Diversity, dynamic and abundance of Geobacteraceae species in paddy soil following slurry incubation

European Journal of Soil Biology 56 (2013) 11e18

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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Diversity, dynamic and abundance of Geobacteraceae species in paddy soil following slurry incubation Weijie Yi a, c, Jiaohua You b, Chao Zhu b, Baoli Wang b, Dong Qu a, * a

College of Natural Resource and Environment, Northwest A&F University, Yangling 712100, PR China College of Life Sciences, Northwest A&F University, Yangling 712100, PR China c Agricultural College, Guizhou University, Guiyang 550025, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 June 2012 Received in revised form 17 December 2012 Accepted 31 January 2013 Available online 19 February 2013 Handling editor: Christoph Tebbe

Microbially-mediated Fe(III) reduction is of environmental significance in wetland ecosystems such as rice fields. Despite a number of incubation experiments showing the dynamic structure and activity of microbial communities in rice paddy soils amended with different substrates, little is known regarding the succession of Fe(III)-reducing bacterial populations in non-amended, natural paddy soils upon flooding. In this study, a 30-d laboratory incubation experiment was conducted to examine the diversity, dynamic and abundance of representative Fe(III)-reducing bacterial family Geobacteraceae in anaerobic natural paddy soil slurry incubations. The Logistic model showed that the microbial Fe(III) reduction rate in paddy slurry reached the highest level (1.36 mg g
1 d
1) after 3.3-d flooding, and the accumulated Fe(II) level stabilized at 8.14 mg g
1 on day 20. Quantitative, real-time PCR assay showed that the absolute abundances of Geobacteraceae and total bacterial populations varied in similar trends. Both decreased from day 1e10 and peaked on day 20 (13.98  106 and 5.29  108 copies of 16S rDNA g
1 dry soil, respectively), followed by large decreases on day 30 (1.94  106 and 0.62  108 copies of 16S rDNA g
1 dry soil, respectively). The relative abundance of Geobacteraceae, i.e., the proportion of Geobacteraceae to total bacteria reached the highest level (w4%) following 5-d flooding, and then slightly fluctuated at 2.6%e3.9% till the end of the experiment. Clone library construction and sequencing analysis showed that the Geobacteraceae mainly consisted of Geobacter spp. which promoted bacterial Fe(III) reduction in paddy slurries upon flooding. UniFrac principal coordinate analysis revealed the succession of Geobacteraceae species, with the highest diversity observed in the initial stage (1 he1 d) and the dominant successional members in the late stage (20e30 d). These results indicated that Geobacteraceae species contributed to Fe(III) reduction in flood paddy soils, and that the structure of Geobacteraceae population was maintained through the common occurrence of generalized species and the succession of specialized species. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Geobacteraceae Paddy soil Abundance Population succession Flooding time Ecological niche

1. Introduction The bacterial family Geobacteraceae represents a group of microorganisms that commonly occur in Fe(III)-reducing environments [1], particularly wetlands sediments associated with contamination/enrichment of organic matter. To date, all cultured members of the Geobacteraceae family have been shown capable of dissimilatory Fe(III) reduction [2]. As their Fe(III) reduction activity can remove heavy metals and organic pollutants from the environment [2], there has been great interest in the ecological and

* Corresponding author. Tel.: þ86 29 87082624;fax: þ86 29 87092603. E-mail address: [email protected] (D. Qu). 1164-5563/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejsobi.2013.01.004

physiological functions of Geobacteraceae in the environment as well as relevant applications in biotechnology [3,4]. Rice fields have served as a managed model of wetland biogeochemistry, in which periodic redox changes can significantly affect the structure and activity of microbial communities, further influencing the elemental cycling of several major nutrient elements as well as trace metals. Unlike naturally saturated sediments or wetland soils, paddy soils, especially in the eastern Asia, experiencing regular specific management practices such as submergence and drainage. A number of studies have focused the dynamic of microbial communities such as total bacteria [5], methaneoxidizing bacteria [6], nitrate- and sulfate-reducing bacteria [7,8], and methane-producing Achaea [9] in flooded paddy soil incubations. A previous study reported that the structure of bacterial communities in bulk paddy soil dramatically shifted within 2 d

