Characterization And Phylogenetic Analysis Of Ferric Reducing Microorganism

Bacterial Fe(III) reduction is an important pathway of bioenergy metabolism in the process of life evolution. Many kinds of archaebacteria and eubacteria are capable of reducing Fe(III) to conserve energy. Fe(III) in environment undergoes reduction under anaerobic condition. And this process is a microbial dissimilatory reduction, namely an anaerobic respiration with Fe(III) as the terminal electron acceptor. Dissimilatory iron reduction occurs generally in the natural anaerobic environment, where dissimilatory ferric reducing microorganism exists universally. It is considered as one of the first microbial respiration. Since it could be inherited in some degree in the evolution process, there must be much more microbial are capable to dissimilatory reduce Fe(III). Iron oxidation and reduction plays an important role in paddies, the model system in studying the redox process in natural eco-systems. So to strengthen the study on dissimilatory iron reduction process could help to understand the evolution process and the energy metabolism in early stages, theoretically. Study on characteristics of ferric reducing microorganism, might help to understand microbial reducing mechanism of iron oxides in paddy soil, to recognize the role of dissimilatory iron reduction in paddy ecosystem, could also help to elucidate the remediation and transformation of contamination in paddy soils.In this paper, two paddy soil samples collected from Sichuan, Jiangxi were used and the soil solutions were incubated under anoxic atmosphere. The methods of separation and purification of ferric reducing microorganism in paddy soils were established. The characteristics of the cell and colony morphology, and physiological characterization were obtained. And the effect of strains on Fe(OH)₃ reduction used with different carbon sources in pure culture was also analyzed. Through 16S rDNA sequence analysis of Fe(III)-reducting microorganisms, the postion of them in phylogenic taxonomy could be ascertained. The main results obtained are listed as follows:1. Using enrichment culture methods, four ferric reducing microorganisms (P4, 8₁₃, 8₁₈, 8₁₉) were isolated from Sichuan paddy soil, and three (4₀₃, 4₁₀, 4₂₀) of them were isolated from Jiangxi paddy soil. Their general characteristics of the cell and colony morphology were small, white, apophysis, round, opacification, smooth surface and diameter 3mm. They were all anaerobic, gram-opsitive, rod-shaped, round-end. Measuring the curve of growth, it attained a logarithms growth period within 10³0h. So, maximum of strains could be achieved by propagation with 50% LB only one day in 30℃.2. The reaction rate of Fe(III) reduction could reflect the Fe(III) reduction stability of microorganisms. Through three generations of 7 ferric reducing strains were grown in Fe(OH)₃ liquid medium, the reaction rates of them were all in 70%⁹0%. This showed the Fe(III) reducing capacities of them were relatively stable.3. To reflect the effect of different strains on Fe(OH)₃ reduction used with carbon sources, the paper showed 60 mmol/L carbon source was an optimal concentration to partition different strains, by compared the effect and logistic model of different strains on Fe(OH)₃ reduction used with 9.6 mmol/L and 60 mmol/L two carbon sources.4. The paper displayed glucose and pyruvate be the optimal carbon sources of the 7 strains. When used with glucose, the Fe(III) reduction and reactive rate of them were maximum, and the maximal potential of Fe(III) reduction was in proper order for: strain 8₁₈>4₂₀>4₀₃>8₁₃>P4>4₁₀>8₁₉. The reaction rate of Fe(III) reduction of strain 818 was maximal to 92.11% ,while the minimum of strain 8₁₉ just was 24.52%. At the initial stage of culture used with glucose and pyruvate, the pH of the whole systerm declined to the minimum in 4.5⁵.5 rapidly, and then picked up to 6.5 slowly. Other Fe(III) reductions were slow by used with other carbon sources, and the reaction rates were very low. The pH was always in 7.0⁷.5. So the lower pH was good for Fe(III) reduction.5. Adopting the PCR acquired parts of 16S rDNA sequences of Fe(III)-reducting strain P4 with 1325 bp. Through NCBI MegaBlast homology analysis and Clustal and Phylip software, 16S rDNA phylogenetic tree was constructed. The tree showed that strain P4 belonged to Clostridium butyricum, most to several uncultured bacterium (DQ441334, DQ083720, DQ083732, AM176850, AY953222) with 98% similarity, and to Clostridium butyricum (AY604563, AM176850, DQ831126, X77834, AF005092) with 98% similarity.