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Quantification and characterization of mercury resistant bacteria in sediments contaminated by artisanal small-scale gold mining activities, Kedougou region, Senegal
Journal of Geochemical Exploration 205 (2019) 106353
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Quantification and characterization of mercury resistant bacteria in sediments contaminated by artisanal small-scale gold mining activities, Kedougou region, Senegal
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Birane Nianea, , Naresh Devarajanb, John Potéc, Robert Moritza a
Department of Earth Sciences, University of Geneva, rue des Maraîchers 13, CH-1205 Geneva, Switzerland Bren School of Environmental Science and Management, University of California Santa Barbara, USA c Department F.-A. Forel for Environmental and Aquatic Sciences, University of Geneva, boulevard Carl-Vogt 66, CH-1211 Geneva 4, Switzerland b
A R T I C LE I N FO
A B S T R A C T
Keywords: Mercury resistant bacteria Artisanal small-scale gold mining Bioremediation
This study describes Hg-resistant bacterial present in the aquatic sediments artisanal small-scale gold mining ASGM activities along Gambia River Kedougou, Senegal. Mercury (Hg) is used for gold amalgamation in artisanal small-scale gold mining (ASGM) activities. The level of total Hg in sediment samples was determined by automatic mercury analyser. Bacterial (colony-forming units) susceptibility to Hg was evaluated by minimal inhibitory concentrations. The phylogenetic diversity analysis of the Hg-resistant bacteria was performed by PCR amplification of 16S rDNA on isolated bacterial strains, followed by restriction fragment length polymorphism, cloning and sequencing. The results documented high concentrations of Hg in ASGM activity areas ranging from 2.4 to 6.2 mg kg-1. Population densities of heterotrophic bacteria in wet sediment ranging from 3.7 × 106 to 4.6 × 108 CFU g−1. The isolated bacterial strains from highly Hg-contaminated sites can grow to medium containing up to 17 mg L−1 of Hg2+. In this study, bacterial strains resistant to Hg are Stenotrophomonas maltophilia, Dyella ginsengisoli, Arthrobacter defluvi, Arthrobacter pascens, Bacillus firmus and Pseudomonas moraviensis. Our results demonstrate the occurrence the presence of diverse groups of bacterial strains resistant to metal (Hg) under tropical conditions. The isolated strains are particularly interesting for further studies to evaluate their role in bioremediation of Hg in contaminated aquatic ecosystems.
1. Introduction Mercury (Hg) is a well-known environmental pollutant, and its emission to the environment can have serious effects on human and animal health. In aquatic systems, Hg exists in various forms: elemental (Hg0), which is the only metal in liquid form at room temperature, inorganic (Hg2+) and organic forms. According to Young (1992), the most toxic is organic Hg, because it accumulates in the food chain and has affinity with sulfhydryl groups in the proteins of living organisms. Several studies on Hg in water, sediment, fish and human have been carried out (e.g., Berzas Nevado et al., 2010; Garcia-Bravo et al., 2011; Guedron et al., 2009), and have identified the principal sources of Hg contamination in the aquatic environment, including gold mining activities, atmospheric deposition, erosion, urban discharge, agricultural material, combustion and industrial discharge. In sub-Saharan Africa, (such as Burkina Faso, Tanzania, Zimbabwe, Ghana and Senegal), limited studies have been conducted on the assessment of mercury
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contamination in artisanal small-scale gold mining areas, as summarized by the recent studies (Gerson et al., 2018; Niane et al., 2014, 2015, 2019; Ouédraogo and Amyot, 2013; Rajaee et al., 2015). These studies reveal that the amalgamation process using Hg is the most widely used technique to recover gold during artisanal activities. During this process a large amount of Hg is transferred to both terrestrial and aquatic ecosystems. The deposited mercury, can react with various organic compounds in the aquatic environment by biotic (sulfur-reducing bacteria) and abiotic (sunlight) pathway, resulting in conversion of organic mercury (Wang et al., 2004). Even small amounts of Hg (esp. inorganic mercury) in water can be toxic for all organisms (Mirzaei et al., 2008). According to Canadian interim marine sediment quality guidelines, inorganic Hg accumulates in sediments and may be a hazard to sediment-dwelling organisms at concentrations above 0.13 mg kg−1 (CCME, 1999). Below this concentration there can be toxic effects if MeHg is present. The impacts of Hg on human health and the environment are well documented around the world (Spiegel and Veiga,
Corresponding author at: DMG/Ministère des Mines et de la Géologie, Sphères Ministérielles Diamniadio-Bâtiment B 5eme Etage, Dakar, Senegal. E-mail addresses: [email protected], [email protected] (B. Niane).
https://doi.org/10.1016/j.gexplo.2019.106353 Received 30 September 2018; Received in revised form 21 July 2019; Accepted 3 August 2019 Available online 05 August 2019 0375-6742/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Location map of the gold mining sites and sampling location. (Modified from Niane et al., 2014)
2017). Sediments are complex habitats, densely colonized by diverse groups of microorganisms, which play key roles in biogeochemical cycling, aquatic food webs, remobilization of Hg, as well as Hg speciation (Garcia-Bravo et al., 2011; Poté et al., 2010; Wang et al., 2004). Some anaerobic bacteria and Archaea have evolved resistance mechanisms that function to degrade organic Hg compounds to inorganic Hg by reduction to gaseous Hg(0). Mercury resistance consisting the Mercuric reductase (MerA, B) protein, the core enzyme in the microbial mercury detoxification system, catalyzes the reduction of Hg(II) to volatile Hg(0) (Li et al., 2010; Susana et al., 2011). The studies performed in sub-Saharan African region were mainly focused on the quantification of mercury contamination in the aquatic environments (water, soil, sediment, fish) and human (hairs) (Gerson et al., 2018; Niane et al., 2014, 2015). To our knowledge, there is a lack of information to assess the impact of Hg pollution on bacterial communities under tropical conditions, such as the study region in Kedougou, eastern Senegal. The objective of this study was to isolate and characterize Hg-resistant bacterial communities in the aquatic sediments of Gambia River Kedougou in the Gambia River Kedougou, Senegal, located in the vicinity of ASGM activities. The total mercury (THg) concentrations, were measured in contaminated and uncontaminated sediment samples from Gambia River and Hg-resistant bacteria were quantified and characterized in the samples.
2010). Methylmercury can attack the human nervous system through the bloodstream, and inhalation of Hg vapor at high levels can result in acute, corrosive bronchitis and interstitial pneumonitis (Gupta et al., 2012; Rasmussen et al., 2008). Due to its persistence in the environment, remediation is necessary to in contaminated Hg sites, such as soils and aquatic systems in the vicinity of ASGM activities. According to Wang et al. (2004), methods of Hg remediation/detoxification in contaminated sites include capping, dredging, precipitation, filtration, ion exchange resin absorption using carbon and other techniques. However, these technologies are relatively expensive, not environmentally friendly, and can in turn produce new environmental problems (Wagner-Döbler, 2013). Alternately, bioremediation using Hg-resistant bacteria has become a fast growing effective technology with many advantages over physicochemical methods (Dhankher et al., 2002). Bacteria are the most ubiquitous organisms found in various environments, including soil and sediment. Several studies have been performed to understand the role of microorganisms for Hg speciation in the aquatic environment (e.g., Devereux et al., 1996; Fleming et al., 2006; Bravo et al., 2015; Osborn et al., 1997; Raposo et al., 2008). They revealed that Hg methylation is known to be favored in suboxic to anoxic environments (e.g., river sediment) and is mostly driven by sulfate-reducing bacteria (SRB). Bridou et al. (2011) have aimed to identify the SRB strains were responsible for Hg methylation by testing their Hg-methylating potential in pure cultures. Some bacterial communities are capable to overcome such toxicity and developed mechanisms that allow them to be resistant to Hg. According to Nascimento and Chartone-Souza (2003), Hg removal by bacteria from contaminant sources is a challenge for the environmental, but may provide a promising technology for bioremediation. Bioremediation of Hg in contaminated environments has been discussed in the recent literature (e.g. Dash and Das, 2012; McCarthy et al.,
2. Materials and methods 2.1. Study sites and sampling The Kedougou region is the major gold province of Senegal. Its geology is dominated by volcano-sedimentary terrains of Birimian age. Approximately 30,000 to 60,000 persons are currently involved in ASGM activities spread across several villages in Eastern Senegal 2
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ultracentrifuge). A white layer of bacterial cells was obtained at the interface between the Nycodenz-soil mix particles and the overlying aqueous layer. This white layer was carefully recovered, and mixed with an equal volume of sterile ultrapure water, then centrifuged at 7500 ×g for 20 min at 10 °C. To remove traces of Nycodenz, pelleted bacteria were resuspended in 20 ml sterile ultrapure water and centrifuged for 20 min at 7500 ×g. The pelleted bacteria were resuspended in sterile molecular biology grade water and preserved in glycerol (10% v/v) at −80 °C until used.
