The differentiation of iron-reducing bacterial community and iron-reduction activity between riverine and marine sediments in the Yellow River estuary
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Rivers are the primary contributors of iron and other elements to the global oceans. Iron-reducing bacteria play an important biogeochemical role in coupling the iron and carbon redox cycles. However, the extent of changes in community structures and iron-reduction activities of iron-reducing bacteria in riverine and coastal marine sediments remains unclear. This study presents information on the spatial patterns and relative abundance of iron-reducing bacteria in sediments of the Yellow River estuary and the adjacent Bohai Sea. High-throughput sequencing of bacterial 16S rRNA found that the highest relative abundances and diversities were from the estuary (Yellow River–Bohai Sea mixing zone). Pseudomonas, Thiobacillus, Geobacter, Rhodoferax, and Clostridium were the most abundant putative iron-reducing bacteria genera in the sediments of the Yellow River. Vibrio, Shewanella, and Thiobacillus were the most abundant in the sediments of the Bohai Sea. The putative iron-reducing bacterial community was positively correlated with the concentrations of total nitrogen and ammonium in coastal marine sediments, and was significantly correlated with the concentration of nitrate in river sediments. The riverine sediments, with a more diverse iron-reducing bacterial community, exhibited increased activity of Fe(III) reduction in enrichment cultures. The estuary-wide high abundance of putative iron-reducing bacteria suggests that the effect of river–sea interaction on bacterial distribution patterns is high. The results of this study will help the understanding of the biogeochemical cycling of iron in riverine and coastal marine environments.
KeywordsIron-reducing bacteria River Coastal sea River–sea interaction
Iron(III)-reducing bacteria (IRB) play a pivotal biogeochemical role in the iron redox cycle, which is coupled with anaerobic organic matter degradation (Lovley 2006) and ammonium oxidation (Li et al. 2015). IRB are widely distributed in many freshwater and marine environments, as well as across a broad range of chemical and physical conditions. However, different communities may play different roles in different habitats. Studies have shown that IRB are usually restricted to specific habitats; for instance, the well-known clade of IRB in the genus Geobacter comprises a larger proportion of iron-reducing populations in river sediments (Ding et al. 2015; Kim et al. 2012; Zheng et al. 2015), whereas Shewanella is mostly found in deep-sea environments (Bowman et al. 1997; Gao et al. 2006; Roh et al. 2006; Stapleton et al. 2005). The Fe(III) reducers, together with the Fe(II) oxidizers, contribute to Fe redox cycling in hypersaline aquatic environments (Halobaculum gomorrense, Desulfosporosinus lacus, etc.) (Emmerich et al. 2012), in a groundwater seep environment (Rhodoferax, Aeromonas, etc.) (Roden et al. 2012) and marine sediments (Shewanella, Deferribacter, Geoglobus, etc.) (Laufer et al. 2015). However, the diversity and community composition of marine and riverine IRB remain uncharacterized, particularly in areas, where rivers and sea interact.
The estuarine–marine environment is an area, where freshwater and marine waters mix (i.e., a brackish water zone). Here, there are steep gradients in iron, nitrogen, sulfur, oxygen, and other elements (MacDonald et al. 2014). The variable river flow may result in these environments having a high variability in the concentrations of electron acceptors and electron donors (Zhang et al. 2017), which are the main controlling factors of IRB community structure. These gradients, which are associated with water movement, may affect the iron-reducing bacterial community structure and their biogeochemical processes.
The Yellow River estuary is one of the most active areas of river–sea interaction in the world (Li et al. 2009). The Yellow River has a very high concentration of suspended particulate matter that contains nitrogen, iron, and other elements. It is also has the second largest sediment load flux to the ocean of any river (Poulton and Raiswell 2002; Pan et al. 2013). Previous studies (Liu et al. 2003; Qiao et al. 2007; Gong et al. 2015; Sheng et al. 2015; Zhang et al. 2017) have shown that the concentrations of ferrous iron (Fe(II)), ferric iron (Fe(III)), total iron (Fe), and ammonium (NH4+–N) were higher in the coastal marine sediment than in the Yellow River sediment, whereas the concentration of nitrate (NO3−–N) was higher in the riverine sediment than in coastal marine sediment. Patterns of IRB abundance and diversity can indicate their contribution to the biogeochemical cycles, while changes in community structures are associated with spatial variations in nutrients [i.e., NO3−–N, NH4+–N, Fe(II), Fe(III)] in different habitats. However, there has been less research (Mcbeth et al. 2013) on how shifts in composition and abundance of IRB with ecosystems, i.e., from rivers through to coastal and oceanic environments, relate to environmental factors.
