The diversity of iron reducing bacteria communities in subtropical paddy soils of China

Applied Soil Ecology 101 (2016) 20–27

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Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil

The diversity of iron reducing bacteria communities in subtropical paddy soils of China Qi-an Penga,b , Muhammad Shaabana , Yupeng Wua , Ronggui Hua,* , Buyun Wangb , Jun Wangb a b

College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, PR China School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 October 2015 Received in revised form 6 January 2016 Accepted 15 January 2016 Available online xxx

Iron(III) reducing bacteria (FeRB), involved in C and N cycle, are vital to regulating environmental biogeochemical processes. Although many important FeRB have been isolated and identified, the diversity of FeRB communities in paddy soils remains largely unknown. Four soils (rice–rapeseed rotation and rice–fallow/flooded rotation collected from Qianjiang, QR and QF soil, respectively, and rice–rapeseed rotation and rice–fallow/flooded rotation collected from Xianning XR and XF soil, respectively) that varied with respect to crop rotation and soil properties, were used in the current study. Incubation experiments were conducted under flooding at 25
C for the evaluation of FeRB and Fe2+ production. The diversity of FeRB community in each soil was evaluated. The composition of the FeRB community of each soil (QR, QF, XR and XF soil) was determined using enrichment culturing techniques under anaerobic conditions and high-resolution bar-coded reversible terminators. The dominant groups (5% of all sequences) were Proteobacteria and Firmicutes, and some rare phyla were also identified. At the genus level, the dominant composition of the clone libraries suggested that known FeRB genera were well represented (e.g. Brevundimonas,Pseudomonas, Burkholderia, Stenotrophomonas and Sporomusa). The analysis also identified novel enrichment culture bacteria genera, such as Sphingomonas, Pandoraea and Azospira, which might be involved in Fe3+ reduction in paddy soils. The diversity of FeRB communities were greatly affected by crop rotation, as well as soil properties such as parent material, pH and C/N ratio. Additionally, the Fe2+production in four soils were significantly different on 40-day incubation. These results indicate that the variation in soil properties and crop rotation have significant effects on the diversity of FeRB community which regulated soil Fe+3 reducing processes. ã 2016 Elsevier B.V. All rights reserved.

Keywords: Iron(III) reducing bacteria High-throughput barcode sequencing Fe3+ reduction Crop rotation

1. Introduction Paddy soils are the largest type of anthropogenic wetlands and are known to be an important source of greenhouse gases (GHGs) in terrestrial ecosystems, especially CH4 and N2O under flooding conditions with heavy nitrogen (N) fertilizer applications (Ding et al., 2014a; Ferre et al., 2012). Various studies have demonstrated that paddy soil microbial communities play a vital role in driving soil organic matter (SOM) accumulation, transformation, and mineralization, as well as N leaching (Ding et al., 2014b; Straub et al., 1996; Zhang et al., 2014). Additionally, many microbial functions involved in C and N cycling are coupled to other minerals (e.g., Fe). However, these biogeochemical processes are still not

* Corresponding author. Fax: +86 27 87396057. E-mail address: [email protected] (R. Hu). http://dx.doi.org/10.1016/j.apsoil.2016.01.012 0929-1393/ ã 2016 Elsevier B.V. All rights reserved.

clearly understood (Bongoua-Devisme et al., 2012; Dubinsky et al., 2010; Yang et al., 2012; Zhang et al., 2014). Iron(III) reducing bacteria (FeRB) are regarded as crucial mediators of C and N processes in paddy soils (Bongoua-Devisme et al., 2013; Ding et al., 2014b; Tan et al., 2006; Wang et al., 2009). Ferric iron can be reduced through respiration (as the electron acceptor) as well as fermentation (as an electron sink) by FeRB under anaerobic conditions (Bongoua-Devisme et al., 2012; Lin, 2006). Previous studies have demonstrated that short-chain fatty acids (e.g., acetate, formate and propionate) produced during anaerobic conditions are oxidized by FeRB and other anaerobes (e.g., methanogens), thereby affecting C mineralization (BongouaDevisme et al., 2012). Additionally, the Feammox pathway, whereby anaerobic NH4+ oxidation is coupled to Fe3+ reduction with either N2, nitrite or nitrate as the end-product, is a potentially important pathway for nitrogen loss in paddy soils (Ding et al., 2014b). Based on these key FeRB-mediated biogeochemical