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after flooding, whereas the microbial substantially decreased from day 2e21 and then stabilized from day 21e168 [10]. However, few studies have been reported on the dynamic of Fe(III)-reducing bacterial pollution in rice paddy soils. Knowledge is lacking regarding the response of these organisms to agricultural management activities such as submergence and drainage. Laboratory incubation experiments have shown that Geobacteraceae play an important role in iron reduction in paddy soils. The Geobacteraceae, which accounted for w5% of the total bacterial community in the 16S rRNA gene clone libraries retrieved from rice roots and the rhizosphere [11,12], were the dominant Fe(III)reducing bacteria in ferrihydrite-enriched cultures from a paddy soil in Hunan, China [13]. Using isotope labeling, the Geobacteraceae participated in acetate metabolism in a ferrihydriteenriched paddy soil, accounting for w85% of the total bacterial community in the corresponding 16S rRNA gene clone library [14]. To date, microbiological studies of Geobacteraceae have largely focused on their community structure in polluted subsurface environments [15e17]. Little is known regarding the succession of Geobacteraceae species in paddy soil slurry, and it is uncertain whether the succession of Geobacteraceae species can be used to predict the changes in Fe(III) reduction capacity. In addition, physiological studies on environmentally important microorganisms are typically conducted in batch cultures, with microbes grown at the maximum growth rate and supplied with abundant substrate. These conditions do not mimic natural conditions, where microorganisms commonly occur with substantially low growth rates. In order to improve the understanding of the succession of Geobacteraceae species in natural paddy soil, this work investigated the succession of Geobacteraceae species in anaerobic paddy slurry incubations through a 30-d experiment. Dissolved Fe(II) content in paddy slurry were simultaneously determined and then analyzed using the Logistic model. The abundance, activity and structure of target organisms were examined at specific time intervals using real-time PCR and 16S rRNA-based gene clone library construction and amplified ribosomal DNA restriction analysis (ARDRA). In addition, the diversity variations and niche breadth of the predominant members of Geobacteraceae that occurred at different time points in the slurry incubations were evaluated. These provide experimental data for studying the ecological function of Geobacteraceae in rice paddy soils with varying Fe(III) availability. 2. Materials and methods 2.1. Soil sampling and incubating conditions The paddy soil was taken from a double- and single-cropping rice field on the Middle-Lower Yangtze Plain in Ningbo, China (29.7 N, 121.4 E). Approximately 2 kg soil was collected from the top 20 cm layer at 5 sites along an S-line track and then mixed to obtain a composite sample. In order to mimic the succession of Geobacteraceae species in rice paddy soil upon flooding, the soil was airdried and then stored in a dry state at room temperature [19]. Major soil characteristics were measured using the standard methods [18]: organic carbon (C) 31.7 g kg
1 dry soil, amorphous Fe(III) oxide 6.2 g kg
1 dry soil, and free Fe oxide content 10.4 g kg
1 dry soil. The soil was crushed with a sterile mortar and sieved through a 1 mm mesh. Small aliquots were immediately mixed with sterile deionized water at a ratio of 1:1 (w/v) in 5 mL glass vials. The vials were capped with butyl rubber septa, sealed with aluminum lid, and then flushed with filtered-sterilized N2 in the headspace. The flooding experiment was carried out at 30  C in the dark for 30 d. Samples were taken from triplicate slurry incubations for Fe(II) measurement on days 0, 1, 3, 5, 8, 12, 16, 20, 25, and 30, and those for molecular biological analyzes were taken at 1 h and on days 1, 5,