(PASMI, 2009), where the region of Kedougou is considered as the major ASGM center. Alluvial gold and gold-bearing quartz veins hosted by shear zones are the two types of gold ore mining in Kedougou (Sylla and Ngom, 1997). There are increasing activities of ASGM activities throughout the region and other areas along the African gold belt since 1995, as a result of the rising price of gold and low cost of ASGM technologies. About 10 years ago, mercury (Hg) has been introduced into the ore dressing process by the mining communities to improve recovery of gold using Hg-amalgamation. Niane et al. (2015) have described the effect of Hg contamination in surface river sediments, the aquatic fauna (mainly fishes) and mining communities of the Kedougou region, using hair samples. Sediment samples were collected during a field campaign, at the beginning of the wet season in June 2013 at four selected sampling sites. Two sites were sampled along Gambia River at Samekouta and Bantako, and two sites at local water ponds at Sabodala and Tinkoto (Fig. 1). Samekouta is defined as the control site, because it is located in an up-stream location and is devoid of any ASGM activities. By contrast, intense ASGM activities are developed at Tinkoto, Bantako and Sabodala. About 20 cm-long sediment cores were collected manually, below a water depth of about 50 cm, and using acrylic tubes with a diameter of 8–10 cm of. Twenty composite samples were collected. Five individual sub samples were thoroughly homogenized from each sampling site to achieve a composite sample. After sampling, the core sediments were stored at 4 °C and transported to the Institute F.A. Forel at the University of Geneva (Switzerland) for bacteriological analysis within 72 h.
2.4. Heterotrophic plate count and isolation of Hg-resistant bacteria The assessment of Hg-resistant bacteria can be performed in different medium including Tryptic Soy Agar (TSA), Tripton Iron Agar (TIA), MS Agar, Luria Bertani Agar, or other ones as summarized by Mirzaei et al. (2008). In this study the heterotrophic plate count (HPC) was performed using the method described by Poté et al. (2010) to determine the Colony-Forming Units (CFU) using tryptic soy agar (TSA) (OXOID, LTD, Basingstoke, England) medium supplemented with the antifungal (Nystatin 50 mg ml−1, Sigma Aldrich, GmbH, Germany). Serially diluted bacterial suspension from the Nycodenz method were spread platted on the medium and incubated at 28 °C for 72 h. The results were expressed as CFU units per g of wet sediment (CFU g−1). Bacterial strains resistant to Hg were isolated on TSA medium supplemented with serial concentrations (10, 12, 15, 17, 18 and 20 mg L−1) of Hg2+. The minimum concentration of Hg2+ that inhibits bacterial growth with no visible colonies in the plates after the 72 h incubation (at 28 °C) was recorded as Minimum Inhibitory Concentration (MIC) values. All analyses were conducted in triplicate for each set of conditions.
2.2. Sediment Hg analysis Sediment samples were freeze-dried before analysis, and ground to a fine homogeneous powder (< 63 μm). Analysis of total Hg in sediment samples was carried out using the Atomic Absorption Spectrometry (AAS) with an Advanced Mercury Analyser (AMA 254, Altec s.r.o, Czech Rep.), following the method described by Hall and Pelchat (1997) and Ross-Barraclough et al. (2002). This method is based on sample combustion, gold amalgamation and AAS. The accuracy of the measurements was obtained by repeated analyses of the certified reference material MESS-3-concentrations found (0.089 ± 0.006 mg kg−1, N = 3) agreed with the certified concentration (0.091 ± 0.008 mg kg−1). The detection limit defined as three times the standard deviation of the blank was 0.005 mg kg−1 and the reproducibility was better than 95%.
2.5. 16s rDNA amplification After enumerating Hg-resistant bacteria several single colonies were picked for DNA extraction using Ultraclean soil DNA Kit (Mo Bio Labs, Solana Beach, CA 92075). Phenotypic characterization of the bacterial species was conducted based on morphology, 16s rDNA amplification, restriction fragment length polymorphism (RFLP) and sequencing. Briefly, morphologically different colonies were picked from each plate at their nearest MIC values. A single colony from the overnight culture was suspended in 50 μL of polymerase chain reaction (PCR) grade water, incubated at 94 °C for 10 min and centrifuged. Five microliters of the supernatant were used as template for the PCR reaction to amplify the 16s rDNA. Amplification of the 16s rDNA was performed using the universal bacterial primer set (Wang et al., 2003) 27f (AGAGTTTGAT CMTGGCTCAG) and 1492r (TACGGYTACCTTGTTACGACTT). PCR was carried out in 50 μL reaction volume containing 1× PCR buffer, 2 mM MgCl2, 200 μM of each dNTP, 200 nM of each primer, 5 units of Taq DNA polymerase (Takara Ex Taq, Hot Start Version, Takara Bio INC, Japan) and template DNA (100 ng). PCR was performed in a Biometra thermocycler (BIOLABO, France) with the following sequence: an initial denaturation at 94 °C for 5 min; 35 cycles of denaturation (94 °C for 45 s), annealing (58 °C for 45 s), extension (72 °C for 45 s); and a final extension for 5 min at 72 °C. PCR products were separated and visualized using electrophoresis on 0.8% agarose gel stained with SYBR safe DNA gel stain (Invitrogen).
2.3. Bacterial cell extraction from sediments samples Density gradient centrifugation was used for separation of complexes based on their molecular masses. In this study, the separation of bacteria and sediment matrix was performed using the Nycodenz™ density gradient centrifugation as described by Bertrand et al. (2005), and slightly modified by Poté et al. (2010). Briefly, 100 g (wet weight) of sediments and 500 ml of sterile 0.2% sodium hexametaphosphate (Na2(PO3)6, E. Lotti S.A, Switzerland) were dispersed in 1 L sterile plastic bottles and mixed for 1 h using the agitator rotary printing-press Watson-Marlow 601 controller (SKAN, Switzerland) at room temperature. The sample was then centrifuged at 750 ×g for 15 min at 15 °C in a 3 K-1 (Sigma, centrifuge). The supernatant (S2) were collected and filtered with sterile gauze. The supernatant was then centrifuged at 7500 ×g for 30 min at 10 °C. The pellet containing the microbial cell fraction was resuspended in sterile 0.8% sodium chloride solution by vortexing. Twenty-five milliliters of the homogeneous solution was transferred to an ultracentrifuge tube containing 11 ml of Nycodenz solution (Axis-Shield, Oslo, Norway) with a density of 1.3 g/ml density (8 g of Nycodenz to 10 ml of sterile ultrapure water). Bacterial cells and sediment particles were separated by high-speed centrifugation (15,000 ×g for 1 h at 10 °C) in Centrikon T-1080 (Kontron, Instrument,
2.6. RFLP, cloning and sequencing The PCR products were analyzed by Restriction Fragment Length Polymorphism (RFLP) as described by Poté et al. (2009). RFLP was performed on PCR products using 3 (HaeIII, AluI and RsaI) endonucleases (FastDigest, Fermentas). Fragments with distinct RFLP patterns were cloned to pGEM-T easy vectors following the manufacture protocol and grown in competent E. coli cells (DHα). Plasmids containing the fragment of the 16s rDNA, extracted from an overnight 3
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culture using plasmid extraction kit (GENELUTE HP PLASMID MINIPREP, Sigma Aldrich, Germany) were submitted to sequencing at StarSEQ GmbH (Germany). Sequences were compared at NCBI data bank using the BLASTN algorithm (Altschul et al., 1990). On the basis of the results of database analyses, sequences were aligned with representative bacterial sequences from GenBank (www.ncbi.nlm.nih. gov/blast) and used for Phylogenetic tree construction by NeighbourJoining method using MEGA 6.05 (Tamura et al., 2013).
