Isolation Of Iron-Reducing Bacteria In Paddy Soil And Its Potential Identification

Dissimilatory Iron Reducing Microorganisms is a kind of bacteria which can use Fe(III) as the terminal electron acceptor and oxidize organic matter to conserve energy. As Fe(III) geochemical abundance is relatively high, we can find the Dissimilatory Iron Reducing happen in all kinds environments which have anaerobic conditions. So for the same reason, we can find the Dissimilatory Iron Reducing Microorganisms exist in all kinds'anaerobic environments. Heretofore, the scientist has isolated many kinds of Fe(III)-reducing microorganisms from freshwater aquatic sediments, pristine deep aquifer, petroleum-contaminated shallow aquifer and marine sediments,etc. Now people have known bacterial Fe(III) reduction is an important pathway of bioenergy metabolism in the process of life evolution and 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, different paddy soil samples from Sichuan and Jiangxi have been incubated and tested, and use anaerobic culture, primarily purified some microorganisms with iron-reducing functions from paddy soils, and bacterial strain, the colony morphology and physiological function identification and 16S rDNA sequence analysis were completed to some bacteria strains . The main results obtained are listed as follows:1. Isolation of sorts of Fe(III)-reducing bacteria from Sichuan paddy soil and Jiangxi paddy soil. These Microorganisms are two kinds of different bacteria. One is the anaerobic Fe(III)-reducing bacteria which is gram-opsitive, rod-shaped, round-end.The colony morphology were small, white, apophysis, round, opacification, smooth surface and diameter 3mm. Another one is the facultative anaerobic Fe(III)-reducing bacteria which is gram-negative bacilus. The colony morphology was small, grey round and rough of edge.2. The methods of isolation and purification of Fe(III)-reducing microorganism in paddy soils were established and design the Fe(III)-citrate medium which can be used to isolate Fe(III)-reducing bacterium.3. Comparisons were done between different iron-reducing bacteria under the conditions with glucose as the sole carbon source, synthetic Fe (OH) 3 (ferrihydrite) and citric acid iron reduction features. For the citric acid soluble iron, facultative anaerobic and strict anaerobic iron reducing strains have shown high efficiency, system trivalent iron reduction rate is between 84.3% and 93.8%. For Fe (OH) 3 , which is difficult to dissolve in water, is strictly anaerobic iron-reducing bacteria have a higher reduction rate, the system of three-valent iron reduction rate can exceed 90 percent, facultative anaerobic bacteria of the iron hydroxide Reduction rate of more than 80%, but the response rate is lower than strictly anaerobic iron reduction4. The paper displayed glucose and pyruvate are the optimal carbon sources of the 6 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 SC-a18>JX-a20>JX-a3>SC-a13>JX-a10>SC-a19. The reaction rate of Fe(III) reduction of strain SC-a18 was maximal to 92.11% ,while the minimum of strain SC-a19 just was 24.52%. Other Fe(III) reductions were slow by used with other carbon sources, and the reaction rates were very low.5. Passage of different strains in the process of analysis and the stability of species preservation methods studies show that, strictly anaerobic iron reducing strain show more stability than anaerobic bacteria strains in the process of passage. Strictly anaerobic iron-reducing bacteria that 80% of the strains can be stabilized passage, and facultative anaerobic bacteria strains can stabilize the passage less than 50 percent. Citric acid in the medium propagation of iron bacteria in 4℃stable can be stored for more than 6 months6. Rejuvenation at the strains in LB medium has relatively short period of stagnation, 30℃cultured for 10 h strain about the growth curve to be able to enter the phases in the citric acid in the liquid medium iron, to train 15 hours or so to enter the growth curve few phases. However, citric acid medium iron into the microbiology of several periods, the growth rate is obviously faster than that in LB medium, and ultimately system microbial biomass is much greater than in LB medium microbial biomass.7. Under anaerobic conditions, take synthetic Fe (OH) 3 as the only electronic receptors, glucose as electron donor, and determination of anaerobic and strict anaerobic iron-reducing bacteria characteristic curve of iron reduction, and of anaerobic bacteria strain in cultured for 24 h after the Fe (III) reduction into the rapid growth period, 108 h after the Fe (III) reduction rate of basic achieve stability. In addition to the JX - o6 strains ferric hydroxide reduction rate at 80 percent, the other three strains are more than 90%. The strict anaerobic bacteria strains iron reducing process, the JX - a10 and JX - a8 in training 36 hours after iron reduction rate increases rapidly than the SC - a15 and SC - a17 strain early 12 h, 72 h after the reduction of ferric hydroxide all reached more than 90 per cent, growth in the number of strict anaerobic bacteria in 12 hours, which makes 80% of the Fe (III) reduction, and responded very quickly. At the same time culture system quickly lower the pH value. In the training system, the control of iron reduction rate almost as a straight line, explaining that it did not participate in the microbiology and when microbial growth are restricted, Fe (OH) 3 reduction reaction almost does not occur.8. Through 16S rDNA sequence homology analysis and comparison found that strains JX - a20, SC - a13, SC - a18 and SC - a19 between the evolutionary relationships are more close to the river with the heat from the Asia-kun of permanganate oxidation (Uncultured bacterium Hot Creek) in the evolutionary relationship in a branch, and strains SC - a17's differences with the former larger. Iron reducing strain JX - a20, SC - a13, SC - a17 and SC - a18 and a variety of acid Clostridium spp (Clostridium butyricum) strains have higher homology, and in the evolution of relations between close relatives of , SC - a19 and strains typical iron reducing strain Clostridium sp. FRB1 the homology of the largest.