CN107254421B - Biological agent for repairing oil-containing soil, repairing method and application - Google Patents

Biological agent for repairing oil-containing soil, repairing method and application Download PDF

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CN107254421B
CN107254421B CN201710506179.1A CN201710506179A CN107254421B CN 107254421 B CN107254421 B CN 107254421B CN 201710506179 A CN201710506179 A CN 201710506179A CN 107254421 B CN107254421 B CN 107254421B
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oil
soil
biological agent
containing soil
repairing
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CN107254421A (en
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王威
张丽丽
郭姝怡
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Nankai University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes

Abstract

The invention discloses a biological agent for repairing oil-containing soil, a repairing method and application. The method comprises the steps of mixing the soil of three different regions with three oils to form oil-containing soil with the oil content of 10%, detecting the pH value of the soil, enabling the pH value of the soil to be alkaline, and adjusting the pH value of the soil to 6-8. The biological agent is prepared by mixing microorganism strains a, b and c according to the bacterial quantity ratio of 1:1: 1. Diluting with water at different ratio, and treating oil-containing soil with pH of 6-8 and original pH at room temperature and 40 deg.C. After 48h-4 weeks of treatment, the oil content in the sample soil was measured. The method has the advantages of simple process, simple required equipment, low cost and environmental friendliness, and the biological agent has high efficiency of repairing the oil-containing soil, and can have high oil reduction rate in a short time. The method is an efficient method for repairing the oil-containing soil by using the biological agent, and has a good application prospect.

Description

Biological agent for repairing oil-containing soil, repairing method and application
Technical Field
The invention belongs to the technical field of environmental bioremediation, and relates to a method for restoring oil-containing soil by using a biological agent and application thereof.
Background
During the process of petroleum extraction, refining, storage and transportation, a large amount of petroleum-contaminated soil is produced, including drilling mud, oil sludge falling to the ground, tank cleaning oil sludge and the like. The petroleum-polluted soil is very complex in components, contains a large amount of toxic and pathogenic carcinogenic substances such as benzene, phenol, anthracene, pyrene and the like, can seriously pollute the environment and harm the health of human bodies. Therefore, development of an effective and harmless treatment method is necessary.
At present, the technologies for treating petroleum-contaminated soil are roughly divided into three categories: physical treatment method, chemical treatment method and biological treatment method. In the actual process, a comprehensive treatment mode is usually adopted. The physical and chemical treatment method has the limitations of complex treatment process, high treatment cost, expensive equipment, high maintenance difficulty and the like, and has secondary pollution of different degrees in the treatment process. The biological treatment method has the characteristics of simple treatment process, low treatment cost, no secondary pollution and the like. Therefore, the way of treating petroleum-contaminated soil by using microorganisms is attracting more and more attention and becomes the focus of domestic and foreign research.
China researches on the microbial remediation of petroleum-polluted soil from the end of the 70 th 20 th century, and the last decade is the golden period of research, but the technology for biologically treating sludge is not put into large-scale application at home. The key of the biological treatment technology lies in the screening and transformation of microbial strains, and the determination of various single-strain proportions and bacterial concentrations in the composite microbial inoculum. Because the components of the petroleum-polluted soil are complex, and a single strain cannot meet the requirement of efficient degradation of the petroleum-polluted soil, the method for selecting the strains capable of efficiently degrading different components and developing the composite microbial inoculum is an effective way for improving the degradation efficiency of the petroleum-polluted soil.
Disclosure of Invention
The invention aims to provide a method for restoring oil-containing soil by using a biological agent. The method for treating the oil sludge has the advantages of low cost, simple process and environmental protection, and has high utilization value. The method is characterized in that: through simulation experiments, various conditions of the biological agent for carrying out petroleum polluted soil remediation experiments are researched, the optimal method and parameters are finally determined, and data support and method guidance are provided for field application experiments.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a biological preparation for restoring oil-containing soil is characterized in that the biological preparation is prepared by mixing microbial strains a, b and c according to the ratio of the bacterial amount to 1:1: 1; wherein the strain a refers to: the strain is preserved in China general microbiological culture Collection center (CGMCC) 11442;
the strain b refers to: thermotoga neoformans-E [ DSM 4359] (Thermotoga neocolitana, which was purchased from American type culture Collection under ATCC number 49049);
the strain c refers to: the microorganism is a small sized strain of oleophilus thermophilus (Thermoleophilum minutum, which is purchased from American type culture Collection and has a deposit number of ATCC No. 35268).
