| According to the different pathway of capturing energy for growth,Fe???reducers can be divided into two distinct types of respiratory?or obligate?Fe???reducers and fermentative?or facultative?Fe???reducers.The unique mechanisms and communities of respiratory Fe???reducers,such as Geobacteraceae and Anaeromyxobacter,have been extensively investigated,because they are considered to be responsible for most of the reduction of Fe???in sedimentary environments.However,due to the limitations of the earlier research methods,the research basis for the fermentative Fe???reducers is relatively weak.Fermentative microorganisms,such as Clostridium and Bacillus,are the ubiquitous and dominant groups in flooded paddy soils,especially during the early stage of incubation,and they may play an important role in the microbial hydrogen production and Fe???reduction in paddy soils.Therefore,it is of great theoretical and practical significance to investigate the effect mechanism and contribution of fermentative microorganisms in the process of Fe???reduction in flooded paddy soils.In the present study,to investigate the effect of organic matter,pH and temperature changes on the community of fermentative microorganisms and microbial Fe???reduction,flooded paddy soils were incubated in different temperature,or amended with glucose and pH regulators,respectively.The responses of metabolically active Clostridium and Bacillus communities to the changes of environmental effects were measured by a combination of reverse transcription quantitative PCR,denaturing gradient gel electrophoresis and high-throughput sequencing approaches.Concurrently,the relationship between the communities of Clostridium and Bacillus and the process of Fe???reduction was assessed to revealed the effect mechanism and contribution of fermentative microorganisms in the process of Fe???reduction.In addition,to verify the effect mechanism of microbial hydrogen production in the process of Fe???reduction,gene encoding the hydrogenase was deleted or overexpressed in Escherichia coli.The major results and conclusions are listed below:?1?In flooded paddy soil,increased glucose concentrations resulted in great difference in the structure of metabolically active bacterial community.Compared with the control treatment,Firmicutes replaced Proteobacteria to become the dominant group when paddy soils were amended with glucose.The glucose additions greatly stimulated the growth of Clostridium and Bacillus and led to a higher relative abundances of Clostridium and Bacillus.Moreover,the additions of glucose significantly altered the community structures of metabolically active Clostridium and Bacillus.The addition of a low concentration of glucose increased the diversity of Clostridium community during the later stage of anaerobic incubation,but decreased the Bacillus diversity.However,both the community diversities of Clostridium and Bacillus were inhibited by the addition of a higher concentration of glucose.During the anaerobic incubation,most of metabolically active Clostridium spp.and Bacillus spp.were closely related to H2-producing,organic acids-producing and Fe???-reducing bacteria.These results indicated that the additions of glucose showed significant influences on the communities of metabolically active Clostridium,Bacillus and total bacteria in flooded paddy soil.?2?During the anaerobic incubation,decreased the initial pH of alkaline paddy soil and increased the initial pH of acidic paddy soil led to significant decreases in the 16S rRNA copy numbers of Clostridium and Bacillus,indicating that microbial optimum pH for growth in paddy soils was closely related to the inherent soil pH,and sudden change in initial pH could greatly inhibit the growth activity of indigenous microorganisms.Additionally,shifted the initial pH of paddy soils led to significant differences in the community structures of metabolically active Clostridium and Bacillus,and the differences between different pH-treatments increased with the anaerobic incubation.Shifted the initial pH showed different effects on the diversities of Clostridium and Bacillus,which increased the Clostridium diversity and decreased the Bacillus diversity.?3?The abundances and relative abundances of metabolically active Clostridium and Bacillus communities at 15°C were lower than those at 30°C during the early stage of incubation,but they were significantly higher during the middle and later stages.The community structures of Clostridium and Bacillus at 30°C shifted rapidly during the early stage,while the stability of microbial community structures at 15°C decreased during the later stage of incubation.In addition,the diversity indices of Clostridium and Bacillus communities increased with increasing temperatures.These results revealed that adjusting the incubation temperature not only affected the metabolic activities of Clostridium and Bacillus,but also caused significant changes in their community structures.?4?The adjustment of organic carbon concentration,initial pH or incubation temperature showed significant effect on the processes of microbial hydrogen production and Fe???reduction.Moreover,the dynamics of the abundances,relative abundances and structures of metabolically active Clostridium and Bacillus communities in response to different environmental factors was closely related to the process of microbial Fe???reduction in flooded paddy soils.When paddy soil was amended with glucose,the significant increase in the maximum accumulation of Fe???may be attributed to the fact that glucose addition enhanced the process of Fe???reduction mediated by Clostridium and Bacillus;on the other hand,glucose addition could provide more electron donors to Fe???reduction by promoting the process of fermentative hydrogen production.As the initial pH of alkaline paddy soil was adjusted to acidic level,the process of