400g of soil, adding the aqueous solutions 1, 2 and 3 to the soil, and uniformly mixing, wherein the dosage of the stenotrophomonas and the acinetobacter leads the amount of the mixed microbial inoculum in the soil to be shown in table 2. And (3) adding 16g of n-hexadecane into the uniformly mixed soil, and uniformly mixing. Supplying water every two days, keeping the water content of the soil at 15-25 wt%, and turning the soil 3 times every week. The petroleum hydrocarbon content of the soil and the total number of soil bacteria were measured every week, and the results are shown in tables 1 and 2, respectively.
Example 7
Weighing 6g of polyacrylic acid to be dissolved in 200g of deionized water to obtain an aqueous solution 1, weighing 0.29g of urea to be dissolved in 100g of deionized water to obtain an aqueous solution 2, weighing 45g of sawdust to be mixed in 2400g of soil, adding the aqueous solutions 1, 2 and 3 to the soil, and uniformly mixing, wherein the dosage of micrococcus luteus and acinetobacter causes the amount of the mixed microbial inoculum in the soil to be shown in table 2. And (3) adding 16g of n-hexadecane into the uniformly mixed soil, and uniformly mixing. Supplying water every two days, keeping the water content of the soil at 15-25 wt%, and turning the soil 3 times every week. The petroleum hydrocarbon content of the soil and the total number of soil bacteria were measured every week, and the results are shown in tables 1 and 2, respectively.
Example 8
Weighing 6g of polyacrylic acid to dissolve in 200g of deionized water to obtain an aqueous solution 1, weighing 0.96g of urea to dissolve in 80g of deionized water to obtain an aqueous solution 2, weighing 45g of micrococcus luteus, Micrococcus dietus and stenotrophomonas (1:1:1) to mix in 200g of deionized water to obtain an aqueous solution 3, weighing 2520g of sawdust to mix in soil, adding the aqueous solutions 1, 2 and 3 to the soil, and mixing uniformly, wherein the dosage of Micrococcus luteus, Dietzia and stenotrophomonas is shown in Table 2. And (3) adding 16g of n-hexadecane into the uniformly mixed soil, and uniformly mixing. Supplying water every two days, keeping the water content of the soil at 15-25 wt%, and turning the soil 3 times every week. The petroleum hydrocarbon content of the soil and the total number of soil bacteria were measured every week, and the results are shown in tables 1 and 2, respectively.
Example 9
Weighing 6g of polyacrylic acid to dissolve in 200g of deionized water to obtain an aqueous solution 1, weighing 0.96g of urea to dissolve in 200g of deionized water to obtain an aqueous solution 2, weighing 45g of sawdust to mix in 2280g of soil, adding the aqueous solutions 1, 2 and 3 to the soil, and uniformly mixing, wherein the dosage of the mixed microbial inoculum in the soil is shown in table 2. And (3) adding 16g of n-hexadecane into the uniformly mixed soil, and uniformly mixing. Supplying water every two days, keeping the water content of the soil at 15-25 wt%, and turning the soil 3 times every week. The petroleum hydrocarbon content of the soil and the total number of soil bacteria were measured every week, and the results are shown in tables 1 and 2, respectively.
Comparative example 1
Petroleum hydrocarbons were degraded as in example 1 except that the inoculum was selected only from stenotrophomonas.
Comparative example 2
Petroleum hydrocarbons were degraded as in example 1 except that the inoculum was selected from Detz alone.
Comparative example 3
Petroleum hydrocarbons were degraded as in example 1, except that the microbial inoculum was selected from Microbacterium estericum.
Comparative example 4
Petroleum hydrocarbons were degraded as in example 1 except that the inoculum was selected from Micrococcus luteus.
Comparative example 5
Petroleum hydrocarbons were degraded as in example 1, except that Acinetobacter only was selected as the microbial inoculum.
Comparative example 6
The petroleum hydrocarbon was degraded as in example 1, except that no microbial inoculum was added.
Comparative example 7
The petroleum hydrocarbon was degraded as in example 1 except that no water retaining agent was added.
Comparative example 8
The petroleum hydrocarbon was degraded as in example 1 except that no nutrients were added.
Comparative example 9
The petroleum hydrocarbon was degraded as in example 1 except that no clay conditioning agent was added.