10, 20, and 30. For molecular biological analysis, samples were stored at
20  C prior to use. 2.2. Fe(II) content assay The Fe(II) content was extracted from in soil slurry and then measured as previously described [20]. Before each sampling, the soil slurry was shaken well and 0.4 mL of soil slurry was pipetted into 4.6 mL of 0.5 mol L
1 HCl for 24 h extraction under N2 atmosphere [21]. The extracts were centrifuged and the Fe(II) content was measured for the supernatant using the 1,10-orthophenanthroline method. The Logistic model, which reflects the growth kinetics of related microorganisms [22], was used to fit the microbiallymediated dissimilatory Fe(III) reduction process. 2.3. Bacterial DNA extraction and quantitative, real-time PCR (qPCR) assays At each time interval, 0.3 g of soil slurry was pipetted into liquid nitrogen for grounding and then transferred to a clean tube. This process was repeated for 10 times until 3 g of slurry was available for DNA extraction following the protocol of Zhou et al. [23] with slight modifications. To remove potential PCR inhibitors such as humid, the raw DNA extract was purified by running 0.8% agarose gel electrophoresis at 70 V for 30 min. The purified DNA extract was recovered from the gel using the TIANgel Midi Purification Kit (Tiangen Biotech Co. Ltd., Beijing) following the manufacture’s instructions. The qPCR assays of Geobacteraceae and total bacterial populations were conducted using the oligonucleotide primers Geo494f/Geo825r and 27f/1492r, respectively [24]. The qPCR reactions (25 mL) were prepared in triplicates with 12.5 mL of SYBR Green 2 Buffer, 2 mL of template DNA, 1 mL of each primer (10 mM), and 8.5 mL of ddH2O. PCR amplification was performed on the BioRad CFX96 system using the following program: 3 min 94  C, followed by 40 cycles of 30 s 94  C, 20 s 55  C, and 30 s 72  C (melting temperature 65e95  C). A final melting curve was plotted to check the product specificity. To prepare a calibration curve [7], the 16S rRNA gene of Geobacter surfulreducens was amplified with primers Geo494f/Geo825r (for Geobacteraceae) and 27f/1492r (for total bacteria), inserted into pMD19-T vector, transformed into competent cells JM109, and finally extracted from the plasmid of positive clones. The standard plasmid DNA was 10-fold serially diluted from 1000 pg to 0.001 fg and then quantified in each qPCR run. The calibration curves were generated using the CFX-Manager software (version 4.6, Bio-Rad). For each standard, the concentration of target DNA copy number was plotted against the cycle number at which the fluorescence signal exceeded the background or threshold value (Ct). The slope of each calibration curve was used in the following equation to determine the reaction efficiency: efficiency ¼ 10
1/slope
1. Accordingly, an efficiency of 1 means a doubling of product in each cycle. Based on the calibration curve, the initial target copy number of the sample was calculated using the CFX-Manager software, and results were used to calculate the copy numbers of target gene per microliter using the following formula: Copy number (copies/mL) ¼ DNA concentration  6.022  efficiency/[(the number of DNA bases in the vector þ the number of DNA bases in a target gene)  660]. 2.4. Generation of amplified ribosomal DNA restriction analysis (ARDRA) profiles 16S rRNA-based PCR assay of Geobacteraceae was carried out using the reverse primer Geo825r [24] in combination with two forward primers Geo163f1 (50 -TAA TAC CGR ATA AGC CYA CG-30 )

W. Yi et al. / European Journal of Soil Biology 56 (2013) 11e18

The electrophoretogram was digitized with NTsys 2.10 using the UPGMA method. The clones with identical ARDRA profile were defined as one phylotype. The online tool of the phylotype richness estimator was used to estimate the coverage size of the clone library and to predict the number of the phylotypes [http://www. aslo.org/lomethods/free/2004/0114a.html, [25]]. To evaluate the diversity of Geobacteraceae species, the ShannoneWiener index (H0 ) and Menhinick index (R2) [26,27] were calculated based on the number of relevant phylotypes and total clone numbers in each library. In addition, the Levins’ niche breadth index (Bi) [28] was calculated for the dominant phylotypes at different time intervals using Equation (1). This index helps to understand the role of Geobacteraceae species (e.g., generalist or specialist) in different certain niches, as well as their environmental frequencies.

