Bioaccumulation of Mercury by Bacteria Isolated from Small Scale Gold Mining Tailings in Lombok, Indonesia

The activity of small scale gold mining (ASGM) in Indonesia has expanded to Lombok Island, West Nusa Tenggara. The use of mercury (Hg) and the traditional mining process cause that the ASGM tailings waste still contains Hg. The tailings waste is piled up near paddy fields and residential areas so that it has the potential to pollute the environment. This study aimed at isolating the bacteria that can accumulate Hg from the ASGM tailings in Lombok. The tailing samples were taken from Sekotong, Pujut, and Jonggat Districts, Lombok. The bacteria that have been isolated from the tailing samples were tested for their ability of Hg hemolysis resistance, and Hg bioaccumulation. Three bacteria with the highest Hg bioaccumulation ability were identified using the 16S rDNA gene squad. The results showed that out of 32 bacterial isolates, 27 isolates were found to be tolerant to Hg. Two bacterial isolates that were able to accumulate the highest amount of Hg and were identified as Fictibacillus nanhainensis (SKT-B) 82.25% and Bacillus toyonensis (PJM-F1) 81.21%. This study proved that the bacteria isolated from ASGM tailings have a great potential to be used as Hg bioaccumulation agents.


INTRODUCTION
Small Scale Gold Mining (ASGM) activities are increasing in many low and middle-income countries, one of which is Indonesia (Basu et al., 2015). One of the islands in Indonesia that is the location of ASGM is Lombok, West Nusa Tenggara (Krisnayanti et al., 2012). The gold mining in Lombok is carried out using the amalgamation and cyanidation methods (Muddarisna et al., 2013). However, in some areas in Lombok, such as Sekotong, miners use the amalgamation method to separate gold from tailings (Utomo et al., 2014). In the amalgamation method, gold is extracted from gold ore using 'gelondong' (small iron cylindrical tubes) and mercury (Hg) (Krisnayanti, 2018). The traditional mining process causes an increase of the Hg contamination from tailings to the environment. Every gram of gold produced through the amalgamation process releases 1-3 g of Hg into the environment through the tailings that are piled around the mining sites (Krisnayanti et al., 2012). Hg is one of the toxic elements that negatively affect the quality of the soil environment. The presence of Hg in the soil environment is associated with the presence of this metal in the lithosphere. Hg is present in very small amounts, but it can pose a threat to the animal and human organisms (Małuszyński et al., 2019). The human activities are increasing the heavy metals concentration in the ecosystem (Tjahjono and Suwarno, 2018).
Bioremediation is one of the remediation techniques that are considered to be more environmentally friendly than others (Ojuederie and Babalola, 2017). Besides, microbes are known to be able to live under extreme conditions. The community of bacteria reflects the relationship between diversity and environmental factors (Fagorzi et al., 2019). The bacteria that live in tailings have various mechanisms to protect themselves from the Hg toxicity, one of which is accumulation. Through the accumulation mechanism, the concentration of Hg in the bacterial cell tissue is higher than that in the environment in which it grows. This is because the metabolism and excretion of Hg from the bacterial body are slower than the absorption of Hg. As a result, Hg accumulates in the body of bacteria (Puspitasari, 2007).
Several studies have been conducted to explore the types of bacteria that can live in the soils with high Hg concentrations. Irawati et al., (2017) (Ekyastuti and Rima, 2015). This study aimed at elucidating the accumulation of Hg by the bacteria isolated from the ASGM tailings in Lombok.

Tailing Samples
The tailings samples for bacterial isolation were collected from three gold processing sites in Lombok (Table 1) at a depth of 0-5 cm (Kuterbekov, 2019). The tailing samples were analyzed for pH (Moore, 1968), organic carbon content (Walkley and Black, 1934), organic matter content (Waksman and Stevens, 1930), available P content (Olsen et al., 1954), total N content (Kjeldahl, 1883), and total K content (Affinih et al., 2014). The results of the sample analysis are presented in Table 3.

