Effect Of Flooding Time On Community Structure And Abundance Of Cultured Clostridium And Bacillus In Paddy Soil

Microbial dissimilatory Fe(III) reduction are capable of oxidizing organic or inorganicelectron donors with ferric iron Fe(III) as electron acceptors. Dissimilatory Fe(III) reducingphenomena exist in almost natural anaerobic environment, as well as iron reducingmicroorganisms. Microbial Fe(III) reduction coincided with a strong suppression ofmethanogenesis because of competition for hydrogen and acetate in anoxic rice paddy soil.Therefore, it has been an effective pathway to control methanogenesis by regulatingmicrobial iron reduction processes in rice field soil. Studies found that some Clostridium andBacillus strains with reduced iron function. Microbial ecology studies showed thatClostridium and Bacillus play an important role in community structure of bacteria in paddysoil. But few investigations have been focused on the relationship between iron-reducingmicroorganisms and iron reduction potentiality of flooding rice paddy soil.The dynamic characteristics of community structure and relative abundance ofClostridium and Bacillus were investigated in order to understand their response to microbialiron(III)-reducing by simulate the flooded paddy soil. Soil was taken from drained paddyfield after harvesting, located in GZ (GuiZhou province, moist single and double croppingrice area in Southwest) and JL (JiLin province, half-moist single cropping rice area inNortheast), respectively. Clostridium and Bacillus community structure characteristics wereobtained by denaturing gradient gel electrophoresis(DGGE) analysis from the anoxic paddysoil microcosms at1h,1d,5d,10d,15d,20d,30d and40d after flooding. Relative abundanceof Clostridium and Bacillus were determined by real-time PCR. We retrieved partial DGGEpreponderant type of Clostridium and Bacillus16S rDNA sequences which was used toconstruct phylogenetic tree. Reduction of Fe(III) in rice paddies was determined withanaerobic slurry incubation method and Logistic equation was used to fit this data. The mainresults were as follows:1. Community structure and relative abundance of cultured Clostridium in GZ and JLpaddy soil(1) Microbial reduction of iron(III) changed greatly in early time and was stable afterincubated for25d in JL paddy soil and the largest iron reduction potential was7.44mg/g with a Vmaxof0.79mg/(g.d) at the time of4.71d whereas this process achieved plateau after25days flooding, while in GZ paddy soil iron(III) reduction potential was4.07mg/g and theVmaxwas0.35mg/(g.d) at the time of3.97d and achieved plateau after20days flooding.(2) Similarity analyzed by Quantity One Software based on DGGE profiles. The resultsas follow: In GZ paddy soil, similarity coefficient of1h and1d treatments was0.93indicatedthat the community structure of Clostridium were similar; while community structure of thesamples30d and40d were quite different since similarity coefficient was only0.56. In JLpaddy soil, simililarity coefficient of5d and10d was0.76indicated that their communitystructure differences smaller, while1h and other samples were quite different.(3) α diversity indices analysis illuminated that there were distinct differences amongeight flooding treatments. In GZ paddy soil, the trends of abundance index(dMa) were40d>10d>15d>5d>20d>30d>1h>1d; Shannon-Wiener index (H’) were5d>10d>40d>15d>30d>20d>1d>1h; Simpson index (Ds) were1h>1d>20d>40d>10d>15d>30d>5d; Evenness index(E) were5d>30d>15d>10d>40d>20d>1d>1h. In JL paddy soil,the trends of dMawere30d>1h>40d>10d>20d>5d>15d>1d; H’ were30d>10d>40d>1h>15d>5d>20d>1d; Ds were1d>20d>15d>5d>40d=1h>10d>30d and Ewere30d>10d>40d>1h>15d>5d>20d>1d.(4) PCA analysis of clostridium in paddy soil of different flooding time by using UnifracSoftware, the results showed that the community structure composition of dominant bacteriaof the samples1h and1d,10d and20d,5d and15d were similar in GZ paddy soil, while thedifferences between30d and other samples were higher. In JL paddy soil, the communitycomposition of dominant bacteria of5d and10d,30d and40d were similar, while1d and20dwere quite different compared with others.(5) In samples of GZ and JL paddy soil,15DGGE-based preponderant types were found.These predominant patterns were belonged to Clostridium in anoxic paddy soil and dividedinto2groups (group1and group2) based on16S rRNA gene phylogenetic analysis.(6) The relative abundance of Clostridium was the lowest in sample30d (0.006‰) andthe highest in sample1d (0.563‰) in GZ paddy soil, while in JL paddy soil, the lowest(0.011‰) and the highest (0.913‰) appear in1h and1d respectively.2. Community structure and relative abundance of cultured Bacillus in GZ and JL paddysoil.(1) Similarity analyzed by Quantity One Software based on DGGE profiles. The resultsas follow: In GZ paddy soil, similarity coefficient of15d and20d treatments was0.96indicated that community structure differences of Bacillus were smallest; while thedifferences of the samples1h and1d compared to others were higher since similaritycoefficient was only0.66. In JL paddy soil, simililarity coefficient of5d and20d was0.84 indicated that their community structure were similar, while1h and other samples were quitedifferent illuminated changes in community structure were higher.(2) α diversity indices analysis illuminated that there were certain regular among eightflooding treatments in GZ and JL paddy soil. In GZ paddy soil, the trends of abundanceindex(dMa) were1d>1h>5d>10d>30d>15d>40d>20d; Shannon-Wiener index(H’)were1h>1d>5d>10d>20d>40d>15d>30d; Simpson index (Ds) were30d=15d>40d>20d>1d>5d>10d>1h and evenness index(E) were20d>1h>10d>5d>40d>15d>30d>1d. In JL paddy soil, the trends of dMawere1d>1h>30d>40d>5d>10d>15d>20d; H’ were1h>1d>30d>20d>15d>10d>5d>40d; Ds were40d>5d>10d>15d>20d>30d>1d>1h and E were1h>1d>20d>30d>15d>10d>5d>40d.(3) PCA analysis of Bacillus in paddy soil of different flooding time by using UnifracSoftware, the results showed that the community structure composition of dominant bacteriaof the samples1h and1d,5d and10d,15d and20d were similar in GZ paddy soil, while thedifferences between30d and other samples were higher. In JL paddy soil, the samples1h,1dand30d were close together;5d,10d and20d were also close together indicated that theircommunity composition were similar, while40d was quite different from others.(4) In samples of GZ and JL paddy soil,9and12DGGE-based preponderant types werefound respectively. These predominant patterns were belonged to Bacillus in anoxic paddysoil and divided into2groups based on16S rRNA gene phylogenetic analysis. The resultsindicated that group2and group3are both closely related to the genomic DNA of Bacillus.(5) The relative abundance of Bacillus was the lowest in sample5d (26.26%) and thehighest in sample15d (62.19%) in GZ paddy soil, while in JL paddy soil, the lowest (18.45%)and the highest (49.15%) appear in1h and10d respectively.By our study, we extended and deepened the knowledge about the community structureand abundance of cultured Clostridium and Bacillus in different paddy soils under differentflooding time, discussed the correlation among community structure, abundance andmicrobial Fe(Ⅲ) reduction. This work provided a theoretical basis for the furtherunderstanding of the distribution, phylogenetic classification and ecological function, as wellas discussing the difference of Fe(Ⅲ) reduction between various paddy soils.