Influences Of Associated Pollution Of Cd～(2+) And Acetochlor On The Community And Bioactivities Of Free-living Diazotrophs In Upland Soil

In this thesis, the effects of Cd²⁺, acetochlor and their association on the number and nitrogenase activity of nitrogen-fixing bacteria were studied in upland soil samples. Moreover, a bacterium capable of fixing nitrogen efficiently was isolated, identified and phylogenetically analyzed. The growth and nitrogenase activity of Azotobacter beijerinckii C4 (G^-) under the pollution was studied. The activity response of the antioxidant enzymes superoxide dismutase, catalase, ATP enzyme activities of A.beijerinckii C4 following exposure to the pollution was also investigated. The results will be valuable to discuss the influence mechanism of strain C4 with pollution and to build up alert index systems and environmental quality evaluation in upland soil polluted by Cd²⁺ and atecholor. Here the main results of this study are presented as the follows:1. The influences of Cd²⁺, acetochlor and their association on the cultural nitrogen-fixing bacteria in upland soil and the intensity of N₂-fixation were investigated using traditional methods. The results showed that the aerobic nitrogen-fixing bacteria were sensitive to Cd²⁺。The low concentration of Cd²⁺ could increase the numbers of aerobic nitrogen-fixing bacteria, while the CFU of N₂-fixing bacteria were strongly inhibited by higher concentration of Cd²⁺. Acetochlor had no essential effect on aerobic nitrogen-fixing bacteria. Anaerobic nitrogen-fixing bacteria were stimulated by Cd²⁺ at the first 8 days, and then restrained. Finally, its numbers were remarkably restrained by the treatment of 5.0 mg kg^-1 dry soil, while others were nearly equal to that of the control. Acetochlor had no evident influence on the numbers of anaerobic nitrogen-fixing bacteria during the whole experiment phase except the group of 2.0 mg kg^-1 dry soil. Association of Cd²⁺ and acetochlor demonstrated joint inhibitory effects on both aerobic and anaerobic nitrogen-fixing bacteria. The inhibition of nitrogenase activity became stronger with the increase of Cd²⁺ concentration, showing a striking dose-response relationship. The concentrations of acetochlor used in this experiment had a relatively mild effect on the intensity of N₂-fixation in a short term. However, the inhibition to that was found at the end of experiments. The nitrogenase activity under the combined pollution accumulated originally, but declined ultimately.2. A bacterium, named strain C4, capable of fixing nitrogen efficiently, was isolated from a kailyard and identified as Azotobacter beijerinckii based on morphology, physio-biochemical properties and a partial sequencing of 16S rDNA. Strain C4 was sensitive to gentamicin, ampicillin, amikacin and erythromycin, but insensitive to penicillin, cephalothin and chloramphenicol. The nitrogenase activity of strain C4 was highest after growth for 24h, with the acetylene reduction activity (ARA) of 1203.779 nmol C2H4 per h per ml culture. The nitrogenase activity declined subsequently, and was only 75.66 nmol C2H4 per h per ml culture grown for 90h.3. The impacts of growth and nitrogenase activity of strain C4 isolated from garden was studied to illuminate the toxic effect of compound pollution on strain C4. Results obtained indicated that the growth and nitrogenase activity of strain C4, especially after growing for 12h, was strongly restrained by Cd2+, and that treated with 0.5 mg L"1 was inhibited evidentIy(p<0.05). Strain C4 demonstrated good tolerance to acetochlor, especially the nitrogenase activity of strain C4 growing for 12h could reach the level of the control even exposed to concentration of 1000 mg/L. The higher sensitivity of strain C4 growing for 12h to the association of Cd2+ and acetochlor was also observed. The growth and nitrogenase activity of strain C4 could only retain at a low level under the pollution stress of the two compounds. It indicated that Cd2+ is the key factor while acetochlor has no marked influence on strain C4 in the associated pollution of compounds. However, notable interaction between Cd2+and acetochlor was not detected.4. The activity response of the antioxidant enzymes of Azotobacter beijerinckii C4 (G) following exposure to Cd2+, acetochlor and their association was investigated. Results showed that activities of SOD, CAT and ATPase in the bacterium induced by Cd2+ or acetochlor were positive and obvious, especially in shorter time after treatments. The associated pollution of Cd2+ and acetochlor had different impacts on the activity of the antioxidant enzymes with different concentrations and treatment times. SOD and ATPase activities of all association pollution appeared stronger than those of a single factor, but the activities recovered finally to that of the control. It demonstrated that various concentration association of the two pollutants caused the inducing or inhibiting actions primarily, but inhibited effects ultimately on CAT activity.