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Mercury methylation-related microbes and genes in the sediments of the Pearl River Estuary and the South China Sea
Ecotoxicology and Environmental Safety 185 (2019) 109722
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Mercury methylation-related microbes and genes in the sediments of the Pearl River Estuary and the South China Sea
T
Ke Yuana, Xin Chena, Ping Chenb, Yongshun Huangc, Jie Jiangd, Tiangang Luana,b, Baowei Chena,∗, Xiaowei Wanga,∗∗ a
Southern Marine Science and Engineering Guangdong Laboratory, School of Marine Sciences, Sun Yat-Sen University, Zhuhai, 519082, China State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China c Guangdong Provincial Hospital for Occupational Diseases Prevention and Treatment, Guangzhou, 510300, China d Shenzhen Center for Disease Control and Prevention, Shenzhen, 518055, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hg-methylating communities hgcAB cluster The Pearl River Estuary The South China Sea
Methylmercury (MeHg) is a toxicant that mainly originates from in situ microbial methylation of inorganic mercury (Hg) in the environment and poses a severe health risk to the public. However, the characteristics of the Hg-methylating microbial community and its relationship with MeHg production in various environments remain to be understood. In the present study, Hg-methylating microbial communities and genes (hgcAB cluster) in the sediments of the Pearl River (PR), Pearl River Estuary (PRE) and South China Sea (SCS) were investigated at a large spatial scale using high-throughput sequencing-based approaches. The results showed that sulfur-reducing bacteria (SRB) and iron-reducing bacteria (IRB) were consistently the dominant microbial strains responsible for the methylation of inorganic Hg in all three regions investigated. The abundance and diversity of Hg-methylating communities and genes were both found to be higher in the PR sediments compared to that in the PRE and SCS sediments, and in good agreement with the spatial distribution of MeHg. Furthermore, a significant correlation was observed between the MeHg concentration and the abundance of both hgcA and hgcB genes in the sediments of the PR, PRE and SCS regions. Overall, the present study suggested that there was the presence of a close link between MeHg and Hg-methylating communities or genes in the ambient aquatic environment, which could be used to reflect the potential of in situ MeHg production.
1. Introduction Mercury (Hg) and its derivatives are a group of global and persistent contaminants with high toxicity (Kessler, 2013). Hg can be released into the environment from both natural and anthropogenic sources in the inorganic forms (namely, Hg2+ and Hg0), and may be subsequently transformed in situ into methylated species (Colombo et al., 2013; Hu et al., 2013). Conversion between Hg forms can lead to a great variation in the toxicity, environmental behavior and bioavailability of these compounds. As a potent neurotoxin, methylmercury (MeHg) has attracted the most attention, and it is mainly derived from Hg methylation processes in various environments. MeHg may be accumulated and biomagnified through the food chain of aquatic systems (Clarkson and Magos, 2006; Grandjean, 2007). It has also been reported that the MeHg concentration detected in fish was approximately 106 times higher than that in the water phase, and greater than 85% of Hg found
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in fish is in the form of MeHg (Hight and Cheng, 2006). In aquatic environments, MeHg may be produced via both biotic and abiotic processes (Hsu-Kim et al., 2013; Li and Cai, 2013). Microbes are considered as the primary producers of MeHg in the environment, predominantly in anaerobic sediment, soil, and bottom water (Hintelmann, 2010). Sulfate-reducing bacteria (SRB) (Gilmour et al., 2011; Zeng et al., 2016) and iron-reducing bacteria (IRB) (Fleming et al., 2006; Kerin et al., 2006), as well as methanogens (Hamelin et al., 2011; Yu et al., 2013), have been identified as the main microbial methylators of Hg. 47 confirmed Hg-methylating strains have been documented, including 31 SRBs, 8 IRBs and 3 methanogens (Gilmour et al., 2013). Among these microbial strains, 27 have been demonstrated to carry the hgcAB gene cluster, which is currently considered to be the only gene cluster responsible for Hg methylation in microbes (Parks et al., 2013). In this cluster, hgcA gene encodes a corrinoid-dependent protein that functions as the methyltransferase similar to the
Corresponding author. Corresponding author. E-mail addresses: [email protected] (B. Chen), [email protected] (X. Wang).
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https://doi.org/10.1016/j.ecoenv.2019.109722 Received 15 July 2019; Received in revised form 14 September 2019; Accepted 23 September 2019 Available online 29 September 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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in the dark before subsequent treatment and analysis.
corrinoid iron-sulfur protein in the reductive acetyl-CoA pathway, while hgcB is able to express an associated ferredoxin protein that potentially reduces the corrinoid center of the HgcA protein (Parks et al., 2013). Therefore, the above Hg-methylating genes could be used as good indicators to evaluate in situ Hg methylation potentials of microbial communities in various environmental settings. To date, the abundance of Hg-methylating communities and hgcAB genes has been quantified in numerous environmental samples. In a previous study, analysis of the hgcA gene in soils collected from the northern Everglades demonstrated that this gene could be carried by diverse bacterial phyla (Bae et al., 2014). Furthermore, a significant correlation was observed between hgcA gene abundance and MeHg concentration along an Hg gradient in paddy soils (Liu et al., 2014a, 2018a), and in the soils and sediments of the Three Gorges Reservoir region (Du et al., 2017). It has been reported that the diversity and distribution of hgcA were apparently discerned among wetland soils, even between different portions of a swamp, suggesting that different bacterial strains may be responsible for MeHg production under these conditions (Schaefer et al., 2014). The Hg methylation abilities of phylogenetically-related bacteria are known to vary greatly (Gilmour et al., 2011). Therefore, elaborately profiling the composition and abundance of the Hg-methylating microbial community would be of great importance for understanding the Hg methylation potential in the aquatic environment. In the past decades, due to the rapid economic development and urbanization, a huge body of various contaminants, such as heavy metals, are being released into the Pearl River (PR), the Pearl River Estuary (PRE) and nearby coastal areas (Chen et al., 2013; Wang et al., 2014; Yuan et al., 2015; Zhao et al., 2018). Analysis of the total Hg concentration in the sediment column has demonstrated that the Hg pollution was aggravated in the PRE over the past 150 years (Shi et al., 2007). It has been estimated that more than 17 tons of Hg is annually discharged from the Pearl River Delta (PRD) into the nearby aquatic environments via sewage discharge or atmospheric deposition (Zheng et al., 2011). The total Hg concentration in the fish of the PRD were reported to be in the range of 2.81–208.5 ng g−1, while the MeHg concentration (1.63–287.0 ng g−1) in these fish was associated with approximately 84% of the MeHg intake by the local population in the region (Shao et al., 2013). Therefore, MeHg production in the aquatic environment may be potentially associated with public health. Detailed knowledge regarding the diversity and abundance of Hg-methylating bacteria and genes is crucial to understanding in situ MeHg production in the PR, PRE and the South China Sea (SCS) areas. Therefore, the objective of the present study was to investigate the spatial distribution of Hg-methylating bacteria and genes in the sediments collected from the PR, PRE and SCS using metagenomic approaches. Furthermore, the study aimed to understand the relationship of these bacteria and genes with the in situ production of MeHg.
