Significance of Fe(II) and environmental factors on carbon-fixing bacterial community in two paddy soils

Ecotoxicology and Environmental Safety 182 (2019) 109456

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Significance of Fe(II) and environmental factors on carbon-fixing bacterial community in two paddy soils

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Sarfraz Hussaina, Zhang Minb, Zhu Xiuxiua, Muzammil Hassan Khana, Li Lifenga, Cao Huia,* a b

College of Life Sciences/Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, 210095, China College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China

ARTICLE INFO

ABSTRACT

Keywords: Illumina MiSeq sequencing cbbM gene Fe(II) Paddy soil Chemotrophic Phototrophic

The seasonal flooding and drainage process affect the paddy soils, the existence of the iron state either Fe(III) or Fe(II) is the main redox system of paddy soil. Its morphological transformation affects the redox nature of paddy soils, which also affects the distribution of bacterial community diversity. This study based on molecular biological methods (qPCR, Illumina MiSeq sequencing technique) to investigate the effect of Fe(II) and environmental factors on cbbM genes containing carbon fixing microbes. Both Eh5 and pH were reduced with Fe(II) concentrations. The Fe(II) addition significantly affects the cbbM gene copy number in both texture soils. In loamy soil, cbbM gene copy number increased with high addition of Fe(II), while both low and high concentrations significantly reduced the cbbM gene copy number in sandy soil. Chemotrophic bacterial abundance significantly increased by 79.7% and 54.8% with high and low Fe(II) addition in loamy soil while in sandy soil its abundance decreased by 53% and 54% with the low and high Fe(II) accumulation. The phototrophic microbial community increased by 37.8% with low Fe(II) concentration and decreased by 16.2% with a high concentration in loamy soil, while in sandy soil increased by 21% and 14.3% in sandy soil with low and high Fe(II) addition. Chemoheterotrophic carbon fixing bacterial abundance decreased with the Fe(II) accumulation in both soil textures in loamy soil its abundance decreased by 5.8% and 24.8%, while in sand soil 15.7% and 12.8% with low and high Fe(II) concentrations. The Fe(II) concentration and soil textures maybe two of the major factors to shape the bacterial community structure in paddy soils. These results provide a scientific basis for management of paddy soil fertility and it can be beneficial to take measures to ease the greenhouse gases effect.

1. Introduction Paddy field is a typical farmland ecosystem in China (Hong-zhao, 1981). China's paddy soil accounts for one-fourth of the total world paddy soil area and can be divided into hundreds of varieties. In paddy soil under flooding conditions, ferric iron is reduced to ferrous iron, and as sequestrated on iron oxide is then released to soil pore water (Turner and Patrick, 1968). Iron is the twenty-five percent major content in the Earth layers and it also exists in paddy soil. Iron respiration may be the first microbial metabolic mechanism prior to oxygen, nitrogen and sulfur respiration (Kashefi and Lovley, 2003; Liu et al., 1997). The seasonal flood and drainage process of paddy soil affects the presence of iron elements, Ferric/Ferrous oxide as the main redox system of paddy soil, and its morphological transformation affects the redox properties of paddy soil and the distribution of bacterial diversity in paddy soil (Liesack et al., 2000). In addition to being chemically reduced, iron oxide can be biologically reduced, such as an iron-reducing bacterium,

*

which uses ferric oxide as an electron receptor to obtain energy from the phosphorylation process to restore the refractory trivalent ferric oxide to a soluble Fe (II) (Lloyd et al., 2000). There is a competitive relationship between biological and chemical oxidation, usually under low pH Fe(II) is not easy to oxidize; however, in neutral and nearneutral conditions Fe(II) oxidizing bacteria need to compete with the rapid change in chemical oxidation, therefore, there is more diversity in the aerobic and anaerobic interface (Emerson and Moyer, 1997). Fe has an importance not only because it can be used as an electron acceptor or electron donor by considerable microbial communities to gain energy, also plays an important role in many other elementary cycles, such as the C and N cycles (Boyd et al., 2007; Canfield, 1989; Jensen et al., 2003; Martin et al., 1991; Straub et al., 1996; Widdel et al., 1993). Microbial-mediated iron cycling cannot only significantly affect the mineralization of organic matter, dissolution, and weathering of minerals, but also participate in carbon cycling. Previous studies showed that 75% organic matter in marine sediments is oxidized by

Corresponding author. E-mail address: [email protected] (C. Hui).

https://doi.org/10.1016/j.ecoenv.2019.109456 Received 27 April 2019; Received in revised form 15 July 2019; Accepted 18 July 2019 Available online 30 July 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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coupling trivalent iron (Weber et al., 2006b). The Placidus study found that Fe could restore carbon dioxide by returning a prototype of acetyl coenzyme A to the metabolic pathway and dechloromonas aromatica genome sequencing. It also found to reduce the rate of amyl dihydrogen phosphate, a two phosphate-phosphorylated enzyme (RUBISCO), using nitrate, chlorinated salts, etc. as electron receptors to restore carbon dioxide (Buchanan and Arnon, 1990). Autotrophic Carbon-sequestration bacteria (CO2) are the only carbon source that, transformed from the environment to CO2 through a series of metabolic reactions. Currently, there are five carbon fixation pathways (Benson et al., 1950; Elsaied and Naganuma, 2001; Herter et al., 2002; Ragsdale, 1991; Thauer, 2007). Calvin cycle is the first carbon sequestration pathway, and it is also the main carbon sequestration pathway for autotrophic nitrogen-fixing microorganisms. The catalytic nucleoside −1,5-two phosphoric acid is involved in the immobilized CO2 of 1,5-two phosphate (RuBisCO), which is the key enzyme in Calvin cycle fixation CO2. There are two main types RuBisCOI and RubiscoII in autotrophic carbon-sequestration bacteria. In recent year's cbbL and cbbM genes have been used by many researchers to study the diversity of carbon sequestration microorganisms in different ecological environments (Nanba et al., 2004a; Xu and Tabita, 1996). At present, with the help of modern molecular biology techniques, molecular mechanisms of carbon sequestration in autotrophic microbes and their responses and feedbacks to different habitats have become the research hotspots in this field by analyzing the diversity of functional genes in environmental samples. RubisCO plays an important role in soil carbon fixation. Microbial community influenced by many physiological and chemical factors such as precipitation, temperature, soil pH, soil chemistry, soil mineralogy, and plants. Across ecosystems, metabolic capacities, biomass chemical composition and types of organic matter significantly affect the community composition of microbes (Liang et al., 2011; Waldrop and Firestone, 2006). Therefore, the present study was designed to explore the relationship between Fe(II), environmental factors and carbon sequestration microbial community in typical paddy soils.

constant temperature incubator. Samples were taken at 3, 8, 15, 25, 40 and 60 days after the trial setting. Later on, used for DNA extraction and determination of soil basic physical and chemical properties. Calibrated HACH HQ30d pH meter was used to measure the pH of soil samples (BANTE, Shanghai, China). Soil pH was determined at a soil-to-H2O ratio of 1:2.5 (w/v). The mixture was first shaken for 5 min and then left undisturbed for 20 min for equilibration. Soil factors (total reduced substances, Eh5, Fe (II), soil organic matter) were determined by using a standard method (Tan, 2005). 2.3. DNA extraction Extraction of DNA was done from 5 g of soil using a high temperature/salt/SDS- based lysis method (Zhou et al., 1996). The DNA was purified by MoBioPowerClean® DNA Clean-Up Kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. Genomic DNA concentration and purity were measured using a Nano Drop ND-2000 (Nano Drop Technologies, Wilmington, DE) spectrophotometer. 2.4. Amplification of the cbbM gene and cloning Amplification of cbbM gene along with genomic DNA was done by using primer cbbM F (5′-TTCTGGCTGGGBGGHGAYTTYATYAARAAYGACGA-3′) and cbbM R (5′-CCGTGRCCRGCVCGRTGGTARTG-3′). A total of 20 μl of the reaction mixture was prepared by using 1.25 U of Taq DNA polymerase (Takara, Japan). Amplification conditions for polymerase chain reaction (PCR) were: an initial denaturation was done at 95 °C for 5 min, denaturation 95 °C for the 40s, annealing at 57 °C for 20s and extension at 72 °C for 30s; the process repeated for 30 cycles, followed by a final extension step at 72 °C for 10 min. PCR product was quantified by gel electrophoresis. The PCR products were purified by gel-extraction kit (Omega, USA) according to the manufacturer's instructions. After purification, products of PCR were cloned into pMD19T vectors (Takara, Japan) and transmuted into the competent DH5α cells of Escherichia coli. The colony PCR was then performed to determine the presence of positive clones with vector-specific primers M13 F (5' -GTA AAA CGA CGG CCAG-3′) and M13 R (5' -CAG GAA ACA GCT ATGAC-3′).