Table 2 The bacterial profiles analyzed by RFLP. Sampling sites
Bacterial profile type
Samekouta Bantako Tinkoto Sabodala
B C, D A, B E
Number of bacterial isolates with the same profile type 4 5 5 4
A, B, C, D, and E indicate the type of RFLP profile observed.
2.7. Data analysis The Kolmogorov–Smirnov test was used to evaluate the normality of the data. The Kruskal–Wallis test was used for multiple sample comparison (H test). All the statistical tests were performed using STATISTICA 12.0 software, and a p < 0.05 was chosen to indicate the statistical significance.
3. Results and discussion 3.1. Mercury concentration in sediment The THg concentration in surface sediments is reported in Table 1. The concentration of THg are 4.2, 2.4, 6.2 mg kg−1, for the sites of Bantako, Tinkoto and Sabodala, respectively. The highest concentration of THg is recorded in Sabodala and the lowest at Samekouta site. According to Niane et al. (2015), the strong positive correlations between THg concentrations and organic matter (OM) in sediment sampled at Bantako during the wet season, suggests that OM in this case is a major carrier phase of Hg in the Gambia River sediments. However, sediments in Sabodala and Tinko are characterized by a strong positive correlation between OM and Thg for both seasons, suggesting that in area where OM is abundant, Hg is mainly bound to OM. Total mercury concentrations recorded in the gold mining study sites (Bantako, Tinkoto, Sabobala) were compared with the sediment quality criteria for freshwater ecosystems published by MacDonald et al. (2000). The sediment Quality Guidelines (SQGs) is based in two thresholds concentrations: a Probable Effect Concentration (PEC) above which toxic effects are likely to be induced, and a threshold effect level (TEL) below which no harmful effect is produced by contaminated sediments. The THg in the ASGM sites are higher than the SQGs-PEC values of 1.06 mg kg−1 defined by MacDonald et al. (2000). Without sediments toxicity survey, the comparison of THg concentrations values from ASGM sites with the SQGs-PEC, may indicates potential toxic effects of sediment contamination for biota and significant human health risks. The contamination of sediments by Hg can be attributed to mining activities, waste and tailings discharged into the river receiving systems. According to Niane et al. (2015), the level of Hg contamination of sediments already has an effect on wildlife with Hg levels in fish being below the guideline of World Health Organization, but dangerous for humans at risk, such as children and pregnant women.
Fig. 2. Gel picture illustrating the RFLP pattern observed in this study. Lane: 1–100 bp ladder (New England biolabs), lane-2: pattern A, lane-3, 5, 7, 8, 10: pattern B, lane-4: pattern C, lane-6: pattern D, lane-9: pattern E.
3.2. Mercury-resistant bacteria in sediment The total numbers of heterotrophic cultivable bacteria range between 3.7 × 106 and 4.6 × 108 CFU g−1 (Table 1). The resistance level of the isolated bacterial cells to Hg was evaluated by determining MIC values. Except for the site of Sabodala site (MIC 18 mg L−1 of Hg2+, Table 1) other sites sampled for bacterial suspensions present MIC values < 10 mg L−1. As expected, the greatest number of Hg-resistant bacteria was found at the Sabodala site which is characterized by a high concentration of THg in sediments of 6.2 mg kg−1 in sediment. The lowest number of Hg-resistant bacteria was detected at the Samekouta site which has the lowest THg concentration. This finding indicates that the abundance of Hg-resistant bacteria increased with increasing concentrations of mercury in the studied sediment samples (Table 1), this is
Table 1 Total mercury (THg) concentration of the host sediment, heterotrophic plate count and Hg-resistant bacteria isolated from contaminated and uncontaminated sediments of Gambia River. Sampling site
THg in sediment (mg kg−1)
Total bacteria (CFU g−1)
Hg resistant bacterial count expressed in colony forming units per gram (CFU g−1) 9a
Samekouta Bantako Tinkoto Sabodala
0.06 4.2 2.4 6.23
4.65 × 108 1.23 × 108 3.7 × 106 4.9 × 107
10a 0.6 × 102 10.1 × 102 3.3 × 102 7.2 × 103
0 0 0 5.6 × 103
12a 0 0 0 4.8 × 103
15a 0 0 0 1.2 × 103
17a 0 0 0 6.1 × 102
18a 0 0 0 0
a Concentration of Hg (mg L−1) amended solid medium to determine the MIC. In bold the MIC values. MIC (Minimum Inhibitor Concentration). In bold MIC for all studied sites.
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Fig. 3. Phylogenetic tree representing the neighbour-joining trees relationship for the 16s rDNA sequences (Neighbour-joining with bootstrapping). Values in red represent bootstrap values. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(Mathema et al., 2011). The reduction of Hg2+ to Hg0 is a way to remove oxidized Hg and to reduce Hg dissolved in a medium (Chowdury et al., 2012).
in line with previous contributions (Ball et al., 2007; Müller et al., 2001; Osborn et al., 1997). Previous studies reported that bacterial species isolated from aquatic environment have a high tolerance with respect to elevated concentrations of Hg2+ between 10 and 120 mg L−1 (e.g. Deng and Wang, 2012). In this study, no bacteria species were able to grow in a solid culturing medium contaminated with Hg2+ at a final concentration of 18 mg L−1. According to Canstein et al. (2002), the bacterium isolate that can grow on synthetic media with a minimum of 5 ppm HgCl2 is a highly Hg-resistant bacterium strain. According to Nascimento and Chartone-Souza (2003) bacterial resistance to Hg is the first step of the detoxification process. They also show that the detoxification of Hg, especially MeHg, is generally preceded by demethylation. Methyl mercury is demethylated into Hg2+, which is then later reduced to Hg0, which the volatile form and less toxic.
4. Conclusions This research presents the first assessment of Hg-resistant bacteria in contaminated and uncontaminated sediments from the Gambia River in eastern Senegal. The results indicate that ASGM activities are responsible for the Hg pollution of the river. We also demonstrated the presence of diverse groups of freshwater bacteria under tropical conditions capable of high tolerance to Hg. The isolates with Hg-resistant bacteria are higher in contaminated areas than in other uncontaminated sites. Moreover, for further research, the isolated strains should be evaluated with particular interest for testing the bioremediation in Hg-contaminated soil and sediments under tropical conditions.