2.2. Analysis of MeHg The concentration of MeHg in the sediment samples was determined according to the method described by Ma et al. (2014). A portion of sediment collected from each of the sampling sites was freeze-dried at −50 °C prior to being ground. A solution of 0.2 mL Me198Hg, serving as a species-specific internal standard, was added into approximately 0.5 g of sediment in 50-mL centrifuge tubes. Next, 2 mL CuSO4 (1 mol L−1) and 10 mL KBr [1 mol L−1 in 5% (v/v) H2SO4] were added to the centrifuge tubes, which were then sealed and shaken for 6 h, followed by the addition of 10 mL CH2Cl2 and further shaking for 4 h. The solution was centrifuged at 6000 rpm for 15 min, the organic phase was then transferred into a 50-mL centrifugation tube, and 30 mL Milli-Q water were added. Subsequently, the organic solvent was removed under gentle nitrogen flow. The sample in the water matrix was transferred to a 100-mL glass flask along with 300 μL acetic acid-acetate buffer (pH = 4.9), followed by addition of 100 mL NaBEt4 (1% in 2% KOH). The volatile organomercury compounds were purged and trapped on a Tenax adsorber, thermodesorbed onto a GC column, and isothermally separated at 36 °C. Finally, the Hg content was analyzed by ICP-MS, and the operation parameters of this technique are provided in Table S2 of the SM. Equilibration of the spiked Hg198 with the natural Hg202 in the sample alters the isotope ratio that is measured and used for calculation. 2.3. DNA extraction and high-throughput sequencing Total bacterial DNA extraction and high-throughput sequencing were conducted according to the method reported in our previous study (Yuan et al., 2017). In brief, the total bacterial DNA was extracted from each sediment sample using a Fast DNASPIN Kit for soil (MP Bio medicals, Santa Ana, CA, USA), according to the kit instructions. DNA extraction was performed multiple times due to the low concentration of DNA present in marine sediments, and the resulting DNA was also combined to avoid the potential bias caused by a single DNA extraction. Subsequently, DNA sequencing was performed using an Illumina Hiseq 2000 device, supplied by Beijing Genomics Institute (Shenzhen, China). The raw reads (100 bp) in all datasets were trimmed to discard the lowquality reads that contained ambiguous nucleotides or a quality value of < 20 using a customized script prior to conducting further bioinformatics analysis. 2.4. Recognition of bacterial community structure and Hg-methylating bacteria The 16S rRNA gene-like reads were recognized by aligning our sequencing datasets against the SILVA Small Subunit (SILVA_119_SSURef_Nr99_tax_silva) database using BLASTn with a cutoff e-value of 1e-20. The 16S rDNA gene-like reads from the BLASTn results were then assigned to NCBI taxonomies using the MEGAN program (version 4.70.4) with the lowest common ancestor algorithm, where the absolute cutoff was a BLAST bitscore of 50 and the relative cutoff was 10% of the top 50 bits. For the identification of Hg-methylating bacteria, a database containing the 16S rRNA genes of Hg-methylating bacteria reported in previous publications was constructed (Gilmour et al., 2013). Subsequently, each of the sequencing datasets of the sediment samples was blasted against this database, and the reads with an identity that was > 97% and an alignment length of at least 100 bp were considered to represent the 16S rRNA genes of Hg-methylating bacteria. the relative abundances of each phylum and Hgmethylating phylum were obtained from dividing the number of 16S rDNA-like reads and Hg methylation-related 16S rDNA-like reads by that of the total reads in the same dataset.
2. Material and methods 2.1. Study area and sampling In total, 12 sediment samples were collected from 8 sites in the PR, the PRE and SCS between 2011 and 2013, and detailed information regarding the sampling sites is presented in Table S1 of the Supplementary Materials (SM). The sites CHHE, LUBA, SHDE and NANS are located in the Beijiang River, which is a main tributary of the PR. The site CHHE is situated at the origin of Beijiang River, and NANS is located at one of the main outlets of the PR. Three sites (E106, E208 and E406) are located in the SCS. Among these, site E208 is near the coast of Southern China, the sites E406 is located in the northern SCS near the Philippines, while site E106 is situated far from the mainland. Moreover, triplicate sediments were also obtained from site A8 in the PRE, which is a transitional zone from PR to SCS. Following sampling, all sediment samples were wrapped in aluminum foil and stored at −80 °C 2
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2.5. Construction of Hg methylation protein (HgcAB) database and hgcAB annotation A Hg methylation protein database was constructed by manually downloading the amino acid (aa) sequences of Hg-methylating enzymes (HgcAB) from the nr database that is compiled by the NCBI. This database included a total of 23 aa sequences encoded by hgcA and 23 aa sequences encoded by hgcB, and these genes were confirmed to be Hgmethylating genes by previous research (Gilmour et al., 2013). Each of the sequencing datasets was interrogated against our self-constructed database using BLASTx with a cutoff e-value of 1e-5. When the best hit presented a similarity of > 75% and an alignment length of at least 25 aa, the read was annotated as an Hg-methylating gene-like sequence.
3. Results and discussion 3.1. Composition and abundance of the total and Hg-methylating microbial communities in sediment samples
Fig. 2. Relative abundance of Hg-methylating phyla in the PR, PRE and SCS sediments. “ppm” represents one 16S rDNA-like reads in one million sequencing reads.
The 16S rRNA genes were determined by aligning our sequencing datasets against the SILVA Small Subunit database, and the microbial communities were further analyzed using MEGAN. A total of 23 bacteria and 2 archaea phyla were identified in the sediment samples obtained from the PR, PRE and SCS regions. The relative abundance of the top 10 phyla in these sediments is displayed in Fig. 1. The top 10 phyla accounted for approximately 88.4–94.7% of the total 16S rRNA genes identified in the sediments. Distinct differences in the relative composition of the microbial community were observed among the sediments of the PR, PRE and SCS. Proteobacteria were identified as the most abundant phylum in the sediments of the PRE and SCS (48.8–57.5%). By contrast, Firmicutes were the most enriched among all phyla in the PR sediments (13.2–51.7%), whereas they only accounted for 2.0–3.2% of the microbial community in the PRE and SCS sediments. The relative percentages of Proteobacteria in the PR sediments (21.0–40.2%) were substantially lower compared with those present in the PRE and SCS sediments. Fig. 2 shows the occurrence and abundance of Hg-methylating phyla in the PR, PRE and SCS sediments, which included two bacterial phyla (Firmicutes and Proteobacteria) and one archaea phylum (Euryarchaeota) (Gilmour et al., 2013). In all samples from our study region, the Hg-methylating strains assigned to bacteria were more popular compared with those of archaea. In addition, Hg-methylating strains in the PR sediments (49.0–136.7 ppm) were more abundant than those in the PRE and SCS sediments (6.2 ppm and 2.3–7.1 ppm, respectively). The total abundance of Hg-methylating strains at the site of