Changes in iron-reducing bacterial community between riverine and marine sediments
The relative abundance of putative IRB at the genus level is summarized in Fig. 2. These show that significant changes in community composition occurred with changes in the redox conditions in the riverine to marine ecosystem gradient. Twenty genera of IRB were present in the samples from YR1 and YR2, 22 genera were present in the samples from the mixing zone (YR3 and BHB02), and 14 genera were present in the samples from P1 and P2. Pseudomonas (Naganuma et al. 2006) (1.70% of total reads), Thiobacillus (Sand 1989) (0.81%), Geobacter (Coates et al. 1996) (0.19%), Rhodoferax (Kim et al. 2012) (0.11%), and Clostridium (Xu et al. 2014) (0.11%) were the most abundant genera in the sediments from the Yellow River estuary, while Vibrio (Jones et al. 1984) (0.76%), Shewanella (Bowman et al. 1997) (0.27%), Thiobacillus (0.14%), Clostridium (0.06%), and Bacillus (Kanso et al. 2002) (0.05%) were most abundant in the sediments from the Bohai Sea ecosystem.
Effects of sedimentary variables on the community structure of IRB
Identification of IRB in iron(III)-reducing enrichment cultures
Iron(III) reduction in iron(III)-reducing enrichment cultures
The decrease of acetate was more rapid in the enrichment samples from the Yellow River than in the samples from the Bohai Sea. The rate of decrease in acetate corresponded to the rate of increase in ferrous iron during the enrichment period (Fig. 6b).
The sequence data related to putative IRB at the genus level was estimated in accordance with published reviews, acknowledging that this estimation method might have led to an overestimation of OTUs for IRB because of the limitation of sequence length from the MiSeq platform and the lack of universal functional gene markers for IRB. Furthermore, limited knowledge of IRB communities may cause an underestimation of the diversity of IRB. The results presented here show that the relative abundance and diversity of IRB in sediments vary with the aquatic ecosystem. More OTUs of IRB were generally found in riverine sediments than in marine sediments. Greater abundances were also observed in the samples obtained from YR3 and BHB02.
Illumina high-throughput sequencing verified the occurrence of substantial changes in the community structure of putative IRB in sediments in the transect from the Yellow River to the sea. The average relative abundance and diversity in the river system were much greater than those in the marine system; this is in agreement with results found in the sediments from other river and coastal marine zones of Bohai Sea (data not shown). This could potentially be attributed to the high concentration of sulfate in marine sediments. Iron oxides are the most important anaerobic electron acceptors in freshwater sediments (Roden and Wetzel 1996), while sulfate reduction is the most important anaerobic pathway for organic matter degradation in marine sediments (Henrichs and Reeburgh 1987).
At the genus level, the dominant communities of putative iron-reducing bacteria in the riverine sediments were distinct from those in the marine sediments. The high relative abundances of Pseudomonas, Thiobacillus, Geobacter, Rhodoferax, and Clostridium are thus proposed as the most important mediators of iron reduction in the riverine sediments of the Yellow River estuary; this is consistent with results obtained in the previous studies of freshwater systems (Haaijer et al. 2012; Kim et al. 2012; Peng et al. 2016; Weber et al. 2006b).
The dominant IRB genera in marine sediments were Vibrio, Shewanella, and Thiobacillus. As previously discussed, these genera are commonly detected in the marine system (Emerson et al. 2010; Esther et al. 2015), suggesting their participation in the biogeochemical process of iron reduction in coastal marine environments. The abundance of Shewanella in marine sediments here is consistent with the data from the Black Sea (Nealson et al. 1991; Perry et al. 1993).
Among of the most significant changes found was the increase in the relative abundance of putative iron-reducing bacteria in the samples from YR3 and BHB02, which are located near to the estuary and markedly affected by the river–sea interaction. This is consistent with estuarine sediments providing an intermediate salinity zone, where freshwater and seawater iron-reducing bacteria mingled (Mcbeth et al. 2013), and suggests that IRB communities can be affected by river–sea interactions.