3.3. Characterization of Hg-resistant bacteria Results from the RFLP analysis show considerable polymorphism and diversity of bacteria (Table 2). The profiles obtained by digestion with restriction enzymes HaeIII, AluI and RsaI 16S rDNA (Fig. 2) served for the selection of similar isolates (Poté et al., 2009). The 16S rDNA sequencing (gene bank accession number: LK871644 - LK871649) and blast analysis indicate the presence of different Hg-resistant bacteria, including Stenotrophomonas maltophilia, Dyella ginsengisoli, Arthrobacter defluvi, Arthrobacter pascens, Bacillus firmus and Pseudomonas moraviensis (Fig. 3). Previous studies performed in similar environmental settings demonstrated that cultivable heterotrophic bacterial species including both gram-negative and gram-positive bacteria such as, Pseudomonas, Alcaligenes, Brevibacterium, E. coli, Arthrobacter, Serratia marcescens, Acinetobacter, Bacillus and Micrococcus, spp., were identified to be highly resistant to Hg and other toxic metals (Chasanah et al., 2018; De and Ramaiah, 2007; Kotala et al., 2014; Mahbub et al., 2016; Mirzaei et al., 2008; Nascimento and Chartone-Souza, 2003; Petrova et al., 2002). Interestingly, in this research project, many of these bacterial species were to be able to develop a resistance to Hg. Our results indicate that Bacillus firmus and Pseudomonas moraviensis are able to grow in medium containing 17 mg L−1 of Hg2+. This is in accordance with previous studies which reported that Bacillus spp., and Pseudomonas moraviensis was found as a mercury resistant bacteria (Chasanah et al., 2018; Irawati et al., 2012). Irawati et al. (2012) have reported that Brevundimonas spp., accumulated up to 1.09 and 2.7 mg g−1 of Hg2+ in dry weight of cells, and removed 64.38 and 57.1% Hg2+ from a medium containing 50 and 100 ppm HgCl2 respectively. Recently in Indonesia, Chasanah et al. (2018) have reported that the highest Hg accumulation from nutrient broth liquid media containing 10, 20 and 30 ppm Hg was observed for Brevundimonas vesicularis. According to Gupta et al. (2012), Hg-resistant bacteria are through to have complex enzyme Hg reductase (MerA) and organo-Hg lyase (MerB), which allows the Hg incorporation through the cytoplasmic membrane into the cell, where it and accumulates. The combined action of MerA and MerB genes generates a broad spectrum Hg detoxification mechanism
Acknowledgements The authors thank Sida-UNESCO project 503RAF2000, and Lombard Foundation and Schmidheiny Fondation, Geneva, Switzerland for partial support of financing the study. The Department of Mines and Geology of Senegal (DMG), the Randgold Company Senegal, and University Cheikh Anta Diop (Dakar) for his logistic help. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Ball, M.M., Carrero, P., Castro, D., Yarzabal, L.A., 2007. Mercury resistance in bacterial strains isolated from tailing ponds in a gold mining area near El Callao (Bolivar State, Venezuela). Curr. Microbiol. 54, 149–154. Bertrand, H., Poly, F., Van, V.T., Lombard, N., Nalin, R., Vogel, T.M., Simonet, P., 2005. High molecular weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction. J. Microbiol. Methods 62, 1–11. Berzas Nevado, J.J., Rodríguez Martín-Doimeadios, R.C., Guzmán Bernardo, F.J., Jiménez Moreno, M., Herculano, A.M., do Nascimento, J.L.M., Crespo-López, M.E., 2010. Mercury in the Tapajós River basin, Brazilian Amazon: a review. Environ. Int. 36, 593–608. Bravo, A.G., Bouchet, S., Guédron, S., Amouroux, D., Dominik, J., Zopfi, J., 2015. High methylmercury production under ferruginous conditions in sediments impacted by sewage treatment plant discharges. Water Resources 80, 245–255. Bridou, R., Monperrus, M., Gonzalez, P.R., Guyoneaud, R., Amouroux, D., 2011. Simultaneous determination of mercury methylation and demethylation capacities of various sulfate-reducing bacteria using species-specific isotopic tracers. Environmental Toxicology Chemistry 30, 337–344. Canstein, H.V., Kelly, S., Li, Y., Wagner-Dobler, I., 2002. Species diversity improves the efficiency of mercury-reducing biofilm under changing environmental conditions. Appl. Environ. Microbiol. 68 (6), 2829–2837. CCME (Canadian Council of Ministers of the Environment), 1999. Canadian sediment quality guidelines for the protection of aquatic life: summary tables. In: Canadian environmental quality guidelines. Canadian Council of Ministers for the Environment, Winnipeg. Chasanah, U., Nuraini, Y., Handayanto, E., 2018. The potential of mercury-resistant bacteria isolated from small-scale gold mine tailings for accumulation of mercury. Journal of Ecological Engineering 19 (2), 236–245. Chowdury, S., Bala, N.N., Dhauria, P., 2012. International Journal of Pharmaceutical, Chemical, and Biological Science 2 (4), 600–611.
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river sediment: case of Gambia River, Kedougou region, southeastern Senegal. J. Geochem. Explor. 144, 517–527. Niane, B., Guédron, S., Moritz, R., Cosio, C., Ngom, P.M., Deverajan, N., Pfeifer, H.-R., Poté, J., 2015. Human exposure to mercury in artisanal small-scale gold mining areas of Kedougou region, Senegal as a function of occupational activity and fish consumption. Environ. Sci. Pollut. Res. 22 (9), 7101–7111. Niane, B., Guédron, S., Feder, F., Legros, S., Ngom, P.M., Moritz, R., 2019. Impact of recent artisanal small-scale gold mining in Senegal: mercury and methylmercury contamination of terrestrial and aquatic ecosystems. Sci. Total Environ. 669, 185–193. Osborn, A.M., Bruce, K.D., Strike, P., Ritchie, D.A., 1997. Distribution, diversity, and evolution of the bacterial mercury resistance (mer) operon. FEMS Microbiology Review 19, 239–262. Ouédraogo, O., Amyot, M., 2013. Mercury, arsenic and selenium concentrations in water and fish from sub-Saharan semi-arid freshwater reservoirs (Burkina Faso). Sciences of the Total Environment 444, 243–254. Petrova, M.A., Mindlin, S.Z., Gorlenko, Zh.M., Kaliaeva, E.S., Soina, V.S., Bogdanova, E.S., 2002. Mercury-resistant bacteria from permafrost sediments and prospects for their use in comparative studies of mercury resistance determinants. Genetika 38, 1569–1574. Poté, John, Mavingui, Patrick, Navarro, Elisabeth, Rosselli, Walter, Wildi, Walter, Simonet, Pascal, Vogel, Timothy M., 2009. Extracellular plant DNA in Geneva groundwater and traditional artesian drinking water fountains. Chemosphere 75, 498–504. Poté, J., Garcia Bravo, A., Mavingui, P., Ariztegui, D., Wildi, W., 2010. Evaluation of quantitative recovery of bacterial cells and DNA from different lake sediments by Nycodenz density gradient centrifugation. Ecol. Indic. 10, 234–240. Programme d'Appui au Secteur Minier PASMI, 2009. Cartographie géologique du Sénégal au 1/500000. Rapport Final. Projet 9 ACP SE 009. Rajaee, M., Obiri, S., Green, A., Long, R., Cobbina, S., Nartey, V., Buck, D., Antwi, E., Basu, N., 2015. Integrated assessment of artisanal and small-scale gold mining in Ghana—part 2: natural sciences review. Int. J. Environ. Res. Public Health 12 (8), 8971–9011. Raposo, J.C., Ozamiz, G., Etxebarria, N., Tueros, I., Munoz, C., Muela, A., Arana, I., Barcina, I., 2008. Mercury biomethylation assessment in the estuary of Bilbao (North of Spain). Environ. Pollut. 156, 482–488. Rasmussen, L.D., Zawadsky, C., Binnerup, S.J., Oregaard, G., Sorensen, S.J., Kroer, N., 2008. Cultivation of hard-to-culture subsurface mercury-resistant bacteria and discovery of new merA gene sequences. Appl. Environ. Microbiol. 74, 3795–3803. Ross-Barraclough, F., Givelet, N., Martinez-Cortizas, A., Goodsite, M.E., Biester, H., Shotyk, W., 2002. An analytical protocol for the determination of total mercury concentrations in solid peat samples. Sci. Total Environ. 292, 129–139. Spiegel, S.J., Veiga, M.M., 2010. International guidelines on mercury management in small-scale gold mining. J. Clean. Prod. 18 (4), 375–385. Susana, S., Dias, T., Ramalhosa, E., 2011. Mercurymethylation versus demethylation: main processes involved. In: Clampet, A.P. (Ed.), Methylmercury: Formation, Sources and Health Effects. Nova Science Publishers, New York, pp. 1–24. Sylla, M., Ngom, P.M., 1997. Le gisement d'or de Sabodala (Sénégal Oriental): une minéralisation filonienne d'origine hydrothermale remobilisée par une tectonique cisaillante. J. Afr. Earth Sci. 25, 183–192. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Wagner-Döbler, I., 2013. Bioremediation of Mercury: Current Research and Industrial: Applications. Horizon Scientific Press, Germany. Wang, X., Heazlewood, S.P., Krause, D.O., Florin, T.H.J., 2003. Molecular characterization of the microbial species that colonize human ileal and colonic mucosa by using 16S rDNA sequence analysis. J. Appl. Microbiol. 95, 508–520. Wang, Q., Kim, D., Dionysiou, D.D., Sorial, G.A., Timberlake, D., 2004. Sources and remediation for mercury contamination in aquatic systems – a literature review. Environ. Pollut. 131, 323–336. Young, R.A., 1992. Toxicity Summary for Methyl Mercury Oak Ridge Reservation Environmental Restoration Program.