2. Materials and methods 2.1. Site description and sample collection

2.5. Quantification of cbbM gene

Two soils were obtained in December 2015 from two rice fields loamy soil (L) collected from 30°50＇8.4＂N 118°36is＇21.9＂E and sandy soil (S) were collected from 30°49＇33＂N 118°54＇47＂E, respectively located in Xuancheng city, Southeast of Anhui Province of China. Multiple soil samples were randomly collected from 0 to 15 cm depth in each site and were taken by a soil corer device. About 10 kg of soil from each rice field was collected, samples were pooled and homogenized. Genomic DNA extraction and chemical analysis were immediately performed according to experimental design.

The abundance of cbbM genes was determined by an ABI 7500 quantitative PCR instrument using an SYBR Premix Ex Taq TM kit (TaKaRa Biotechnology Co. Ltd) and the same primers above mentioned. Each reaction was performed in a 20 μL volume containing 2 μL template DNA (2–20 ng), 10 μL SYBR Premix Ex Taq TM, 0.4 μL ROX Dye II (50 × ) (TaKaRa Biotech, Dalian, China). The PCR conditions were as follows: pre-denaturation at 95 °C for 2 min one cycle, denaturation at 95 °C for 15s, annealing at 57 °C for the 20s, extension 72 °C for 34s and repeated for total 40 cycles to make the melting curve. Melting curve analysis and agarose gel electrophoresis were done at the end of qPCR to check the specificity of amplification.

2.2. Experiment design and chemical analysis Samples (soil) were assimilated by proper mixing and then immediately put in storage at 4 °C for further processing. Then, the soil samples were air-dried for 48 h, after drying samples were passed through a filter (2-mm) to eliminate plant roots, leaves, and other material. After sieving 200 g of soil were taken into culture bottles (9 cm diameter and 12 cm height) for the experiment. Sandy (S) and loamy (L) soils were prepared for low (100 mg/kg) addition of FeSO4·7H2O as S1 and L1, for high (1000 mg/kg) addition of FeSO4·7H2O as S2 and L2. S1-1, S1-2, S1-3 were the three replicates representing sandy soil with low Fe addition, L1-1, L1-2, L1-3 were the three replicates representing loamy soil with low Fe addition. S2-1, S22, S2-3 and L2-1, L2-2, L2-3 were the three replicates representing sandy and loamy soil with high Fe addition respectively. The mixed homogeneous test specimens were placed in 25 °C

2.6. Library construction and sequencing All samples were sent to the SBC company (Shanghai, China) for library construction and high throughput sequencing. Library construction and sequencing processes were as followed: DNA was extracted from 18 soil samples and the functional gene cbbM was amplified using cbbM F (5′-TTCTGGCTGGGBGGHGAYTTYATYAARAAYGACGA-3′) and cbbM R (5′-CCGTGRCCRGCVCGRTGGTARTG-3′). For library construction and sequencing purified PCR products were quantified by Qubit®3.0 (Life Invitrogen) and every twenty-four amplicons whose barcodes were different were mixed equally. The pooled DNA product was used to construct the Illumina Pair-End library following Illumina's genomic DNA library 2

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preparation procedure. Then the amplicon library was paired-end sequenced (2 × 250) on an Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd) according to the standard protocols. 2.7. Sequencing and phylogenetic analysis Sequences were analyzed with the QIIME software package (Quantitative Insights into Microbial Ecology) (Caporaso et al., 2010). The reads were first filtered by QIIME quality filters, default settings for Illumina processing in QIIME were used. The sequences were compared with known cbbM gene sequences from the GenBank (NCBI) database using BLAST. Then the obtained qualified reads were spliced in MOTHUR software (Schloss et al., 2009). The sequences were clustered into operational taxonomic units (OTUs) at a similarity level of 97%, to generate rarefaction curves and to calculate the richness and diversity indices (Colwell and Coddington, 1994). Unweighted UniFrac distances were also performed in Mothur. CCA (Canonical correspondence analysis) was done to quantify the physicochemical parameter. To compare the association and structure of communities in different samples, heat maps generated with OTUs using R package software.

Fig. 1. The abundance of cbbM genes in the two texture soils under two different treatments, loamy soil without treatment (LCK), Low addition of Fe(II) in loamy soil (L1) and High addition of Fe(II) in loamy soil (L2). Sandy soil without addition of Fe(II) (SCK) Low addition of Fe(II) in sandy soil (S1) and High addition of Fe(II) in sandy soil (S2). Vertical bars indicate standard errors of the means (n = 3). Bars with the same letter are not significantly different at P < 0.05.

3.2. cbbM fluorescence quantification

2.8. Statistical analyses

The number of cbbM gene copies was determined by the quantitative PCR method (shown in Fig. 1). In loamy soil, there was a tendency to increase the number of cbbM gene copies. As the Fe(II) addition increased, the copy number increased from 1.05 × 106 g−1 in LCK to 1.09 × 106 g−1, 1.15 × 106 g−1 in L1 and L2 respectively. Meanwhile, the number of cbbM genes copies were significantly reduced in the sandy soil regardless added Fe(II) concentrations i.e. the number of cbbM gene copies decreased from 1.27 × 106 g−1 in SCK to 0.45 × 105 g−1, 0.44 × 105 g−1in S1 and S2, respectively.

Comparison between gene copy numbers was determined using oneway ANOVA followed by Duncan's multiple range test. Different samples were analyzed by using UniFrac to check the similarities among the microbial communities. Weighted and unweighted UniFrac can be calculated by QIIME. On the basis of the protocol that was previously published by (Kuczynski et al., 2012) unweighted and weighted UniFrac was used to conduct the Principal Component Analysis (PCA) and unweighted pair group method with arithmetic mean (UPGMA) clustering. The effect of physical and chemical parameters on microbial communities was measured by Canonical Correspondence Analysis (CCA).

3.3. The diversity and community structure of the bacterial carbon-fixing cbbM gene

3. Results

3.3.1. Alpha diversity The Illumina MiSeq analysis produced 89,024 high-quality reads from two paddy soil textures (loamy and sandy). At 3% sequence dissimilarity level, the OTU numbers in two soil textures vary from 129 to 204. The number of OTUs in both textures indicated different trends, specifically, the OTU number in loamy soil decreased while increased in sandy soil along with Fe(II) concentration. The α diversity results of two soil textures under different treatment showed in Table 2. The microbial diversity index of the two soil textures showed different trends after the addition of Fe (II). In loamy soil, ACE, Shannon, and Chao index continued to decrease along with Fe (II) concentration, while the Simpson index increased. In sandy soil, changes of bacterial diversity index present a reverse, except for the ACE index.