In the present study, the RDA analysis showed that concentrations of TN and NH4+–N significantly and positively influenced IRB communities in coastal marine sediments. By comparison, the IRB community of riverine sediments was closely related to the concentration of NO3−–N. This implies that sediment NH4+–N and NO3−–N contents are important parameters in determining the IRB in riverine and coastal marine sediments. This relationship may be attributable to the coupled redox cycling of iron and nitrogen. For example, some members of the dominant genera Pseudomonas and Thiobacillus (e.g., Pseudomonas stutzeri, Thiobacillus ferrooxidans) are also known to be capable of nitrate-dependent Fe(II) oxidation (Coby et al. 2011; Straub et al. 1996; Sugio et al. 1994). Some Geobacter species are also capable of nitrate-reducing Fe(II) oxidation with the reduction of NO3− to NH4+ (Coby et al. 2011; Weber et al. 2006b). Shewanella can also reduce nitrate (Gao et al. 2009). In addition, ammonium is a major end product of anaerobic nitrate-dependent Fe(II) oxidation and might participate in Fe(III) reduction as an electron donor.
To increase the in situ availability of IRB and its differentiation between the two ecosystems in culture, it is necessary to add AmoFe in relatively large amounts to the anaerobic iron(III)-reducing bacteria, although this experimental result may lead to a discrepancy arising from the field conditions. A total of 16 genera of well-known iron-reducing bacteria were identified in the iron(III)-reducing enrichment cultures; these corresponded with the putative iron-reducing bacterial communities found in the in situ samples. Notably, Deferrisoma spp. that were not detected in the in situ samples exhibited the highest relative abundance in iron(III)-reducing enrichment cultures of sediments from P1 and P2. Thus, some iron-reducing populations with low abundance that could not be detected in the field samples could be stimulated by the presence of AmoFe in enrichment cultures. However, the Fe(II) production of riverine sediments was higher than that of marine sediments (but not BHB02 from the mixing zone). Thus, the samples from river and BHB02, which have a greater IRB diversity, exhibited increased activity of Fe(III) reduction in enrichment cultures. Deferrisoma spp., which have been previously observed in shallow-water or deep-sea hydrothermal vents (Pérez-Rodríguez et al. 2016; Slobodkina et al. 2012), had the highest relative IRB abundance in riverine sediments’ enrichment cultures (YR1 and YR2). Ethanol is an important intermediate metabolite of acetate and can also be used as an electron donor for IRB. Thus, Fe(II) is still produced even when acetate is exhausted in the enrichment cultures (Fig. 6b).
16S rRNA gene analysis showed the differences in iron-reducing bacterial community compositions and indicated that the diversity and distribution of IRB in sediments, from typical coastal areas with river–sea interaction, were mainly determined by hydrology and habitat type. Shifts in the diversity of iron-reducing bacteria indicated a novel distribution pattern in the Yellow River estuary and the adjacent Bohai Sea. High relative abundances and diversities of iron-reducing bacteria were observed at the coastal transitional sites. The results of the enrichment culture studies indicate that IRB in marine sediment can be stimulated by Fe(III) reduction activity in the presence of AmoFe. In addition, TN, NH4+–N, and NO3−–N concentrations contributed to variations in iron-reducing bacterial communities, demonstrating the relationship between iron redox biogeochemical cycling and nitrogen cycling. The results presented here show that there is a variation in the structure of iron-reducing bacterial communities and this indicates the presence of a biogeographic pattern in coastal ecosystems.
Materials and methods
Description of study sites and sediment samples
The Yellow River sediment samples were collected in August 2014 from the Yellow River to the Bohai Sea (37°45′N–38°5′N, 118°48′E–119°30′E) (Fig. 1), which is located in the most active river–sea interaction coastal zone. Six sites were selected based on the distance from the river mouth, including 3 sites (YR1, YR2, and YR3) in the Yellow River (YR) and 3 other sites (BHB02, P1, and P2) in the coastal marine system (Bohai Sea, BH). YR3 is near the river mouth, and BHB02 is a transitional site. Each site contained 3 duplicate plots, and 18 (6 sites × 3 replicates) sediment samples were collected.
