The chemical species of mercury accumulated by Pseudomonas idrijaensis, a bacterium from a rock of the Idrija mercury mine, Slovenia.
Introduction
Although mercury deposits are globally distributed in 26 mercury mineral belts, nearly three quarters of the total world’s production has originated from just five mercury belts (Rytuba, 2003). Almadén, the most important mercury belt comprising 11 mercury deposits in central Spain, has produced over one-third of the world’s mercury. The second largest mercury mine in the world was Idrija, Slovenia. Idrija is located about 50 km west of the Slovenian capital Ljubljana. The mine is 1500 m long and 300–600 m wide and extends below the surface of the Idrija valley in the NW – SE direction. It has produced more than 12.7 Mt of ore with 145,000 t Hg (average content of 1.13% Hg) since 1490 (Mlakar, 1974). The production of mercury stopped in 1988 and the mine finally closed in 1995. Idrija still contains 10% of the world’s known mercury reserves (Brinck and van Wambeke, 1975). Idrija was considered for centuries to be the center of scientific and technological progress in the region. Mining operations in more than 150 orebodies extended vertically over 360 m (+330 to - 33 m) on fifteen levels (Lavrič and Spangenberg, 2003). Ore minerals are cinnabar, metacinnabar, native mercury, pyrite and scarce barite (Palinkaš et al., 2008). The host rocks and ore itself contain organic matter as disseminated kerogen, black solid hydrothermal bitumen, and greenish idrialite, a complex mixture of polycyclic aromatic hydrocarbons containing nitrogen and sulfur-bearing compounds (Lavrič and Spangenberg, 2001). Half a millennium of Hg production has resulted in elevated mercury content in all environmental compartments (Gosar et al., 2016). The Soca River (Isonzo River in Italy) and its tributary, the Idrija River, have drained for centuries the Hg-enriched deposits of the Idrija mine, resulting in trapping of Hg in the sediments of the Gulf of Trieste, that in turn constitute a net source of MeHg (Faganeli et al., 2003). On the banks of the Idrija River, the total Hg and MeHg concentrations in soils are ranging from 0.25 to 1650 mg/kg and not detected to 0.44 mg/kg, respectively (Tomiyasu et al., 2017). Hg levels in foodstuffs from the Idrija mine area were higher compared to those in food from non-contaminated areas (Miklavčič et al., 2013). Retired Idrija mine workers and Idrija residents occupationally unexposed contained much higher Hg concentrations in their tissues (sampled by autopsy) than control individuals (Falnoga et al., 2000). It was shown that the long-term occupational exposure to Hg endured by the Idrija mine workers caused renal dysfunction (Franko et al., 2005). In 2012, Almadén and Idrija were included to the Mercury World Heritage list of the UNESCO. Total Hg concentration in contaminated soils has been reported to vary between 8.4 and 415 mg/kg (Kocman et al., 2004) and between 0.3 and 973 mg/kg (Gosar et al., 2006).
Hg reduces the diversity of bacterial communities in soil, and this decrease is directly proportional to the Hg concentration in soil (Rasmussen et al., 2000). To survive such a hostile environment bacteria are endowed with the mer operon. This operon provides bacterial resistance to inorganic and organic Hg through the action of the mercuric reductase encoding merA gene and the organomercurial lyase encoding merB gene (Barkay et al., 2003; Mathema et al., 2011). Genes of the mer operon (merA and merB) have been previously detected in water samples downstream of the mine, not upstream (Hines et al., 2000). The Hg-resistant bacterium Acinetobacter idrijaensis has been isolated from a soil on the Idrija site. It contains in its genome the merB, merA, merP, merT, and merR genes (Campos-Guillén et al., 2014). This strain is able to grow in the presence of high concentrations of HgCl2 (200 mg/L, 737 μM) and catalyzes the volatilization of Hg (merA gene).
Here, we document a new bacterial species named Pseudomonas idrijaensis. It has been recovered from a rock of the Idrija mine situated approximately 104 m below the surface and containing as much as 2952 mg Hg/kg. We show that this bacterial strain can thrive in such a toxic environment because it contains the mer operon in its genome. Another goal of the present study was to address the question related to the chemical forms of the Hg atoms within the bacterial cells after exposure to inorganic Hg and the possible influence of the mer operon on these chemical forms.
Section snippets
Sampling and characterization of the rock
A piece of black shale/siltstone from the Upper Ladinian formation was sampled aseptically in April 2017 at the third mine level, 110 m below the entrance to Borba Shaft situated 226 m above sea level (Fig. S1). The rock was put in a sterile glass bottle and transferred to the laboratory within 4 h. We chose this rock because it contained both native elemental mercury and organic matter. The air temperature in mine was 13 °C and the air humidity 84% at the time of sampling.
The chemical
Characterization of the rock
The pH value of the rock powder was 3.67. Quartz and mica are the main constituents (Table S1), in agreement with chemical analysis (Table 1, Table 2). They are followed by kaolinite and gypsum. Pyrite, goethite and rozenite are minor (Table S1). Goethite, rozenite and gypsum are weathering products of pyrite. No mercury-bearing phases such as cinnabar and metacinnabar were identified in the sample. Yet, this rock contains a high level of mercury, 2952 mg/kg, and several other toxic metals and
Discussion
The rock sample contains four times less Hg (2952 mg/kg) than the Idrija deposit on average (Mlakar, 1974), and almost seventeen times less than the Almadén mine on average (5%, Higueras et al., 2011). The Hg content of 2952 mg/kg is, however, two times higher than the highest Hg content of 1650 mg/kg reported in soil near the Idrija mine (Tomiyasu et al., 2017).
Comparison of the amounts of Hg added to the living bacteria and measured in the biomass after 195 min exposure shows that bacteria
Conclusion
While the Tn5041 element occurs most commonly on plasmids, it is inserted in the chromosome of P. idrijaensis like for the Pseudomonas strain KHP41, Pseudomonas sp. strain 1239 and P. putida DRA525 (Kholodii et al., 1997; Crovadore et al., 2018; Drace et al., 2018). This mobile element is old since it has been found in P. fluorescens bacteria isolated from Siberian permafrost dated to the Pleistocene, about 15,000 to 40,000 BP (Petrova et al., 2002; Mindlin et al., 2005). The permafrost soil
CRediT authorship contribution statement
Jean-Paul Bourdineaud: Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration, Funding acquisition. Goran Durn: Methodology, Validation, Investigation, Writing - review & editing, Visualization. Bojan Režun: Resources. Alain Manceau: Methodology, Validation, Investigation, Writing - original draft, Writing - review & editing, Visualization, Funding acquisition. Jasna Hrenović:
Declaration of competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work has been supported in part by the Croatian Science Foundation (project no. IP-2014-09-5656). We thank S. Kazazić, Ruđer Bošković Institute for MALDI-TOF MS identification and B. Hunjak, Croatian Institute of Public Health for enabling the use of Vitek system. The French National Research Agency (ANR) under grant ANR-12-BS06-0008-01 provided support to J-P.B. Support was provided to A.M by the French National Research Agency (ANR) under grant ANR-10-EQPX-27-01 (EcoX Equipex). We thank
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