Shunde (136.7 ppm) in the PR was highest among all sampling sites, whereas that the total abundance at the site E106 in the SCS was the lowest. The Hg-methylating communities in the PRE and SCS sediments were characterized by the predominance of Proteobacteria, e.g., the PRE (91.6%) and the SCS (86.1–94.1%). In contrast to the aforementioned locations, Hg-methylating communities in the PR sediments were relatively equally constituted by Proteobacteria (35.5–64.5%) and Firmicutes (34.9–64.4%). The present study further examined the relationships between the abundance of Hg-methylating communities and that of the total bacteria sorting to the same phylum, and the results are presented in Fig. S1 of the SM. A significant positive correlation was only observed between the abundance of Hg-methylating Firmicutes and the total abundance of this phylum across the PR, PRE and SCS sediments, instead of Proteobacteria and Euryarchaeota. This result implies that Hgmethylating bacterial strains may be not a stable composition in the total bacterial communities in sediments and vary with sampling sites, which could contribute to the difference in MeHg production among different locations. The Hg-methylating bacteria detected in the sediment samples obtained from the PR, PRE and SCS belonged to four microbial classes, including Methanomicrobia (Euryarchaeota), Deltaproteobacteria (Proteobacteria), and Clostridia and Negativicutes (Firmicutes), as shown in Fig. S2 of the SM. The Deltaproteobacteria consistently comprised a dominant Hg-methylating class in all sediment samples, including 91.6% in the PRE, 86.1–94.1% in the SCS, and 35.5–64.4% in the PR. Besides Deltaproteobacteria, the Hg-methylating Clostridia (33.4–61.7%) were also predominant over other classes in the PR sediments; for instance, the relative percentage of this class over the total Hg-methylating classes in the sediment at the site Shunde was up to 61.7%. Furthermore, the relative percentages of Hg-methylating Clostridia in the sediment samples obtained from the PRE (6.2%) and SCS (3.6–10.1%) were always low. As shown in Fig. 3, there were 11 Hg-methylating genera classified to the class of Deltaproteobacteria in the PR, PRE and SCS sediments. Among them, 10 genera (including Desulfacinum, Desulfobacter, Desulfobacterium, Desulfobulbus, Desulfococcus, Desulfomicrobium, Desulfonatronospira, Desulfosarcina, Desulfovibrio, and Syntrophus) were identified as SRB (Bridou et al., 2011; Gilmour et al., 2013; Lin and Jay, 2007; Sievert and Kuever, 2000), whereas only the genus Geobacter was identified as IRB (Fleming et al., 2006). Geobacter and Desulfovibrio were the major Hg-methylating genera in the PR sediments, with a relative percentage of 68.7–76.0% over the total Hg-methylating Deltaproteobacteria. Regarding the Clostridia class, 4 Hg-methylating
Fig. 1. Microbial communities at the phylum level in sediment samples obtained from the PR, PRE and SCS regions. The top 10 phyla are demonstrated separately, while the remaining phyla are combined and presented as “Other phyla”. 3
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Fig. 3. Abundance of Hg-methylating genera that belong to the classes of Deltaproteobacteria (A) and Clostridia (B) in the PR, PRE and SCS sediments.
hgcB in the PR sediments was 5.11 and 1.07 ppm, respectively; both of these values are much greater than those obtained in the PRE (3.53 and 0.77 ppm, respectively) and SCS samples (1.65 and 0.26 ppm, respectively). The spatial patterns of Hg-methylating genes within three study regions were consistent with that of Hg-methylating community. In addition, the Hg-methylating genes in the PR sediments of the site Nansha (6.42 ppm) were the most abundant among all sampling sites, exhibiting a number that was approximately 20 times higher than that at site E106 (0.32 ppm) in the SCS. A significant correlation was observed between the abundance of hgcA and that of hgcB in our samples (Fig. 4B), suggesting the coexistence of the two genes in the genomes of Hg-methylating communities. It also renders an additional implication that the in situ generation of MeHg in the aquatic environment requires the biological functions of both of hgcAB genes. The hgcA genes in the self-constructed database were sorted into three groups, according to the phyla of annotated microbial hosts, including the Proteobacteria (14), Firmicutes (7), and Euryarchaeota (2). As shown in Fig. 5, the hgcA gene detected in the sediments of the PR (18–21 subtypes) and PRE (21 subtypes) regions was more diverse when compared with that detected in the SCS sediments (7–19 subtypes). The hgcA gene (75.6–100%) in the PR, PRE and SCS sediments was predominantly carried by bacterial strains belonging to the phyla of Proteobacteria and Firmicutes, which was generally in accordance with the predominance of these two phyla in the Hg-methylating microbial communities. A significant difference in the relative composition of hgcA genes in the sediments was found among the PR, PRE and SCS areas. The composition patterns of the hgcA genes in the PR and PRE sediments featured a high abundance of Methanospirillum hungatei JF-1 (ABD40626.1), Desulfosporosinus youngiae (EHQ91195.1), Syntrophus aciditrophicus SB (ABC78804.1), and Geobacter metallireducens GS-15 (ABB31476.1). Nevertheless, the hgcA subtypes of Desulfococcus multivorans 1be1 “Gottingen” (EPR42565.1) and Desulfovibrio desulfuricans ND132 (EGB14269.1) were prevalent in the SCS sediments. Regarding the hgcB genes, a total of 21 subtypes assigned to three phyla [including the Proteobacteria (12), Firmicutes (7) and Euryarchaeota (2)] were found in the sediments of the study regions (Fig. S3 of the SM). Similar to the hgcA genes, the SCS could be differentiated from the PR and PRE regions in terms of the diversity and composition of hgcB genes in the sediments. The diversity of hgcB genes detected in the sediments of the PR (10–17 subtypes) and PRE (15 subtypes) were higher than that detected in SCS sediments (0–10 subtypes). Furthermore, the hgcB subtypes identified in the PR and PRE sediments were associated with all of the three phyla. Most of the hgcB genes detected in the sediments of the three regions (77.8–100%) were consistently carried by Proteobacteria. For instance, Desulfovibrio
genera were identified in the sediments of the PR, PRE and SCS (Fig. 3B), including Dethiobacter alkaliphilus, Ethanoligenens harbinense, Desulfosporosinus, and Desulfitobacterium. The genus of Desulfosporosinus, as an SRB (Lee et al., 2009), was found to be the most abundant (10.1–50.6 ppm; 59.9–61.8% over the total Hg-methylating Clostridia) in the PR sediments among these 4 genera. However, the abundance of the 4 genera was consistently low in the sediment samples obtained from the PRE (0.02–0.19 ppm) and SCS (0–0.22 ppm). The Hg-methylating communities were investigated using various culturable and non-culturable approaches. Certain Hg-methylating bacteria (including the Geobacter bemidjiensis sp nov., Geobacter psychrophilus sp nov., and Desulfacinum hydrothermale sp nov.) were isolated from the environmental samples and further characterized (Nevin et al., 2005; Sievert and Kuever, 2000). Following the amplification of 16S rRNA genes, high-throughput sequencing was applied for unculturable Hg-methylating communities (Liu et al., 2018b; Yang et al., 2018). In addition to 16S rRNA, some functional genes, such as dsrB [encoding a β-subunit of dissimilatory (bi)sulfite reductase] and mcrA (encoding a methyl-coenzyme M reductase of methanogens), were used to indicate the abundance and diversity of SRB and methanogens in a number of previous studies, since these strains were thought to serve key roles in Hg methylation in the environment (Bae et al., 2014; Liu et al., 2014b). Moreover, the Hg-methylating community structure could be monitored by amplification and sequencing of hgcA genes (Liu et al., 2014a). All these previous studies revealed the importance of SRB, IRB and methanogens in the in situ production of MeHg in various environmental settings. Although SRB are typically considered as the most important methylators of inorganic Hg, other microbial strains (such as IRB that belong to the Geobacteriaceae family) could substantially contribute to the MeHg generation under insufficient supply of sulfate (Bae et al., 2014). This present study investigated all the identified Hg-methylating bacteria rather than focusing on SRB alone, and thus more comprehensive information on the Hg-methylating communities could be obtained. Moreover, our results also demonstrated that the composition and abundance of microbes related to Hg methylation were substantially different among aquatic environments, which provided a good proof to explain the fair variation in the in situ MeHg production at these locations. 3.2. Abundance and diversity of Hg-methylating genes in sediments In order to analyze the Hg-methylating genes in the sediments, our sequencing datasets were aligned against the self-constructed HgcAB database and normalized by the total number of sequencing reads in the same sample. As displayed in Fig. 4A, the mean abundance of hgcA and 4
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Fig. 4. Abundance (A) and correlation (B) of Hg-methylating genes in sediment samples obtained from the PR, PRE and SCS regions.
