The Diversity And Abundance Of Fe-Hydrogenase Microbe In Paddy Soil During Flooding Incubation

Clostridium microorganisms widely existed in the anaerobic environment of natural flooded paddy soil, especially the Fe-hydrogenase microbial that belong to Clostridium owning high hydrogen-production ability that attracted much attention on the fermentative hydrogen study. The Fe-hydrogenase of hydrogen-production microorganisms was the key enzymes for H2producing, played an important role in the biochemical metabolic chain, soil reducing power transferation of Fe reduction and CH4production process. Therefore, to study the diversity of Fe-hydrogenase for revealing the Fe-hydrogenase microorganisms and its communities and exploring the production microbial biochemical metabolic mechanism and the ecological balance of the paddy soil during the paddy soil flooding incubation.In this paper, we discussed the diversity and abundance changes of Fe-hydrogenase gene in1h,1d,5d,10d,20d,30d,40d and60d waterlogged incubation under the non-cultivation of paddy soil anaerobic mode, through the research to reveal Fe-hydrogenase microbial species, community structure and succession variation by applying PCR-DGGE(PCR-denatured gradient gel electrophoresis) and Real-time quantitative techniques. The main conclusions of the study are as follows:The results of Guizhou paddy soil(1) The band number of Fe-hydrogenase denatured gradient gel electrophoresis fingerprints indicated Fe-hydrogenase microbes’structure varied significantly during the completely flooding incubation. With the growth of the flooding incubation time Fe-hydrogenase microbial community structure became relatively stable and convergence.(2) The Principal Component Analysis (PCA) showed that the highly similar community of Fe-hydrogenase microbial was divided into three groups:1d and20d, the5d,30d and40d.(3) a diversity index analysis found that the richness index (R), Shannon-Weaver index (H’), Simpson index (Ds) numerical values of Id and10d were lower compared with other points, indicated the two points of low Fe-hydrogenase diversity, simple Fe-hydrogenase microbial community structure and successive variation of community structure during the whole flooding incubation. After20d a diversity index of Fe-hydrogenase became relatively steady.(4) After sequencing15Fe-hydrogenase preponderant bands (labeled by GZ-H1-GZ-H15), the phylogenetic tree of Fe-hydrogenase showed that the preponderant bands all had high similarity with the Clostridium Fe-hydrogenase in earlier flooding incubation stage and non-Clostridium Fe-hydrogenase in the later stage. All the Clostridium Fe-hydrogenase species were:Clostridium perfringens, Clostridium saccharolyticum, Clostridium thermocellum, Clostridium botulinum, and the non-Clostridium Fe-hydrogenase were:Thermoanaerobacter ethanolicus, Ignavibacterium album, Marinilabilia salmonicolor.The results of Ningxia paddy soil(1) The Fe-hydrogenase denatured gradient gel electrophoresis fingerprints bands indicated Fe-hydrogenase microbes structure had significant variation during the completely flooding incubation. However, there are four bands existed on all time point. With the growth of the flooding incubation time Fe-hydrogenase microbial community structure became relatively stable and convergence after20d.(2) The Principal Component Analysis (PCA) showed the highly similar community of Fe-hydrogenase microbial was divided into three groups:Ih and Id, the5d,20d and30d, the10d,40d and60d.(3) a diversity index analysis found that the richness index (R), Shannon-Weaver index (Hr), Simpson index (Ds) numerical values of Id and10d were lower compared with other points, indicated the two time points of low Fe-hydrogenase diversity, simple Fe-hydrogenase microbial community structure and successive variation of community structure during the whole flooding incubation, a diversity index of5d increased significantly as well as the20d. After20d a diversity index of Fe-hydrogenase became relatively steady.(4) After sequencing13Fe-hydrogenase preponderant bands (labeled by NX-H1-NX-H13), the phylogenetic tree of Fe-hydrogenase showed that the preponderant bands had6high similarity with the Clostridium Fe-hydrogenase and2non-Clostridium Fe-hydrogenase. All the Clostridium Fe-hydrogenase species were:Clostridium thermocellum, Clostridium butyricum, Clostridium perfringens, Clostridium papyrosolvens, Clostridium pasteurianum, Clostridium sartagoforme, and the non-Clostridium Fe-hydrogenase were: Odoribacter splanchnicus and uncultured bacteria.The most of Fe-hydrogenase genes detected in Guizhou and Ningxia paddy soils were Clostridium Fe-hydrogenase, but Non-Clostridium Fe-hydrogenase genes were also discovered. During the whole of flooding incubation of Ningxia paddy soil appeared the same type of Fe-hydrogenase genes, the map of Fe-hydrogenase denaturing gel electrophoresis and most of detected Clostridium Fe-hydrogenases were diverse compared with Guizhou paddy soil.The results of Guizhou, Tianjin, Jilin, Jiangxi, Ningxia paddy soil Real-time PCR:(1) The Fe-hydrogenase quantitative PCR results of5kinds of paddy soil showed:the Fe-hydrogenase gene copy number in Guizhou and Jiangxi paddy soil was106.g-1dry soil, Tianjin, Jilin, and Ningxia Fe-hydrogenase gene copy number was105·g-1dry soil. Except Jiangxi paddy soil, in the early incubation period the Fe-hydrogenase copy number basically had gone through a process of high and low volatility, and the Fe-hydrogenase gene copy number stabilized higher in the late stage of culture with only a small Fe-hydrogenase copy number fluctuations, The Fe-hydrogenase gene copy number trend upward at the flooding incubation end. Overall relative to Fe-hydrogenase gene copy number, Clostridium16S rRNA gene copy number was107and the bacterial16S rRNA gene copy number was109.(2) Combining the analysis Fe-hydrogenase copy number and Fe-reduction capacity of the five paddy soil, the results indicated that the Fe reducing capacity of Guizhou and Jiangxi with Fe-hydrogenase106copy number was faster than other three paddy soil with Fe-hydrogenase105copy number during the flooding incubation process, The higher of Fe-hydrogenase copy number, the shorter time of Fe reduction equilibrium, indicating that the Fe-hydrogenase and its microbials had correlation with the Fe reduction during the paddy soil flooding incubation.Conclusion:The Fe-hydrogenase denatured gradient gel electrophoresis fingerprints bands of Guizhou and Ningxia indicated Fe-hydrogenase microbes’structure had significant variation in the early stage, with the growth of the flooding incubation time Fe-hydrogenase microbial community structure became relatively stable and convergence. The Fe-hydrogenase we found in the study showed that most of Fe-hydrogenase belonged to Clostridium and non-Clostridium also existed. Compared with Guizhou and Ningxia paddy soil, the map of Fe-hydrogenase denaturing gel electrophoresis was diverse and most of detected Fe-hydrogenases were different.The Fe-hydrogenase quantitative PCR result of5kinds of paddy soil was105-106.g-1dry soil. The Fe-hydrogenase and its microbials had correlation with the Fe reduction during the paddy soil flooding incubation.