Bioremediation Methods And Toxicity Risk Inhibition Effects Of Oil-contaminated Soil

As a cost-effective and non-secondary pollution remediation method for oil-contaminated soil,bioremediation mostly adopts single or combined remediation methods,but has limited effect on the removal of petroleum hydrocarbons in contaminated soil.The ecotoxicity of petroleum hydrocarbons in soil is an important basis for studying the remediation effect and controlling polluted soil,but most of the current literature studies evaluate the remediation effect of petroleum-contaminated soil based on the removal efficiency of petroleum hydrocarbons in soil.Based on the above problems,this paper takes the removal efficiency and ecotoxicity of petroleum hydrocarbons in soil by bioremediation as the evaluation index,and explores the deep removal method of petroleum hydrocarbon residues in soil,which provides a scientific basis for the treatment of oil-contaminated soil.This paper takes soil with different levels of oil pollution as the research object,uses different bioremediation methods to treat it,explores the effects of different remediation methods on the content and physicochemical properties of total petroleum hydrocarbons（TPHs）in soil,and studies the occurrence of carbon and nitrogen conversion functional genes by q PCR technology,explores soil ecotoxicity by earthworm toxicity test and wheat seed germination experiment,and then selects suitable multi-stage remediation methods for remediation.The removal law of soil TPHs and the inhibitory effect of multi-stage remediation on soil ecotoxicity were explored.The following conclusions were drawn from the study:（1）Nine restoration methods were used to add chicken manure organic fertilizer（YJ）,cattle and sheep manure organic fertilizer（YNY）,NH₄Cl（NH）,KNO₃（NO）,inoculation of L2 degrading bacteria（L2）,inoculation of F4 degrading bacteria（F4）,planting corn（ZY）,planting wheat（ZM）,and natural degradation（CK）to the contaminated soil for 60 days.In general,the degradation rate of TPHs in the soil with three pollution levels was from high to low:organic fertilizer>inorganic nitrogen was applied>planted plants were>natural degradation≈inoculation of degrading bacteria.The content of TPHs in the soil after 60 days was reduced from 7087,14426 and 30580mg·kg^-1to 3818,7142 and 14812 mg·kg^-1,respectively,and the degradation rates were2.33,2.89 and 2.43 times that of natural degradation.When inorganic nitrogen source remediation was applied,the effect of adding NH₄Cl to 7087 mg·kg^-1（low pollution level soil）and 14426 mg·kg^-1（medium pollution level soil）contaminated soil was better than KNO₃,with degradation rates of 34.66%and 32.41%,respectively,and there was no significant difference in 30580 mg·kg^-1（high pollution level soil）.Planting maize had a better effect on removing TPHs than growing wheat.Inoculation of degrading bacteria had a poor effect on the removal of TPHs.（2）The overall downward trend of ammonium nitrogen,available phosphorus and pH values in the soil physicochemical properties of a single remediation method showed a downward trend,while nitrate nitrogen showed a downward trend first and then an upward trend.The content of ammonia nitrogen in NH,YNY and YJ treatment gradually decreased,and the contaminated soil of 30580 mg·kg^-1was the most obvious,from 65.47,44.84 and 55.13 mg·kg^-1to 30.33,10.27 and 3.46 mg·kg^-1,respectively.The nitrate content in NO treatment gradually decreased,and the contaminated soil of30580 mg·kg^-1was the most obvious,from 3869.17 mg·kg^-1to 2287.60 mg·kg^-1,and the nitrate content in NH,YYN and YJ treatment showed a trend of first decreasing and then rising.The available phosphorus content in YNY and YJ treatment decreased significantly from low pollution level to high level soil,and the available phosphorus content in YNY treatment decreased from 215.28,211.90 and 207.46 mg·kg^-1to 133.78,128.46 and 125.64 mg·kg^-1,respectively,and the available phosphorus content in YJ treatment decreased from 166.06,169.31 and 170.12 mg·kg^-1to 122.27,115.93 and115.26 mg·kg^-1,respectively。All treatments had pH values greater than 7.00 and showed a decreasing trend.In general,the physical and chemical properties of contaminated soil treated with 30580 mg·kg^-1under YJ,YNY,NN and NO remediation were significantly changed.