3.1. Physical and chemical analysis The physical and chemical properties of different treated soils, as shown in Table 1, pH values and Eh5 of each treated soil significantly decreased along with added Fe(II) concentration, while reducing substances increased correspondingly in sandy and loamy soil. The data showed that soil pH in LCK was 6.0 which decreased to 5.15 and 3.95 after added low and high concentrations of Fe(II), while in SCK pH values decreased from 5.54 to 4.37 and 3.32 with low and high Fe(II) concentrations. Compared with sandy soil, the loamy soil had a small change in pH after adding the same concentration of Fe(II), indicated that the loamy soil had strong buffering capacities. There was no significant difference in the organic matter among different treated soils. Table 1 The physical and chemical properties of different treated soils. Treatments

pH

Eh5

Fe (II) (mg kg−1)

RS (cmol kg−1)

LCK L1 L2 SCK S1 S2

6.0 ± 0.75a 5.14 ± 0.54abc 3.95 ± 1.19cd 5.54 ± 0.68ab 4.37 ± 0.08bcd 3.32 ± 0.77d

543 ± 18a 400 ± 51b 231 ± 38c 529 ± 18a 245 ± 6c 120 ± 21d

192 ± 30c 235 ± 16c 580 ± 110b 18 ± 6d 70 ± 1d 555 ± 46b

4.51 4.81 8.18 1.11 1.58 8.01

± ± ± ± ± ±

0.6b 0.26b 0.82a 0.31c 0.21c 0.47a

OM: organic matter, Eh5: redox potential, RS: reducing substances, Fe(II): ferrous oxide. Values in a column for each class superscript with different letters are significantly different from each other at (P < 0.05). 3

OM (g kg−1) 13.29 ± 0.43a 14.12 ± 0.31a 13.28 ± 0.50a 7.87 ± 0.20c 8.49 ± 0.33c 7.85 ± 0.33c

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Table 2 The alpha diversity results of two soil textures under different treatment. Sample

Reads

OTUs*

Coverage

Richness Diversity ACE*

Chao LCK L1 L2 SCK S1 S2

15012 15993 16112 15074 13660 13173

184 165 129 168 204 228

0.996 0.996 0.997 0.993 0.994 0.992

a

223 ± 6 207 ± 29a 175 ± 15a 225 ± 8a 226 ± 17a 246 ± 8a

Shannon a

219 ± 11 219 ± 37a 182 ± 30a 241 ± 11a 221 ± 18a 248 ± 7a

3.47 2.90 1.81 2.18 4.48 4.58

± ± ± ± ± ±

Simpson b

0.17 0.60b 0.07a 0.36a 0.05b 0.01b

0.0685 0.1648 0.3302 0.2626 0.0179 0.0173

± ± ± ± ± ±

0.0265a 0.1341a 0.0112b 0.0616b 0.0016a 0.0005a

OTU: Operational Taxonomic Unit, ACE: Abundance-based Coverage Estimator *Note: Values in a column for each class superscript with different letters are significantly different from each other at (P < 0.05).

Fig. 2. Principal Component Analysis (PCA) plot created on the cbbM gene sequencing (Illumina MiSeq analysis) from differently treated samples and the scatter plot is of principal coordinate (PC1) verses (PC2).

Actinobacteria was considered as the second most abundant phylum in all samples. Cyanobacteria were the least abundant phylum in all samples. Classification of bacterial community at the class level on the basis of average relative abundance total of 8 classes were dominated. The abundant classes were Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria in LCK and SCK. Acidithiobacillia abundance increased with treatment of low Fe(II) in loamy soil while significantly increased in sandy soil treated with low or high Fe(II). Ferrous iron is an energy substance for autotrophic bacteria, and also a toxic chemical whose toxicity is determined by concentration. The dominant genera (≥1%) within different samples are shown in Table 3. As far as loamy soil, the relative abundance of some genera such as Thiobacillus, Halothiobacillus, and Bradyrhizobium significantly decreased with Fe(II) addition, while the others such as Acidihalobacter and Leptothrix increased. Surprisingly, we found that other genera abundance stimulated by low Fe(II) concentration but inhibited by high Fe(II) concentration, these genera include Rhodopseudomonas, Acidithiobacillus, Rhodovulum, Thiohalomonas, and Sulfuritalea. The relative abundance of these bacteria account for 36.7% in the LCK while increased to 79.5% in the L1, but decreased to 14.5% in L2. For sandy soil, the relative abundance of genus Magnetospirillum sharp decreased with the addition of Fe(II), from 73.6% in SCK to 8.6% and 4.3% in S1and S2, respectively. Other genera such as Sulfuricella and Thiocystis, their relative abundance significantly increased in low Fe(II) concentration. Furthermore, high Fe(II) addition significantly increased the relative abundance of genera including Acidihalobacter, Halothiobacillus, Rhodobacter, Leptothrix, Thiohalorhabdus, and Nocardia.

3.3.2. Beta diversity The PCA analysis based on OTU level is shown in Fig. 2. Using PCA analysis, it is possible to extract two coordinate axes that reflect the difference between samples. The more similar the community composition of the samples, the closer they are in the PCA plot. LCK is close to L1, but far from L2, indicating that LCK and L1 have similar bacterial community composition, but they differ greatly from L2 bacterial community composition, indicating that the addition of high Fe (II) in loamy soil has a greater impact on the composition of the autotrophic carbon-fixing bacterial community. S1 and S2 can be clustered together, but they are far away from SCK, indicating that Fe(II) concentration affects autotrophic carbon-fixing bacterial community composition in sandy soil, but this effect does not correlate with Fe(II) concentrations. The weighted Unifrac distance shown in Fig. 3 to evaluate the similarity coefficient among treated soils. The figure showed that the community structure of cbbM sequences in LCK and L1 had similarities compared to L2. The community structure of carbon-fixing bacteria in S1 and S2 was similar and there was no similarity with SCK. 3.3.3. Phylogenetic analysis of the microbial community After sieving chimeras all valid reads of 18 trials, low-quality reads and trimming the primers, barcodes, and adapters. QIIME default setting was used to classify all valid reads from phylum to genus. The taxonomic distribution from phylum level to genus includes 3 phyla, 8 classes, 17 orders, 20 families and 26 genera. Proteobacteria was the most abundant phylum in all samples, its percentage content is 96% or more in each sample. With an average relative abundance, 4

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Fig. 3. Weighted Unifrac distance matrix showed that the color level is proportional to the value of the dissimilarity between observations.

The dominants chemotrophic carbon-fixing bacterial abundance in control soils account for 49.0% and 87.4% in loamy and sandy soil respectively. Chemotrophic cbbM encoding bacterial abundance significantly increased from 49.0% to 54.8% and 79.7%with low and high addition of Fe(II) in loamy soil, while its abundance in sandy soil decreased by 87.4%–53.0% and 54.1% with low and high addition of Fe (II), respectively. Chemoautotrophic bacterial such as Acidihalobacter abundance increased significantly with high Fe(II) addition in both soil textures, which increased 49.4% in the loamy soils and increased 10.3% in the sandy soils. Thioehalomonas and Sulfuricella abundance with low Fe(II) addition increased in both soil textures, each of them increased by 0.3% and 2.5%. In sandy soil, chemoautotrophic bacterial Thiohalorhabdus abundance increased by 2.1% with high Fe(II) addition. Only in loamy soil, chemoautotrophic bacteria Sulfuritalea abundance increased by 17.9% with low Fe(II) addition. Halothiobacillus abundance decreased 1.3% in loamy soils treated with Fe(II), while with high addition of Fe(II) increased abundance by 5.1% in sandy soils. Chemoautotrophic Acidithiobacillus abundance was increased 18.2% in loamy soils. Chemoheterotrophic bacterial Thiobacillus and

Magnetospirillum abundance with high Fe(II) addition decreased 27.6% and 31% in loamy soil, 65% and 69.3% in sandy soil. Bradyrhizobium abundance decreased 0.5% and 0.9% respectively with low and high Fe (II) addition in loamy soil while in sandy soil significantly not affected. Leptothrix abundance with high Fe(II) addition in loamy soils 23.1% and 22.7% increased and in sandy soils with low Fe(II) addition 1.5% increased. 3.3.4. Dominant genera and environmental factors analysis The relationship between the dominant genera and environmental factors was analyzed by CCA method (shown in Fig. 4). Fe(II) addition was positively correlated with Acidihalobacter and Rhodobacter. There was a positive correlation between Eh5 and Bradyrhizobium. Rhodovulum, Rhodopseudomonas were positively related to OM. Thiobacillus was positively related to pH. Acidithiobacillus seems to be positively correlated with L1 and LCK. It is worth mentioning that Magnetospirillum associate with SCK.

