Figure 1 shows the mercury recovery by various surfactants as a function of their concentration. The enrichment, foam volume and recovery percentage of mercury were investigated. The concentration of mercury in the solution was 10 mg L⁻¹, and the foam was collected after 3 min of initiation of airflow (1 L min⁻¹). The concentration of the surfactants varied by orders of 0.5, 1, 2, 5, 5, 10, and 30 times the critical micelle concentration (CMC) of the surfactants investigated. The CMC values of the surfactants used in this study are 7.5 × 10⁻³, 8.2 and 1.2 × 10⁻² mM for surfactin, SDS, and Tween-80, respectively [39–41]. The recovery of mercury was found to be sensitive to the surfactant concentration. No foam was accumulated in the stipulated time of 3 min in the collection flask when 0.5 and 1 × CMC surfactin and SDS were used; this could be due to insufficient volume of the foam generated in the solution, and the small amount of foam generated did not travel through the J-tube to reach the collector flask. Similarly, in case of Tween-80, the foam did not reach the collector flask, even with surfactant with 2.5 × CMC. Figure 2(a) shows that the highest mercury recovery was obtained by surfactin at 10 × CMC, while SDS and Tween-80 recovered highest mercury recovery at <10× and >10 × CMC, respectively. Recovery of the mercury by the surfactants at given concentrations was increased with the increase of foam volume (Figure 2(b)). However, the enrichment of mercury ions in the foam was superior with surfactin compared with chemical surfactants and was highest at lower concentrations (2.5 × CMC). The anionic surfactants (surfactin and SDS) recovered higher mercury at low concentrations compared to Tween-80; this may due to the electrostatic interaction between negatively charged functional groups (carboxylates in surfactin and sulfate in SDS) and mercury ions (Hg²⁺) resulting in the formation of a complex in the form of micelle [42]. The maximum recovery of mercury ions (10.4%) using surfactin was observed at 10 × CMC, whereas that of SDS at the same concentration was 8.14%; the highest mercury recovery for SDS (8.8%) was obtained at 5 × CMC. The highest recovery rate overall, i.e., 14.2%, was observed with Tween-80, but this required a concentration of 30 × CMC. The mercury enrichment value corresponding to the highest metal recovery by surfactin (10.4%) was 1.53; this may be due to the presence of two carboxylate functional groups of surfactin, which can bind to the metal ions, whereas only one sulfate group is present in the SDS molecule. It is also known that, as the concentration of surfactant increases in multiples of the CMC, vesicle structures outnumber the micelles; in addition the structure and aggregate numbers also change. The difference in the recovery and enrichment values at the given surfactant concentration may be due to variable liquid concentration in the foam that varies significantly with the nature of the surfactants used. Hence, a higher concentration of Tween-80 was required for mercury recovery compared anionic surfactants. These results indicated that the complexation efficiency and separation of surfactin along with metal ions strongly depend on the nature and concentration of the surfactant. Surfactin was found to form micelles at very low concentrations, but resulted in a high mercury removal (10.4% at 10 × CMC) [39,40]. The following experiments were also performed using surfactin at a concentration of 10 × CMC, in which various parameters were altered in order to investigate the factors affecting the recovery of mercury from the solution.

Figure 2 presents the effect of the mercury concentration on its recovery using surfactin at a flow rate of 1 L min⁻¹ and a foaming time of 3 min. Recovery was performed at mercury concentrations of 2, 5, 10, 20, 50, and 100 mg L⁻¹ and in a 10 × CMC surfactin solution. The metal recovery was found to decrease with increasing mercury concentration. The highest recovery observed was 36.4% from 2 mg L⁻¹ mercury solution and 2.29% from100 mg L⁻¹ solution. It has already been reported that the percentage recovery of mercury by foam fractionation was better at low concentrations [24]. The effective complexation ratio of surfactin to metal decreased with the increase of mercury concentration in the solution. Theoretically, in the absence of interfering ions, the molar ratio of surfactin to Hg²⁺ is expected to be 0.5 due to complexation of two Hg²⁺ ions with one surfactin molecule (each surfactin molecule contains two carboxylate groups); however, the surfactin to Hg²⁺ ratio in 2 mg L⁻¹ solution was found to be 3.75 that decreased to 0.075 for a 100 mg L⁻¹ solution. This is probably limited by the micelle formation in surfactin solution (10 × CMC), which was able to form a complex effectively from lower concentration of mercury ions carry to the foam collector.