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processes, investigating FeRB community composition is essential to understanding C and N cycles in paddy soils. In recent decades, various FeRB belonging to different phylogenetic groups have been described by both culturedependent and culture-independent methods. Isolating FeRB using culture-dependent approaches was advocated in the early years (Shaaban et al., 2014). It is more common for studies to report the abundance and diversity of FeRB using Most Probable Number (MPN) analysis (Petrie et al., 2003), MPN–PCR (Petrie et al., 2003), denaturing gradient gel electrophoresis (DGGE) analysis (Cahyani et al., 2008) or terminal restriction fragment length polymorphism (T–RFLP) technologies (Wang et al., 2009). Although many studies have been conducted on FeRB, including bacteria, fungi and archaea in various environments (Lin, 2006), the lack of a universal functional gene marker makes it difficult to track FeRB (North et al., 2004), especially those of rare taxa. Despite the use of these conventional molecular biology approaches, FeRB remain to be fully described. Therefore, to evaluate the diversity of FeRB in paddy soils we utilized a high resolution technique. High-throughput microbial community analysis on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) is an effective method for studying environmental microbial communities (Caporaso et al., 2012). This platform is able to analyze V2 or V4 variable regions from microbial 16S rRNA (Baker et al., 2003; Broadhurst et al., 2012; Roden et al., 2012). We hypothesized that soils undergone different crop rotations with distinctive properties contain unique FeRB communities which have significant influence on Fe3+ reduction processes. Therefore, using high-resolution high-throughput technique, we evaluated whether the diversity of FeRB communities for anaerobic enrichment coincides with (i) crop rotation and/or (ii) soil properties. To assess FeRB community diversity, an exploratory comparative study based on analysis of the 16S rRNA metagenome of four paddy soils was conducted using Illumina MiSeq reversible terminators. 2. Materials and methods 2.1. Sample collection and preparation Soils were collected from cultivated paddy fields in the cities of Qianjiang and Xianning, Hubei Province, China. Both cities have a typical subtropical monsoon climate. In March 2012, two soils from Qianjiang city, a rice–rapeseed (Brassica napus) crop rotation (QR) and a rice–fallow/flooded rotation (QF), were sampled at flowering stage of rapeseed and fallow/flooded period, respectively. Both soils are classified as calcareous alluvial soil and cambisol in FAO system (FAO, 1974). Similarly, two soils from Xianning city, a rice– rapeseed rotation (XR) and a rice–fallow/flooded rotation (XF), were also sampled at flowering stage of rapeseed and fallow/ flooded period, respectively. These two soils are classified as Quaternary red clay (Peng et al., 2015), and Ferralsols in FAO system (FAO, 1974). Soil samples (0–15 cm) at 10 subsamples were sampled with an auger and the samples were thoroughly mixed to obtain a single homogeneous sample for soil analysis. After manual removal of visible plant residues and roots, the sample was placed in airtight plastic bags that had been purged with N2. A subsample from each replication fresh soil sample (total 10 subsamples) was stored at 4
C for high-throughput sequencing analysis, and the remainder was air-dried and ground to pass through a 2 mm sieve for incubation experiments. Soil physical and chemical properties were analyzed as described previously (Peng et al., 2015; Shaaban et al., 2014). The main characteristics of these soils are given in Table 1.