TABLE 1
| Initial | 7d | 14d | 21d | 28d | 35d | 42d | 49d | |
| Example 1 | 5326 | 4862 | 4397 | 3155 | 1655 | 1034 | 776 | 591 |
| Example 2 | 5326 | 4914 | 4551 | 3362 | 2068 | 1344 | 1190 | 1034 |
| Example 3 | 5326 | 4811 | 4189 | 2793 | 1344 | 880 | 673 | 439 |
| Example 4 | 5326 | 5121 | 4914 | 3931 | 3104 | 2327 | 2121 | 1565 |
| Example 5 | 5326 | 4604 | 3879 | 2483 | 1034 | 673 | 414 | 310 |
| Example 6 | 5326 | 4758 | 4086 | 2690 | 1241 | 827 | 621 | 388 |
| Example 7 | 5326 | 5017 | 4758 | 3672 | 2587 | 1810 | 1551 | 1248 |
| Example 8 | 5326 | 4966 | 4604 | 3465 | 2224 | 1448 | 1241 | 1190 |
| Example 9 | 5326 | 4707 | 4034 | 2587 | 1190 | 776 | 517 | 362 |
| Comparative example 1 | 5326 | 5169 | 4975 | 4185 | 3494 | 2545 | 2305 | 1986 |
| Comparative example 2 | 5326 | 5118 | 5000 | 4142 | 3412 | 2504 | 2389 | 1942 |
| Comparative example 3 | 5326 | 5123 | 5023 | 4106 | 3453 | 2542 | 2354 | 1999 |
| Comparative example 4 | 5326 | 5178 | 4989 | 4172 | 3401 | 2567 | 2340 | 2012 |
| Comparative example 5 | 5326 | 5186 | 5015 | 4100 | 3450 | 2576 | 2304 | 2003 |
| Comparative example 6 | 5326 | 5225 | 5065 | 4707 | 4397 | 3982 | 3465 | 3305 |
| Comparative example 7 | 5326 | 5121 | 4914 | 4655 | 3985 | 3446 | 3197 | 2846 |
| Comparative example 8 | 5326 | 5264 | 5120 | 4801 | 4420 | 4031 | 3510 | 3400 |
| Comparative example 9 | 5326 | 5289 | 5189 | 4867 | 4490 | 4054 | 3569 | 3457 |
Note: the petroleum hydrocarbon content is in mg/kg soil.
TABLE 2
Note: the total number of bacteria is 106CFU/g soil.
As can be seen from the above table, when the scheme of the invention is adopted for soil remediation, the microorganisms can reach the peak of the growth curve relatively quickly and degrade pollutants. When the scheme in the comparative examples 7-9 is adopted for restoration, the soil condition is relatively poor, and the degradation condition of pollutants is determined by the growth of microorganisms; compared with the scheme of comparative examples 1-5 for restoration, the mixed microbial inoculum of the invention can show more excellent symbiotic synergistic effect, the microorganism grows rapidly, and the degradation effect of soil petroleum hydrocarbon is good; in addition, when the repair was performed using the scheme of comparative example 6, the initial degradation rate of petroleum hydrocarbon was slow because no additional petroleum hydrocarbon-degrading bacteria were added.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (9)
1. The soil remediation composition is characterized by comprising a water-retaining agent, a soil conditioner, a nutrient substance and a mixed microbial inoculum;
wherein the mixed microbial inoculum is a combination of stenotrophomonas and Microbacterium estericum, a combination of Micrococcus luteus and Microbacterium estericum, a combination of stenotrophomonas and Acinetobacter, or a combination of Microbacterium estericum, Dietzia, stenotrophomonas and Micrococcus luteus;
wherein the Stenotrophomonas is Stenotrophomonas (Stenotrophoromonas) CGMCC1.6393, and the Dietzia is Dietzia (Dietzia) CGMCC1.6332TMicrococcus luteus is Micrococcus luteus (CGMCC 1.10554), Acinetobacter is Acinetobacter (Acinetobacter) CGMCC1.10395TThe Microbacterium estericum is Microbacterium estericum (CGMCC 1.10674);
wherein the water retaining agent is selected from one or more of acrylic acid polymer, acrylamide polymer and starch-acrylic acid copolymer;
wherein based on 100 parts by weight of the water retention agent, the content of the soil property regulator is 1500 parts by weight, the content of the nutrient substance is 10-500 parts by weight, and the content of the mixed microbial inoculum is 1010-1013CFU。
2. The soil remediation composition of claim 1, wherein the soil conditioning agent is selected from one or more of sawdust, straw, zeolite powder.
3. The soil remediation composition of claim 1 or 2, wherein the nutrient is a soil nutrient containing elemental carbon, elemental nitrogen, and elemental phosphorus.
4. The soil remediation composition of claim 3, wherein the nutrient is urea and/or corn steep liquor.
5. Use of a soil remediation composition as claimed in any one of claims 1 to 4 for remediation of soil.
6. A method for bioremediation of soil, the method comprising: soil remediation using the soil remediation composition of any one of claims 1 to 4.
7. The remediating method as set forth in claim 6, wherein the soil remediation composition is used in an amount such that the water retaining agent is contained in an amount of 0.01 to 1 part by weight, the soil conditioner is contained in an amount of 0.1 to 5 parts by weight, and the nutrients are contained in a carbon-nitrogen ratio in soil of C: n (5-15):1, and the content of the mixed microbial inoculum is 108-1012CFU。
8. A remediation method according to claim 6 or claim 7 wherein the soil water content is maintained during remediation in the range of 15 to 25% by weight.
9. The remediation method of claim 8 wherein the soil is petroleum hydrocarbon contaminated soil.
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