The invention further discloses a method for restoring the oil-containing soil by the biological agent for restoring the oil-containing soil, which is characterized by comprising the following steps:
1) preparing culture medium, culturing biological bacteria, diluting the mixed bacteria with water, mixing the bacteria with waterThe mass ratio is 1: 10-1000 parts; the culture medium comprises: na (Na)2HPO4·12H2O 0.08％ KH2PO40.04％；NaNO30.06%；FeSO4·7H2O 0.002％；CaCl2·2H2O 0.002％；MgSO47H2O 0.04%; 0.1% of yeast powder;
3% of glucose; water: the balance;
2) diluting the mixed microbial inoculum according to the proportion of 1:1, mixing with oil-containing soil; fully stirring for 10-30 minutes at normal temperature/40 ℃ to form slurry, standing for 48h-4 weeks, and sampling to detect the oil content of the soil; wherein the oil-containing soil is 10% of soil with pH of 6-9.5.
The invention further discloses an application of the biological agent for repairing the oil-containing soil in the aspects of degrading the oil content in the oil-containing soil, improving the degradation efficiency of oil sludge and enabling the soil to reach the emission standard; the oil-containing soil refers to 10% oil-containing soil. The experimental results show that: the degradation rate of the biological agent for treating 10% oil-containing soil almost reaches 100%.
The soil and oil used in the experiment originated from three areas in hong Kong (seaside area soil, hole shop area soil, Wang guan Tun soil) and three corresponding oil products (Hongkong Datai oil, Yangsanmu Dai oil, guan Datai crude oil). The invention is used as a typical example to detect the pH of soil in three regions, and the soil is mixed with oil products to prepare an oil-containing soil sample with the oil content of 10%.
Preparing a culture medium: na (Na)2HPO4·12H2O 0.08％；KH2PO40.04％；NaNO30.06%；FeSO4·7H2O0.002％；CaCl2·2H2O 0.002％；MgSO47H2O 0.04%; 0.1% of yeast powder; 3% of glucose; the balance of water.
And (3) detecting the pH of the soil in the three regions, and mixing the soil and an oil product to prepare an oil-containing soil sample with the oil content of 10%. Preparing a culture medium, culturing a biological agent, mixing the biological agent with water according to different proportions, treating three kinds of oil-containing soil, and fully stirring for about 10 minutes under the conditions of pH 6-8/original pH and normal temperature/40 ℃ to enable the three kinds of oil-containing soil to be in a slurry state. Standing for 48h, 1 week, 2 weeks, 3 weeks and 4 weeks, and sampling to detect the oil content in the soil.
The invention mainly considers the following contents:
(1) the pH value of the oil-containing soil is different, the pH range of the application of the biological agent is 6-9.5, and the biological agent has effects on different oil product pH values.
(2) Because the temperature change caused by climate influences the activity of microorganisms, the biological agent of the invention can be applied to a wider temperature range and has obvious effect at normal temperature of-40 ℃.
(3) The microbial agent has extremely low petroleum degrading efficiency, the bacterial strain has high petroleum degrading efficiency, the higher the concentration of the bacteria is, the higher the degrading efficiency is, and the higher the degrading efficiency is when the microbial agent is diluted by 5000 times.
The method for restoring the oil-containing soil by using the biological agent has the characteristics that:
(1) the pH range of the biological agent is pH 6-9.5, and the biological agent has effects on different oil product pH values.
(2) The biological agent of the invention can be applied to a wider temperature range and has obvious effect in different temperature ranges.
(3) The biological preparation has high oil degrading efficiency, can have obvious effect in a short time, has obvious effect when the dilution ratio of the liquid medicine is 1/10-1/1000, and has shorter acting time when the dilution ratio is smaller under the condition of achieving the same degrading efficiency.
(4) The microbial agent used in the invention has no toxicity, and has no corrosion to related equipment when in use;
(5) the method has the advantages of simple process, simple required equipment and low cost.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the invention are commercially available; wherein the bacterial species
a means: the strain is preserved in China general microbiological culture Collection center (CGMCC) 11442; the source is as follows: see patent, application No. 201610012840.9.
b refers to: thermotoga neoformans-E [ DSM 4359] (Thermotoga neocolitana, which was purchased from American type culture Collection under ATCC number 49049);
c means: the microorganism is a small sized strain of oleophilus thermophilus (Thermoleophilum minutum, which is purchased from American type culture Collection and has a deposit number of ATCC No. 35268).