Bi ¼ 1=

r X j¼1

p2ij

(1)

where r refers to the incubation time. The ratio between the number of the dominant phylotype i in the succession of the j-th period and the total number of the dominant phylotype i in the whole succession (r) was calculated as follows: Pij ¼ nij =Ni . To assess the similarity of resource usage and ecological adaptation among the dominant phylotypes, the mean Pianka’s index of niche overlap (aij) was computed [29,30]. For the dominant phylotypes 1 and 2 with resource uses p1i and p2i, the Pianka’s overlap index (a12) was calculated using the following equation:

a12 ¼ a21 ¼

r X

P2i P1i qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi: Pr 2 2 i¼1 i ¼ 1 ðP2i Þ ðP1i Þ

(2)

2.6. Sequencing and phylogenetic analyzes

3. Results 3.1. Fe(III) reduction in the anaerobic incubations During the 30-d flooding experiment, dissolved Fe(II) content substantially increased in the paddy soil slurry incubations within the first 10 days, and gradually stabilized at the highest level on day 20 (Fig. 1). According to the Logistic model, the maximum Fe(II) accumulation rate was 8.1 mg g
1 dry soil, with the highest reaction rate of 1.4 mg g
1 d
1 on day 3.3. 3.2. Abundance of Geobacteraceae in flooded paddy soil The abundances of Geobacteraceae and total bacterial populations showed similar fluctuations in the flooded paddy soil slurries (Fig. 2). Both decreased from day 0 (6.30  106 and 4.74  108 copies g
1 dry soil, respectively) to day 10 (1.66  106 and 0.42  108 copies g
1 dry soil, respectively), and then peaked on day 20 (13.98  106 and 5.29  108 copies g
1 dry soil, respectively). Thereafter, the abundances of Geobacteraceae and total bacterial populations declined by w7.2e8.5 times to relatively low levels on day 30 (1.94  106 and 0.61  108 copies g
1 dry soil, respectively). As for the relative abundance of Geobacteraceae, the proportion of Geobacteraceae to total bacteria (G/B ratio, %) in flooded paddy soil remained low from 1 h to 1 d (0.4e1.3%), increased by 10-fold following 5-d flooding (4.11%), and then slightly fluctuated at 2.6%e3.9% over the following period.

8

ZJ

6

-1

2.5. Calculation of the diversity and niche indices

species, 60 representative clones were selected for sequencing at Shanghai Sangon using an ABI 3730xl sequencer with the BigDye terminator cycle sequence kit (Applied Biosystem). The obtained sequences were checked for chimera using the Mothur tool (http://www.mothur.org/wiki/Chimera.check), and then analyzed using the RDPII Classifier and Sequence Match (Cole et al., 2005) in combination with the NCBI BLASTN search program aligned with the sequences of the closest relatives using MEGA 4.0 [31]. Phylogenetic trees were constructed using the neighborjoining method and the Kimura 2-parameter matrix model [32]. Robustness of derived groupings was tested by bootstrap using 1000 replications. The Newick file of the phylogenetic tree was submitted to the UniFrac site [http://bmf.colorado.edu/unifrac, [33e35]] for the UniFrac principal coordinate analysis (PCA). Nucleotide sequences generated in this study were submitted to GenBank under the following accession numbers JN091569 to JN091629.

Fe (II) content (mg g dry soil)

and Geo163f2 (50 -TAA TAC CTG ATA AGC CCA CG-30 ), respectively. The Geo163f1 and Geo163f2 were designed with the 16S rDNA sequence of Escherichia coli DH1 as the reference. The PCR reaction (50 mL) contained 2 mL of template DNA, 25 mL of Premix Taq DNA polymerase (TaKaRa Biotechnology Co. Ltd., Dalian), 1 mL of each primer (10 mM) and 21 mL of ddH2O. PCR amplification was performed as described previously [24]. The amplicons were checked by running 0.8% agarose gel electrophoresis, purified with the TIANgel Midi Purification Kit (Tiangen Biotech Co. Ltd., Beijing), and then inserted into the pGEM-T vector (Promega, Beijing). Ligation products were transformed into E. coli JM109 competent cells. The transformants were spread on LB agar containing ampicillin (100 mg mL
1), X-Gal (8 mg mL
1), and IPTG (320 mg mL
1). A total of 200 positive, white clones were randomly picked from each library, and then checked for inserts via colony PCR with the primers M13f (50 -CGC CAG GGT TTT CCC AGT CAC GAC-30 ) and M13r (50 -GAG CGG ATA ACA ATT TCA CAC AGG-30 ). A total of 6 libraries were constructed, corresponding to 6 sampling intervals. Amplicons of positive colonies were digested by Msp I and Mnl I restriction enzymes at 37  C for 6 h. The enzyme-digested products were checked by 12% polyacrylamide gel electrophoresis and then visualized by silver staining. The obtained sequences were grouped into 10 phylotypes based on a threshold of 98%.