Table 3 The dominant genera abundance of different treated soils. Genus Thiobacillus Rhodopseudomonas Uncultured Acidithiobacillus Acidihalobacter Halothiobacillus Rhodovulum Magnetospirillum Rhodobacter Bradyrhizobium Thiohalomonas Sulfuricella Sulfuritalea Leptothrix Thiocystis Thiohalorhabdus Nocardia

LCK

L1 a

0.317 ± 0.11 0.273 ± 0.04b 0.149 ± 0.05c 0.058 ± 0.03a 0.039 ± 0.01de 0.024 ± 0.01b 0.024 ± 0.01b 0.019 ± 0.00bc 0.015 ± 0.00b 0.01 ± 0.00a 0.009 ± 0.00b 0.007 ± 0.01bc 0.003 ± 0.00c 0.002 ± 0.00c 0.001 ± 0.00c 0.001 ± 0.00c 0.000 ± 0.00c

0.041 0.317 0.060 0.240 0.040 0.007 0.044 0.011 0.017 0.005 0.012 0.001 0.182 0.006 0.002 0.001 0.000

L2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

b

0.03 0.11a 0.01d 0.33a 0.02d 0.00c 0.02a 0.00bc 0.00ab 0.00ab 0.00a 0.00c 0.13a 0.00bc 0.00c 0.00c 0.00c

SCK b

0.007 ± 0.00 0.139 ± 0.00dc 0.030 ± 0.00d 0.002 ± 0.00b 0.533 ± 0.00a 0.011 ± 0.00c 0.003 ± 0.00c 0.008 ± 0.01c 0.02 ± 0.00ab 0.001 ± 0.00c 0.000 ± 0.00c 0.000 ± 0.00c 0.001 ± 0.00c 0.233 ± 0.01a 0.001 ± 0.00c 0.000 ± 0.00c 0.000 ± 0.00c

0.023 0.059 0.055 0.003 0.030 0.005 0.001 0.736 0.002 0.003 0.000 0.004 0.036 0.002 0.028 0.002 0.002

S1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

b

0.01 0.02e 0.02d 0.00b 0.01f 0.00c 0.00c 0.09a 0.00c 0.00bc 0.00c 0.00bc 0.01bc 0.00c 0.01b 0.00c 0.00c

0.051 0.171 0.210 0.020 0.112 0.048 0.008 0.086 0.031 0.006 0.004 0.029 0.057 0.019 0.074 0.013 0.011

* Values in a column for each class superscript with different letters are significantly different from each other at (P < 0.05). 5

S2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

b

0.00 0.02c 0.05b 0.00b 0.00c 0.00a 0.00bc 0.02b 0.00a 0.00ab 0.00c 0.00a 0.00bc 0.00b 0.01a 0.00b 0.00b

0.068 0.095 0.267 0.031 0.133 0.056 0.015 0.043 0.033 0.003 0.002 0.013 0.096 0.017 0.038 0.023 0.018

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01b 0.00de 0.02b 0.00ab 0.00b 0.00a 0.00bc 0.00bc 0.01a 0.00c 0.00c 0.00b 0.01b 0.01b 0.01b 0.00a 0.00a

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Fig. 4. Canonical correspondence analysis (CCA) of dominant genera and environmental factors.

4. Discussion

autotrophic bacteria are able to oxidize Fe(II) and reduce nitrate. In order to maintain metabolic cycle by anaplerotic reactions, several heterotrophic microorganisms can also fix CO2 for new sugars formations for cell wall synthesis and excrete organic acids for nutrient mobilization (Feisthauer et al., 2008; Miltner et al., 2005; Šantrůčková et al., 2005). Microbial oxidation of Fe(II) to Fe(III) can be accelerated by a wide range of Fe(II) oxidizing bacteria under both acidic or neutral pH and oxic or anoxic conditions. The biological oxidation of Fe in bacteria particularly they accomplish the formation of mineral deposits within their ultrastructure are yet not fully understood. Studies showed that the unavailability of universal functional gene markers, the composition of the chemoautotrophic FeOB community to fix CO2 in mixed cultures or environmental soils have been relatively limited. Our results show that carbon fixation microbial community in paddy soil (loamy and sandy soil) altered with the addition of Fe(II), depicted by the previous study that drainage and flooding cycles subjected to periodic changes into oxic and anoxic conditions in (Sun et al., 2015). Based on thermodynamic theory oxygen, nitrate, iron oxides, sulfate, and carbon dioxide reduced sequentially after flooding (Zehnder, 1988). Our study showed that the effect of Fe(II) on the diversity of cbbM gene encoding bacterial community in paddy soils offers significant potential for the microbial assimilation of atmospheric CO2. Our study found that at the cbbM gene copy number level, there was a significant increase in loamy soil treated with high Fe(II) addition, while both low and high Fe(II) addition could significantly reduce the cbbM gene copy number in sandy soil. Therefore, we speculate that this might be due to the stable composition of loamy soil and its good buffering ability to change the external environment, while for sandy soil, its sand content is high and its buffering ability to change the external environment is poor, so the addition of iron has a significant impact on the copy number of cbbM gene. Acidihalobacter is aerobic and strictly chemoautotrophic, grows on ferrous iron, elemental sulfur, on sulfidic ores like sphalerite, pyrite, chalcopyrite, galena, arseno-pyrite and H2S (Pablo et al., 2015). Acidihalobacter can grow in acidic conditions of high salt and high osmotic pressure and can utilize Fe(II) as a nutrient (Nicolle et al., 2009). Acidihalobacter abundance increased with the addition of Fe(II) in both

Fe (iron) is the main redox-active element on the soil, so the microbial Fe redox cycle plays an important role in environmental biogeochemistry (Weber et al., 2006a). The iron reduction is a vital geobiochemical route in paddy soils, and limited data is available about the association between iron reduction and microbial community composition. Microbes are exposed to a number of metallic ions in their surroundings and act together with them but it depends on the physiochemical state and nature of metal ions either they are favorable or detrimental (Shrivastava et al., 2004). One of the essential nutrients for living agents is iron attributable to its noticeable activity in electron transport reactions in biological systems. However, its insolubility and reactivity lead to problems of poor availability and toxicity, respectively (Andrews, 1998). In wetlands and oceans the autotrophic bacteria can assimilate carbon dioxide through Calvin– Benson–Bassham (CBB) cycle shown that significantly improve the net uptake of atmospheric CO2 (Cannon et al., 2001; Stanley et al., 2003). A study showed that soils reveal a similar potential and in rice paddy soils carbon fixation rate may increase up to 0.36% of the total Carbon and 0.19% in upland soils over in eighty days of incubation (Yuan et al., 2012). RubisCO is the most important carboxylting enzyme that fixes CO2 for obligate and facultative chemotrophic or photoautotrophic microorganisms highly abundant in cultivated soil, forestry land, and volcanic soils (Nanba et al., 2004b; Selesi et al., 2007; Tolli and King, 2005). Chemoautotrophs microorganisms can use inorganic molecules as electron donors to alter Carbon dioxide into carbon-based compounds in soils (Li et al., 2016). Under low oxygen conditions chemotrophic bacteria that can use Fe(II) as an energy source and converting CO2 into biomass are known as Fe(II)-oxidizing bacteria (FeOB), able to couple Fe and C cycles (Field et al., 2015; Kato et al., 2015). Paddy soil denotes as the main atmosphere for Fe and C cycling. The elemental abundance of iron, seasonal redox reactions, micro-aerobic oxidation of ferrous iron around rice rhizosphere and soil-water interface are considered as a dominant geobiochemical process in water-logged paddy soils (Emerson et al., 2010). In anoxic and neutral pH, the mixotrophic and 6