Bacterial Isolation
Isolation was carried out on Nutrient Agar (NA) medium using serial dilution and Pour Plate methods up to 10 -7 dilution series. The culture was incubated for 48 hours. The bacterial colonies that grew on each Petri were counted. The total bacterial population was calculated using the following formula (Husen et al., 2007): Total bacteria (CFU g −1 ) = total colony x fp soil weight (1) where: fp = dilution factor in Petri dishes whose colonies are counted  Morphology of the bacterial colonies was identified based on the Bergey's Manual Determinative of Bacteria system.

Bacterial Tolerance Test
The bacterial tolerance test was carried out in Nutrient Agar (NA) enriched with HgCl 2 (Irawati et al., 2013) according to treatment, namely 0 ppm (control), 25, 50, 75, 100, and 125 ppm using the streak method plates. The bacterial isolates were incubated for 14 days. The colonies that are resistant to Hg will grow and form new colonies on the streak line (Ekyastuti and Setyawati, 2015).

Bacterial Pathogenicity Test
The bacterial pathogenicity test was used to determine the potential of bacterial pathogenicity through its ability to dissolve blood. The pathogenicity test was carried out on the Blood Agar medium (Tryptic Soy Agar (TSA) medium enriched with 5% sheep blood) using the streak plates method and incubated for 24 hours. The observation of the ability of bacterial hemolysis was carried out over 24 hours. The bacteria are capable of hemolyzing blood if a clear zone (β hemolysis) or media discolouration (α hemolysis) around the colony turns to green or brown (Buxton, 2005). The bacteria that are unable to hemolyze blood (γ hemolysis) were used in further tests.

Bacterial growth curve
The bacterial isolates that were unable to hemolyze blood were tested for their growing ability on the Luria Bertani (LB) medium enriched with HgCl 2 . The bacteria that have been made inoculum stock were transferred to 25 mL of LB + HgCl 2 medium with a new dose of 125 ppm with a concentration of 10% (v / v). The culture was incubated for 24 x 5 hours in the shaker (Irawati et al., 2013;Ekyastuti and Setyawati, 2015). The bacterial growth was observed every 24 hours by measuring the cell density using a spectrophotometer at a wavelength of 600 nm. The bacterial growth test in enriched media was repeated three times. The changes in the colour of bacterial colonies grown on HgCl 2 enriched medium and without HgCl 2 were also observed (Irawati et al., 2013).

Hg accumulation test
The Hg accumulation test was carried out on 25 mL of LB medium enriched with 125 ppm HgCl 2 . 2.5 mL of bacterial inoculum stock was added to 25 ml LB. The accumulation test of each bacterial isolate was repeated three times (Irawati et al., 2013). The culture was incubated for 24 x 5 hours at room temperature. The culture was observed on the fifth day or at the 120 th hour. The culture inoculum was shaken using a centrifuge

Identification of potential bacterial species from tailings
Three selected bacterial isolates were identified through the biochemical and molecular tests using the 16S rDNA gene squad (Irawati et