13

4

2

0

Clones were randomly selected and checked for the correct insert size via standard, targeted PCR and agarose gel electrophoresis. To determine the phylogenetic identity of major Geobacteraceae

0

5

10

15

20

25

Incubation time (d) Fig. 1. Dissolved Fe (II) content in the flooded paddy soil.

30

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W. Yi et al. / European Journal of Soil Biology 56 (2013) 11e18

1x10 9x10 8x10

2.86E6±1.0E5 7x10

-6

6x10 5x10

1.4E7±2.6E5

-7

6.3E6±2.0E5

4x10 6.0x10

1.9E6±2.7E5 -8

2.46E6±2.1E5

Proportion of G/B (%)

-1

Copies of 16S rDNA g dry soil

5 4 3 2 1 0

Geobacteracea Bacteria

1.7E6±8.0E4

4.0x10 -9

2.0x10 0.0 -2.0x10

-10 1h

1d

5d

10 d

20 d

30 d

Flooding time Fig. 2. The abundances of Geobacteraceae and total bacteria in the flooded paddy soil.

3.3. Diversity of Geobacteraceae in flooded paddy soil The phylotype-richness estimates reached an asymptotic maximum at or near the correct number of phylotypes, and the capacity value C of each treatment was between 65% and 74%. These indicated that the constructed libraries were large enough to yield stable and unbiased estimates of phylotype richness for accurate evaluation of changes in the population structure of soil Geobacteraceae (Supplementary material). There were 71, 74, 75, 72 and 73 phylotypes observed in the clone libraries of incubations after 1 h to 20 d flooding, respectively, with 61 phylotypes only in the clone library of the 30-d incubation. The proportion of single clones with low frequency of band pattern (observed only once) was largest in the 1-h incubation (35.3%), and gradually decreased to 26% in the 30-d incubation. The a diversity indices were the highest on 1 d (H0 ¼ 3.99, R2 ¼ 6.28), and declined to the minimum on day 30 after incubation (H0 ¼ 3.50, R2 ¼ 4.93). 3.4. Succession of dominant Geobacteraceae phylotypes in flooded paddy soil During the 30-d incubation, 10 dominant phylotypes of Geobacteraceae (AeJ) were observed in the flooded paddy slurries, accounting for 6e16% of the Geobacteraceae population, whereas other minor phylotypes accounted for less than 3% of the Geobacteraceae population (Fig. 3). Of these, the type A, type B, type C and type D widely occurred in all incubations, with the largest proportion of type B found in soil slurries during days 20e30 (13e 16%). With prolonged incubation, the proportion of type E first increased to 6% on day 1 and then gradually disappeared on day 30. Type F only appeared in paddy slurries on day 1 as the dominant specie (6%), while type H initially occurred in a small proportion (<1%) and gradually became dominant during days 20e30. The proportions of type G and type J were significantly higher in incubations during days 5e10 compared with those at other time points. The values of niche breadth (Table 1) indicated that the Geobacteraceae species adapted to the environmental variations in terms of resource use, further reflecting the competition and coexistence of diverse Geobacteraceae species. The dominant