3.3. Relationship between hgcAB and MeHg production in the sediments
africanus str. Walvis Bay (EGJ48475.1), Geobacter metallireducens GS-15 (ABB31477.1), Geobacter bemidjiensis Bem (ACH38203.1), Geobacter daltonii FRC-32 (ACM20835.1) and Desulfomicrobium baculatum X (ACU88502.1) detected in the PR and the PRE sediments, as the main carriers of hgcB genes, were found to all belong to Proteobacteria. In general, analysis of relative abundance of microbial hosts identified as the carriers of hgcA and hgcB was in good accordance with the results of Hg-methylating communities based on analysis of 16S rRNA genes. It explained that hgcAB and 16S rRNA genes were both used as suitable analytical targets to reflect the diversity and composition of microbial communities related to MeHg production in the aquatic environment.
As displayed in Fig. 6a, MeHg concentration in the sediments was much higher in the PR and PRE regions in comparison with that in the SCS, with a distinct gradient in the MeHg level observed from the PR to the PRE, and on to the SCS. These results implied that significant emission of Hg, accompanying with the fair activity of in situ Hg methylation, resulted in more serious MeHg pollution in the PR and PRE regions in comparison to the SCS. Moreover, the spatial distribution of MeHg was found to be similar with that of Hg-methylating communities and genes across the PR, PRE and SCS regions. Correlation analysis (Fig. 6b) showed that the MeHg concentration was significantly correlated with both hgcA and hgcB genes in the sediments of the PR, PRE Fig. 5. Bubble chart showing the abundance of hgcA subtypes in the sediments of the PR, PRE and SCS regions ( Proteobacteria; Firmicutes; Euryarchaeota). The size of each bubble indicates the abundance of each hgcA subtype. hgcA grouping was performed according to the phyla of the annotated microbial hosts, and the colors cyan, green and purple represent the Proteobacteria, Firmicutes and Euryarchaeota, respectively. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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supplementary evidence that the strains carried these two genes played key roles in MeHg production consistently in the river, estuary and marine environments. 4. Conclusions The present study revealed that the abundance of Hg-methylating communities and genes in the sediments was obviously decreased from the PR to the PRE, and on to the SCS, which was in good accordance with the spatial pattern of MeHg. A significant correlation was also found between the hgcAB abundance and MeHg concentration in the sediments of the study region. The results suggest that Hg may be transformed in situ to MeHg by microbes carrying hgcAB genes. Although the community structure of Hg-methylating communities varied greatly among the different sampling regions, SRB and IRB were consistently the primary microbial methylators of inorganic Hg, reflecting their significant contribution to MeHg production. Nevertheless, the composition and abundance of Hg-methylating communities may be the determining factors in MeHg pollution in aquatic environments. Acknowledgements The financial support from the National Natural Science Foundation of China (21777198, 21707176 and 21625703) is gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109722. References Bae, H.S., et al., 2014. Syntrophs dominate sequences associated with the mercury methylation-related gene hgcA in the water conservation areas of the Florida Everglades. Appl. Environ. Microbiol. 80, 6517–6526. Bridou, R., et al., 2011. Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using species-specific isotopic tracers. Environ. Toxicol. Chem. 30, 337–344. Chen, B.W., et al., 2013. Metagenomic profiles of antibiotic resistance genes (ARGs) between human impacted estuary and deep ocean sediments. Environ. Sci. Technol. 47, 12753–12760. Clarkson, T.W., Magos, L., 2006. The toxicology of mercury and its chemical compounds. Crit. Rev. Toxicol. 36, 609–662. Colombo, M.J., et al., 2013. Anaerobic oxidation of Hg(0) and methylmercury formation by Desulfovibrio desulfuricans ND132. Geochem. Cosmochim. Acta 112, 166–177. Du, H.X., et al., 2017. Mercury-methylating genes dsrB and hgcA in soils/sediments of the Three Gorges Reservoir. Environ. Sci. Pollut. Control Ser. 24, 5001–5011. Fleming, E.J., et al., 2006. Mercury methylation from unexpected sources: molybdateinhibited freshwater sediments and an iron-reducing bacterium. Appl. Environ. Microbiol. 72, 457–464. Gilmour, C.C., et al., 2011. Sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 as a model for understanding bacterial mercury methylation. Appl. Environ. Microbiol. 77, 3938–3951. Gilmour, C.C., et al., 2013. Mercury methylation by novel microorganisms from new environments. Environ. Sci. Technol. 47, 11810–11820. Grandjean, P., 2007. Methylmercury toxicity and functional programming. Reprod. Toxicol. 23, 414–420. Hamelin, S., et al., 2011. Methanogens: principal methylators of mercury in lake periphyton. Environ. Sci. Technol. 45, 7693–7700. Hight, S.C., Cheng, J., 2006. Determination of methylmercury and estimation of total mercury in seafood using high performance liquid chromatography (HPLC) and inductively coupled plasma-mass spectrometry (ICP-MS): method development and validation. Anal. Chim. Acta 567, 160–172. Hintelmann, H., 2010. Organomercurials. Their formation and pathways in the environment. Organometallics Environ. Toxicol. 7, 365–401. Hsu-Kim, H., et al., 2013. Mechanisms regulating mercury bioavailability for methylating microorganisms in the aquatic environment: a critical review. Environ. Sci. Technol. 47, 2441–2456. Hu, H.Y., et al., 2013. Oxidation and methylation of dissolved elemental mercury by anaerobic bacteria. Nat. Geosci. 6, 751–754. Kerin, E.J., et al., 2006. Mercury methylation by dissimilatory iron-reducing bacteria. Appl. Environ. Microbiol. 72, 7919–7921. Kessler, R., 2013. The minamata convention on mercury: a first step toward protecting
Fig. 6. MeHg concentration in sediments collected from the PR, PRE and SCS regions (A) and its correlation with hgcA and hgcB genes (B).