Fe???reduction was inhibited greatly.This was mainly due to the fact that suddenly decreased initial pH inhibited the growth activities and altered the structures of microorganisms associated with Fe???reduction,such as Clostridium and Bacillus.However,although the growth of Clostridium and Bacillus was also inhibited by increased initial pH of acidic paddy soil,the effect of increased initial pH on Fe???reduction was not obvious as a result of stimulation of hydrogen utilization ability of Fe???reducers.Compared with the treatment at 15°C,raised the incubation temperature greatly enhanced the metabolic rate of Clostridium and Bacillus and the hydrogen utilization efficiency of Fe???reducers,which jointly promoted the process of Fe???reduction in flooded paddy soils.?5?As the organic carbon concentration,initial pH or incubation temperature was adjusted,the relative abundances and structures of metabolically active Geobacteraceae and Anaeromyxobacter communities,as representatives of obligate Fe???-reducing bacteria,showed significant changes.The addition of glucose greatly decreased the relative abundance of obligate Fe???-reducing bacteria and its ratio to facultative Fe???-reducing bacteria?Clostridium and Bacillus?during the early and middle stages of incubation,and the inhibitory effects increased with increasing glucose concentrations.These results indicated that glucose addition could significantly increase the contribution of facultative Fe???-reducing bacteria;especially when the carbon source was sufficient,facultative Fe???-reducing bacteria may play a dominant role in the process of Fe???reduction.Decreased the initial pH of alkaline paddy soil greatly reduced the ratio of obligate Fe???-reducing bacteria to facultative Fe???-reducing bacteria,indicating that the contribution of facultative Fe???-reducing bacteria to Fe???reduction was increased;however,increased the initial pH of acidic paddy soil showed little effect on the ratio of these two types of microorganisms,and facultative Fe???-reducing bacteria were always in the dominant position in different treatments.In BD paddy soil,facultative Fe???-reducing bacteria could have a higher contribution at 15°C,while increasing incubation temperature was beneficial to the function of obligate Fe???-reducing bacteria;in addition,elevated incubation temperature also showed a few positive effect on obligate Fe???-reducing bacteria in NC paddy soil.The response sensitivities of obligate and facultative Fe???-reducing bacteria to environmental changes were quite different,and the diversity of obligate Fe???-reducing bacteria showed a higher differences among different treatments.Compared with obligate Fe???-reducing bacteria,facultative Fe???-reducing bacteria showed a stronger adaptability to environmental changes,which could enable them to perform stable functions in different environmental conditions.?6?Addition of different concentrations of phosphate to paddy soil significantly inhibited the process of Fe???reduction during the early stage of anaerobic incubation,and decreased the maximum Fe???reduction rate.However,phosphate addition had no significant effect on the Fe???accumulation during the later stage of incubation.Phosphate addition resulted in significant decreases in the abundances and great differences in the structures of Clostridium and Geobacteraceae communities.Moreover,the dynamics of the abundances and structures of Clostridium and Geobacteraceae were closely correlated with the process of Fe???reduction in flooded paddy soil.During the early state of incubation,the addition of phosphate could inhibit Fe???reduction by decreasing the abundances and changing the structures of Clostridium and Geobacteraceae communities;however,the inhibition could be eliminated with increased incubation time,as a result of the decreasing inhibition of phosphate addition on the growth and function of Clostridium and Geobacteraceae.?7?E.coli was used as a model strain for investigating the relationship between microbial hydrogen production and Fe???reduction.The deletion of the large subunit gene?hycE?of hydrogenase-3?Hyd-3?completely abolished the hydrogen production,accompanied by a significant decrease in the capacity of Fe???reduction;moreover,the hydrogen production and Fe???reduction could be restored to the level of the wild strain in the Hyd-3 knockout mutant by re-expressing hycE from a plasmid.It showed that fermentative hydrogen production regulated by Hyd-3 was responsible for microbial Fe???reduction in E.coli.The single deletion of the large subunit gene of hydrogenase-1?Hyd-1?or hydrogenase-2?Hyd-2?showed little effect on the hydrogen production and Fe???reduction,which may be due to a compensation effect between these two hydrogenases;however,the highly efficient expression of Hyd-1 or Hyd-2 related genes in the wild strain greatly promoted the capacities of Fe???reduction and hydrogen utilization.These results indicated that E.coli could couple hydrogen oxidation to Fe???reduction,and hydrogen-dependent Fe???reduction was closely related to uptake hydrogenases?Hyd-1 and Hyd-2?.This study deepened the knowledge of the dynamics of fermentative microorganism in response to environmental factors and the relationship between fermentative microorganism and Fe???reduction in flooded paddy soils,and revealed that fermentative microorganism could play an important role in Fe???production via direct and indirect effects during the process of organic matter fermentation.The results in this study revealed the response mechanism of obligate and facultative Fe???-reducing bacteria to environmental factors and the effect mechanism of microbial hydrogen production on the Fe???reduction,and provided important theoretical basis for revealing the mechanism of Fe???reduction in flooded paddy soil. |
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