phylotypes A, C, and D had the highest values of niche breadth than the others, whereas the types E, F, G, H and I had substantially lower values of niche breadth and spread over specific flooding periods. The ecological niche overlap values were relatively high between types G and J (0.98) as well as B and I (0.96), followed by those among types A, C, D and other major types (0.50e0.91) (Table 1). By comparison, the ecological niche overlap values were relatively lower between types G and other species, particularly types H and I (0.04e0.14, Table 1). 3.5. Phylogenetic analysis Environmental sequences of the 10 major phylotypes (AeJ) were chosen for phylogenetic analysis (Fig. 4). Result shows that these sequences fall into three distinct phylogenetic clades, designated as ‘Clade 1’, ‘Clade 2’, and ‘Clade GM’ (Geobacter metallireducens). Clade 1 includes types A, B, E and I, as well as the Fe(III)-reducing Geobacter bemidjiensis, Geobacter humireducens, Geobacter daltonii FRC-32 and Geobacter uraniumreducens commonly found in petroleum- or uranium-contaminated aquifer or sediments. Clade 2 includes types D, G, F, and J as well as Geobacter chapelleii and Geobacter sp. Ply1 respectively isolated from deep pristine aquifer sediment and subsurface environment treated with calcium magnesium acetate as the deicing agent. Clade GM mainly harbored type C, which occurred in all paddy slurries during the flooding period. The type C is closely related to Geobacter sulfurreducens, G. metallireducens and Geobacter hydrogenophilus isolated from hydrocarbon-contaminated soil, freshwater sediment, and petroleum-contaminated sediment, respectively. PCA analysis by UniFrac (Fig. 5) allowed a simultaneous comparison of the phylogenetic composition of Geobacteraceae in all 6 incubations. The sequences of 1-h incubation were found most similar to the environmental clone sequences from 1-d incubation, whereas those of days 5 and 10 were relatively similar. A distance matrix was used to plot the n samples in the n-dimensional space. The vector through the space that described the largest variation was principal coordinate 1. Orthogonal axes were subsequently assigned to explain as much of the variation not yet explained by previously assigned axes as possible. The first two principal coordinates described 54% of the variation, suggesting that many

W. Yi et al. / European Journal of Soil Biology 56 (2013) 11e18

15

16

A F

B G

C H

D I

E J

Proportion (%)

12

8

4

0 1h

1d

5d

10 d

20 d

30 d

Incubation time (d) Fig. 3. Succession of the major Geobacteraceae phylotypes in the flooded paddy soil.

is discussed in correspondence with the three-stage Fe(III) reduction process (Fig. 2). In the early flooding period, microbial Fe(III) reduction was rapidly initialized, leading to accumulation of dissolved Fe(II). This could be related to the presence of a diversity of Fe(III)-reducing Geobacteraceae, of which the phylotypes AeF (mainly Clade 1 and Clade GM) were identified as the dominant members. Together the relatively high copy number of Geobacteraceae (Fig. 2) indicated that the Fe(III)-reducing bacterial population was diverse and active immediately after flooding. The relative abundance of Geobacteraceae decreased from 1.3% to 0.4% during this period, possibly due to substrate consumption and competition with other (facultative) (an)aerobic soil bacteria, e.g., Clostridium [36,37] and Bacillus [38,39]. In the transitional stage (1e10 d), Fe(III) reduction rapidly occurred along with the increased G/B ratio after 5-d flooding (4.1%). Correspondingly, the proportion of Geobacteraceae phylotypes D, G and J (Clade 2) increased (Fig. 3), suggesting their important role in Fe(III) reduction during this period. The qPCR assay showed that the absolute abundance of Geobacteraceae continuously decreased from day 1e10, likely due to the lack of bioavailable substrates and/or accumulation of toxic byproduct(s). Surprisingly, the abundance of Geobacteraceae as well as total bacterial population fluctuated and peaked on day 20 during the late stage (day 10e30 d). Probably the microbially-mediated sulfate reduction and methane production occurred in this stage,

independent factors caused variation between samples (as might be expected for such diverse environments). Strikingly, plots of the principal components produced biologically meaningful clusters of samples, even though the individual components accounted for little of the variation. The second principal coordinate explained 24.89% of the variation and clearly separated the incubations of 1 he10 d from those after 20 d (Fig. 5). This suggested that succession of Geobacteraceae after flooding could be divided into three stages, the early stage (1 he1 d), the transitional stage (1e10 d), and the late stage (10e30 d).