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Table 1 The properties of the fresh soils. Soils

Fe(II+III)

pH

BD

TC

TN

C/N

QR QF XR XF

0.53 c 0.86 c 2.97 b 7.18 a

7.14 a 7.39 a 5.54 b 5.63 b

1.29 b 1.10 b 1.40 a 1.20 b

1.88 b 1.91 a 1.41 c 1.89 ab

0.20 a 0.16 b 0.15 b 0.20 a

9.40 11.94 9.40 9.45

Notes: Different letters in a column indicate significant differences among soils (Tukey’s test, p < 0.05); BD, Bulk density (g cm3); TC (%), soil total carbon; TN (%), soil total nitrogen; Fe(II) and Fe(III) in fresh soil, mmol kg1, extracted by 0.5 mol L1 HCl. QR and QF: rice–rapeseed rotation and rice–fallow/flooded rotation soils were collected from Qianjiang; XR and XF: rice–rapeseed rotation and rice–fallow/ flooded rotation soils were collected from Xianning

2.2. FeRB enrichment and reversible terminators Fresh soil of four different locations (QR, QF, XR, and XF soils) was used for anaerobic enrichment with three replicates. First, 90 mL anoxic water was added to 10.0 g fresh soil followed by the addition of 3 mmol Fe(OH)3 and the cultures were incubated at 25
C in the dark. After 1 week, a 20 mL suspension was added to 180 mL enrichment medium in a 250 mL serum bottle (Wang et al., 2009), and incubated at 25
C in the dark. Next the culture was added to mineral medium containing 10 mmol L1 acetate and 30 mmol L1 ferrihydrite and incubated under anaerobic conditions (90:10 N2:CO2, v/v). The synthetic ferrihydrite method was performed as described by Straub et al. (2005), and confirmed using Bruker X-ray diffraction (data not shown). Extraction, purification, and sequencing of genomic DNA from anaerobic enrichment were conducted at the Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). Partial sequences of 16S rRNA, including the V4 hypervariable region, were amplified by PCR using the forward primers V4 F(50 -AYTGGGYDTAAAGNG-30 ) and reverse primer V4 R(50 -TACNVGGGTATCTAATCC-30 ) (GarciaPichel et al., 2013). Sequencing was performed on an Illumina MiSeq sequencer (Illumina). Sequences with an average phred score lower than 25, containing ambiguous bases, max homopolymer run exceeding 6, having mismatched primers, or sequence lengths shorter than 200 bp were discarded. For V4 pair-end reads, only sequences with overlaps longer than 10 bp and without any mismatches were assembled according to their overlap sequence. 2.3. FeRB diversity and richness analysis The Quantitative Insights Into Microbial Ecology (QIIME) suite of analysis tools was used to filter and analyze the sequence data (Broadhurst et al., 2012). Sequences were assigned to operational taxonomic units (OTUs) with a threshold of 97% pair-wise identity and then classified taxonomically using Ribosomal Database Project (RDP) classifiers (Broadhurst et al., 2012). The RDP classification assignments were randomly confirmed using BLASTN by comparing sequences of strains reported in the NCBI 16S rRNA database. Rarefaction curves were plotted for each sample to ensure adequate coverage. To further characterize FeRB community diversity and relative abundance, 16S rRNA was classified taxonomically at the genus level. Mothur was also used to estimate FeRB diversity and richness (Schloss et al., 2009). The Shannon and Simpson indices were used to calculate community diversity for the chosen OTUs (Bowman et al., 2012). The Chao1 and ACE estimators were used to calculate community richness, and heat map was utilized to community structure (Dong et al., 2014). BioEnv and canonical correspondence analysis (CCA) were used to identify the abiotic factors that were most important to bacterial community composition (Liu et al., 2014).