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textures of soil. All members of the genus Halothiobacillus, Thiohalomonas and Thiohalorhabdus are halophilic or halotolerant obligate chemoautotrophs, assimilating carbon dioxide via the CBB cycle at the expense of the oxidation of reduced Sulfur. Halothiobacillus species are capable of oxidizing sulfide, elemental sulfur, thiosulfate, and tetrathionate to sulfur, sulfite, polythionates, and sulfate (Kelly and Wood, 2000; Shi et al., 2011; Sievert et al., 2000). Previous studies showed that members of Ectothiorhodospiraceae (Thiohalomonas and Thiohalorhabdus) have different types of metabolisms, e.g. photolithotrophic, photoheterotrophic, chemoheterotrophic, methylotrophic and chemolithotrophic using nitrite, sulfur compounds, arsenite and iron as inorganic electron donors (Hallberg et al., 2011; Hoeft et al., 2007; Imhoff, 2006; Sorokin et al., 2007). A study data showed that dominance phylotypes of Gammaproteobacteria (Thiohalomonas, Allochromatium, Chromatium, Thiomicrospira) associated with carbon and sulfur cycling (Yousuf et al., 2014). The other study showed that the cbbL and cbbM genes present in members of the genus Thiohalorhabdus in sediment from a hypersaline lake in Kulunda Steppe (Russia) (Tourova et al., 2010). Acidithiobacillus is an autotrophic aerobic Fe oxidizing bacteria that requires ferrous iron as only energy and electrons source for carbon fixation. Acidithiobacillus is an important environmental microbe that generates energy by oxidation of ferrous iron along with the reduction of oxygen (White et al., 2016). Carbon fixing genes cbbL and cbbM phenotypes categorized into genera Sulfuricella, Sideroxydans, and Acidithiobacillus (Herrmann et al., 2015), which points the strong link between autotrophy and the oxidation of reduced sulfur compounds, as previously reported for groundwater environments (Alfreider et al., 2009). However, given the low concentrations of Fe(II) in the groundwater along with the prevailing oxic conditions the availability of Fe(II) is likely too low to support a considerable contribution of iron oxidation to autotrophy. The dominance of this genus species S. denitrificans may be explained by its versatile lithotrophic metabolism, as it is able to oxidize elemental sulfur and thiosulfate to sulfate aerobically or coupled to denitrification under anoxic conditions (Kojima and Fukui, 2010). Sulfur-oxidizing bacteria Sulfuritalea can oxidize elemental sulfur, hydrogen, and thiosulfate as the only source of energy for growth (Okamura et al., 2009). Sulfuritalea exploits substitute electron acceptors alike manganese or iron have not been recognized yet (Schippers and Jørgensen, 2001). Thiobacillus thiooxidans (sulfur-oxidizing bacterium) was initially isolated from the soil in 1921 (Waksman and Joffe, 1921). Hinkle and Colmer testified the primary isolation of Thiobacillus ferrooxidans responsible for the oxidation of inorganic Sulfur compounds and iron from the acid mine drainage of bituminous coal mines (Colmer and Hinkle, 1947). Magnetospirillum grows chemo-organo-hetero-trophically require organic acids as an electron acceptor and carbon sources (Bazylinski and Williams, 2007), some species of genera are chemolithoautotrophically grow using reduced sulfur compounds as an electron source(Geelhoed et al., 2009, 2010). A study conducted by (Chen et al., 2017) indicating that the genera Bdellovibrio, Magnetospirillum, Azospirillum and Dechloromonas (belongs to Proteobacteria), had noticeably higher abundances in the bacterial communities populations play a significant role in microaerobic Fe(II) oxidation in connection with CO2 assimilation in paddy soil. The presence of this gene in Magnetospirilla may indicate the ability for autotrophic growth; however, autotrophic growth of Magnetospirillum species has not been demonstrated (Bazylinski et al., 2004; Schüler et al., 1999). Rhizobia bacteria Bradyrhizobium either free-living soil microorganism or an endosymbiont within the legume root nodule cells. Iron plays an important role in rhizobia-legume symbioses for the nitrogenase enzymes (Brear et al., 2013). Some species of Bradyrhizobium are known to assimilate CO2 via the CBB cycle such as Bradyrhizobium japonicum, Bradyrhizobium strain BTAi1. They required ferric iron for aerobic growth and symbioses for nitrogenase enzymes, our findings suggested that Fe (II) had an adverse effect that might be due to non-utilization of Fe(II) by Bradyrhizobium. Acidophilic mixotrophic Fe-oxidizing bacteria

Leptothrix in acidic environments had been previously studied well, where biotic Fe oxidation plays a significant role in Fe cycling (Arkesteyn, 1980; Leduc and Ferroni, 1994). Leptothrix abundance in and the requirement for Fe rich waters indicate that it requires high concentrations of Fe(II) for growth (Canfield and 404 Teske, 1996; Kappler et al., 2004; Tender et al., 2002) The phototrophic carbon-fixing microbial abundances account for 31.2% and 6.2% in loamy and sandy soil under control treatments, respectively. Phototrophic cbbM encoding bacterial abundance significantly increased with low addition of Fe(II) by 37.8% while high Fe (II) addition decreased by 16.2% in loamy soil while in sandy soil carbon fixing phototrophic bacterial abundance increased by 21.0%–14.3% with low and high Fe(II) concentrations. Rhodovulum abundance 2.0% increased with low Fe(II) addition in this experiment photoheterotrophic bacterial genus Rhodopseudomonas 4.4% increase in loamy soils. The Rhodobacter abundance increased by 3.1% with a high concentration of Fe(II) in sandy soil. A study showed that the Fe(II) oxidation rate of marine phototrophic iron oxidizing Rhodovulum species are dependent not only on light intensity but also on initial Fe(II) concentration (Swanner et al., 2014), we also found that 2.0% abundance of cbbM containing Rhodovulum increased with the low concentration of Fe(II). A study conducted by (Liu et al., 2016) indicated that members of the Proteobacteria (Thiobacillus, Allochromatium, Rhodovulum, Caenispirillum, Acidithiobacillus, Rhodospirillum) were the predominant ones with the ability to fix CO2. The purple non-sulfur bacteria that grow photoheterotrophically in the anoxic environment includes Rhodobacter, Rhodospirillum, and Rhodopseudomonas contains RubisCO form II carbon-fixing enzyme (Kusian and Bowien, 1997; Long et al., 2015; Tabita et al., 2007). Genetic studies of the phototrophic showed Fe-oxidizing Rhodobacter sp. strain SW2 (Ehrenreich and Widdel, 1994) and Rhodopseudomonas palustris strain TIE-1 (Jiao et al., 2005) identified genes (fox and pio operons, respectively) encoding proteins specific for iron oxidation. Another study showed that under anaerobic conditions Cu(II) and ferrous iron act together to delay the growth of various bacteria including Rhodopseudomonas (Bird et al., 2013). The diversity analysis results (Table 2) clearly indicated the putative chemoautotrophs possessing cbbM genes in the loamy soils have higher abundance than those in the sandy soils. The species richness and the abundance of each species can influence ecosystem functioning (Cornwell et al., 2008; Niklaus et al., 2006). Soil microbial functional diversity is linked with the stability of soil microbial communities and levels of soil biodiversity (Theuerl et al., 2010). cbbM gene copy numbers differ significantly between the different treatments, indicating a significant difference in carbon fixing microbial community in samples. The microbial community in loamy soil had a higher abundance than sandy soil and strongest carbon sequestration ability. A study showed that during iron bio-mineralization, some toxic metals such as arsenic can be efficiently precipitated, however, the addition of Fe(II) might have an adverse effect on microbial community (Hohmann et al., 2009). Overall, our results demonstrated that with respect to the degree of the response to environmental parameters and Fe(II) concentrations the bacterial community structure and function were not consistent. Generally, more available nutrients in small soil fractions favor the accumulation of microbial biomass as well as the development of gene diversity (Balesdent et al., 2000; Kandeler et al., 1999). Fine-textured loamy paddy soils tend to have more microbial biomass than coarsetextured sandy paddy soils. Many studies showed that soil physicochemical and biological properties play a significant role in determining the structure of microbial communities, such as the amount of organic matter (Crecchio et al., 2007; Nüsslein and Tiedje, 1999; Zhou et al., 2002), redox potential (Braker et al., 2001) and pH (Fierer and Jackson, 2006). Iron (Fe) is the most abundant redox-active element on the Earth, thus microbial iron redox cycling has a fundamental role in environmental biogeochemistry (Weber et al., 2006a). physicochemical conditions 7

