Chemicals utilized included mercuric chloride (HgCl₂) for mercury stock solution preparation, sodium hydroxide (NaOH), and hydrochloric acid (HCl) for pH adjustments. Bacterial cultures were grown using Nutrient Agar (NA) for revival and sub-culturing, and Lactose Broth (LB) as the growth medium for experimental bioremediation. Key instruments employed were a spectrophotometer (for OD₆₀₀ measurements), a Cold Vapor Atomic Absorption Spectrophotometer (CV-AAS) (for mercury quantification), an incubator (for maintaining temperature), and a rotary shaker (for agitation). All glassware and equipment were sterilized prior to use to prevent contamination.

This study adopted an experimental laboratory design to evaluate the bioremediation potential under controlled conditions. The experimental setup comprised eight distinct treatment groups for each of two pH conditions: neutral (6.9–7.0) and alkaline (10.0). The treatment groups included: (1) a control group without bacterial inoculation; (2) single cultures of B. subtilis inoculated at 1 mL (BS1) and 2 mL (BS2) volumes; (3) single cultures of P. aeruginosa inoculated at 1 mL (PA1) and 2 mL (PA2) volumes; and (4) mixed cultures containing both B. subtilis and P. aeruginosa (each at 1 mL for MC1 and 2 mL each for MC2). The design aimed to comprehensively assess the influence of pH and inoculum volume on mercury reduction efficiency and bacterial population dynamics, comparing single versus mixed culture performance.

The research procedure involved several key steps:

1). Bacterial Culture Preparation: Pure cultures of B. subtilis and P. aeruginosa were revived on NA media. Subsequently, they were sub-cultured into LB and incubated. Bacterial growth kinetics were monitored hourly for the first seven hours by measuring turbidity at 600 nm (OD₆₀₀) using a spectrophotometer. This allowed for harvesting the inoculum during the exponential growth phase, crucial for optimal activity ((Wibowo et al., XXXX); (Imron et al., 2019)). 2). Preparation of Mercury-Contaminated Wastewater: A simulated contaminated wastewater was prepared by dissolving HgCl₂ to create a 100 mg/L Hg²⁺ stock solution. This stock solution was then diluted to a final working concentration of 2 mg/L Hg²⁺ for all experimental treatments. The pH of each solution was precisely adjusted to either neutral (6.9–7.0) or alkaline (10.0) using NaOH or HCl as required; 3). Bioremediation Experiment Setup: Each treatment group was prepared in 200 mL of LB medium containing 2 mg/L Hg²⁺. The inoculated and control reactors were incubated for seven days at a temperature of 30–35 °C with continuous agitation at 150 rpm. This constant agitation ensured homogeneity of the culture medium and adequate oxygenation throughout the incubation period(Titah et al., 2018).

1). Mercury concentration was quantified on days 0 (baseline), 3, and 7 using Cold Vapor Atomic Absorption Spectrophotometry (CV-AAS), strictly adhering to the SNI 19-6964.2-2003 standard protocol (Wibowo et al., XXXX). 2). Bacterial population dynamics were assessed by determining colony-forming units per milliliter (log CFU/mL) using the pour plate technique. Colony counts were recorded on days 0, 1, 2, 3, and 7. 3). Environmental parameters, specifically pH and temperature of the culture media, were monitored daily throughout the seven-day incubation period. 4). Data Analysis: All collected data were subjected to descriptive analysis. The percentage reduction of mercury was calculated by comparing concentrations on days 3 and 7 against the baseline (day 0). Bacterial growth trends were interpreted in relation to variations in pH and inoculum volume to assess their influence on the overall efficacy of the bioremediation process (Imron et al., 2019). This systematic framework was designed to identify optimal operational parameters with a view toward potential field application.

Pseudomonas have a high level of resistance to heavy metals, including mercury. For instance, research by Vasanthi et al. (2020) demonstrated the significant mercury resistance of Bacillus cereus isolated from contaminated soil, while work by Hassan et al. (2018) identified specific resistance genes (mer operon) in a Pseudomonas species that allow it to thrive in mercury-rich environments. This finding strengthens the suspicion that these two bacteria can be optimized as efficient and sustainable bioremediation agents for handling mercury pollution on a wider scale.

This study used B. subtilis and P. aeruginosa with two culture methods: single inoculum and mixed culture). The number of bacterial colonies was calculated using the Total Plate Count (TPC) method with a range of 30-300 CFU/mL to ensure statistical validity. Measurements were taken on days 0, 1, 2, 3, and 7, and the results were converted to log CFU/mL for more accurate bacterial growth analysis.

Figure 3 and Figure 4 illustrate the effectiveness of sterilization, with control reactors lacking inoculum consistently showing bacterial colony counts of less than 30 CFU/mL (too few to count, TFTC). For single bacterial cultures, optimal growth was observed by day 2 or 3 across both neutral and alkaline pH conditions, indicating sufficient initial nutrient availability and supportive environmental factors. Specifically, Bacillus subtilis and Pseudomonas aeruginosa reached peak counts averaging 9.8–10.4 log CFU/mL and 9.9–10.2 log CFU/mL, respectively, depending on pH and inoculum volume. However, by day 7, the colony counts for single cultures dramatically decreased to TFTC, suggesting a significant depletion of nutrient sources and/or the accumulation of mercury-related toxicity within the media, which subsequently inhibited bacterial proliferation.

A distinct and crucial finding emerged from the mixed cultures, particularly under alkaline pH conditions (Figure 4). While single cultures plummeted to TFTC by day 7, the mixed inoculum maintained a robust population of 7.4 log CFU/mL. This sustained viability strongly suggests synergistic interactions or mutualistic protection between B. subtilis and P. aeruginosa in the presence of mercury under alkaline conditions, enabling them to survive longer than when grown individually. The observed differences in peak growth timing between neutral and alkaline pH also indicate that pH significantly influences mercury bioavailability, which, in turn, impacts bacterial growth dynamics and potentially the overall efficiency of bioremediation on a broader scale.

The underlying mechanisms contributing to the observed mercury detoxification capabilities of B. subtilis and P. aeruginosa are largely attributed to their mercury resistance (mer) operon. This operon comprises genes such as merA and merB, which are crucial for converting toxic mercury species into less harmful forms. merA encodes mercuric reductase, an enzyme responsible for reducing highly toxic inorganic mercuric ions (Hg²⁺) to elemental mercury (Hg⁰), while merB encodes organomercurial lyase, which cleaves organic mercury compounds to release Hg²⁺ for subsequent reduction (Barkay et al., 2003). Understanding potential genetic variations within the mer operon of strains isolated from ASGM sites is vital, as these variations could explain their unique adaptation to high mercury concentrations and inform strategies for genetic engineering to further enhance their bioremediation capacity (Hsu-Kim et al., 2013).