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QR 1000

QF XF

OTUs

800

600

XR 400

200

0 0

5000 10000 15000 20000 25000 30000 35000 40000 45000

Sequences Per Sample Fig. 1. Rarefaction curves at the 97% similarity level. Notes: QR and QF: rice–rapeseed rotation and rice–fallow/flooded rotation soils were collected from Qianjiang; XR and XF: rice–rapeseed rotation and rice–fallow/ flooded rotation soils were collected from Xianning

2.4. Iron(III) reduction experiments Iron(III) reduction experiments were conducted in 1 L glass bottles with 200 g air-dried soil under flooded conditions created by adding distilled water (1:1, soil to water). The experiment was carried out under anaerobic slurry conditions at 25  1
C for 40 days. At days 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 16, 19, 25, 30, and 40 the soil Fe(II) concentrations were measured. Triplicate subsamples were collected over time for 0.5 M HCl extraction and Fe2+ concentration was determined using the ferrozine assay (Peretyazhko and Sposito, 2005). 2.5. Statistical analysis The experiment had a completely randomized block design with three replications that had the following treatments: four different soils (QR, QF, XR, and XF soils, as described above). Oneway analysis of variance (ANOVA) and Tukey’s HSD tests were applied to differentiate significant differences among the FeRB and the Fe(II) concentrations of four soils. 3. Results 3.1. FeRB richness and diversity To evaluate FeRB diversity and richness, rarefaction curves were constructed for each of the four soils (Fig. 1). The XR sample displayed a saturated trend while the QR, QF and XF samples had upward trends. Similarly, the diversity and richness of the XR sample was significantly (p < 0.05) lower than the other samples. Additionally, the rarefaction curves indicated that sufficient

sequencing depths were achieved, which was supported by the sequencing coverage of the different samples. The coverage in all samples ranged from 0.98 to 0.99 (Table 2). We obtained total 151,386 quality sequences from enrichment samples of four soils (each soil containing triplicate samples), and ranged from 25,935 to 44,993 sequences (Table 2). Optimized reads were detected at an average length of 224 bp by the MiSeq platform, and sequences were assigned to 553–1056 OTUs at a 97% similarity level (Table 2). Species richness was evaluated using Chao1 and ACE indices, both of which indicated that the XR soil richness was significantly (p < 0.05) lower than the other soils. The Shannon and Simpson indices indicated that the species diversity of QF and XR was less than QR and XF (Table 2). A similar trend was observed in the OTU variations between the samples. The number of OTUs in QR, QF, XR and XF was 1056, 986, 553, and 903, respectively. Venn diagrams indicated that the FeRB communities of the four soils shared many OTUs (Fig. 2). There were total 91 OTUs detected in all four soils. Soils from the same parent material showed considerable overlap, with calcareous alluvial (QR and QF) and quaternary red clay (XR and XF) soils having 223 and 186 shared OTUs, respectively. Similarly, samples from the same crop rotation (but different soil parent material) had considerable overlap, with rice-rapeseed (QR and XR) and rice-fallow/flooded (QF and XF) soils having 178 and 255 shared OTUs, respectively (Fig. 2). 3.2. FeRB community composition and structure There were 25 phyla, 52 classes, 81 orders, 136 families, and 280 genera in the FeRB communities of the four soils. At the phylum level, the distribution of FeRB in QR, QF, XR, XF samples was 93.0%, 92.1%, 96.3% and 79.4%, respectively; for Proteobacteria, and 1.3%, 4.8%, 2.3% and 15.2%, respectively; for Firmicutes, the rare FeRB of all the soils were classified as Bacteroidetes, Actinobacteria, Acidobacteria, Cyanobacteria, Spirochaetes, Nitrospira and Verrucomicrobia (Fig. 3a). For the class Proteobacteria, substantial differences in community composition among these samples were observed (Fig. 3b). Alphaproteobacteria and Gammaproteobacteria (53.3% and 34.9% of total reads, respectively) dominated the QR community, while Gammaproteobacteria and Betaproteobacteria dominated the QF (72.9% and 19.2%, respectively) and XR (87.4% and 7.9%, respectively) communities. The XF community was dominated by Betaproteobacteria. The main composition of the FeRB communities is presented in Table 3. In the QR community, the dominant FeRB genera were Brevundimonas, Stenotrophomonas and Sphingomonas, respectively. Pseudomonas and Azospira were dominant genera in the QF community. These genera also dominated the XR community, but another FeRB genus (Stenotrophomonas) comprised a large proportion of the community. The XF community did not have a single dominant genus and instead was largely comprised of the genera Burkholderia, Pandoraea, Azospira and Sporomusa, which were not well represented in the other soils. Additionally, there were many unclassified FeRB in all taxa, which increased from phylum to genus.