Total bacterial population
The three gold processing districts have different bacterial populations. The highest bacterial population was obtained from Jonggat District (JGT) tailings with a total of 8.50 x 10 7 CFU, followed by Sekotong District (SKT) tailings of 1.93 x 10 6 CFU, and Pujut District (PJT) tailings of 1.46 x 10 6 ( Figure 1). The difference in bacterial populations is related to the different condition of the tailings in the three districts (Table 1) The chemical properties of soil affect the total bacterial population (Rao, 1977). The results of chemical analysis of tailings from the three districts showed different results. The tailings from Jonggat (JGT) has organic carbon (C-organic), total nitrogen (N), phosphorus (P), and exchangeable potassium (K) higher than others (Table 3). Carbon, nitrogen, and phosphorus are important elements that support the bacterial growth and metabolism, so the presence of these elements greatly affects the bacterial population (Lay and Hastowo, 1992; Subandi, 2014). Organic carbon is a source of carbon and energy for bacteria. The decomposition of complex organic materials into simpler organic materials or inorganic materials will produce energy in the form of ATP. The energy produced by the bacteria from the decomposition process will be used in the synthesis of macromolecules, such as nucleic acids, lipids, and polysaccharides (Lay and Hastowo, 1992). The formation of nucleic acids (RNA and DNA) in cells requires nitrogen and phosphorus. Nitrogen also plays a role in the formation of proteins responsible for the activity of cell metabolism and phosphorus needed in the synthesis of highenergy organic compounds such as ATP (adenosine triphosphate) (Subandi, 2014).
Cellular metabolism in bacteria also requires other elements in the form of minerals, such as potassium (Subandi, 2014). Potassium is the main intracellular cation in bacteria as well as in eukaryotic cells. The potassium absorption in cells is regulated by the ion and turgor concentrations. Potassium acts as a signal giver in the cytoplasm, activating and/or inducing the enzymes and transportation systems that allow cells to adapt to increased osmolarity (Epstein, 2003). Potassium is obtained by bacteria from basic compounds in the soil, so its availability greatly affects the bacterial population (Subandi, 2014).
The Hg concentration (Table 2) is directly proportional to the bacterial population ( Figure 1). The Jonggat District tailing with the highest concentration of Hg has the highest bacterial population and vice versa. The results of this study differed from the previous studies that have been conducted. Most of the results showed that the bacterial population was negatively affected by the Hg concentration. The bacterial population and protozoa will decrease with the increase of the Hg concentration (Muller et al., 2001). Hg has been shown to negatively affect the conjugate transfer to plasmid (Rasmussen et al., 2008). However, considering the long history of the Hg contamination and the low concentration of the bioavailable Hg, a plausible explanation for the high diversity of the Hg-resistant population is a horizontal transfer of mobile genetic elements carrying the mer-operon (Rasmussen et al., 2008). This results in the formation of bacteria that are tolerant of Hg, contributing to high bacterial populations in contaminated soil.

The tolerance ability of isolates to Hg
There were 27 isolated bacterial isolates that could tolerate Hg at a test dose of 125 ppm (Figure 2). The ability of bacteria to tolerate Hg is because bacteria have the mer-operons that contain the encoding genes that can make the bacteria tolerant and have different mechanisms for adapting to the Hg stress (Ianieva, 2009).
The genes encoded by the mer-operons can be found in plasmids, chromosomes, transposomes, and integrins. Cellular elements (plasmids, transposons, and integrons) play an important role in the spread of mercury resistance throughout the microbial community through horizontal transfer. In addition, the resistant bacteria can also effectively transfer phenotypes (Hg resistant genes) to potentially pathogenic species (Morel et al., 1998;Kahl, 2015). This causes the mechanism of resistance that can also be found in the pathogenic bacteria.
The structure of mer-operons is different for each type of bacteria (Barkay et al., 2003). The key genes of the mer-operon are merA (gene reductase), merT (transport protein gene), merP (extracellular mercury-binding protein gene) and merR (regulatory protein gene). However, operons can contain additional genes, such as merB (organomercurial lyase gene), merC (transport protein gene), merD (regulatory protein gene), merЕ (transport protein gene), merF (transport protein gene) and merG (a gene that provides resistance to phenyl mercury) (Ianieva, 2009). The bacteria that possess only the mercury reductase (merA) gene are called narrow spectrum mercury bacteria. Some bacteria that have genes other than merA or merB are called broad-spectrum mercury resistant bacteria. The MerA protein has the function of reducing the toxic mercury ions to mercury (Hg (0)) which is less toxic and volatile at room temperature. The MerB protein has the function of catalyzing the breaking of mercury-carbon bonds to produce organic compounds and Hg (II) ions (Barkay et al., 2003).
The ability of bacteria to transmit the resistance-coding genes or tolerance to the pathogenic bacteria causes a high potential for obtaining these bacteria during isolation. Reducing the potential use of pathogenic bacteria for the next stage of this stydy was carried out by testing the ability of blood hemolysis or pathogenicity tests. The results of the pathogenicity tests showed that 15 of the 27 isolates obtained were capable of the blood hemolysis. The ability of hemolysis is demonstrated by the formation of clear zones (β hemolysis) or changes in the color of the media (α hemolysis) to green or brown around the bacterial colony (Buxton, 2016). There were 12 bacterial isolates that were unable to hemolyze blood (γ hemolysis) (Figure 3). Escherichia coli is a pathogenic bacterium that is known to be resistant to Hg. E. coli is known to be able to store the Hg resistant genetic markers that make this bacterial species a promising alternative to the bioremediation process (Costa et al., 2013). In addition, E. coli also passed the test using it as a Hgresistant transgenic bacteria (Irawati et al., 2013).