4. Discussion 4.1. Impact of flooding treatment on Fe(III) reduction and associated Geobacteraceae This study demonstrated that flooding treatment stimulated the microbial Fe(III) reduction process and significantly affected Geobacteraceae in paddy soil slurries, which accounted for 0.4%e4.1% of the total bacterial population (G/B ratio, Fig. 2), in agreement with previous findings (<5%) [11,12]. Sequence analysis showed that the Geobacteraceae clones mainly consisted of Geobacterrelated species and were grouped into three clades (Fig. 5). Considering that the accumulation of Fe(III) in flooded paddy soil experienced three stages (Fig. 1), the succession of Geobacteraceae

Table 1 Niche breadth and overlap of major Geobacteraceae phylotypes in the flooded paddy soil. Major types

Niche breadth

Niche overlap A

B

C

D

E

F

G

H

I

J

A B C D E F G H I J

5.56 3.99 4.95 5.61 4.25 4.19 2.49 2.19 3.37 2.88

1

0.85 1

0.83 0.86 1

0.91 0.73 0.83 1

0.76 0.42 0.63 0.86 1

0.7 0.68 0.89 0.78 0.77 1

0.6 0.28 0.5 0.71 0.58 0.29 1

0.5 0.74 0.82 0.56 0.33 0.82 0.04 1

0.74 0.96 0.84 0.64 0.4 0.73 0.14 0.75 1

0.66 0.36 0.54 0.74 0.61 0.32 0.98 0.04 0.25 1

16

W. Yi et al. / European Journal of Soil Biology 56 (2013) 11e18 5d-6-G (JN091591)

95 100

10d-47-J (JN091603) 5d-44-J (JN091593)

42

1d-13-D (JN091581)

100 16

10d-35-G (JN091601) Uncultured delta Proteobacterium P-R33 (JN038820)

27

22

Geobacte chapellei 172 (U41561)

100

Geobacter psychrophilus P35 (AY653549)

11

1d-86-F (JN091582)

Clade 2

1h-62-F (JN091577)

18 47

Geobacter_argillaceus G12 (DQ145534) 1h-31-D (JN091574)

96

12

1d-77-C (JN091583) Geobacter pelophilus Dfr2 (U96918)

97

21

Uncultured Geobacter sp.KB-12 (AY780557) 1d-160-H (JN091589)

86 21

Uncultured Banisveld landfill bacterium BVA59 (AY013609) Geobacter thiogenes (AF223382) Iron-reducing bacterium enrichment culture clone (FJ269083) 42 99

1h-39-E (JN091578) 1d-27-E (JN091580)

93 32

10d-7-I (JN091608) 99 67

45

1h-29-B (JN091570) 1d-60-A (JN091584)

59

21

1d-100-B (JN091587)

25

Clade 1

1h-14-A (JN091569) 20d-11-I (JN091612)

15

Geobacter .daltonii FRC-32 (EU660516)

98

Geobacter uraniireducens Rf4 (EF527427)

64

Uncultured Clostridiales bacterium (JN540227) 63

37

Geobacter bremensis Dfr1 (U96917)

99 50

Geobacter bemidjiensis Bem (AY187307) Uncultured delta Proteobacterium (GQ342321)

Geobacter pickeringii G13 (DQ145535) 100 62

Geobacter sulfurreducens PCA (U13928) Uncultured bacterium d006 (AF422626)

72

Uncultured Geobacter sp.IPL 25 (EU037913)

92

Geobacter_hydrogenophilus H2 U28173)

92 80

Clade GM

Geobacter grbiciae TACP-5 (AF335183) Geobacter metallireducens GS-15 (L07834) 5d-159-H (JN091600)

1h-47-C (JN091571) Rummeliibacillus pycnus (AB271739)

0.02

Fig. 4. Phylogenetic tree of major Geobacteraceae species based on the 660-bp partial sequence of 16S rDNA amplified with primers Geo825r/Geo163f1 or Geo825r/Geo163f2 and their close relatives retrieved from the RDP database (bootstrap value 1000). Bar represents 1% variance. Bold aebec: a, the flooding periods; b, the clone number; and c, the phylotypes of Geobacteraceae.