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could stimulate the abundance of different microbial communities in the soil environment. The important findings of the observed trend indicate that soil organic matter (SOM), redox potential (Braker et al., 2001) and pH (Fierer and Jackson, 2006) promotes biological activity. The iron redox cycling in paddy soils pH influenced by periodic changes of pH and Eh (Li et al., 2006) and pH seems to be one of the utmost significant environmental factors. It is commonly recognized that pH has an important influence on the general composition and diversity of bacterial populations in a range of aquatic and terrestrial environments (Kuang et al., 2013; Lauber et al., 2009; Nicol et al., 2008; Wang et al., 2012). The pH values in loamy soils L1 and L2 decreased by 0.86 with low and 2.05 with high Fe(II) additions in sandy soil S1 and S2 pH values decreased by 1.17 with low and 2.22 with high Fe(II) addition. Soil pH mainly depends on water content and the concentration of sodium chloride. It is also reported in the previous study that water content increased pH value of soil while sodium chloride decreased its value (Li, 1992a). Reduction of iron oxides required hydrogen ions thus causing pH deviations. Biodegradation of organic matters in the soil environment can influence the pH values either consumption or production of hydrogen ions (Li, 1992a). The seasonal variation of redox potential (Eh) is one of the most exceptional physical characteristics of paddy soil. These variations of paddy soil are a direct reflection of dramatic changes in a number of redox-sensitive compounds such as oxygen, iron, manganese, N, sulfur (S), and carbon. The Eh5 of each treated soil significantly decreased along with added Fe(II) concentration which is remarkable that iron oxidation/reduction reactions play an important role, particularly in paddy soil especially those containing high Fe(II) concentration and long flooding periods (Li, 1992b). In the current study, we found that there is no significant difference in the organic matter among Fe(II) treated soils. Carbon dioxide is the main causative agent for global warming and the concentration of CO2 increased in the atmosphere (Singh et al., 2010). Paddy soil in the monsoonal area is the main source of global greenhouse gases such as methane and carbon dioxide(Lee et al., 2010; Solomon et al., 2007). Carbon emissions from paddy soil are probable to be a long-term contributor to greenhouse gases, conceivably even more over the past 5000 years (Ruddiman and Thomson, 2001). Calvin cycle is the first carbon sequestration pathway, and also the main carbon sequestration pathway for autotrophic nitrogen-fixing microorganisms. Recent studies proved that soil CO2 concentration and microbial biomass can be altered with the adaptation or change of microbial communities (Carney et al., 2007). Autotrophic prokaryotes assimilate carbon dioxide as the principal source of cellular carbon while chemotrophic microbes assimilate CO2 in a dark environment (Neuhard, 1996; van der Meer et al., 2001). As far as our results concerned, Fe(II) addition have influenced the carbon dioxide sequestration rate of different paddy soil textures by the photoautotrophic and chemoautotrophic bacterial community. Our findings suggested that Fe (II) addition influence carbon-fixing bacterial community and the cbbM gene copy numbers in both paddy soils, which imply that it can be helpful to improve soil microbial carbon sequestration capacity by generating a controlled ferric iron environment in the paddy soils.

community of photoautotrophic and chemoautotrophic can be altered with the treatment of Fe(II) that affects the diversity of autotrophic carbon-fixing cbbM containing the bacterial community in paddy soil, it can be beneficial to take measures to ease the greenhouse effect and improve soil utilization. Acknowledgment This work was financially supported by National Natural Science Foundation of China (41371261). References Alfreider, A., et al., 2009. Distribution and diversity of autotrophic bacteria in groundwater systems based on the analysis of RubisCO genotypes. Syst. Appl. Microbiol. 32, 140–150. Andrews, S.C., 1998. Iron storage in bacteria. Adv. Microb. Physiol. 40, 281–351. Arkesteyn, G.J.M.W., 1980. Pyrite oxidation in acid sulphate soils: the role of miroorganisms. Plant Soil 54, 119–134. Balesdent, J., et al., 2000. Relationship of soil organic matter dynamics to physical protection and tillage. Soil Tillage Res. 53, 215–230. Bazylinski, D., Williams, T., 2007. Ecophysiology of magnetotactic bacteria. In: Schüler, D. (Ed.), Magnetoreception and Magnetosomes in Bacteria. vol. 3 Springer, Berlin, Heidelberg. Bazylinski, D.A., et al., 2004. Chemolithoautotrophy in the marine, magnetotactic bacterial strains MV-1 and MV-2. Arch. Microbiol. 182, 373–387. Benson, A., et al., 1950. The path of carbon in photosynthesis. V. Paper chromatography and radioautography of the products1. J. Am. Chem. Soc. 72, 1710–1718. Bird, L.J., et al., 2013. Iron and copper act synergistically to delay anaerobic growth of bacteria. Appl. Environ. Microbiol. 79, 3619–3627. Boyd, P.W., et al., 2007. Mesoscale iron enrichment experiments 1993-2005: synthesis and future directions. Science 315, 612–617. Braker, G., et al., 2001. Community structure of denitrifiers, Bacteria, and Archaea along redox gradients in Pacific Northwest marine sediments by terminal restriction fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes. Appl. Environ. Microbiol. 67, 1893–1901. Brear, E.M., et al., 2013. Iron: an essential micronutrient for the legume-rhizobium symbiosis. Front. Plant Sci. 4, 359. Buchanan, B.B., Arnon, D.I., 1990. A reverse KREBS cycle in photosynthesis: consensus at last. Photosynth. Res. 24, 47–53. Canfield, D.E., 1989. Reactive iron in marine sediments. Geochem. Cosmochim. Acta 53, 619–632. Canfield, D.E., Teske, A., 1996. Late Proterozoic rise in atmospheric oxygen concentration inferred from phylogenetic and sulphur-isotope studies. Nature 382, 127. Cannon, G.C., et al., 2001. Microcompartments in prokaryotes: carboxysomes and related polyhedra. Appl. Environ. Microbiol. 67, 5351–5361. Caporaso, J.G., et al., 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335. Carney, K.M., et al., 2007. Altered soil microbial community at elevated CO2 leads to loss of soil carbon. Proc. Natl. Acad. Sci. U.S.A. 104, 4990–4995. Chen, Y., et al., 2017. Microaerobic iron oxidation and carbon assimilation and associated microbial community in paddy soil. Geochim. Acta. 36, 502–505. Colmer, A.R., Hinkle, M., 1947. The role of microorganisms in acid mine drainage: a preliminary report. Science 106, 253–256. Colwell, R.K., Coddington, J.A., 1994. Estimating terrestrial biodiversity through extrapolation. Philos. Trans. R. Soc. Lond. B Biol. Sci. 345, 101–118. Cornwell, W.K., et al., 2008. Plant species traits are the predominant control on litter decomposition rates within biomes worldwide. Ecol. Lett. 11, 1065–1071. Crecchio, C., et al., 2007. Soil microbial dynamics and genetic diversity in soil under monoculture wheat grown in different long-term management systems. Soil Biol. Biochem. 39, 1391–1400. Ehrenreich, A., Widdel, F., 1994. Anaerobic oxidation of ferrous iron by purple bacteria, a new type of phototrophic metabolism. Appl. Environ. Microbiol. 60, 4517. Elsaied, H., Naganuma, T., 2001. Phylogenetic diversity of ribulose-1, 5-bisphosphate carboxylase/oxygenase large-subunit genes from deep-sea microorganisms. Appl. Environ. Microbiol. 67, 1751–1765. Emerson, D., et al., 2010. Iron-oxidizing bacteria: an environmental and genomic perspective. Annu. Rev. Microbiol. 64, 561. Emerson, D., Moyer, C., 1997. Isolation and characterization of novel iron-oxidizing bacteria that grow at circumneutral pH. Appl. Environ. Microbiol. 63, 4784–4792. Feisthauer, S., et al., 2008. Differences of heterotrophic 13CO2 assimilation by Pseudomonas knackmussii strain B13 and Rhodococcus opacus 1CP and potential impact on biomarker stable isotope probing. Environ. Microbiol. 10, 1641–1651. Field, E.K., et al., 2015. Genomic insights into the uncultivated marine zetaproteobacteria at loihi seamount. ISME J. 9, 857. Fierer, N., Jackson, R.B., 2006. The diversity and biogeography of soil bacterial communities. Proc. Natl. Acad. Sci. U.S.A. 103, 626–631. Geelhoed, J.S., et al., 2010. Reduced inorganic sulfur oxidation supports autotrophic and mixotrophic growth of Magnetospirillum strain J10 and Magnetospirillum gryphiswaldense. Environ. Microbiol. 12, 1031–1040. Geelhoed, J.S., et al., 2009. Microbial sulfide oxidation in the oxic–anoxic transition zone of freshwater sediment: involvement of lithoautotrophic Magnetospirillum strain J10.