（3）Soil DNA of 27 sample species was extracted after single bioremediation to 60days,and the functional gene abundance was studied by Qpcr technology.The abundance of alkane degradation genes in soil was between 2.21×10⁵～3.99×10⁹copies·g^-1,the abundance of aromatic genes was between 1.40×10¹⁰～1.34×10²¹copies·g^-1,the abundance of alkane degradation genes in soil was lower than that of aromatic degradation genes,and the degradation potential of soil microorganisms to aromatics was higher than that of alkanes when it was repaired to 60 days.The results of nitrogen conversion functional genes showed that the genes with higher abundance were mainly genes encoding membrane-bound nitrate reductase(1.09×10⁸～8.95×10¹¹copies·g^-1)and nitrite reductase(2.47×10⁶～8.07×10⁹copies·g^-1),so the nitrogen cycle after 60 days of repair was mainly based on denitrification process.（4）Earthworm toxicity test and wheat seed germination growth test were carried out after 60 days of single bioremediation,and the earthworm lethality rate,earthworm body weight inhibition rate,wheat germination rate,wheat growth index,earthworm enzyme activity and wheat leaf enzyme activity were determined.The lethality rate of earthworms treated with F4 in soil with low pollution level before and after single bioremediation was 100%,the lethality rate of earthworms treated with NH and NO in soil with medium and high pollution levels was 100%,and the lethality rate of earthworms in soil treated with natural degradation,application of 15%organic fertilizer and planting plants was lower than 0d in contaminated soil.The results showed that the application of inorganic nitrogen sources according to C/N=10:1 would increase soil ecotoxicity,and the application of 15%organic fertilizer and planting plants could inhibit the ecotoxicity of TPHs to earthworms.After 60 days of soil remediation with different pollution levels by NH and NO,the germination rate and growth indexes of wheat in contaminated soil had a significant inhibitory effect compared with CK treatment,and the rest of the remediation had no significant inhibitory effect on the germination rate and growth indexes of wheat.The MDA enzyme activity in the biological treatment group of earthworms and wheat leaves was lower than CK,indicating that biological treatment could reduce the stress effect of TPHs on wheat in soil.The content of soil TPHs with low and medium pollution levels was significantly positively correlated with the germination rate of wheat,and the content of soil TPHs in medium and high pollution levels was significantly positively correlated with the number of primary roots of wheat.In general,the application of inorganic nitrogen sources according to C/N=10:1 increased soil ecotoxicity.（5）The soil contaminated by 25253 mg·kg^-1TPHs was remediated in three stages for 90 days by natural degradation（CK）,15%chicken manure organic fertilizer（F1）,twice 15%chicken manure organic fertilizer（F2）and 15%chicken manure organic fertilizer+planting corn+NH₄Cl（F3）according to C/N to 10/1.The content of TPHs in soil decreased from 25253 mg·kg^-1to 19067,8679,9818 and 8048 mg·kg^-1,respectively,and the third-order treatment of F3 had the best effect on the removal of petroleum hydrocarbons in soil,which was 2.78 times that of natural degradation.The abundance and distribution of functional genes showed that nitrogen conversion was mainly reduced by nitrite,the abundance of alkane degradation genes was between6.41×10³～3.99×10⁷copies·g^-1,and the abundance of aromatic degradation genes was between 7.63×10²～3.74×10¹²copies·g^-1,so the degradation potential of aromatic hydrocarbons by microorganisms was higher than that of alkanes.In the earthworm toxicity test after 90 days of remediation,the body weight inhibition rates of earthworms in F1 treatment for 7 d and 14 days were-11.06%and-18.60%,respectively,compared with the lowest remediation,the body weight inhibition rates of F2 and F3 earthworms were higher than those in contaminated soil and CK treatment at0 d.The content of TPHs in soil treated with CK and F1 treatment was positively correlated with the inhibition rate of earthworm body weight,indicating that the natural degradation of contaminated soil and the application of 15%chicken manure organic fertilizer treatment were related to the content of TPHs in soil.The correlation between TPHs content and genes showed that the degradation of alkanes and biphenyls,ammonia oxidation and nitrite reduction were mainly carried out in the early stage of repair,and the degradation of toluene and phenolic substances and nitrate dissimilation reduction were carried out in the later stage.In summary,considering the removal rate of TPHs and soil ecotoxicity,the application of chicken manure organic fertilizer can be used as a better remediation technology for oil-contaminated soil.