and SCS (p < 0.05). The abundance of genes related to Hg methylation is always closely associated with the in situ production of MeHg although great difference in the composition and diversity of Hg-methylating communities and genes are observed among different environmental niches. A positive correlation between hgcA gene abundance and MeHg content was previously reported in paddy fields (Liu et al., 2014a). Based on the analysis of dsrAB gene, the MeHg content was also significantly correlated to the abundance of SRB in paddy soils (Liu et al., 2014b). Du et al. also demonstrated the positive correlations between MeHg concentration and the genes hgcA or dsrB in three soil/sediment samples from the Three Gorges Reservoirs, which indicated the potential contribution of SRB to MeHg production (Du et al., 2017). In comparison to the above studies, the present study was conducted in a region with a larger scale, across the river, the estuarine and the open sea. Intrinsic relationships between MeHg concentration and Hg methylation-related genes (hgcA and hgcB) were still observed in the sediments examined in the current study, providing supplementary evidence on the irreplaceable roles of microbes in MeHg generation in aquatic environments. Furthermore, by contrast to a single gene investigated in previous studies, our study demonstrated that the abundance of hgcA and hgcB genes, which have different function in Hg methylation process, were both significantly correlated with MeHg concentration. It provided a 6
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Mercury methylation-related microbes and genes in the sediments of the Pearl River Estuary and the South China Sea
T
Ke Yuana, Xin Chena, Ping Chenb, Yongshun Huangc, Jie Jiangd, Tiangang Luana,b, Baowei Chena,∗, Xiaowei Wanga,∗∗ a
Southern Marine Science and Engineering Guangdong Laboratory, School of Marine Sciences, Sun Yat-Sen University, Zhuhai, 519082, China State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China c Guangdong Provincial Hospital for Occupational Diseases Prevention and Treatment, Guangzhou, 510300, China d Shenzhen Center for Disease Control and Prevention, Shenzhen, 518055, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hg-methylating communities hgcAB cluster The Pearl River Estuary The South China Sea
Methylmercury (MeHg) is a toxicant that mainly originates from in situ microbial methylation of inorganic mercury (Hg) in the environment and poses a severe health risk to the public. However, the characteristics of the Hg-methylating microbial community and its relationship with MeHg production in various environments remain to be understood. In the present study, Hg-methylating microbial communities and genes (hgcAB cluster) in the sediments of the Pearl River (PR), Pearl River Estuary (PRE) and South China Sea (SCS) were investigated at a large spatial scale using high-throughput sequencing-based approaches. The results showed that sulfur-reducing bacteria (SRB) and iron-reducing bacteria (IRB) were consistently the dominant microbial strains responsible for the methylation of inorganic Hg in all three regions investigated. The abundance and diversity of Hg-methylating communities and genes were both found to be higher in the PR sediments compared to that in the PRE and SCS sediments, and in good agreement with the spatial distribution of MeHg. Furthermore, a significant correlation was observed between the MeHg concentration and the abundance of both hgcA and hgcB genes in the sediments of the PR, PRE and SCS regions. Overall, the present study suggested that there was the presence of a close link between MeHg and Hg-methylating communities or genes in the ambient aquatic environment, which could be used to reflect the potential of in situ MeHg production.
1. Introduction Mercury (Hg) and its derivatives are a group of global and persistent contaminants with high toxicity (Kessler, 2013). Hg can be released into the environment from both natural and anthropogenic sources in the inorganic forms (namely, Hg2+ and Hg0), and may be subsequently transformed in situ into methylated species (Colombo et al., 2013; Hu et al., 2013). Conversion between Hg forms can lead to a great variation in the toxicity, environmental behavior and bioavailability of these compounds. As a potent neurotoxin, methylmercury (MeHg) has attracted the most attention, and it is mainly derived from Hg methylation processes in various environments. MeHg may be accumulated and biomagnified through the food chain of aquatic systems (Clarkson and Magos, 2006; Grandjean, 2007). It has also been reported that the MeHg concentration detected in fish was approximately 106 times higher than that in the water phase, and greater than 85% of Hg found
∗
in fish is in the form of MeHg (Hight and Cheng, 2006). In aquatic environments, MeHg may be produced via both biotic and abiotic processes (Hsu-Kim et al., 2013; Li and Cai, 2013). Microbes are considered as the primary producers of MeHg in the environment, predominantly in anaerobic sediment, soil, and bottom water (Hintelmann, 2010). Sulfate-reducing bacteria (SRB) (Gilmour et al., 2011; Zeng et al., 2016) and iron-reducing bacteria (IRB) (Fleming et al., 2006; Kerin et al., 2006), as well as methanogens (Hamelin et al., 2011; Yu et al., 2013), have been identified as the main microbial methylators of Hg. 47 confirmed Hg-methylating strains have been documented, including 31 SRBs, 8 IRBs and 3 methanogens (Gilmour et al., 2013). Among these microbial strains, 27 have been demonstrated to carry the hgcAB gene cluster, which is currently considered to be the only gene cluster responsible for Hg methylation in microbes (Parks et al., 2013). In this cluster, hgcA gene encodes a corrinoid-dependent protein that functions as the methyltransferase similar to the
Corresponding author. Corresponding author. E-mail addresses: [email protected] (B. Chen), [email protected] (X. Wang).
∗∗
https://doi.org/10.1016/j.ecoenv.2019.109722 Received 15 July 2019; Received in revised form 14 September 2019; Accepted 23 September 2019 Available online 29 September 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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in the dark before subsequent treatment and analysis.
corrinoid iron-sulfur protein in the reductive acetyl-CoA pathway, while hgcB is able to express an associated ferredoxin protein that potentially reduces the corrinoid center of the HgcA protein (Parks et al., 2013). Therefore, the above Hg-methylating genes could be used as good indicators to evaluate in situ Hg methylation potentials of microbial communities in various environmental settings. To date, the abundance of Hg-methylating communities and hgcAB genes has been quantified in numerous environmental samples. In a previous study, analysis of the hgcA gene in soils collected from the northern Everglades demonstrated that this gene could be carried by diverse bacterial phyla (Bae et al., 2014). Furthermore, a significant correlation was observed between hgcA gene abundance and MeHg concentration along an Hg gradient in paddy soils (Liu et al., 2014a, 2018a), and in the soils and sediments of the Three Gorges Reservoir region (Du et al., 2017). It has been reported that the diversity and distribution of hgcA were apparently discerned among wetland soils, even between different portions of a swamp, suggesting that different bacterial strains may be responsible for MeHg production under these conditions (Schaefer et al., 2014). The Hg methylation abilities of phylogenetically-related bacteria are known to vary greatly (Gilmour et al., 2011). Therefore, elaborately profiling the composition and abundance of the Hg-methylating microbial community would be of great importance for understanding the Hg methylation potential in the aquatic environment. In the past decades, due to the rapid economic development and urbanization, a huge body of various contaminants, such as heavy metals, are being released into the Pearl River (PR), the Pearl River Estuary (PRE) and nearby coastal areas (Chen et al., 2013; Wang et al., 2014; Yuan et al., 2015; Zhao et al., 2018). Analysis of the total Hg concentration in the sediment column has demonstrated that the Hg pollution was aggravated in the PRE over the past 150 years (Shi et al., 2007). It has been estimated that more than 17 tons of Hg is annually discharged from the Pearl River Delta (PRD) into the nearby aquatic environments via sewage discharge or atmospheric deposition (Zheng et al., 2011). The total Hg concentration in the fish of the PRD were reported to be in the range of 2.81–208.5 ng g−1, while the MeHg concentration (1.63–287.0 ng g−1) in these fish was associated with approximately 84% of the MeHg intake by the local population in the region (Shao et al., 2013). Therefore, MeHg production in the aquatic environment may be potentially associated with public health. Detailed knowledge regarding the diversity and abundance of Hg-methylating bacteria and genes is crucial to understanding in situ MeHg production in the PR, PRE and the South China Sea (SCS) areas. Therefore, the objective of the present study was to investigate the spatial distribution of Hg-methylating bacteria and genes in the sediments collected from the PR, PRE and SCS using metagenomic approaches. Furthermore, the study aimed to understand the relationship of these bacteria and genes with the in situ production of MeHg.