Bioaccumulation of Hg by isolated bacteria
The bacterial isolate with the highest Hg bioaccumulation ability was JGT-F2 (89.56%), followed by SKT-B (82.25%) and JGT-F1 (81.21%) ( Figure 4). Two bacteria with the highest Hg bioaccumulation ability were obtained from the Jonggat District tailings having the highest Hg concentration. One bacterial isolate with the highest ability to accumulate Hg was obtained from the Sekotong district tailings having the secondhighest Hg concentration. In addition, the three highest accumulator bacteria also had the best ability to adapt to Hg in the test media. Figure 5 shows that JGT-F2, SKT-B, and JGT-F1 isolates were able to adapt quickly, and their growth was quite stable on LB + Hg media compared to other isolates. This shows that the higher the Hg concentration in a soil, the greater the ability of bacteria to adapt to toxic metals.
The differences in the ability of bacteria to accumulate Hg are caused not only by the differences in the Hg concentrations in their habitats but also by the differences in the response mechanisms. The mechanism of bacterial response to high Hg in the environment is done in three ways, namely: (1) by inhibiting cell metabolism so that cells grow slowly, even dying, (2) inducing an operon system that plays a role in Hg resistance so that cells remain alive under stress, and (3) having a plasmid containing the Hg resistance genes (Ekyastuti and Setyawati, 2015). In the Hg resistant and Hg accumulator bacteria, Hg in the soil is transported across the lipid membrane that surrounds the cell. The absorption of Hg by bacteria is a crucial step in bioaccumulation. Most metals enter cells through special transmembrane cation transporters or through other metal transporters. Hg at high concentrations is transported by bacteria through protein transport or MerT, whereas at low concentrations, Hg is absorbed through the lipid membrane in the form of water-soluble Hg. The chemical bonds Fat solubility is generally measured by the octanol-water partition coefficient, which measures the relative solubility of a compound in octanol and water and ranging from almost zero for very hydrophilic molecules to 108 for highly hydrophobic (Morel et al., 1998).
The pattern of bacterial cell growth rate is influenced by the characteristics of individual bacterial cells. The bacterial growth on the media containing heavy metals is influenced by the type of bacteria and the effect of heavy metals on bacterial cells. Different bacterial species have various tolerance abilities, which are caused by the toxic properties of Hg and the strength of bacterial affinity for chelating agents in cells such as proteins, lipids and nucleic acids (Ekyastuti and Setyawati, 2015).

Identification of Hg bioaccumulator bacteria
The results of bacterial identification showed that the JGT-F1 is Basillus toyonensis (Figure 6), and the SKT-B is Fictibacillus nanhaiensis ( Figure 7). However, the identification results also showed that the JGT-F2 was not a pure isolate, so the isolate was eliminated from the list of bacteria with the best ability. The two best bacteria were identified as gram-negative bacteria in the form of rod (bacilli) ( Table 3).
The operons in Gram-negative bacteria can encode some proteins. This is not found in the   gram-positive bacterial operons. Some of them are membrane proteins or MerC, as well as MerF and MerE. MerE and MerF, which replace MerC in several operons, can work alone in the absorption of Hg (II), except mer on Thiobacilli, which is always known to be replaced by MerT. Periplasmic protein (MerG) is also found in some Gram-negative bacteria. This protein gives a slight increase in the organomercurial resistance, even in strains that lack MerB. This causes that in many Gram-negative bacteria there is an additional protein called MerD, which appears to be hostile to the MerR activation for the transcription of mer-operons (Barkay et al., 2003).

CONCLUSION
The bacteria isolated from Pujut, Sekotong, and Jonggat Districts in Lombok Island, West Nusa Tenggara, were able to accumulate Hg with bioaccumulation capabilities of 53.94-89.56%. The bacteria found can be used as remediation agents for tailings so that the contamination of Hg from tailings can be reduced. Exploration of the ability of bacteria needs to be carried out further to find out the extent of the ability of bacteria to accumulate Hg and its mechanism.