Example 1
The method for repairing the oil-containing soil by using the biological agent comprises the following steps:
(1) the microbial inoculum applied in the method is a mixed microbial inoculum, and is prepared by mixing strains a, b and c according to the ratio of the bacterial amount to 1:1: 1. Diluting with water at ratios of 1/10, 1/100, 1/1000 and 1/5000.
(2) The 10% oil-containing soil used in the method is respectively seaside area soil + hongkong Datai oil, hole shop area soil + Yangsanmu Datai oil, Wangnoutun soil + guandao crude oil.
(3) The pH of the 10% oil-containing soil used by the method is respectively as follows: the original pH of the side region soil + the hongkongdong grand station oil was 8.31, the original pH of the hole shop region soil + the tsunami Yangsu grand station oil was 9.22, and the original pH of the Wangnong Tungtu + the Yangtong grand station crude oil was 8.91; the three oil-bearing soils were adjusted to pH6, 7, 8, respectively.
(4) The temperatures used in the process were ambient temperature and 40 ℃.
(5) The treatment process and the reaction conditions of the method are as follows:
preparing a culture medium, culturing a biological agent, diluting with water, treating 12 kinds of 10% oil-containing soil with the original pH value of 6, the original pH value of 7 and the original pH value of 8 at the normal temperature and the temperature of 40 ℃, treating for 48 hours, and detecting the oil content of the soil. Through determination, the pH value and the temperature (40 ℃ C. \ normal temperature) have no obvious influence on the degradation rate of oil in soil. Probably because the pH and temperature adaptation range of the biological agent is wide, the biological agent has better action effect in a certain pH and temperature range. After the biological agent treats the sample for 48 hours, the degradation rate of oil in the soil reaches about 90% under the condition that the liquid medicine ratio is 1/10; the degradation rate of the oil in the soil is over 20 percent under the condition that the liquid medicine ratio is 1/100; the degradation rate of oil in soil is mostly over 10% under the condition that the liquid medicine ratio is 1/1000; the liquid medicine is much larger than the oil in the soil under the condition of 1/5000 without obvious degradation.
The culture medium comprises: na (Na)2HPO4·12H2O 0.08％；KH2PO40.04％；NaNO30.06%；FeSO4·7H2O0.002％；CaCl2·2H2O 0.002％；MgSO47H2O 0.04%; 0.1% of yeast powder; 3% of glucose; the balance of water.
Example 2
The method for repairing the oil-containing soil by using the biological agent comprises the following steps:
(1) the microbial inoculum applied in the method is a mixed microbial inoculum, and is prepared by mixing strains a, b and c according to the ratio of the bacterial amount to 1:1: 1. Diluting with water at ratios of 1/10, 1/100, 1/1000 and 1/5000.
(2) The 10% oil-containing soil used in the method is respectively seaside area soil + hongkong Datai oil, hole shop area soil + Yangsanmu Datai oil, Wangnoutun soil + guandao crude oil.
(3) The pH of the 10% oil-containing soil used by the method is respectively as follows: the original pH of the side region soil + the hongkongdong grand station oil was 8.31, the original pH of the hole shop region soil + the tsunami Yangsu grand station oil was 9.22, and the original pH of the Wangnong Tungtu + the Yangtong grand station crude oil was 8.91; the three oil-bearing soils were adjusted to pH6, 7, 8, respectively.
(4) The temperatures used in the process were ambient temperature and 40 ℃.
(5) The treatment process and the reaction conditions of the method are as follows:
preparing a culture medium, culturing a biological agent, diluting with water, treating 12 kinds of 10% oil-containing soil with original pH value of 6, pH value of 7 and pH value of 8 respectively at normal temperature and 40 ℃, treating for one week, and detecting the oil content of the soil. Through determination, the pH value and the temperature (40 ℃ C. \ normal temperature) have no obvious influence on the degradation rate of oil in soil. The degradation rate of oil in soil is almost 100% under the condition that the liquid medicine ratio is 1/10; the degradation rate of oil in the soil is mostly over 70 percent under the condition that the liquid medicine ratio is 1/100, wherein the degradation rate of oil in the seaside area soil and the Hongkong-Dong-Datai station is over 90 percent; the degradation rate of oil in soil is mostly over 60% under the condition that the liquid medicine ratio is 1/1000, wherein the degradation rate of oil in seaside areas soil and Hongkong great station is over 85%; the liquid medicine ratio is 1/5000, and most of the oil in the soil is more than 10%.