2.2. Analysis of MeHg The concentration of MeHg in the sediment samples was determined according to the method described by Ma et al. (2014). A portion of sediment collected from each of the sampling sites was freeze-dried at −50 °C prior to being ground. A solution of 0.2 mL Me198Hg, serving as a species-specific internal standard, was added into approximately 0.5 g of sediment in 50-mL centrifuge tubes. Next, 2 mL CuSO4 (1 mol L−1) and 10 mL KBr [1 mol L−1 in 5% (v/v) H2SO4] were added to the centrifuge tubes, which were then sealed and shaken for 6 h, followed by the addition of 10 mL CH2Cl2 and further shaking for 4 h. The solution was centrifuged at 6000 rpm for 15 min, the organic phase was then transferred into a 50-mL centrifugation tube, and 30 mL Milli-Q water were added. Subsequently, the organic solvent was removed under gentle nitrogen flow. The sample in the water matrix was transferred to a 100-mL glass flask along with 300 μL acetic acid-acetate buffer (pH = 4.9), followed by addition of 100 mL NaBEt4 (1% in 2% KOH). The volatile organomercury compounds were purged and trapped on a Tenax adsorber, thermodesorbed onto a GC column, and isothermally separated at 36 °C. Finally, the Hg content was analyzed by ICP-MS, and the operation parameters of this technique are provided in Table S2 of the SM. Equilibration of the spiked Hg198 with the natural Hg202 in the sample alters the isotope ratio that is measured and used for calculation. 2.3. DNA extraction and high-throughput sequencing Total bacterial DNA extraction and high-throughput sequencing were conducted according to the method reported in our previous study (Yuan et al., 2017). In brief, the total bacterial DNA was extracted from each sediment sample using a Fast DNASPIN Kit for soil (MP Bio medicals, Santa Ana, CA, USA), according to the kit instructions. DNA extraction was performed multiple times due to the low concentration of DNA present in marine sediments, and the resulting DNA was also combined to avoid the potential bias caused by a single DNA extraction. Subsequently, DNA sequencing was performed using an Illumina Hiseq 2000 device, supplied by Beijing Genomics Institute (Shenzhen, China). The raw reads (100 bp) in all datasets were trimmed to discard the lowquality reads that contained ambiguous nucleotides or a quality value of < 20 using a customized script prior to conducting further bioinformatics analysis. 2.4. Recognition of bacterial community structure and Hg-methylating bacteria The 16S rRNA gene-like reads were recognized by aligning our sequencing datasets against the SILVA Small Subunit (SILVA_119_SSURef_Nr99_tax_silva) database using BLASTn with a cutoff e-value of 1e-20. The 16S rDNA gene-like reads from the BLASTn results were then assigned to NCBI taxonomies using the MEGAN program (version 4.70.4) with the lowest common ancestor algorithm, where the absolute cutoff was a BLAST bitscore of 50 and the relative cutoff was 10% of the top 50 bits. For the identification of Hg-methylating bacteria, a database containing the 16S rRNA genes of Hg-methylating bacteria reported in previous publications was constructed (Gilmour et al., 2013). Subsequently, each of the sequencing datasets of the sediment samples was blasted against this database, and the reads with an identity that was > 97% and an alignment length of at least 100 bp were considered to represent the 16S rRNA genes of Hg-methylating bacteria. the relative abundances of each phylum and Hgmethylating phylum were obtained from dividing the number of 16S rDNA-like reads and Hg methylation-related 16S rDNA-like reads by that of the total reads in the same dataset.
2. Material and methods 2.1. Study area and sampling In total, 12 sediment samples were collected from 8 sites in the PR, the PRE and SCS between 2011 and 2013, and detailed information regarding the sampling sites is presented in Table S1 of the Supplementary Materials (SM). The sites CHHE, LUBA, SHDE and NANS are located in the Beijiang River, which is a main tributary of the PR. The site CHHE is situated at the origin of Beijiang River, and NANS is located at one of the main outlets of the PR. Three sites (E106, E208 and E406) are located in the SCS. Among these, site E208 is near the coast of Southern China, the sites E406 is located in the northern SCS near the Philippines, while site E106 is situated far from the mainland. Moreover, triplicate sediments were also obtained from site A8 in the PRE, which is a transitional zone from PR to SCS. Following sampling, all sediment samples were wrapped in aluminum foil and stored at −80 °C 2
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2.5. Construction of Hg methylation protein (HgcAB) database and hgcAB annotation A Hg methylation protein database was constructed by manually downloading the amino acid (aa) sequences of Hg-methylating enzymes (HgcAB) from the nr database that is compiled by the NCBI. This database included a total of 23 aa sequences encoded by hgcA and 23 aa sequences encoded by hgcB, and these genes were confirmed to be Hgmethylating genes by previous research (Gilmour et al., 2013). Each of the sequencing datasets was interrogated against our self-constructed database using BLASTx with a cutoff e-value of 1e-5. When the best hit presented a similarity of > 75% and an alignment length of at least 25 aa, the read was annotated as an Hg-methylating gene-like sequence.
3. Results and discussion 3.1. Composition and abundance of the total and Hg-methylating microbial communities in sediment samples
Fig. 2. Relative abundance of Hg-methylating phyla in the PR, PRE and SCS sediments. “ppm” represents one 16S rDNA-like reads in one million sequencing reads.
The 16S rRNA genes were determined by aligning our sequencing datasets against the SILVA Small Subunit database, and the microbial communities were further analyzed using MEGAN. A total of 23 bacteria and 2 archaea phyla were identified in the sediment samples obtained from the PR, PRE and SCS regions. The relative abundance of the top 10 phyla in these sediments is displayed in Fig. 1. The top 10 phyla accounted for approximately 88.4–94.7% of the total 16S rRNA genes identified in the sediments. Distinct differences in the relative composition of the microbial community were observed among the sediments of the PR, PRE and SCS. Proteobacteria were identified as the most abundant phylum in the sediments of the PRE and SCS (48.8–57.5%). By contrast, Firmicutes were the most enriched among all phyla in the PR sediments (13.2–51.7%), whereas they only accounted for 2.0–3.2% of the microbial community in the PRE and SCS sediments. The relative percentages of Proteobacteria in the PR sediments (21.0–40.2%) were substantially lower compared with those present in the PRE and SCS sediments. Fig. 2 shows the occurrence and abundance of Hg-methylating phyla in the PR, PRE and SCS sediments, which included two bacterial phyla (Firmicutes and Proteobacteria) and one archaea phylum (Euryarchaeota) (Gilmour et al., 2013). In all samples from our study region, the Hg-methylating strains assigned to bacteria were more popular compared with those of archaea. In addition, Hg-methylating strains in the PR sediments (49.0–136.7 ppm) were more abundant than those in the PRE and SCS sediments (6.2 ppm and 2.3–7.1 ppm, respectively). The total abundance of Hg-methylating strains at the site of
Shunde (136.7 ppm) in the PR was highest among all sampling sites, whereas that the total abundance at the site E106 in the SCS was the lowest. The Hg-methylating communities in the PRE and SCS sediments were characterized by the predominance of Proteobacteria, e.g., the PRE (91.6%) and the SCS (86.1–94.1%). In contrast to the aforementioned locations, Hg-methylating communities in the PR sediments were relatively equally constituted by Proteobacteria (35.5–64.5%) and Firmicutes (34.9–64.4%). The present study further examined the relationships between the abundance of Hg-methylating communities and that of the total bacteria sorting to the same phylum, and the results are presented in Fig. S1 of the SM. A significant positive correlation was only observed between the abundance of Hg-methylating Firmicutes and the total abundance of this phylum across the PR, PRE and SCS sediments, instead of Proteobacteria and Euryarchaeota. This result implies that Hgmethylating bacterial strains may be not a stable composition in the total bacterial communities in sediments and vary with sampling sites, which could contribute to the difference in MeHg production among different locations. The Hg-methylating bacteria detected in the sediment samples obtained from the PR, PRE and SCS belonged to four microbial classes, including Methanomicrobia (Euryarchaeota), Deltaproteobacteria (Proteobacteria), and Clostridia and Negativicutes (Firmicutes), as shown in Fig. S2 of the SM. The Deltaproteobacteria consistently comprised a dominant Hg-methylating class in all sediment samples, including 91.6% in the PRE, 86.1–94.1% in the SCS, and 35.5–64.4% in the PR. Besides Deltaproteobacteria, the Hg-methylating Clostridia (33.4–61.7%) were also predominant over other classes in the PR sediments; for instance, the relative percentage of this class over the total Hg-methylating classes in the sediment at the site Shunde was up to 61.7%. Furthermore, the relative percentages of Hg-methylating Clostridia in the sediment samples obtained from the PRE (6.2%) and SCS (3.6–10.1%) were always low. As shown in Fig. 3, there were 11 Hg-methylating genera classified to the class of Deltaproteobacteria in the PR, PRE and SCS sediments. Among them, 10 genera (including Desulfacinum, Desulfobacter, Desulfobacterium, Desulfobulbus, Desulfococcus, Desulfomicrobium, Desulfonatronospira, Desulfosarcina, Desulfovibrio, and Syntrophus) were identified as SRB (Bridou et al., 2011; Gilmour et al., 2013; Lin and Jay, 2007; Sievert and Kuever, 2000), whereas only the genus Geobacter was identified as IRB (Fleming et al., 2006). Geobacter and Desulfovibrio were the major Hg-methylating genera in the PR sediments, with a relative percentage of 68.7–76.0% over the total Hg-methylating Deltaproteobacteria. Regarding the Clostridia class, 4 Hg-methylating
Fig. 1. Microbial communities at the phylum level in sediment samples obtained from the PR, PRE and SCS regions. The top 10 phyla are demonstrated separately, while the remaining phyla are combined and presented as “Other phyla”. 3
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Fig. 3. Abundance of Hg-methylating genera that belong to the classes of Deltaproteobacteria (A) and Clostridia (B) in the PR, PRE and SCS sediments.