Table 2 Richness and diversity at the similarity level of 97% for FeRB in four soils. Soils

Optimized sequence

OTUs

Chao1

ACE

Shannon index

Simpson

Good

QR QF XR XF

44993 a 37336 b 43122 a 25935 c

1056 a 986 a 553 b 903 a

2223 b 2840a 1503 c 1645 bc

3647 ab 4469 a 2644 b 2171 b

1.89 ab 1.55 b 1.24 b 2.52a

0.2879 b 0.5083 a 0.5173 a 0.2039 b

0.99a 0.98a 0.99a 0.98a

Notes: OTUs, The operational taxonomic units were defined with 3% dissimilarity; Good, The Good’s coverage. QR and QF: rice–rapeseed rotation and rice–fallow/flooded rotation soils were collected from Qianjiang; XR and XF: rice–rapeseed rotation and rice–fallow/flooded rotation soils were collected from Xianning. Values within a column followed by the same letter are not significantly different at p  0.05.

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Fig. 2. Venn diagram of the FeRB richness. Notes: QR and QF: rice–rapeseed rotation and rice–fallow/flooded rotation soils were collected from Qianjiang; XR and XF: rice–rapeseed rotation and rice–fallow/flooded rotation soils were collected from Xianning

The most abundance of 50 OTUs, which were identified in the four enrichment samples, was used to perform clustering analysis (Fig. 4). The results of heat map showed the FeRB distribution among the samples. Although many OTUs were unclassified beyond the phylum level, the relative abundance and phylogenetic diversity of FeRB is evident. Proteobacteria (28 OTUs) were dominant in all soils, which included Betaproteobacteria (11 OTUs), Alphaproteobacteria (8 OTUs), Gammaproteobacteria (6 OTUs) and unclassified Proteobacteria (3 OTUs). The next-most abundant groups were Firmicutes (11 OTUs), Bacteroidetes (4 OTUs) and unclassified bacteria (4 OTUs) followed by the least abundant

groups of OTUs, which were Nitrospira (1 OTU) and Actinobacteria (2 OTUs). 3.3. Fe2+ production The Fe2+ concentration in all the soils increased rapidly within the first few days after the start of incubation. In general, Fe2+ concentrations increased in all four soils, but in XR and XF soils decreased on day 30, while in QR and QF soils continuously increased and were highest on day 40 of the incubation. Iron(II) concentrations were higher in QR and QF soils than in XR and XF

Fig. 3. Numerically dominant clades in the FeRB communities. (Notes: (a), at the phylum level, the relative abundance >0.1%, Proteobacteria dominated four soils FeRB; (b), at the Class level, Alphaproteobacteria and Gammaproteobacteria dominated the QR, Gammaproteobacteria and Betaproteobacteria dominated the QF and XR, and Betaproteobacteria dominated the XF.)

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Table 3 The dominant FeRB in different phylogenetic OTUs taxa obtained by pyrosequencing of 16S rRNA genes using MiSeq platforms. Taxonomic description

