| jMicrobial Fe(III) reduction are capable of oxidizing organic or inorganic electron donors with ferric iron Fe(III) as electron acceptors. Dissimilatory Fe(III) reducing phenomena exist in almost natural anaerobic environment, as well as iron reducing microorganisms. There has been a continuous increase in the knowledge about microbial iron reduction in paddy soil in the last decade. Nevertheless, people only looked at some typical species of iron reducing microorganisms such as Geobacter and Shewanella, because of the uncertainty on phylogenetic diversity of iron reducing microorganisms and iron reducing ways. Iron redox processes play important roles in paddy rice-upland crop rotation systems. The system attracts plenty of attention as a model of redox research. However, little is known about the dynamic distribution of microbial community under flooded anaerobic condition. Furthermore, the data available in present literature fail to prove the contribution rate of bacteria in iron reduction. Therefore, the study on dominant Fe(III) microflora with different carbon sources in paddy soil not only can deepen the understanding of microbial ecology in paddy soil, but also has great significance for clarifying the mechanism of microbial Fe(III) reduction and the characteristics of community succession in paddy soil.In this study, we used anaerobic incubation with a constant temperature, added amorphous iron oxides with different carbon sources enrichment acclimation, analyzed Fe(III) reducing characteristics. Then we used 16S rDNA-ARDRA molecular biology techniques to analyze the diversity of bacterial strains with a high Fe(III) reducing capacity which were isolated and screened from the incubation system. Typical bacterial strains were determined 16S rDNA sequence. Microorganisms in iron reducing environment were isolated and purified directly, then measured 16S rDNA sequence in order to get some information about their categorization in taxonomy. To identify succession on superior dominant population, we compared the differences of microbial community structure with the domestication of amended carbon sources and iron oxides. Microflora extracted from paddy soil of different rice growing region in different flooded time was inoculated with different carbon sources as the only substrate in iron-rich medium. We measured ferrous concentration, pH and dehydrogenase activity regularly, to explore the maximum and stable period of the activity of microbial iron reduction, and get its fastest recovery time. All above can provide the foundation for the research on difference capacity and influencing factors of microbial iron reduction in different paddy soil. We analyzed the dynamic characteristics of microbial Fe(Ⅲ) reduction and dehydrogenase activity, in order to clarify the internal relationship between dehydrogenase activity and microbial Fe(Ⅲ) reduction and provide the necessary theoretical basis for revealing the mechanism of microbial Fe(Ⅲ) reduction in paddy soil.The main results obtained are as following:(1) The iron reduction tendency in the amended glucose and pyruvate paddy soil with organic matter depleted was similar with that in the natural paddy soil; however, there was a difference in the acetate treatment. These results indicate that iron reducing bacteria used H2 from glucose fermentation resulted in a high-speed iron reduction at earlier stage in flooded paddy soil, while iron reducing bacteria relying on H2 made more contribution to iron reduction in paddy soil than those depending acetate.(2) Iron reducing microbial community composition and structure have an important influence on iron reduction in paddy soil. In mixed cultivation with soil extraction, flooding time had a significant impact on Vmax, Fe(Ⅲ) reducing feature value, Vmax decreased in the order 20 d>30 d>12 d>1 d>5 d. The main reason caused different Fe(Ⅲ) reducing ability was the changes of microorganism population structure in paddy soil. There was a significant difference among iron reducing microbial community with carbon sources in different flooding time. Glucose and pyruvate were the superior carbon sources in the flooded 1-12 day treatments; lactate was used efficiently in flooded 12 days and 20 days treatments. There was a significant increase in using acetate in flooded 30-day treatment.(3) There was a significant difference between biological reduction and chemical reduction of iron citrate, because of the effect of illumination and high temperature on iron citrate chemical reduction. Iron citrate chemical reduction will not happen at 30℃and without light. In this experiment, what made this happen was microorganism. However, microorganism able to reduce iron citrate may not reduce ferrihydrite. Hence, we called those bacterial strains that could reduce more than 50% of ferrihydrire as iron reducing bacteria. In enrichment of different carbon sources, we obtained efficient iron reducing strains 88 from JL paddy soil, 54 from SC paddy soil, 161 from HN paddy soil, 88 from ZJ paddy soil, 33 from TJ paddy soil and 67 from JX paddy soil.(4) Fromαdiversity index, we can see that there was concentrative dominant population under glucose enrichment, while there was rich diversity in short organic acid treatment. The dominant population divided into seven kinds in HN paddy soil, according to the analysis of 16S rDNA-ARDRA. Paenibacillus spp. and Clostridium.spp. appeared in all carbon source treatments. Solibacillus and Lysinibacillus were the dominant population in acetate enrichment treatment, as Bacillus in pyruvate enrichment treatment. Azotobacter was the dominant population in all treatment but glucose treatment. Pseudomonas spp. was the dominant population only in glucose treatment.(5) Microbial Fe(Ⅲ) reducing had a significant effect on dehydrogenase activity, ferrous concentration was 91.0~344.6 mg·L-1 corresponding the maximum of dehydrogenase activity in all treatments. Dehydrogenase activity decreased as the level of Fe(Ⅲ) reduction increased. There was a correlation between dehydrogenase activity and level of Fe(Ⅲ) reduction. There was a positive correlation between the time reaching peak value of dehydrogenase activity and TVmax of Fe(OH)3 reduction, while there was a significantly negative correlation between time and Vmax. Dehydrogenase activity was an important influencing factor for iron reduction in paddy soil. We speculate that the main reason for high-speed iron reduction at earlier stage in paddy soil may be the H2 production from organic metabolism of microorganisms.(6) Under natural flooding conditions, isolates from paddy soils appeared in the early successional habitats adopted r-strategists to have growth and reproduction. They had a relatively rich in species diversity which could be attributed to Lysinibacillus, Staphylococcus, Paenibacillus and Azotobacillus phylogenetic branch. Iron reduction reached to a stable stage in late succession and there was a relatively concentrated group, which was predominated as Bacillus spp.. Microbial community structure had a significant variation when the mixed organic carbon sources and ferrihydrite were amended.Through this study, we got further knowledge of dominant iron reducing bacteria species and microbial community succession under different carbon source enrichment in paddy soil. We discussed the relation between dehydrogenase activity and microbial iron reduction, to provide the foundation for further clarifying the iron-reducing bacteria community structure and phyletic classification in flooded paddy soil incubation, as well as the differences of microbial iron reducing capacity. Furthermore, we put forward the initial ideas of microbial Fe(Ⅲ) reducing mechanism in paddy soil. |
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