leading to the production of favorable organic substrates such as acetate, thereby stimulating acetate-utilizing Geobacteraceae members. This could be supported by Scheid et al. [40], which showed that the addition of sulfate to rice root incubations stimulated the occurrence of several Geobacter species. Thereafter, the organic substrates might largely be depleted, accounting for the remarkable decrease in the abundance of Geobacteraceae on day 30 (Fig. 2). Of note, the relative abundance of type B increased by 2 times in this stage (Fig. 3). Members of this phylotype likely favor small molecule organic carbon sources produced via other coexisting bacterial species [22].

4.2. Relationships of Geobacteraceae species in flooded paddy soil Niche breadth reflects the environmental adaptability of microbial species and their competitive relationships. To some extent, niche breadth also indirectly shows the ecological feature of bacterial species [29]. In the present study, all the major phylotypes of Geobacteraceae occupied their corresponding ecological amplitudes, indicating their different ecological features and environmental adaptabilities. Types AeD occurred as the generalize species with high niche breadth values (Table 1). These phylotypes dominated Geobacteraceae during the entire succession process,

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functions (e.g., Fe(III) reduction) and accounted for the long-lasting stability and succession of Geobacteraceae population. 5. Conclusions This study demonstrated that the Geobacteraceae contributed to Fe(III) reduction in flood paddy soils. The high diversity and abundance of Geobacteraceae were found in the flooded paddy soil in the initial flooding period (1 he1 d) associated with rapid microbial Fe(III) reduction, whereas relatively low diversity and high abundance of Geobacteraceae species occurred in the late period (20e30 d) with a plateaued Fe(III) reduction rate. The structure of Geobacteraceae population was maintained through the common occurrence of generalized species and the succession of specialized species, which further mediate Fe(III) reduction as well as other related processes such as organic matter mineralization. Acknowledgments This study was financially supported by the National Natural Science Foundation (40971158). The authors thank the three anonymous reviewers for quality comments. Fig. 5. PCA analysis based on the phylogenetic information of paddy soil slurries during different flooding periods. The environment abbreviations are the flooding incubations of specific time points where the sequences are retrieved: “F” is flooding, and “h” and “d” are “hour” and “day”, respectively. The percentages in the axis labels represent the percentages of variation explained by the principal coordinates.

possibly due to their high environmental adaptability and wide metabolic pathways. This was consistent with their close phylogenetic relationship with G. bemidjiensis, G. humireducens, G. daltonii FRC-32 and G. uraniumreducens (Fig. 4), which are commonly found in contaminated aquifer or sediments with enriched nutrient sources. By comparison, the types EeJ occurred as specialized species with lower niche breadth values, indicating their low environmental adaptability and narrower resource selection range. These Fe(III)-reducing organisms only occurred as minor competitors and dominated Geobacteraceae at some certain stage (mostly middle and late stages of succession), primarily limited by environmental stresses such as food scarcity. The physiology of these specialized species is confirmed by their close relationship with G. chapelleii and Geobacter sp. Ply1, which were respectively isolated from deep pristine aquifer sediment and subsurface environment associated with commonly low nutrient levels. In soil microbial community, the niche of specific bacterial population forms a basic unit of the niche of the entire community. Microbial populations with common needs of specific resources often overlap in their niches to different degrees [29]. In the present study, Geobacteraceae phylotypes with relatively wide niches (types A, C and D) showed large ecological overlaps with other species and widely occurred throughout the flooding period (Table 1). Despite the high ecological niche overlap values between types G and J as well as types H and I, these organisms co-existed in the middle and late stages of succession without competitive exclusion. Pianka [29] proposed that the species with similar ecological characteristics only compete when the common resources are lacking, and that the ecological niche overlap may not reflect the degree of competition in the presence of sufficient resources. In our case, the coexistence of types G and J as well as types H and I could be attributed their competitive as well as symbiotic relationships. Together these indicate that different Geobacteraceae species performed similar

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