Dash, H.R., Das, S., 2012. Bioremediation of mercury and the importance of bacterial mer genes. Int. Biodeterior. Biodegradation 75, 207–213. De, J., Ramaiah, N., 2007. Characterization of marine bacteria highly resistant to mercury exhibiting multiple resistances to toxic chemicals. Ecol. Indic. 7, 511–520. Deng, X., Wang, P., 2012. Isolation of marine bacteria highly resistant to mercury and their bioaccumulation process. Bioresour. Technol. 121, 342–347. Devereux, R., Winfrey, M.R., Winfrey, J., StahlD, A., 1996. Depth profile of sulfate-reducing bacterial ribosomal RNA and mercury methylation in an estuarine sediment. FEMS Microbiol. Ecol. 20, 23–31. Dhankher, O.P., Li, Y., Rosen, B.P., 2002. Engineering tolerance and hyper accumulation of arsenic in plants by combining arsenate reductase and gamma-glutamylcysteine synthetase expression. Nat. Biotechnol. 20, 1140–1144. Fleming, E.J., Mack, E.E., Green, P.G., Nelson, D.C., 2006. Mercury methylation from unexpected sources: molybdate-inhibited freshwater sediments and an iron-reducing bacterium. Appl. Environ. Microbiol. 72, 457–464. Garcia-Bravo, A., Bouchet, S., Amouroux, D., Poté, J., Dominik, J., 2011. Distribution of mercury and organic matter in particle-size classes in sediments contaminated by a waste water treatment plant: Vidy Bay, Lake Geneva, Switzerland. J. Environ. Monit. 13, 974–982. Gerson, J.R., Driscoll, C.T., Hsu-Kim, H., Bernhardt, E.S., 2018. Senegalese artisanal gold mining leads to elevated total mercury and methylmercury concentrations in soils, sediments, and rivers. Elementa Science of the Anthropocene 6, 11. Guedron, S., Grangeon, S., Lanson, B., Grimaldi, M., 2009. Mercury speciation in a tropical soil association; consequence of gold mining on Hg distribution in French Guiana. Geoderma 153, 331–346. Gupta, S., Goyal, R., Nirwan, J., Cameotra, S.S., Tejoprakash, N., 2012. Bio sequestration, transformation and volatilization of mercury by Lysinibacillus fusiformis isolated from industrial effluent. J. Microbiol. Biotechnol. 22, 684–689. Hall, G.E.M., Pelchat, P., 1997. Evaluation of a direct solid sampling atomic absorption spectrometer for the trace determination of mercury in geological samples. Analyst 122, 921–924. Irawati, W., Patricia, Soraya, Y., Baskoro, A.H., 2012. A study on mercury-resistant bacteria isolated from a gold mine in Pongkor Village, Bogor, Indonesia. Hayati Journal of Bioscience 19 (4), 197–200. Kotala, S., Kawuri, R., Gunam, I.B.W., 2014. The presence of mercury resistant bacteria in sediment of gold processing plant at Waekerta village of Buru district, Maluku province and their activity in reducing mercury. Current World Environment 9 (2), 271–279. Li, Y., Mao, Y., Liu, G., Tachiev, G., Roelant, D., Feng, X., Cai, Y., 2010. Degradation of methylmercury and its effects on mercury distribution and cycling in the Florida Everglades. Environmental Science Technology 44 (17), 6661–6666. MacDonald, D.D., Ingersoll, C.G., Berger, T.A., 2000. Development and evaluation of consensus-based sediment quality guidelines for freshwater ecosystems. Archives of Environmental Contamination of Toxicology 39, 20–31. Mahbub, K.R., Krishnan, K., Megharaj, M., Naidu, R., 2016. Bioremediation potential of a highly mercury resistant bacterial strain Sphingobium SA2 isolated from contaminated soil. Chemosphere 144, 330–337. Mathema, V.B., Thakuri, B.C., Sillanpä, M., 2011. Bacterial mer operon-mediated detoxification of mercurial compounds: a short review. Archives Microbiology 193, 837–844. McCarthy, D., Edwards, G.C., Gustin, M.S., Care, A., Miller, M.B., Sunna, A., 2017. An innovative approach to bioremediation of mercury contaminated soils from industrial mining operations. Chemosphere 184, 694–699. Mirzaei, N., Kafilzadeh, F., Kargar, M., 2008. Isolation and identification of resistant bacteria from Kor River, Iran. J. Biol. Sci. 8, 935–939. Müller, A.K., Rasmussen, L.D., Sorensen, S.J., 2001. Adaptation of the bacterial community to mercury contamination. FEMS Microbiol. Lett. 204, 49–53. Nascimento, A.M.A., Chartone-Souza, E., 2003. Operon mer: bacterial resistance to mercury and potential for bioremediation of contaminated environments. Genet. Mol. Res. 2, 92–101. Niane, B., Moritz, R., Guédron, S., Ngom, P.M., Pfeifer, H.-R., Mall, I., Poté, J., 2014. Effect of recent artisanal small-scale gold mining on the contamination of surface
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Quantification and characterization of mercury resistant bacteria in sediments contaminated by artisanal small-scale gold mining activities, Kedougou region, Senegal
T
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Birane Nianea, , Naresh Devarajanb, John Potéc, Robert Moritza a
Department of Earth Sciences, University of Geneva, rue des Maraîchers 13, CH-1205 Geneva, Switzerland Bren School of Environmental Science and Management, University of California Santa Barbara, USA c Department F.-A. Forel for Environmental and Aquatic Sciences, University of Geneva, boulevard Carl-Vogt 66, CH-1211 Geneva 4, Switzerland b
A R T I C LE I N FO
A B S T R A C T
Keywords: Mercury resistant bacteria Artisanal small-scale gold mining Bioremediation
This study describes Hg-resistant bacterial present in the aquatic sediments artisanal small-scale gold mining ASGM activities along Gambia River Kedougou, Senegal. Mercury (Hg) is used for gold amalgamation in artisanal small-scale gold mining (ASGM) activities. The level of total Hg in sediment samples was determined by automatic mercury analyser. Bacterial (colony-forming units) susceptibility to Hg was evaluated by minimal inhibitory concentrations. The phylogenetic diversity analysis of the Hg-resistant bacteria was performed by PCR amplification of 16S rDNA on isolated bacterial strains, followed by restriction fragment length polymorphism, cloning and sequencing. The results documented high concentrations of Hg in ASGM activity areas ranging from 2.4 to 6.2 mg kg-1. Population densities of heterotrophic bacteria in wet sediment ranging from 3.7 × 106 to 4.6 × 108 CFU g−1. The isolated bacterial strains from highly Hg-contaminated sites can grow to medium containing up to 17 mg L−1 of Hg2+. In this study, bacterial strains resistant to Hg are Stenotrophomonas maltophilia, Dyella ginsengisoli, Arthrobacter defluvi, Arthrobacter pascens, Bacillus firmus and Pseudomonas moraviensis. Our results demonstrate the occurrence the presence of diverse groups of bacterial strains resistant to metal (Hg) under tropical conditions. The isolated strains are particularly interesting for further studies to evaluate their role in bioremediation of Hg in contaminated aquatic ecosystems.