Percent of total sequence QR

Proteobacteria AlphaProteobacteri Caulobacteraceae Brevundimonas

QF

XR

XF

40.3

0.7

0.1

0.2

9.5

0.0

0.0

0.0

0.6

70.0

15.6

0.4

Rhodocyclaceae Azospira

0.1

12.7

6.3

11.5

Burkholderiaceae Burkholderia Pandoraea

0.1 0.0

0.7 0.6

0.8 0.6

34.4 25.7

GammaProteobacteria Xanthomonadaceae Stenotrophomonas

33.7

2.0

69.9

1.0

Firmicutes Clostridia Veillonellaceae Sporomusa

0.0

0.0

0.0

7.1

Sphingomonadaceae Sphingomonas BetaProteobacteria Pseudomonadaceae Pseudomonas

soils (Fig. 5). At the end of 40-day incubation, Fe2+ production in QR, QF, XR and XF soils was 120.46, 90.21, 32.78 and 63.18 mmol kg1, respectively. 3.4. Relationship between FeRB and soil properties The relationships between soil variables and bacterial community were assessed using CCA. Relationships between FeRB and soil properties, including extractable Fe, C/N and pH, are shown in Fig. 6. The eigenvalues of the two axes were 0.776 and 0.635, and explained 45.2% and 36.9% of the variance in FeRB, respectively. Soil-extractable Fe and C/N ratios were positively influenced by the FeRB in XF and QF soils, respectively. In the XR soil, FeRB was positively correlated with the soil pH. 4. Discussion

Notes: QR and QF: rice–rapeseed rotation and rice–fallow/flooded rotation soils were collected from Qianjiang; XR and XF: rice–rapeseed rotation and rice–fallow/ flooded rotation soils were collected from Xianning. Bold numbers indicate the most important percent of total sequence.

Previous studies have characterized the FeRB communities composition using culture-dependent or culture-independent techniques. Earlier culture-based approaches included MPN enrichment that focused on culturable FeRB or the numbers of FeRB (Dubinsky et al., 2010; Lovley, 1991; Petrie et al., 2003). This approach renders quantification of the phylogenetic diversity of FeRB communities difficult, especially since non-culturable bacteria would not be evaluated. Molecular techniques have also been employed to investigate FeRB abundance and phylogenetic community diversity (Lin, 2006). However, these techniques identify microbes based on clone libraries, which could lead to artificially decreased taxa. For example, limited groups of the FeRB taxa in paddy soils have been reported by T-RFLP (Li et al., 2010; North et al., 2004) and PCR-DGGE (Cahyani et al., 2008). In the present study, which used the Illumina MiSeq platform, we

Fig. 4. Heatmap of four soils based on the abundance similarity of the 50 OTUs which contained the most reads. Colour zone represent the OTUs relative abundance. Notes: QR and QF: rice–rapeseed rotation and rice–fallow/flooded rotation soils were collected from Qianjiang; XR and XF: rice–rapeseed rotation and rice–fallow/flooded rotation soils were collected from Xianning.

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QR QF XR XF

-1

production ( mmol kg )

150

ab (p<0.05) a (p<0.05) c (p<0.05) bc (p<0.05)

100

Fe

2+

50

0 0

5

10

15

20

25

30

35

40

Time (days) Fig. 5. Fe(II) production in laboratory incubation experiment. Vertical bars indicate the standard deviation of the mean value (n = 3). Notes: different small letters (a–c) denote that Fe(II) concentration is significant (p < 0.05) in soils. Notes: QR and QF: rice-rapeseed rotation and rice-fallow/flooded rotation soils were collected from Qianjiang; XR and XF: rice-rapeseed rotation and rice-fallow/flooded rotation soils were collected from Xianning.

obtained a number of sequences and OTUs from paddy soils (Table 2). Our approach identified conventional taxa that were present in enrichment cultures (Fig. 3a), and also revealed some rare FeRB community taxa (e.g. Planctomycetes, Spirochaetes, Fusobacteria, Deinococcus-Thermus, Chloroflexi, Gemmatimonadetes, Synergistetes etc.). Additionally, many “Unclassified” sequences were identified in this study, suggesting that these paddy soils contain a quantity of unknown FeRB. According to previous studies, Proteobacteria and Firmicutes are typically the dominant groups among the FeRB communities in paddy soils (Li et al., 2011; Zhang et al., 2013). Previously, members of other phyla, such as Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Nitrospira, Spirochaetes and Verrucomicrobia, have been described in different agricultural soils and may play important roles in C and N cycling. However, these taxa are rarely classified as FeRB, especially in paddy soils (Liu et al., 2014; Lopes et al., 2014; Zhang et al., 2013). Some of these phyla, including Actinobacteria, Bacteroidetes and Verrucomicrobia, have also been found in groundwater samples and other environments and have

Fig. 6. CCA ordination diagram of different enrichment samples associated with environmental variables. The numbers indicate the four soils. Environmental variables are indicated by arrows.