The riverine and marine sediment and pore water samples were collected using a grab sampler, and the surface-layer subsamples (top: 0–5 cm) were sub-cored with a custom-made corer (inner diameter: 1.5 cm) and then homogenized. Core material was transferred into cryovials and then stored immediately in liquid nitrogen for DNA extraction. Sediment subsamples were placed in sealed containers and archived at 4 °C for physicochemical analysis within 7 days.
Salinity and pH in the overlying water at each site were measured with an electronic probe (Hydrolab MS5, HACH, USA). Nitrate (NO3−–N), ammonium (NH4+–N), total organic carbon (TOC) and total nitrogen (TN) in sediment samples, and the concentration of sulfate (SO42−) in the sediment pore waters were determined, as described in Zhang et al. (2017). Dissolved ferrous iron (Fe(II)) was measured using a ferrozine-based assay (Lovley and Phillips 1987; Stookey 1970). Total reactive hydroxylamine-reducible iron (Fe(III)) was extracted from the sediments and then measured in accordance with our previous report (Zheng et al. 2015). Low-frequency magnetic susceptibility (χLF) was measured with a Bartington MS2B sensor. The concentration of acetate was measured by high-performance liquid chromatography, using a 1260 Infinity HPLC (Agilent Technologies, USA) with a Hi-plex H column equipped with a refractive index detector, using 5 mmol L−1 H2SO4 as the eluent.
Iron(III)-reducing enrichment culture
Iron-reducing bacteria in riverine sediments and marine sediments were enriched in both the freshwater and seawater enrichment medium. The basic components of the freshwater and seawater enrichments were prepared according to Lovley and Phillips (1986). Amorphous Fe(III) oxides at a final concentration of 66 mmol L−1 and acetate at a final concentration of 33 mmol L−1 were added into the media, with the former as the electron acceptor and the latter as the electron donor (Lovley and Phillips 1986; Zheng et al. 2015). Amorphous Fe(III) oxides were formed by neutralizing a 0.4 mol L−1 solution of FeCl3 to a pH of 7 with NaOH (Lovley and Phillips 1986).
RNA extraction of enrichment cultures and cDNA synthesis was conducted as described in a previous study (Zheng et al. 2015). The V4–V5 hypervariable regions of 16S rRNA were amplified using the universal primer set 519f (5′-CAGCMGCCGCGGTAATWC-3′) and 907r (5′-CCGTCAATTCMTTTRAGTTT-3′) (Feng et al. 2015) for bacteria.
DNA extraction and amplicon library preparation for deep sequencing
Total DNA was extracted from 0.5 g of sediment using a FastDNA SPIN Kit for soil (MP Biomedicals, Santa Ana, CA, USA) in accordance with the manufacturer’s instructions. PCR amplification was conducted using the universal primer set 519f/907r targeting the V4–V5 hypervariable regions of bacterial 16S rRNA. The purified PCR products with different barcodes were normalized in equimolar amounts and then prepared using TruSeq™ DNA Sample Prep LT Kit and sequenced using MiSeq Reagent Kit (500-cycles-PE) following the protocols provided by the manufacturer.
Deep sequencing data processing
Raw deep sequencing data were processed using the Quantitative Insights Into Microbial Ecology [http://www.qiime.org, QIIME version 1.7.0; (Caporaso et al. 2010)] with the default parameters unless otherwise noted. After the low-quality sequences and chimeric sequences were removed, qualified sequences were clustered into operational taxonomic units (OTUs) at the 97% sequence identity level, and the most abundant sequence from each OTU was chosen as a representative sequence for that OTU. Taxonomic classification of each OTU was assigned using the Ribosomal Database Project classifier (Li et al. 2016).
16S rRNA gene-based sequences related to putative IRB at the genus level were selected according to published reviews (Emerson et al. 2010; Esther et al. 2015; Lovley et al. 2004; Lovley 2006; Weber et al. 2006a) owing to the lack of universal functional gene markers for IRB.
To assess the relationship between the iron-reducing bacterial communities and the environmental factors, redundancy analysis (RDA) was conducted using the CANOCO 4.5 software. A Monte Carlo permutation test (999 random unrestricted permutations) was performed to test the statistical significance of the environmental variables. Meta-analysis of data on the relative abundance of iron-cycling bacteria in riverine and marine sediments was conducted in accordance with the previous study (Luo et al. 2006).