1. Introduction Mercury (Hg) is a well-known environmental pollutant, and its emission to the environment can have serious effects on human and animal health. In aquatic systems, Hg exists in various forms: elemental (Hg0), which is the only metal in liquid form at room temperature, inorganic (Hg2+) and organic forms. According to Young (1992), the most toxic is organic Hg, because it accumulates in the food chain and has affinity with sulfhydryl groups in the proteins of living organisms. Several studies on Hg in water, sediment, fish and human have been carried out (e.g., Berzas Nevado et al., 2010; Garcia-Bravo et al., 2011; Guedron et al., 2009), and have identified the principal sources of Hg contamination in the aquatic environment, including gold mining activities, atmospheric deposition, erosion, urban discharge, agricultural material, combustion and industrial discharge. In sub-Saharan Africa, (such as Burkina Faso, Tanzania, Zimbabwe, Ghana and Senegal), limited studies have been conducted on the assessment of mercury
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contamination in artisanal small-scale gold mining areas, as summarized by the recent studies (Gerson et al., 2018; Niane et al., 2014, 2015, 2019; Ouédraogo and Amyot, 2013; Rajaee et al., 2015). These studies reveal that the amalgamation process using Hg is the most widely used technique to recover gold during artisanal activities. During this process a large amount of Hg is transferred to both terrestrial and aquatic ecosystems. The deposited mercury, can react with various organic compounds in the aquatic environment by biotic (sulfur-reducing bacteria) and abiotic (sunlight) pathway, resulting in conversion of organic mercury (Wang et al., 2004). Even small amounts of Hg (esp. inorganic mercury) in water can be toxic for all organisms (Mirzaei et al., 2008). According to Canadian interim marine sediment quality guidelines, inorganic Hg accumulates in sediments and may be a hazard to sediment-dwelling organisms at concentrations above 0.13 mg kg−1 (CCME, 1999). Below this concentration there can be toxic effects if MeHg is present. The impacts of Hg on human health and the environment are well documented around the world (Spiegel and Veiga,
Corresponding author at: DMG/Ministère des Mines et de la Géologie, Sphères Ministérielles Diamniadio-Bâtiment B 5eme Etage, Dakar, Senegal. E-mail addresses: [email protected], [email protected] (B. Niane).
https://doi.org/10.1016/j.gexplo.2019.106353 Received 30 September 2018; Received in revised form 21 July 2019; Accepted 3 August 2019 Available online 05 August 2019 0375-6742/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Location map of the gold mining sites and sampling location. (Modified from Niane et al., 2014)
2017). Sediments are complex habitats, densely colonized by diverse groups of microorganisms, which play key roles in biogeochemical cycling, aquatic food webs, remobilization of Hg, as well as Hg speciation (Garcia-Bravo et al., 2011; Poté et al., 2010; Wang et al., 2004). Some anaerobic bacteria and Archaea have evolved resistance mechanisms that function to degrade organic Hg compounds to inorganic Hg by reduction to gaseous Hg(0). Mercury resistance consisting the Mercuric reductase (MerA, B) protein, the core enzyme in the microbial mercury detoxification system, catalyzes the reduction of Hg(II) to volatile Hg(0) (Li et al., 2010; Susana et al., 2011). The studies performed in sub-Saharan African region were mainly focused on the quantification of mercury contamination in the aquatic environments (water, soil, sediment, fish) and human (hairs) (Gerson et al., 2018; Niane et al., 2014, 2015). To our knowledge, there is a lack of information to assess the impact of Hg pollution on bacterial communities under tropical conditions, such as the study region in Kedougou, eastern Senegal. The objective of this study was to isolate and characterize Hg-resistant bacterial communities in the aquatic sediments of Gambia River Kedougou in the Gambia River Kedougou, Senegal, located in the vicinity of ASGM activities. The total mercury (THg) concentrations, were measured in contaminated and uncontaminated sediment samples from Gambia River and Hg-resistant bacteria were quantified and characterized in the samples.
2010). Methylmercury can attack the human nervous system through the bloodstream, and inhalation of Hg vapor at high levels can result in acute, corrosive bronchitis and interstitial pneumonitis (Gupta et al., 2012; Rasmussen et al., 2008). Due to its persistence in the environment, remediation is necessary to in contaminated Hg sites, such as soils and aquatic systems in the vicinity of ASGM activities. According to Wang et al. (2004), methods of Hg remediation/detoxification in contaminated sites include capping, dredging, precipitation, filtration, ion exchange resin absorption using carbon and other techniques. However, these technologies are relatively expensive, not environmentally friendly, and can in turn produce new environmental problems (Wagner-Döbler, 2013). Alternately, bioremediation using Hg-resistant bacteria has become a fast growing effective technology with many advantages over physicochemical methods (Dhankher et al., 2002). Bacteria are the most ubiquitous organisms found in various environments, including soil and sediment. Several studies have been performed to understand the role of microorganisms for Hg speciation in the aquatic environment (e.g., Devereux et al., 1996; Fleming et al., 2006; Bravo et al., 2015; Osborn et al., 1997; Raposo et al., 2008). They revealed that Hg methylation is known to be favored in suboxic to anoxic environments (e.g., river sediment) and is mostly driven by sulfate-reducing bacteria (SRB). Bridou et al. (2011) have aimed to identify the SRB strains were responsible for Hg methylation by testing their Hg-methylating potential in pure cultures. Some bacterial communities are capable to overcome such toxicity and developed mechanisms that allow them to be resistant to Hg. According to Nascimento and Chartone-Souza (2003), Hg removal by bacteria from contaminant sources is a challenge for the environmental, but may provide a promising technology for bioremediation. Bioremediation of Hg in contaminated environments has been discussed in the recent literature (e.g. Dash and Das, 2012; McCarthy et al.,
2. Materials and methods 2.1. Study sites and sampling The Kedougou region is the major gold province of Senegal. Its geology is dominated by volcano-sedimentary terrains of Birimian age. Approximately 30,000 to 60,000 persons are currently involved in ASGM activities spread across several villages in Eastern Senegal 2
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ultracentrifuge). A white layer of bacterial cells was obtained at the interface between the Nycodenz-soil mix particles and the overlying aqueous layer. This white layer was carefully recovered, and mixed with an equal volume of sterile ultrapure water, then centrifuged at 7500 ×g for 20 min at 10 °C. To remove traces of Nycodenz, pelleted bacteria were resuspended in 20 ml sterile ultrapure water and centrifuged for 20 min at 7500 ×g. The pelleted bacteria were resuspended in sterile molecular biology grade water and preserved in glycerol (10% v/v) at −80 °C until used.