The culture medium comprises: na (Na)2HPO4·12H2O 0.08％；KH2PO40.04％；NaNO30.06%；FeSO4·7H2O0.002％；CaCl2·2H2O 0.002％；MgSO47H2O 0.04%; 0.1% of yeast powder; 3% of glucose; the balance of water.
Example 3
The method for repairing the oil-containing soil by using the biological agent comprises the following steps:
(1) the microbial inoculum applied in the method is a mixed microbial inoculum, and is prepared by mixing strains a, b and c according to the ratio of the bacterial amount to 1:1: 1. Diluting with water at ratios of 1/10, 1/100, 1/1000 and 1/5000.
(2) The 10% oil-containing soil used in the method is respectively seaside area soil + hongkong Datai oil, hole shop area soil + Yangsanmu Datai oil, Wangnoutun soil + guandao crude oil.
(3) The pH of the 10% oil-containing soil used by the method is respectively as follows: the original pH of the side region soil + the hongkongdong grand station oil was 8.31, the original pH of the hole shop region soil + the tsunami Yangsu grand station oil was 9.22, and the original pH of the Wangnong Tungtu + the Yangtong grand station crude oil was 8.91; the three oil-bearing soils were adjusted to pH6, 7, 8, respectively.
(4) The temperatures used in the process were ambient temperature and 40 ℃.
(5) The treatment process and the reaction conditions of the method are as follows:
preparing a culture medium, culturing a biological agent, diluting with water, treating 12 kinds of 10% oil-containing soil with original pH, pH6, pH7 and pH8 at normal temperature and 40 ℃, treating for two weeks, and detecting the oil content of the soil. Through determination, the pH value and the temperature (40 ℃ C. \ normal temperature) have no obvious influence on the degradation rate of oil in soil. The degradation rate of the oil in the soil is over 90 percent under the condition that the liquid medicine ratio is 1/100; the degradation rate of the oil in the soil is mostly over 80 percent under the condition that the liquid-chemical ratio is 1/1000, and the degradation rate of the oil in the soil is not obviously changed under the condition that the liquid-chemical ratio is 1/5000.
The culture medium comprises: na (Na)2HPO4·12H2O 0.08％；KH2PO40.04％；NaNO30.06%；FeSO4·7H2O0.002％；CaCl2·2H2O 0.002％；MgSO47H2O 0.04%; 0.1% of yeast powder; 3% of glucose; the balance of water.
Example 4
The method for repairing the oil-containing soil by using the biological agent comprises the following steps:
(1) the microbial inoculum applied in the method is a mixed microbial inoculum, and is prepared by mixing strains a, b and c according to the ratio of the bacterial amount to 1:1: 1. Diluting with water at ratios of 1/10, 1/100, 1/1000 and 1/5000.
(2) The 10% oil-containing soil used in the method is respectively seaside area soil + hongkong Datai oil, hole shop area soil + Yangsanmu Datai oil, Wangnoutun soil + guandao crude oil.
(3) The pH of the 10% oil-containing soil used by the method is respectively as follows: the original pH of the side region soil + the hongkongdong grand station oil was 8.31, the original pH of the hole shop region soil + the tsunami Yangsu grand station oil was 9.22, and the original pH of the Wangnong Tungtu + the Yangtong grand station crude oil was 8.91; the three oil-bearing soils were adjusted to pH6, 7, 8, respectively.
(4) The temperatures used in the process were ambient temperature and 40 ℃.
(5) The treatment process and the reaction conditions of the method are as follows:
preparing a culture medium, culturing a biological agent, diluting with water, treating 12 kinds of 10% oil-containing soil with original pH, pH6, pH7 and pH8 at normal temperature and 40 ℃, treating for three weeks, and detecting the oil content of the soil. Through determination, the pH value and the temperature (40 ℃ C. \ normal temperature) have no obvious influence on the degradation rate of oil in soil. The degradation rate of oil in soil reaches or almost reaches 100% under the conditions that the liquid medicine ratio is 1/10 and 1/100; the degradation rate of oil in soil is mostly over 95% under the condition that the liquid medicine ratio is 1/1000; compared with the first two weeks, the degradation rate of the oil in the soil under the condition that the water ratio is 1/5000 has no obvious change.
The culture medium comprises: na (Na)2HPO4·12H2O 0.08％；KH2PO40.04％；NaNO30.06%；FeSO4·7H2O0.002％；CaCl2·2H2O 0.002％；MgSO47H2O 0.04%; 0.1% of yeast powder; 3% of glucose; the balance of water.
