Research On Slow-release Remediation Technology Of Immobilized Microbial Agents In Petroleum Hydrocarbon Contaminated Soil

As an unconventional energy source,shale gas is an important planning target for the country to optimize its energy structure and is one of the five high-end growth industries identified by Sichuan Province.The domestic and foreign experience of shale gas development shows that the ecological environment protection is one of the biggest challenges.The remediation of high oil-containing solid waste and oil contaminated soil which generated from the application of oil-based materials urgently requires technical support.Using petroleum hydrocarbon pollutants as a nutrient medium to achieve its harmlessness through metabolism of microorganisms,especially different degrading bacteria are combined to constitute a highly efficient degrading bacterial population for highly complex mixed hydrocarbon system,is a research hotspot in this field.Synergistic and combined effects of a variety of microorganisms are one of the major research directions for petroleum hydrocarbon contaminated soil restoration in recent years.However adding free microbial agents directly has the disadvantages of poor tolerance to the environment and rapid loss of microbial agents,this paper aimed to develop a type of sustained-release repair agent based on microencapsulation technology,which will increase the activity and density of microorganisms through sustained release of nutrients.In this paper,microorganisms grown with petroleum hydrocarbons as the sole carbon source were obtained through enrichment and gradient acclimation from the soil near the shale gas wellsite in Sichuan;and a consortium with high-efficiency petroleum hydrocarbon degradability were constructed;the optimum nitrogen and phosphorus and its concentration was studied.Next,two kinds of preparation methods for sustained-release bioremediation agents in laboratories were developed,and the application effects of two bioremediation agents were investigated through indoor simulation experiments.The main research contents and results are as follows:?1?Screening and identification of high-efficiency oil-degrading bacteria for restoring oil-contaminated soil in shale gas fieldA total of 12 strains were isolated and obtained,which used crude oil as the sole carbon source.The highest 7d-TPH removal rates of the four strains were:34.98%?MR-01?,32.72%?MR-04?,41.22%?MR-06?and 37.88%?MR-12?.The morphological,physiological characteristic and molecular biology of the four isolated bacteria were identified.The 16S rDNA conclusion was that strain MR-01 was Citrobacter farmeri;strain MR-04 was Acinetobacter johnsonii;MR-06 and MR-12 are both Pseudomonas aeruginosa.At the same time,in order to fully understand the biological characteristics of these four strains,not only their growth curves were drawn,but also the effects of external environmental factors such as temperature,pH.and salinity on the growth characteristics of the isolated strains.?2?Construction of a highly efficient petroleum-degrading consortium and selective study of N and PA combination of strains MR-01,MR-04,MR-06,and MR-12 was used to find the most efficient degrading consortium.According to the 12d-TPH removal rate,a bacterial flora?Mixed 146?consisting of strains MR-01,MR-04 and MR-06 was identified a highly effective strain that TPH removal rate up to 77.49%.The compositional changes before and after degradation of single bacteria and mixed 146 were analyzed by chromatography and GC-MS.The optimum nutrient study showed that both the water-soluble organic nitrogen source yeast powder and the phosphorus source sodium glycerophosphate can significantly promote the growth of four single strains and Mixed 146,when the yeast powder concentration is 50 g/L,and the concentration of sodium glycerophosphate is 4.0 g/L.?N:P=12.5:1?,Mixed 146 grew best.?3?Preparation and performance evaluation of immobilized sustained-release microcapsulesThe sodium alginate was selected as the wall material of the microcapsules,and the microcapsule agents were prepared by direct embedding and adsorption followed by embedding.Taking the yield of the prepared microcapsules,the loading amount of nitrogen and phosphorus,and the carrying amount of bacteria as the evaluation indexes,the orthogonal experiment was used to optimize the process parameters of the two methods.The best technology parameters of direct embedding method:sodium alginate 2.5%,CaCl₂ 3%,2h cross-linking time,and 18G diameter of syringe.Under this process,N and P samples loadings were 45.02%and 12.91%respectively,and the embedding rates were 45.14%and 93.32%respectively,and the bacterial load Log?CFU/g?was 10.63.The optimum process for adsorption followed by embedding was:biochar concentration 0.75%,sodium alginate 3.5%,and 2%CaCl₂.Under this process,N and P samples loadings were 47.06%and 11.56%respectively,and the embedding rates were 52.41%and 93.11%respectively,and the bacteria load was Log?CFU/g?=10.47.The slow-release time of the nutrients of the microcapsule prepared by the two processes can be maintained in water for 24 hours.In addition,SEM shows that the surface of the microcapsules has a rich fold structure,and the pores of the internal network structure are developed.Adsorbents biochar that has developed pore structure and large specific surface area are favorably improved pore properties of the microcapsules.FT-IR showed that the microcapsules prepared by both processes were successfully loaded with nitrogen and phosphorus and were physically adsorbed and embedded.Thermal comprehensive analysis showed that two kinds of microcapsules prepared by two processes have good thermal stability.BET pore diameter analysis showed that the initial pore diameter of the microcapsules was directly expanded by biochar in the first adsorption and embedding process.?4?Study on sustained release repair effect of immobilized microbial agentsDuring the 60 days of soil remediation,the TPH degradation rates of immobilized bacteria?Only embedded bacteria of Direct embedding:BQ and Adsorption followed by embedding:XQ?and immobilized slow-release bacteria?Embedding bacteria and nutrients of Direct embedding:YBQ and Adsorption followed by embedding:YXQ?were all higher than free Mixed 146?35.06%?.While the degradation rate of YBQ?62.45%?is higher than degradation rate of BQ?45.61%?by 16.84%,the degradation rate of YXQ?66.95%?is higher than degradation rate of XQ?48.74%?by 18.21%.This means that immobilized slow-release bacteria can promote the activity of microorganisms and the degradation of pollutants.At the same time,the degradation rate of YXQ was 4.5%higher than that of YBQ,and XQ was 3.13%higher than that of BQ,while the order of microbial activity within the 15 to 50d period was YXQ>YBQ>XQ>BQ>FC>blank control.This shows that the adsorptivity and porosity improve the passage of the immobilized carrier.Two kinds of immobilized release microcapsule remediation agents have good engineering application potential.