(PASMI, 2009), where the region of Kedougou is considered as the major ASGM center. Alluvial gold and gold-bearing quartz veins hosted by shear zones are the two types of gold ore mining in Kedougou (Sylla and Ngom, 1997). There are increasing activities of ASGM activities throughout the region and other areas along the African gold belt since 1995, as a result of the rising price of gold and low cost of ASGM technologies. About 10 years ago, mercury (Hg) has been introduced into the ore dressing process by the mining communities to improve recovery of gold using Hg-amalgamation. Niane et al. (2015) have described the effect of Hg contamination in surface river sediments, the aquatic fauna (mainly fishes) and mining communities of the Kedougou region, using hair samples. Sediment samples were collected during a field campaign, at the beginning of the wet season in June 2013 at four selected sampling sites. Two sites were sampled along Gambia River at Samekouta and Bantako, and two sites at local water ponds at Sabodala and Tinkoto (Fig. 1). Samekouta is defined as the control site, because it is located in an up-stream location and is devoid of any ASGM activities. By contrast, intense ASGM activities are developed at Tinkoto, Bantako and Sabodala. About 20 cm-long sediment cores were collected manually, below a water depth of about 50 cm, and using acrylic tubes with a diameter of 8–10 cm of. Twenty composite samples were collected. Five individual sub samples were thoroughly homogenized from each sampling site to achieve a composite sample. After sampling, the core sediments were stored at 4 °C and transported to the Institute F.A. Forel at the University of Geneva (Switzerland) for bacteriological analysis within 72 h.
2.4. Heterotrophic plate count and isolation of Hg-resistant bacteria The assessment of Hg-resistant bacteria can be performed in different medium including Tryptic Soy Agar (TSA), Tripton Iron Agar (TIA), MS Agar, Luria Bertani Agar, or other ones as summarized by Mirzaei et al. (2008). In this study the heterotrophic plate count (HPC) was performed using the method described by Poté et al. (2010) to determine the Colony-Forming Units (CFU) using tryptic soy agar (TSA) (OXOID, LTD, Basingstoke, England) medium supplemented with the antifungal (Nystatin 50 mg ml−1, Sigma Aldrich, GmbH, Germany). Serially diluted bacterial suspension from the Nycodenz method were spread platted on the medium and incubated at 28 °C for 72 h. The results were expressed as CFU units per g of wet sediment (CFU g−1). Bacterial strains resistant to Hg were isolated on TSA medium supplemented with serial concentrations (10, 12, 15, 17, 18 and 20 mg L−1) of Hg2+. The minimum concentration of Hg2+ that inhibits bacterial growth with no visible colonies in the plates after the 72 h incubation (at 28 °C) was recorded as Minimum Inhibitory Concentration (MIC) values. All analyses were conducted in triplicate for each set of conditions.
2.2. Sediment Hg analysis Sediment samples were freeze-dried before analysis, and ground to a fine homogeneous powder (< 63 μm). Analysis of total Hg in sediment samples was carried out using the Atomic Absorption Spectrometry (AAS) with an Advanced Mercury Analyser (AMA 254, Altec s.r.o, Czech Rep.), following the method described by Hall and Pelchat (1997) and Ross-Barraclough et al. (2002). This method is based on sample combustion, gold amalgamation and AAS. The accuracy of the measurements was obtained by repeated analyses of the certified reference material MESS-3-concentrations found (0.089 ± 0.006 mg kg−1, N = 3) agreed with the certified concentration (0.091 ± 0.008 mg kg−1). The detection limit defined as three times the standard deviation of the blank was 0.005 mg kg−1 and the reproducibility was better than 95%.
2.5. 16s rDNA amplification After enumerating Hg-resistant bacteria several single colonies were picked for DNA extraction using Ultraclean soil DNA Kit (Mo Bio Labs, Solana Beach, CA 92075). Phenotypic characterization of the bacterial species was conducted based on morphology, 16s rDNA amplification, restriction fragment length polymorphism (RFLP) and sequencing. Briefly, morphologically different colonies were picked from each plate at their nearest MIC values. A single colony from the overnight culture was suspended in 50 μL of polymerase chain reaction (PCR) grade water, incubated at 94 °C for 10 min and centrifuged. Five microliters of the supernatant were used as template for the PCR reaction to amplify the 16s rDNA. Amplification of the 16s rDNA was performed using the universal bacterial primer set (Wang et al., 2003) 27f (AGAGTTTGAT CMTGGCTCAG) and 1492r (TACGGYTACCTTGTTACGACTT). PCR was carried out in 50 μL reaction volume containing 1× PCR buffer, 2 mM MgCl2, 200 μM of each dNTP, 200 nM of each primer, 5 units of Taq DNA polymerase (Takara Ex Taq, Hot Start Version, Takara Bio INC, Japan) and template DNA (100 ng). PCR was performed in a Biometra thermocycler (BIOLABO, France) with the following sequence: an initial denaturation at 94 °C for 5 min; 35 cycles of denaturation (94 °C for 45 s), annealing (58 °C for 45 s), extension (72 °C for 45 s); and a final extension for 5 min at 72 °C. PCR products were separated and visualized using electrophoresis on 0.8% agarose gel stained with SYBR safe DNA gel stain (Invitrogen).
2.3. Bacterial cell extraction from sediments samples Density gradient centrifugation was used for separation of complexes based on their molecular masses. In this study, the separation of bacteria and sediment matrix was performed using the Nycodenz™ density gradient centrifugation as described by Bertrand et al. (2005), and slightly modified by Poté et al. (2010). Briefly, 100 g (wet weight) of sediments and 500 ml of sterile 0.2% sodium hexametaphosphate (Na2(PO3)6, E. Lotti S.A, Switzerland) were dispersed in 1 L sterile plastic bottles and mixed for 1 h using the agitator rotary printing-press Watson-Marlow 601 controller (SKAN, Switzerland) at room temperature. The sample was then centrifuged at 750 ×g for 15 min at 15 °C in a 3 K-1 (Sigma, centrifuge). The supernatant (S2) were collected and filtered with sterile gauze. The supernatant was then centrifuged at 7500 ×g for 30 min at 10 °C. The pellet containing the microbial cell fraction was resuspended in sterile 0.8% sodium chloride solution by vortexing. Twenty-five milliliters of the homogeneous solution was transferred to an ultracentrifuge tube containing 11 ml of Nycodenz solution (Axis-Shield, Oslo, Norway) with a density of 1.3 g/ml density (8 g of Nycodenz to 10 ml of sterile ultrapure water). Bacterial cells and sediment particles were separated by high-speed centrifugation (15,000 ×g for 1 h at 10 °C) in Centrikon T-1080 (Kontron, Instrument,
2.6. RFLP, cloning and sequencing The PCR products were analyzed by Restriction Fragment Length Polymorphism (RFLP) as described by Poté et al. (2009). RFLP was performed on PCR products using 3 (HaeIII, AluI and RsaI) endonucleases (FastDigest, Fermentas). Fragments with distinct RFLP patterns were cloned to pGEM-T easy vectors following the manufacture protocol and grown in competent E. coli cells (DHα). Plasmids containing the fragment of the 16s rDNA, extracted from an overnight 3
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culture using plasmid extraction kit (GENELUTE HP PLASMID MINIPREP, Sigma Aldrich, Germany) were submitted to sequencing at StarSEQ GmbH (Germany). Sequences were compared at NCBI data bank using the BLASTN algorithm (Altschul et al., 1990). On the basis of the results of database analyses, sequences were aligned with representative bacterial sequences from GenBank (www.ncbi.nlm.nih. gov/blast) and used for Phylogenetic tree construction by NeighbourJoining method using MEGA 6.05 (Tamura et al., 2013).
Table 2 The bacterial profiles analyzed by RFLP. Sampling sites
Bacterial profile type
Samekouta Bantako Tinkoto Sabodala
B C, D A, B E
Number of bacterial isolates with the same profile type 4 5 5 4
A, B, C, D, and E indicate the type of RFLP profile observed.
2.7. Data analysis The Kolmogorov–Smirnov test was used to evaluate the normality of the data. The Kruskal–Wallis test was used for multiple sample comparison (H test). All the statistical tests were performed using STATISTICA 12.0 software, and a p < 0.05 was chosen to indicate the statistical significance.