Example 5
The method for repairing the oil-containing soil by using the biological agent comprises the following steps:
(1) the microbial inoculum applied in the method is a mixed microbial inoculum, and is prepared by mixing strains a, b and c according to the ratio of the bacterial amount to 1:1: 1. Diluting with water at ratios of 1/10, 1/100, 1/1000 and 1/5000.
(2) The 10% oil-containing soil used in the method is respectively seaside area soil + hongkong Datai oil, hole shop area soil + Yangsanmu Datai oil, Wangnoutun soil + guandao crude oil.
(3) The pH of the 10% oil-containing soil used by the method is respectively as follows: the original pH of the side region soil + the hongkongdong grand station oil was 8.31, the original pH of the hole shop region soil + the tsunami Yangsu grand station oil was 9.22, and the original pH of the Wangnong Tungtu + the Yangtong grand station crude oil was 8.91; the three oil-bearing soils were adjusted to pH6, 7, 8, respectively.
(4) The temperatures used in the process were ambient temperature and 40 ℃.
(5) The treatment process and the reaction conditions of the method are as follows:
preparing a culture medium, culturing a biological agent, diluting with water, treating 12 kinds of 10% oil-containing soil with original pH value of 6, pH value of 7 and pH value of 8 respectively at normal temperature and 40 ℃, treating four weeks, and detecting the oil content of the soil. Through determination, the pH value and the temperature (40 ℃ C. \ normal temperature) have no obvious influence on the degradation rate of oil in soil. The degradation rate of oil in soil reaches 100% under the conditions that the liquid medicine ratio is 1/10 and 1/100; the degradation rate of oil in soil reaches or approaches 100% under the condition that the liquid medicine ratio is 1/1000; compared with the previous three weeks, the degradation rate of the oil in the soil is not obviously increased under the condition that the liquid medicine ratio is 1/5000 and is maintained at 10-15%.
The culture medium comprises: na (Na)2HPO4·12H2O 0.08％；KH2PO40.04％；NaNO30.06%；FeSO4·7H2O0.002％；CaCl2·2H2O 0.002％；MgSO47H2O 0.04%; 0.1% of yeast powder; 3% of glucose; the balance of water.
Example 6
Actual use case
The microbial agent can repair oil sludge with low oil content (about 10 percent), so that the oil sludge reaches the emission standard.
Comparative test
Figure 638646DEST_PATH_IMAGE001
Conventional data comes from (1) patents: jiahanzhong, Majiabobo, Wangzhenyi, bending and bracing hoarding a microbial remediation method of petroleum-contaminated soil, CN106734181A, (2) literature: lihua, Dongxian, screening of oil sludge degrading bacteria in refinery and initial exploration of oil reducing conditions [ J ] chemical and biological engineering, 2012, 29(9): 75-77.

Claims (3)

1. A biological preparation for restoring oil-containing soil is characterized in that a mixed microbial inoculum is prepared by mixing microbial strains a, b and c according to the ratio of the microbial strains to the microbial inoculum of 1:1: 1; wherein the strain a refers to: the strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 11442; the strain b refers to: thermotoga neoformans (Thermotoga neocolitana) NS-E [ DSM 4359], which was purchased from the American type culture Collection under accession number ATCC number 49049;
the strain c refers to: the strain is obtained from the American type culture Collection, and has a deposit number of ATCC No. 35268.
2. A method for remediating oil-contaminated soil using the biological agent for remediating oil-contaminated soil as set forth in claim 1, which comprises the steps of:
1) preparing a culture medium, culturing a biological agent, diluting a mixed microbial agent with water, wherein the mass ratio of the mixed microbial agent to the water is 1: 10-1000 parts; the culture medium comprises: na (Na)2HPO4·12H2O 0.08％ KH2PO40.04％；NaNO30.06%；FeSO4·7H2O0.002％；CaCl2·2H2O 0.002％；MgSO47H2O 0.04%; 0.1% of yeast powder;
3% of glucose; water: the balance;
2) diluting the mixed microbial inoculum according to the proportion of 1:1, mixing with oil-containing soil; fully stirring for 10-30 minutes at normal temperature/40 ℃ to form slurry, standing for 48h-4 weeks, and sampling to detect the oil content of the soil; wherein the oil-containing soil is 10% oil-containing soil with pH of 6-9.5.
3. Use of the biological agent for remediating oily soil as defined in claim 1 for degrading oil in oily soil to bring the soil to emission standards; the oil-containing soil refers to 10% oil-containing soil.
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