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been classified as FeRB (Lin, 2006). Therefore, additional unknown phyla of FeRB could be a subject for future investigations. In the present study, the phyla Proteobacteria and Firmicutes were predominant in the soils with variations in the relative abundance of these groups. For example, Brevundimonas spp. (Alphaproteobacteria) were uniquely over abundant in the QR soil and may have contributed to the observed differences in Fe2+ production due to its ability to use alternate Fe3+ electron acceptors during anaerobic respiration (Ghosh and Sar, 2013). Other notable differences in FeRB include more abundant Gammaproteobacteria (Stenotrophomonas) in rice-rapeseed soils (QR and XR) than in ricefallow/flooded soils (QF and XF). In contrast, Betaproteobacteria were more abundant in rice-fallow/flooded soils (QF and XF) than in rice-rapeseed soils (QR and XR) (Table 3). It is possible that distinct bacterial communities are closely correlated to soil flooding (Kan et al., 2014). Our results also identified higher Firmicutes diversity in rice-fallow/flooded rotation soils than in rice-rapeseed crop rotation soils (Table 3). The Firmicutes were mainly comprised of OTUs in the order Clostridia (mainly Sporomusa) followed by Bacillales (mainly Bacillus). Previous studies have documented abundance of similar anaerobic bacterial communities in long-term flooded paddy soils as in case of our current study (Kan et al., 2014). These results suggest that crop rotation has greatly driving influence on FeRB community variations in paddy soils. Furthermore, FeRB community considerably influenced Fe2+ production in the current study. The difference in Fe2+ production between soils of the same parent material may be attributed to differences in the FeRB community. For example, Brevundimonas spp. comprised a large proportion of the FeRB community in the QR soil. It is possible that Brevundimonas spp. not only participated in Fe3+-reducing processes, but also decomposed labile carbon for other FeRB (Ghosh and Sar, 2013). This may explain the higher Fe2+ production in the QR soil after 20 days of incubation. In contrast to the FeRB in QR soil, the QF soil was composed mostly of Pseudomonas spp, which are not necessarily true Fe3+ reducers having persistent ability, but weak Fe3+ reduction (Naganuma et al., 2006). This may partly explain the lower Fe2+ production in the QF soil during 40 days of incubation. The phylogenetic classification showed that Betaproteobacteria spp. was different in soils with the same crop rotation (e.g., rice–fallow/flooded). At the genera level, Pseudomonas and Azospira dominated the QF soil community, while Burkholderia, Pandoraea and Azospira dominated the XF soil community (Table 3). The Fe3+-reducing ability of Pseudomonas spp. and Burkholderia spp. are significantly different as described by Naganuma et al. (2006) and Roden et al. (2012). Therefore, FeRB has a close relationship with Fe contents. Such as, the results of the CCA analysis showed that the FeRB had strong relationships with Fe contents in XF soil. It has been previously reported that the diversity of bacterial community is greatly influenced by soil properties (BongouaDevisme et al., 2013; Liu et al., 2014; Zhang et al., 2013). In the present study, soil pH was significantly different in two types of parent material (Calcareous alluvial vs Quaternary red clay). Furthermore, soil-extractable Fe(II+III) from these soils also showed significant differences. Implying that the diversity of FeRB in different paddy soils were influenced by soil properties. Soil pH and Fe content may play an important role in distributing of soil FeRB communities. The Canonical Correspondence Analysis (CCA) between the FeRB communities and environmental variables showed that soil pH significantly influenced FeRB communities in XR soil (Fig. 6). However, in case of the same parent material, the diversity of FeRB in XF soil was significantly closely related with soil extractable Fe content. Therefore, other factors might be regulating FeRB distribution. For example, QF soil has higher C/N ratio than other soils, and the diversity of FeRB was positively