3. Results and discussion 3.1. Mercury concentration in sediment The THg concentration in surface sediments is reported in Table 1. The concentration of THg are 4.2, 2.4, 6.2 mg kg−1, for the sites of Bantako, Tinkoto and Sabodala, respectively. The highest concentration of THg is recorded in Sabodala and the lowest at Samekouta site. According to Niane et al. (2015), the strong positive correlations between THg concentrations and organic matter (OM) in sediment sampled at Bantako during the wet season, suggests that OM in this case is a major carrier phase of Hg in the Gambia River sediments. However, sediments in Sabodala and Tinko are characterized by a strong positive correlation between OM and Thg for both seasons, suggesting that in area where OM is abundant, Hg is mainly bound to OM. Total mercury concentrations recorded in the gold mining study sites (Bantako, Tinkoto, Sabobala) were compared with the sediment quality criteria for freshwater ecosystems published by MacDonald et al. (2000). The sediment Quality Guidelines (SQGs) is based in two thresholds concentrations: a Probable Effect Concentration (PEC) above which toxic effects are likely to be induced, and a threshold effect level (TEL) below which no harmful effect is produced by contaminated sediments. The THg in the ASGM sites are higher than the SQGs-PEC values of 1.06 mg kg−1 defined by MacDonald et al. (2000). Without sediments toxicity survey, the comparison of THg concentrations values from ASGM sites with the SQGs-PEC, may indicates potential toxic effects of sediment contamination for biota and significant human health risks. The contamination of sediments by Hg can be attributed to mining activities, waste and tailings discharged into the river receiving systems. According to Niane et al. (2015), the level of Hg contamination of sediments already has an effect on wildlife with Hg levels in fish being below the guideline of World Health Organization, but dangerous for humans at risk, such as children and pregnant women.
Fig. 2. Gel picture illustrating the RFLP pattern observed in this study. Lane: 1–100 bp ladder (New England biolabs), lane-2: pattern A, lane-3, 5, 7, 8, 10: pattern B, lane-4: pattern C, lane-6: pattern D, lane-9: pattern E.
3.2. Mercury-resistant bacteria in sediment The total numbers of heterotrophic cultivable bacteria range between 3.7 × 106 and 4.6 × 108 CFU g−1 (Table 1). The resistance level of the isolated bacterial cells to Hg was evaluated by determining MIC values. Except for the site of Sabodala site (MIC 18 mg L−1 of Hg2+, Table 1) other sites sampled for bacterial suspensions present MIC values < 10 mg L−1. As expected, the greatest number of Hg-resistant bacteria was found at the Sabodala site which is characterized by a high concentration of THg in sediments of 6.2 mg kg−1 in sediment. The lowest number of Hg-resistant bacteria was detected at the Samekouta site which has the lowest THg concentration. This finding indicates that the abundance of Hg-resistant bacteria increased with increasing concentrations of mercury in the studied sediment samples (Table 1), this is
Table 1 Total mercury (THg) concentration of the host sediment, heterotrophic plate count and Hg-resistant bacteria isolated from contaminated and uncontaminated sediments of Gambia River. Sampling site
THg in sediment (mg kg−1)
Total bacteria (CFU g−1)
Hg resistant bacterial count expressed in colony forming units per gram (CFU g−1) 9a
Samekouta Bantako Tinkoto Sabodala
0.06 4.2 2.4 6.23
4.65 × 108 1.23 × 108 3.7 × 106 4.9 × 107
10a 0.6 × 102 10.1 × 102 3.3 × 102 7.2 × 103
0 0 0 5.6 × 103
12a 0 0 0 4.8 × 103
15a 0 0 0 1.2 × 103
17a 0 0 0 6.1 × 102
18a 0 0 0 0
a Concentration of Hg (mg L−1) amended solid medium to determine the MIC. In bold the MIC values. MIC (Minimum Inhibitor Concentration). In bold MIC for all studied sites.
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Fig. 3. Phylogenetic tree representing the neighbour-joining trees relationship for the 16s rDNA sequences (Neighbour-joining with bootstrapping). Values in red represent bootstrap values. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(Mathema et al., 2011). The reduction of Hg2+ to Hg0 is a way to remove oxidized Hg and to reduce Hg dissolved in a medium (Chowdury et al., 2012).
in line with previous contributions (Ball et al., 2007; Müller et al., 2001; Osborn et al., 1997). Previous studies reported that bacterial species isolated from aquatic environment have a high tolerance with respect to elevated concentrations of Hg2+ between 10 and 120 mg L−1 (e.g. Deng and Wang, 2012). In this study, no bacteria species were able to grow in a solid culturing medium contaminated with Hg2+ at a final concentration of 18 mg L−1. According to Canstein et al. (2002), the bacterium isolate that can grow on synthetic media with a minimum of 5 ppm HgCl2 is a highly Hg-resistant bacterium strain. According to Nascimento and Chartone-Souza (2003) bacterial resistance to Hg is the first step of the detoxification process. They also show that the detoxification of Hg, especially MeHg, is generally preceded by demethylation. Methyl mercury is demethylated into Hg2+, which is then later reduced to Hg0, which the volatile form and less toxic.
4. Conclusions This research presents the first assessment of Hg-resistant bacteria in contaminated and uncontaminated sediments from the Gambia River in eastern Senegal. The results indicate that ASGM activities are responsible for the Hg pollution of the river. We also demonstrated the presence of diverse groups of freshwater bacteria under tropical conditions capable of high tolerance to Hg. The isolates with Hg-resistant bacteria are higher in contaminated areas than in other uncontaminated sites. Moreover, for further research, the isolated strains should be evaluated with particular interest for testing the bioremediation in Hg-contaminated soil and sediments under tropical conditions.
3.3. Characterization of Hg-resistant bacteria Results from the RFLP analysis show considerable polymorphism and diversity of bacteria (Table 2). The profiles obtained by digestion with restriction enzymes HaeIII, AluI and RsaI 16S rDNA (Fig. 2) served for the selection of similar isolates (Poté et al., 2009). The 16S rDNA sequencing (gene bank accession number: LK871644 - LK871649) and blast analysis indicate the presence of different Hg-resistant bacteria, including Stenotrophomonas maltophilia, Dyella ginsengisoli, Arthrobacter defluvi, Arthrobacter pascens, Bacillus firmus and Pseudomonas moraviensis (Fig. 3). Previous studies performed in similar environmental settings demonstrated that cultivable heterotrophic bacterial species including both gram-negative and gram-positive bacteria such as, Pseudomonas, Alcaligenes, Brevibacterium, E. coli, Arthrobacter, Serratia marcescens, Acinetobacter, Bacillus and Micrococcus, spp., were identified to be highly resistant to Hg and other toxic metals (Chasanah et al., 2018; De and Ramaiah, 2007; Kotala et al., 2014; Mahbub et al., 2016; Mirzaei et al., 2008; Nascimento and Chartone-Souza, 2003; Petrova et al., 2002). Interestingly, in this research project, many of these bacterial species were to be able to develop a resistance to Hg. Our results indicate that Bacillus firmus and Pseudomonas moraviensis are able to grow in medium containing 17 mg L−1 of Hg2+. This is in accordance with previous studies which reported that Bacillus spp., and Pseudomonas moraviensis was found as a mercury resistant bacteria (Chasanah et al., 2018; Irawati et al., 2012). Irawati et al. (2012) have reported that Brevundimonas spp., accumulated up to 1.09 and 2.7 mg g−1 of Hg2+ in dry weight of cells, and removed 64.38 and 57.1% Hg2+ from a medium containing 50 and 100 ppm HgCl2 respectively. Recently in Indonesia, Chasanah et al. (2018) have reported that the highest Hg accumulation from nutrient broth liquid media containing 10, 20 and 30 ppm Hg was observed for Brevundimonas vesicularis. According to Gupta et al. (2012), Hg-resistant bacteria are through to have complex enzyme Hg reductase (MerA) and organo-Hg lyase (MerB), which allows the Hg incorporation through the cytoplasmic membrane into the cell, where it and accumulates. The combined action of MerA and MerB genes generates a broad spectrum Hg detoxification mechanism
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