5. Conclusion The present study demonstrates that the environmental factors have a significant effect on cbbM containing a microbial community in paddy soil. Different textures of paddy soil have different content of the microbial community. Among the two soil textures, chemoautotrophic bacterial abundance increased with high Fe(II) addition in loamy soil while in sandy soil low and high Fe(II) addition significantly increased. Chemoheterotrophic genera in sandy soil have a significantly negative correlation with Fe(II) addition. Photoautotrophic bacteria with low Fe (II) concentration in loamy soil increased and the proportion with high Fe(II) concentration decreased. Photoautotrophic bacterial abundance in sandy soil increased when treated with Fe(II) concentrations. The 8

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S. Hussain, et al. FEMS Microbiol. Ecol. 70, 54–65. Hallberg, K.B., et al., 2011. Acidiferrobacter thiooxydans, gen. nov. sp. nov.; an acidophilic, thermo-tolerant, facultatively anaerobic iron- and sulfur-oxidizer of the family Ectothiorhodospiraceae. Extremophiles 15, 271. Herrmann, M., et al., 2015. Large fractions of CO2-fixing microorganisms in pristine limestone aquifers appear to be involved in the oxidation of reduced sulfur and nitrogen compounds. Appl. Environ. Microbiol. 81, 2384–2394. Herter, S., et al., 2002. L-Malyl-coenzyme A lyase/β-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus, a bifunctional enzyme involved in autotrophic CO2 fixation. J. Bacteriol. 184, 5999–6006. Hoeft, S.E., et al., 2007. Alkalilimnicola ehrlichii sp. nov., a novel, arsenite-oxidizing haloalkaliphilic gammaproteobacterium capable of chemoautotrophic or heterotrophic growth with nitrate or oxygen as the electron acceptor. Int. J. Syst. Evol. Microbiol. 57, 504–512. Hohmann, C., et al., 2009. Anaerobic Fe (II)-oxidizing bacteria show as resistance and immobilize as during Fe (III) mineral precipitation. Environ. Sci. Technol. 44, 94–101. Hong-zhao, C., 1981. Geographical distribution of paddy soils in China. In: Proceedings of Symposium on Paddy Soils. Springer, pp. 734–740. Imhoff, J.F., 2006. The Family Ectothiorhodospiraceae. The Prokaryotes: Volume 6: Proteobacteria: Gamma Subclass, pp. 874–886. Jensen, M.M., et al., 2003. Rates and regulation of microbial iron reduction in sediments of the Baltic-North Sea transition. Biogeochemistry 65, 295–317. Jiao, Y., et al., 2005. Isolation and characterization of a genetically tractable photoautotrophic Fe(II)-Oxidizing bacterium, Rhodopseudomonas palustris strain TIE-1. Appl. Environ. Microbiol. 71, 4487–4496. Kandeler, E., et al., 1999. Response of soil microbial biomass, urease and xylanase within particle size fractions to long-term soil management. Soil Biol. Biochem. 31, 261–273. Kappler, Andreas Newman, Dianne K, 2004. Formation of Fe (III)-minerals by Fe (II)oxidizing photoautotrophic bacteria. Geochimica et Cosmochimica Acta 68 (6), 1217–1226. Kashefi, K., Lovley, D.R., 2003. Extending the upper temperature limit for life. Science 301 934-934. Kato, S., et al., 2015. Comparative genomic insights into ecophysiology of neutrophilic, microaerophilic iron oxidizing bacteria. Front. Microbiol. 6, 58–61. Kelly, D.P., Wood, A.P., 2000. Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov. Int. J. Syst. Evol. Microbiol. 50, 511–516. Kojima, H., Fukui, M., 2010. Sulfuricella denitrificans gen. nov., sp. nov., a sulfur-oxidizing autotroph isolated from a freshwater lake. Int. J. Syst. Evol. Microbiol. 60, 2862–2866. Kuang, J.-L., et al., 2013. Contemporary environmental variation determines microbial diversity patterns in acid mine drainage. ISME J. 7, 1038. Kuczynski, J., et al., 2012. Using QIIME to analyze 16S rRNA gene sequences from microbial communities. Curr protoc microbiol 27 1E. 5.1-1E. 5.20. Kusian, B., Bowien, B., 1997. Organization and regulation of cbb CO2 assimilation genes in autotrophic bacteria. FEMS Microbiol. Rev. 21, 135–155. Lauber, C.L., et al., 2009. Soil pH as a predictor of soil bacterial community structure at the continental scale: a pyrosequencing-based assessment. Appl. Environ. Microbiol. Leduc, L.G., Ferroni, G.D., 1994. The chemolithotrophic bacterium Thiobacillus ferrooxidans. FEMS Microbiol. Rev. 14, 103–119. Lee, C.H., et al., 2010. Effect of Chinese milk vetch (Astragalus sinicus L.) as a green manure on rice productivity and methane emission in paddy soil. Agric. Ecosyst. Environ. 138, 343–347. Li, C., et al., 2006. Assessing alternatives for mitigating net greenhouse gas emissions and increasing yields from rice production in China over the next twenty years. J. Environ. Qual. 35, 1554. Li, Q., 1992a. Acidity of Paddy Soils: Paddy Soils of China. Science Press, Beijing. Li, Q., 1992b. Soil Environment of Rice Rhizosphere. Paddy Solis of China. Science Press, Beijing, pp. 413–430. Li, X., et al., 2016. Changes in the composition and diversity of microbial communities during anaerobic nitrate reduction and Fe (II) oxidation at circumneutral pH in paddy soil. Soil Biol. Biochem. 94, 70–79. Liang, C., et al., 2011. An Absorbing Markov chain approach to understanding the microbial role in soil carbon stabilization. Biogeochemistry 106, 303–309. Liesack, W., et al., 2000. Microbiology of flooded rice paddies. FEMS Microbiol. Rev. 24, 625–645. Liu, J.F., et al., 2016. Microbial communities responsible for fixation of CO2 revealed by using mcrA, cbbM, cbbL, fthfs, fefe-hydrogenase genes as molecular biomarkers in petroleum reservoirs of different temperatures. Int. Biodeterior. Biodegrad. 114, 164–175. Liu, S.V., et al., 1997. Thermophilic Fe (III)-reducing bacteria from the deep subsurface: the evolutionary implications. Science 277, 1106–1109. Lloyd, J.R., et al., 2000. Direct and Fe (II)-mediated reduction of technetium by Fe (III)reducing bacteria. Appl. Environ. Microbiol. 66, 3743–3749. Long, X.E., et al., 2015. Community structure and soil pH determine chemoautotrophic carbon dioxide fixation in drained paddy soils. Environ. Sci. Technol. 49, 7152–7160. Martin, J.H., et al., 1991. The case for iron. Limnol. Oceanogr. 36, 1793–1802. Miltner, A., et al., 2005. Non-phototrophic CO2 fixation by soil microorganisms. Plant Soil 269, 193–203. Nanba, K., et al., 2004a. Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1, 5-bisphosphate carboxylase/oxygenase. Appl. Environ. Microbiol. 70, 2245–2253. Nanba, K., et al., 2004b. Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate

carboxylase/oxygenase. Appl. Environ. Microbiol. 70, 2245–2253. Neuhard, J., 1996. Biosynthesis and conversion of pyrimidines. Escherichia coli and Salmonella typhimurium. Mol. Cell. Biol. Nicol, G.W., et al., 2008. The influence of soil pH on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria. Environ. Microbiol. 10, 2966–2978. Nicolle, J.L.C., et al., 2009. Ferrous iron oxidation and rusticyanin in halotolerant, acidophilic ‘Thiobacillus prosperus’. Microbiology 155, 1302–1309. Niklaus, P.A., et al., 2006. Effects of plant species diversity and composition on nitrogen cycling and the trace gas balance of soils. Plant Soil 282, 83–98. Nüsslein, K., Tiedje, J.M., 1999. Soil bacterial community shift correlated with change from forest to pasture vegetation in a tropical soil. Appl. Environ. Microbiol. 65, 3622–3626. Okamura, K., et al., 2009. Rhodoplanes serenus sp. nov., a purple non-sulfur bacterium isolated from pond water. Int. J. Syst. Evol. Microbiol. 59, 531–535. Pablo, C.J., et al., 2015. Reclassification of 'Thiobacillus prosperus' Huber and Stetter 1989 as Acidihalobacter prosperus gen. nov., sp. nov., a member of the family Ectothiorhodospiraceae. Int. J. Syst. Evol. Microbiol. 65, 3641. Ragsdale, S.W., 1991. Enzymology of the acetyl-CoA pathway of CO2 fixation. Crit. Rev. Biochem. Mol. Biol. 26, 261–300. Ruddiman, W.F., Thomson, J.S., 2001. The case for human causes of increased atmospheric CH4 over the last 5000 years. Quat. Sci. Rev. 20, 1769–1777. Šantrůčková, H., et al., 2005. Heterotrophic fixation of CO 2 in soil. Microb. Ecol. 49, 218–225. Schippers, A., Jørgensen, B., 2001. Oxidation of pyrite and iron sulfide by manganese dioxide in marine sediments. Geochem. Cosmochim. Acta 65, 915–922. Schloss, P.D., et al., 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541. Schüler, D., et al., 1999. Improved technique for the isolation of magnetotactic spirilla from a freshwater sediment and their phylogenetic characterization. Syst. Appl. Microbiol. 22, 466–471. Selesi, D., et al., 2007. Quantification of bacterial RubisCO genes in soils by cbbL targeted real-time PCR. J. Microbiol. Methods 69, 497–503. Shi, J., et al., 2011. Isolation and characterization of a novel sulfuroxidizing chemolithoautotroph Halothiobacillus from Pb polluted paddy soil. Afr. J. Biotechnol. 10, 4121–4126. Shrivastava, H.Y., et al., 2004. A Schiff base complex of chromium(III): an efficient inhibitor for the pathogenic and invasive potential of Shigella dysenteriae. J. Inorg. Biochem. 98, 387–392. Sievert, S.M., et al., 2000. Halothiobacillus kellyi sp. nov., a mesophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium isolated from a shallow-water hydrothermal vent in the Aegean Sea, and emended description of the genus Halothiobacillus. Int. J. Syst. Evol. Microbiol. 50 Pt 3, 1229. Singh, B.K., et al., 2010. Microorganisms and climate change: terrestrial feedbacks and mitigation options. Nat. Rev. Microbiol. 8, 779. Solomon, S., et al., 2007. Climate Change 2007-the Physical Science Basis: Working Group I Contribution to the Fourth Assessment Report of the IPCC. Cambridge university press. Sorokin, D.Y., et al., 2007. Methylohalomonas lacus gen. nov., sp. nov. and Methylonatrum kenyense gen. nov., sp. nov., methylotrophic gammaproteobacteria from hypersaline lakes. Int. J. Syst. Evol. Microbiol. 57, 27629. Stanley, E.H., et al., 2003. Evaluating the influence of macrophytes on algal and bacterial production in multiple habitats of a freshwater wetland. Limnol. Oceanogr. 48, 1101–1111. Straub, K.L., et al., 1996. Anaerobic, nitrate-dependent microbial oxidation of ferrous iron. Appl. Environ. Microbiol. 62, 1458–1460. Sun, M., et al., 2015. Microbial community analysis in rice paddy soils irrigated by acid mine drainage contaminated water. Appl. Microbiol. Biotechnol. 99, 2911–2922. Swanner, E.D., et al., 2014. Characterization of the physiology and cell–mineral interactions of the marine anoxygenic phototrophic Fe(II) oxidizer Rhodovulum iodosum – implications for Precambrian Fe(II) oxidation. FEMS Microbiol. Ecol. 88, 503–515. Tabita, F.R., et al., 2007. Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs. Microbiol. Mol. Biol. Rev. 71, 576–599. Tan, K.H., 2005. Soil Sampling, Preparation, and Analysis. CRC press. Tender, L.M., et al., 2002. Harnessing microbially generated power on the seafloor. Nature biotechnology 20, 821. Thauer, R.K., 2007. 博文 [science] 碳固定第五种途径——a fifth pathway of carbon fixation. Science 318, 1732–1733. Theuerl, S., et al., 2010. Response of recalcitrant soil substances to reduced N deposition in a spruce forest soil: integrating laccase-encoding genes and lignin decomposition. FEMS Microbiol. Ecol. 73, 166–177. Tolli, J., King, G.M., 2005. Diversity and structure of bacterial chemolithotrophic communities in pine forest and agroecosystem soils. Appl. Environ. Microbiol. 71, 8411–8418. Tourova, T.P., et al., 2010. Ribulose-1,5-bisphosphate carboxylase/oxygenase genes as a functional marker for chemolithoautotrophic halophilic sulfur-oxidizing bacteria in hypersaline habitats. Microbiology 156, 2016–2025. Turner, F., Patrick, W., 1968. Chemical changes in waterlogged soils as a result of oxygen depletion. Int. Soc. Soil Sci. Trans. van der Meer, M.T., et al., 2001. Biosynthetic controls on the 13C contents of organic components in the photoautotrophic bacterium Chloroflexus aurantiacus. J. Biol. Chem. 276, 10971–10976. Waksman, S.A., Joffe, J.S., 1921. Acid production by a new sulfur-oxidizing bacterium. Science 53 216-216. Waldrop, M., Firestone, M., 2006. Seasonal dynamics of microbial community

9

Ecotoxicology and Environmental Safety 182 (2019) 109456

S. Hussain, et al. composition and function in oak canopy and open grassland soils. Microb. Ecol. 52, 470–479. Wang, X., et al., 2012. Pyrosequencing analysis of bacterial diversity in 14 wastewater treatment systems in China. Appl. Environ. Microbiol. AEM 01617-12. Weber, K.A., et al., 2006a. Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction. Nat. Rev. Microbiol. 4, 752. Weber, K.A., et al., 2006b. Anaerobic nitrate-dependent iron (II) bio-oxidation by a novel lithoautotrophic betaproteobacterium, strain 2002. Appl. Environ. Microbiol. 72, 686–694. White, G.F., et al., 2016. Chapter three - mechanisms of bacterial extracellular electron exchange. In: Poole, R.K. (Ed.), Adv. Microb. Physiol #. . Academic Press, pp. 87–138. Widdel, F., et al., 1993. Ferrous iron oxidation by anoxygenic phototrophic bacteria. Nature 362, 834.

Xu, H.H., Tabita, F.R., 1996. Ribulose-1, 5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms. Appl. Environ. Microbiol. 62, 1913–1921. Yousuf, B., et al., 2014. Unravelling the carbon and sulphur metabolism in coastal soil ecosystems using comparative cultivation-independent genome-level characterisation of microbial communities. PLoS One 9, e107025. Yuan, H., et al., 2012. Significant role for microbial autotrophy in the sequestration of soil carbon. Appl. Environ. Microbiol. 78, 2328–2336. Zehnder, A.J.B., 1988. Biology of Anaerobic Microorganisms. John Wiley and Sons Inc. Zhou, J., et al., 1996. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316–322. Zhou, J., et al., 2002. Spatial and resource factors influencing high microbial diversity in soil. Appl. Environ. Microbiol. 68, 326–334.

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