hgcB in the PR sediments was 5.11 and 1.07 ppm, respectively; both of these values are much greater than those obtained in the PRE (3.53 and 0.77 ppm, respectively) and SCS samples (1.65 and 0.26 ppm, respectively). The spatial patterns of Hg-methylating genes within three study regions were consistent with that of Hg-methylating community. In addition, the Hg-methylating genes in the PR sediments of the site Nansha (6.42 ppm) were the most abundant among all sampling sites, exhibiting a number that was approximately 20 times higher than that at site E106 (0.32 ppm) in the SCS. A significant correlation was observed between the abundance of hgcA and that of hgcB in our samples (Fig. 4B), suggesting the coexistence of the two genes in the genomes of Hg-methylating communities. It also renders an additional implication that the in situ generation of MeHg in the aquatic environment requires the biological functions of both of hgcAB genes. The hgcA genes in the self-constructed database were sorted into three groups, according to the phyla of annotated microbial hosts, including the Proteobacteria (14), Firmicutes (7), and Euryarchaeota (2). As shown in Fig. 5, the hgcA gene detected in the sediments of the PR (18–21 subtypes) and PRE (21 subtypes) regions was more diverse when compared with that detected in the SCS sediments (7–19 subtypes). The hgcA gene (75.6–100%) in the PR, PRE and SCS sediments was predominantly carried by bacterial strains belonging to the phyla of Proteobacteria and Firmicutes, which was generally in accordance with the predominance of these two phyla in the Hg-methylating microbial communities. A significant difference in the relative composition of hgcA genes in the sediments was found among the PR, PRE and SCS areas. The composition patterns of the hgcA genes in the PR and PRE sediments featured a high abundance of Methanospirillum hungatei JF-1 (ABD40626.1), Desulfosporosinus youngiae (EHQ91195.1), Syntrophus aciditrophicus SB (ABC78804.1), and Geobacter metallireducens GS-15 (ABB31476.1). Nevertheless, the hgcA subtypes of Desulfococcus multivorans 1be1 “Gottingen” (EPR42565.1) and Desulfovibrio desulfuricans ND132 (EGB14269.1) were prevalent in the SCS sediments. Regarding the hgcB genes, a total of 21 subtypes assigned to three phyla [including the Proteobacteria (12), Firmicutes (7) and Euryarchaeota (2)] were found in the sediments of the study regions (Fig. S3 of the SM). Similar to the hgcA genes, the SCS could be differentiated from the PR and PRE regions in terms of the diversity and composition of hgcB genes in the sediments. The diversity of hgcB genes detected in the sediments of the PR (10–17 subtypes) and PRE (15 subtypes) were higher than that detected in SCS sediments (0–10 subtypes). Furthermore, the hgcB subtypes identified in the PR and PRE sediments were associated with all of the three phyla. Most of the hgcB genes detected in the sediments of the three regions (77.8–100%) were consistently carried by Proteobacteria. For instance, Desulfovibrio
genera were identified in the sediments of the PR, PRE and SCS (Fig. 3B), including Dethiobacter alkaliphilus, Ethanoligenens harbinense, Desulfosporosinus, and Desulfitobacterium. The genus of Desulfosporosinus, as an SRB (Lee et al., 2009), was found to be the most abundant (10.1–50.6 ppm; 59.9–61.8% over the total Hg-methylating Clostridia) in the PR sediments among these 4 genera. However, the abundance of the 4 genera was consistently low in the sediment samples obtained from the PRE (0.02–0.19 ppm) and SCS (0–0.22 ppm). The Hg-methylating communities were investigated using various culturable and non-culturable approaches. Certain Hg-methylating bacteria (including the Geobacter bemidjiensis sp nov., Geobacter psychrophilus sp nov., and Desulfacinum hydrothermale sp nov.) were isolated from the environmental samples and further characterized (Nevin et al., 2005; Sievert and Kuever, 2000). Following the amplification of 16S rRNA genes, high-throughput sequencing was applied for unculturable Hg-methylating communities (Liu et al., 2018b; Yang et al., 2018). In addition to 16S rRNA, some functional genes, such as dsrB [encoding a β-subunit of dissimilatory (bi)sulfite reductase] and mcrA (encoding a methyl-coenzyme M reductase of methanogens), were used to indicate the abundance and diversity of SRB and methanogens in a number of previous studies, since these strains were thought to serve key roles in Hg methylation in the environment (Bae et al., 2014; Liu et al., 2014b). Moreover, the Hg-methylating community structure could be monitored by amplification and sequencing of hgcA genes (Liu et al., 2014a). All these previous studies revealed the importance of SRB, IRB and methanogens in the in situ production of MeHg in various environmental settings. Although SRB are typically considered as the most important methylators of inorganic Hg, other microbial strains (such as IRB that belong to the Geobacteriaceae family) could substantially contribute to the MeHg generation under insufficient supply of sulfate (Bae et al., 2014). This present study investigated all the identified Hg-methylating bacteria rather than focusing on SRB alone, and thus more comprehensive information on the Hg-methylating communities could be obtained. Moreover, our results also demonstrated that the composition and abundance of microbes related to Hg methylation were substantially different among aquatic environments, which provided a good proof to explain the fair variation in the in situ MeHg production at these locations. 3.2. Abundance and diversity of Hg-methylating genes in sediments In order to analyze the Hg-methylating genes in the sediments, our sequencing datasets were aligned against the self-constructed HgcAB database and normalized by the total number of sequencing reads in the same sample. As displayed in Fig. 4A, the mean abundance of hgcA and 4
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Fig. 4. Abundance (A) and correlation (B) of Hg-methylating genes in sediment samples obtained from the PR, PRE and SCS regions.