Accession number of nucleotide sequences
Raw sequence reads of bacterial 16S RNA and 16S rRNA genes have been submitted to the Sequence Read Archive (SRA) with accession number PRJNA342373.
We would like to thank Prof. Jianhui Tang for sharing marine sediment samples. This research was supported by the National Natural Science Foundation of China (nos. 91751112, 41807325, and 41573071); the senior user project of RV KEXUE (no. KEXUE2018G01) and the Key Research Project of Frontier Science (no. QYZDJ-SSW-DQC015) of Chinese Academy of Sciences; the Natural Science Foundation (no. JQ201608 and ZR2018MD011) and the Young Taishan Scholars Program (no. tsqn20161054) of Shandong Province.
HZ, FL, and SZ contributed to the presented idea and design. HZ implemented the computational and statistic analyses and took the lead in writing the manuscript. XZ assisted with data analysis. FL and SZ supervised the findings of this work. All authors provided critical feedback and helped to conduct the research, analysis, and manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Animal and human rights statement
This article does not contain any studies with human participants or animals performed by any of the authors.
- Bowman JP, Mccammon SA, Nichols DS, Skerratt JH, Rea SM, Nichols PD, Mcmeekin TA (1997) Shewanella gelidimarina sp. nov. and Shewanella frigidimarina sp. nov., novel Antarctic species with the ability to produce eicosapentaenoic acid (20:5 omega 3) and grow anaerobically by dissimilatory Fe(III) reduction. Int J Syst Bacteriol 47:1040–1047CrossRefGoogle Scholar
- Coates JD, Phillips EJ, Lonergan DJ, Jenter H, Lovley DR (1996) Isolation of Geobacter species from diverse sedimentary environments. Appl Environ Microbiol 62:1531–1536Google Scholar
- Kanso S, Greene AC, Patel BK (2002) Bacillus subterraneus sp. nov., an iron- and manganese-reducing bacterium from a deep subsurface Australian thermal aquifer. Int J Syst Evol Microbiol 52:869–874Google Scholar
- Lovley D (2006) Dissimilatory Fe(III)- and Mn(IV)-reducing prokaryotes, Theprokaryotes. Springer, New York, pp 635–658Google Scholar
- Lovley DR, Phillips EJ (1986) Organic matter mineralization with reduction of ferric iron in anaerobic sediments. Appl Environ Microbiol 51:683–689Google Scholar
- Lovley DR, Phillips EJ (1987) Rapid assay for microbially reducible ferric iron in aquatic sediments. Appl Environ Microbiol 53:1536–1540Google Scholar
- MacDonald DJ, Findlay AJ, McAllister SM, Barnett JM, Hredzak-Showalter P, Krepski ST, Cone SG, Scott J, Bennett SK, Chan CS (2014) Using in situ voltammetry as a tool to identify and characterize habitats of iron-oxidizing bacteria: from fresh water wetlands to hydrothermal vent sites. Environ Sci Processes Impacts 16:2117–2126CrossRefGoogle Scholar
- Slobodkina GB, Reysenbach AL, Panteleeva A, Kostrikina N, Wagner I, Bonch-Osmolovskaya E, Slobodkin AI (2012) Deferrisoma camini gen. nov., sp. nov., a moderately thermophilic, dissimilatory iron(III)-reducing bacterium from a deep-sea hydrothermal vent that forms a distinct phylogenetic branch in the Deltaproteobacteria. Int J Syst Evol Microbiol 62:2463–2468CrossRefGoogle Scholar
- Straub KL, Benz M, Schink B, Widdel F (1996) Anaerobic, nitrate-dependent microbial oxidation of ferrous iron. Appl Environ Microbiol 62:1458–1460Google Scholar
- Sugio T, Uemura S, Makino I, Iwahori K, Tano T (1994) Sensitivity of iron-oxidizing bacteria, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans, to bisulfite ion. Appl Environ Microbiol 60:722–725Google Scholar
- Zheng S, Zhang H, Li Y, Zhang H, Wang O, Zhang J, Liu F (2015) Co-occurrence of Methanosarcina mazei and Geobacteraceae in an iron(III)-reducing enrichment culture. Front Microbiol 6:941Google Scholar