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correlated with soil C/N ratio. This implies that soil C and N content is another important parameter in determining the FeRB of paddy soils (Burgin et al., 2011; Naganuma et al., 2006). In addition, fresh soil used for FeRB enrichment experiments and air dried soil used for Fe3+ reduction may have directly mediated reductions in Fe3+ (Fig. 5). Ginn et al. (2014) confirmed that these two storage methods neither significantly change FeRB diversity nor significantly decrease the Fe-reducing capacity of the soil. An exciting finding of this study was related to the un-identified or un-classified OTUs in the Proteobacteria phylum. Members of Betaproteobacteria have been reported as being crucial mediators of Fe cycling in soils in Hubei, China (Shaaban et al., 2014), where Pandoraea spp. and Azospira spp. were first reported as FeRB with the ability to reduce Fe3+. Additionally, within Alphaproteobacteria, Sphingomonas spp. were also identified as FeRB. Based on these studies and our results, the phylogenetic diversity of Alphaproteobacteria and Betaproteobacteria should be investigated in future studies. Additionally, among the active FeRB populations detected, Geobacter spp. were perhaps the most familiar (Nevin and Lovley, 2000). In this study, although we detected distinct populations in the enrichment cultures, the relative abundance of Geobacter spp. was less than 5% and was not be identified as a dominant FeRB in the four soils. Future studies are expected to collect more samples across a wide range of paddy soils in China. 5. Conclusions Rare FeRB phyla including Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Nitrospira, Spirochaetes and Verrucomicrobia were identified using high-throughput sequencing analysis. Within the dominant phylum Proteobacteria, the genera Sphingomonas, Pandoraea and Azospira were for the first time reported as FeRB in paddy soils. These results indicate that FeRB community diversity in subtropical paddy soils of China are determined mainly by crop rotation. Additionally, soil pH and C/N ratio contributed to variations in the bacterial communities. Acknowledgements This research work was jointly supported by National Basic Research Program of China (2012CB417106), Natural Science Foundation of China (41171212 and 43007002), Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, South-Central University for Nationalities. References Baker, G.C., Smith, J.J., Cowan, D.A., 2003. Review and re-analysis of domain-specific 16S primers. J. Microbiol. Methods 55, 541–555. Bongoua-Devisme, A.J., Mustin, C., Berthelin, J., 2012. Responses of iron-reducing bacteria to salinity and organic matter amendment in paddy soils of Thailand. Pedosphere 22, 375–393. Bongoua-Devisme, A.J., Cebron, A., Kassin, K.E., Yoro, G.R., Mustin, C., Berthelin, J., 2013. Microbial communities involved in Fe reduction and mobility during soil organic matter (SOM) mineralization in two contrasted paddy soils. Geomicrobiol. J. 30, 347–361. Bowman, J.S., Rasmussen, S., Blom, N., Deming, J.W., Rysgaard, S., Sicheritz-Ponten, T., 2012. Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene. ISME J. 6, 11–20. Broadhurst, M.J., Ardeshir, A., Kanwar, B., Mirpuri, J., Gundra, U.M., Leung, J.M., Wiens, K.E., Vujkovic-Cvijin, I., Kim, C.C., Yarovinsky, F., Lerche, N.W., McCune, J. M., Loke, P.n., 2012. Therapeutic helminth infection of macaques with idiopathic chronic diarrhea alters the inflammatory signature and mucosal microbiota of the colon. PLoS Pathog. 8. Burgin, A.J., Yang, W.H., Hamilton, S.K., Silver, W.L., 2011. Beyond carbon and nitrogen: how the microbial energy economy couples elemental cycles in diverse ecosystems. Front. Ecol. Environ. 9, 44–52. Cahyani, V.R., Murase, J., Ikeda, A., Taki, K., Asakawa, S., Kimura, M., 2008. Bacterial communities in iron mottles in the plow pan layer in a Japanese rice field:

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