3.3. Relationship between hgcAB and MeHg production in the sediments
africanus str. Walvis Bay (EGJ48475.1), Geobacter metallireducens GS-15 (ABB31477.1), Geobacter bemidjiensis Bem (ACH38203.1), Geobacter daltonii FRC-32 (ACM20835.1) and Desulfomicrobium baculatum X (ACU88502.1) detected in the PR and the PRE sediments, as the main carriers of hgcB genes, were found to all belong to Proteobacteria. In general, analysis of relative abundance of microbial hosts identified as the carriers of hgcA and hgcB was in good accordance with the results of Hg-methylating communities based on analysis of 16S rRNA genes. It explained that hgcAB and 16S rRNA genes were both used as suitable analytical targets to reflect the diversity and composition of microbial communities related to MeHg production in the aquatic environment.
As displayed in Fig. 6a, MeHg concentration in the sediments was much higher in the PR and PRE regions in comparison with that in the SCS, with a distinct gradient in the MeHg level observed from the PR to the PRE, and on to the SCS. These results implied that significant emission of Hg, accompanying with the fair activity of in situ Hg methylation, resulted in more serious MeHg pollution in the PR and PRE regions in comparison to the SCS. Moreover, the spatial distribution of MeHg was found to be similar with that of Hg-methylating communities and genes across the PR, PRE and SCS regions. Correlation analysis (Fig. 6b) showed that the MeHg concentration was significantly correlated with both hgcA and hgcB genes in the sediments of the PR, PRE Fig. 5. Bubble chart showing the abundance of hgcA subtypes in the sediments of the PR, PRE and SCS regions ( Proteobacteria; Firmicutes; Euryarchaeota). The size of each bubble indicates the abundance of each hgcA subtype. hgcA grouping was performed according to the phyla of the annotated microbial hosts, and the colors cyan, green and purple represent the Proteobacteria, Firmicutes and Euryarchaeota, respectively. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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supplementary evidence that the strains carried these two genes played key roles in MeHg production consistently in the river, estuary and marine environments. 4. Conclusions The present study revealed that the abundance of Hg-methylating communities and genes in the sediments was obviously decreased from the PR to the PRE, and on to the SCS, which was in good accordance with the spatial pattern of MeHg. A significant correlation was also found between the hgcAB abundance and MeHg concentration in the sediments of the study region. The results suggest that Hg may be transformed in situ to MeHg by microbes carrying hgcAB genes. Although the community structure of Hg-methylating communities varied greatly among the different sampling regions, SRB and IRB were consistently the primary microbial methylators of inorganic Hg, reflecting their significant contribution to MeHg production. Nevertheless, the composition and abundance of Hg-methylating communities may be the determining factors in MeHg pollution in aquatic environments. Acknowledgements The financial support from the National Natural Science Foundation of China (21777198, 21707176 and 21625703) is gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109722. References Bae, H.S., et al., 2014. Syntrophs dominate sequences associated with the mercury methylation-related gene hgcA in the water conservation areas of the Florida Everglades. Appl. Environ. Microbiol. 80, 6517–6526. Bridou, R., et al., 2011. Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using species-specific isotopic tracers. Environ. Toxicol. Chem. 30, 337–344. Chen, B.W., et al., 2013. Metagenomic profiles of antibiotic resistance genes (ARGs) between human impacted estuary and deep ocean sediments. Environ. Sci. Technol. 47, 12753–12760. Clarkson, T.W., Magos, L., 2006. The toxicology of mercury and its chemical compounds. Crit. Rev. Toxicol. 36, 609–662. Colombo, M.J., et al., 2013. Anaerobic oxidation of Hg(0) and methylmercury formation by Desulfovibrio desulfuricans ND132. Geochem. Cosmochim. Acta 112, 166–177. Du, H.X., et al., 2017. Mercury-methylating genes dsrB and hgcA in soils/sediments of the Three Gorges Reservoir. Environ. Sci. Pollut. Control Ser. 24, 5001–5011. Fleming, E.J., et al., 2006. Mercury methylation from unexpected sources: molybdateinhibited freshwater sediments and an iron-reducing bacterium. Appl. Environ. Microbiol. 72, 457–464. Gilmour, C.C., et al., 2011. Sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 as a model for understanding bacterial mercury methylation. Appl. Environ. Microbiol. 77, 3938–3951. Gilmour, C.C., et al., 2013. Mercury methylation by novel microorganisms from new environments. Environ. Sci. Technol. 47, 11810–11820. Grandjean, P., 2007. Methylmercury toxicity and functional programming. Reprod. Toxicol. 23, 414–420. Hamelin, S., et al., 2011. Methanogens: principal methylators of mercury in lake periphyton. Environ. Sci. Technol. 45, 7693–7700. Hight, S.C., Cheng, J., 2006. Determination of methylmercury and estimation of total mercury in seafood using high performance liquid chromatography (HPLC) and inductively coupled plasma-mass spectrometry (ICP-MS): method development and validation. Anal. Chim. Acta 567, 160–172. Hintelmann, H., 2010. Organomercurials. Their formation and pathways in the environment. Organometallics Environ. Toxicol. 7, 365–401. Hsu-Kim, H., et al., 2013. Mechanisms regulating mercury bioavailability for methylating microorganisms in the aquatic environment: a critical review. Environ. Sci. Technol. 47, 2441–2456. Hu, H.Y., et al., 2013. Oxidation and methylation of dissolved elemental mercury by anaerobic bacteria. Nat. Geosci. 6, 751–754. Kerin, E.J., et al., 2006. Mercury methylation by dissimilatory iron-reducing bacteria. Appl. Environ. Microbiol. 72, 7919–7921. Kessler, R., 2013. The minamata convention on mercury: a first step toward protecting
Fig. 6. MeHg concentration in sediments collected from the PR, PRE and SCS regions (A) and its correlation with hgcA and hgcB genes (B).
and SCS (p < 0.05). The abundance of genes related to Hg methylation is always closely associated with the in situ production of MeHg although great difference in the composition and diversity of Hg-methylating communities and genes are observed among different environmental niches. A positive correlation between hgcA gene abundance and MeHg content was previously reported in paddy fields (Liu et al., 2014a). Based on the analysis of dsrAB gene, the MeHg content was also significantly correlated to the abundance of SRB in paddy soils (Liu et al., 2014b). Du et al. also demonstrated the positive correlations between MeHg concentration and the genes hgcA or dsrB in three soil/sediment samples from the Three Gorges Reservoirs, which indicated the potential contribution of SRB to MeHg production (Du et al., 2017). In comparison to the above studies, the present study was conducted in a region with a larger scale, across the river, the estuarine and the open sea. Intrinsic relationships between MeHg concentration and Hg methylation-related genes (hgcA and hgcB) were still observed in the sediments examined in the current study, providing supplementary evidence on the irreplaceable roles of microbes in MeHg generation in aquatic environments. Furthermore, by contrast to a single gene investigated in previous studies, our study demonstrated that the abundance of hgcA and hgcB genes, which have different function in Hg methylation process, were both significantly correlated with MeHg concentration. It provided a 6
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