| Petroleum contaminated soil was caused by crude oil and various petroleum products into the environment when they were exploited, refined, transported and used. With the increasing of oil productions and consumption, environment pollution is gradually severe. Spilled oil in the soil can destruct oil structure and plant roots, affect soil compactness, ecosystems of soil-plant and soil-microbial as well as damage underground water, prevent root respiration, even soil productivity and crops yield. Therefore, remediation of contaminated soil is urgent. It has great significance to human survival and sustainable development of society. In this paper, we carried out bioremediation technology to the different concentrations of petroleum contaminated soil and provided strong technical strategy, research results are as follows:1. Oil components of oily sludge were separated into alkanes, polycyclic aromatic hydrocarbons and non-hydrocarbon by column chromatography method. Then the best conditions for any peak by gas chromatography-mass spectrometry (GC-MS) were optimized. Research on the method for qualitative and quantitative determination of components through GC-MS for the further research was carried out.2. Our laboratories have isolated Bacillus amyloliquefaciens Bz6-1 that can produce lipopeptide biosurfactants, so we conducted a study about culture conditions of Bz6-1:9 hours in the VGPA liquid medium, the surface tension down to 31 mN m-1; the surface tension attain the stable phase after 15 hours. In the medium condition optimization, we find that molasses is the best carbon source and peptone is the best nitrogen source and when pH=8, temperature= 25℃, the surface tension is lowest. In the sludge elution studies, the oil content of 33.9% sludge, by Bz6-1 produced lipopeptide surfactant and electrolyte (NaCl) treatment, the top oil recovery rate of 80.9%, washout rate of 89.7%, residual rate of 3.4%. A series of elution experiments confirmed that the surface-active agent can be used to deal with the oil sludge of high level and had a good effectiveness.3. The oil content of plant disposal sludge was lower than that of the control in phytoremediation experiments for 4 months. At the end of phytoremediation, treatments with different plants lead to unequal results, degradation rate of sorghum, maize, tall fescue, alfalfa, soybeans, respectively, was 13.7%,20.1%,23.1%,26.6%,28.5%,34.2%. Among all the plants, the oil content of tall fescue, alfalfa and soybean significantly was lower than that the control (p <0.05).However, in the later period of remediation (60-120 d), large-scale death were appeared to alfalfa. At the same condition, oil and salt stress of soybean is serious than tall fescue in our further research. Therefore, it is proposed that tall fescue is the appropriate plant for phytoremediation of the sludge with oil contaminated.4. Experiment of the isolation of plant growth-promoting bacteria (PGPR) from oil contaiminated soil of Sheng li Oil field and the root of tall fescue, was carried out with the method of enrichment screening as well as that ACC was used as the sole nitrogen source.60 strains of microorganism were isolated, in which only three bacteria contained 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, respectively, SL-14, SL-20, SL-50. Subsequently, three bacteria were initially identified as Klebsiella oxytoca, Klebsiella, and Pseudomonas by colony morphology, microscopic observation, Biolog, as well as 16S rDNA sequence analysis. In addition, the growth-promoting bacteria can produce a-keto acids, we detected ACC deaminase activity of three growth-promoting bacterials with BioRad protein microdetermination method, the quantitative determination of SL-14, SL-20, SL-50 was 0.5 u mg-1,0.35 u mg-1,0.4 u mg-1. Growth curve showed that the three strains entered to the stationary phrase in TSB liquid medium at the time of 9th hour, and did not show signs of decline after 48 h. Finally, through the petri dish germination experiments, we studied promoting effect of the plant growth-promoting bacteria on oats, ryegrass and tall fescue under different salt conditions (0,0.3,0.5,0.7%NaCl).The results showed that seed germination rate with growth-promoting bacteria (SL-14, SL-20, SL-50) treatment did not change significantly with control at different salt concentrations. This may be caused by seed quality of their own. However, length of sprouts of oats is significant different with that of control (P<0.05), in which shoot length of growth promoting bacteria SL-50 is longest. The same is true to rye grass. However, growth-promoting bacteria and ck have no significant difference to tall fescue in shoot length, but shoot length of growth-promoting bacteria SL-50 is longest, yet. The research above showed that growth promoting bacteria with the ACC deaminase activity can effectively improve the length of sprouts of oats, ryegrass, tall fescue under salt stress condition, to a certain extent. |
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