It is also known that, in the foam fractionation method, foam volume affects the metal ion enrichment by carrying a more number of surfactant–metal complex entities. Hence, foam was collected at intervals of two minutes using different concentrations (2, 5, 10, 20, 50, and 100 mg L⁻¹) of mercury ions in the solution in order to investigate the effect of foaming time on mercury recovery. The concentration of surfactin and the airflow were fixed at 10 × CMC and 1 L min⁻¹, respectively. Figure 3 shows that the foam collected after 6 min enriched with mercury by 2.26% from the surfactin solution containing 2 mg L⁻¹ Hg²⁺. It is presumed that the low enrichment after 3 (1.87%) and 5 (1.78%) min of foam collection was due to the large number of foam bubbles collected in the flask. Thus, foam volume is also an important factor affecting mercury ion enrichment in the foam fractionation method. When the mercury ion concentration was 2 and 5 mg L⁻¹, the enrichment was higher compared to other concentrations, and thereafter remained nearly constant with increased mercury ion concentration up to 100 mg L⁻¹ [43].

The effect of the digestion time, which in turn influences the complexation of the metal ions with the surfactant, is also important in the recovery of heavy metals. The effect of digestion time of mercury in surfactin solution was investigated for mercury removal by conducting experiment with fresh and overnight solutions. Figure 4 presents enrichment of mercury from the surfactin solution (10 × CMC) of different mercury concentrations and foaming time of 7 min. Apparently, there was considerable recovery of mercury ions from the solution digested overnight; the highest recovery was 46%, when the mercury ion concentration was 2 mg L⁻¹, and recovery was higher by a magnitude of four than that observed at the highest mercury ion concentration, i.e., 100 mg L⁻¹. Thus, the removal efficiency of mercury ions increased when mercury ions were digested overnight in the surfactin solution, due to the stable complex formation of mercury ions with surfactin. It was conjectured that, in order for the highest recovery % to be obtained, each functional group must bind a mercury ion. It has previously been reported that a prolonged digestion time of approximately 36 hours for contaminated soil with rhamnolipid solution yielded recovery of heavy metals up to 92% [31].

Figure 5 shows the results of varying the airflow rate to generate foam of differing bubble size and stability. The effect of different flow rates using 10 × CMC surfactin and 2 mg L⁻¹ mercury was studied in order to assess the influence of airflow on the recovery of mercury ions. The airflow rate was varied from 0.6 to 2.5 L min⁻¹. The foaming time and the time required to pass through the J-type column was slow when the airflow rate was 0.6 L min⁻¹, and a large number of bubbles in the foam were broken or remained in the J-type column and didn’t reach the foam collector; hence, this airflow rate resulted in a lower volume of foam in the collector, the analysis of which could be erroneous. Mercury ion enrichment at this low airflow rate was highest at 8.6%, but the mercury ion recovery was only 13.17%. The efficiency of mercury ion enrichment was almost constant when the airflow rate was above 1.0 L min⁻¹, while mercury ion recovery was 43.4% and 42.6% when the airflow rate was 2.0 and 2.5 L min⁻¹, respectively. Qua et al. reported that an increase in airflow rate with SDS decreased the enrichment ratio of Cd²⁺, while the % removal of the metal ions increased [44]. These results can be explained based on the fact that with an increase in airflow rate, a greater amount of the liquid could be transported into the foam and adsorbed onto the bubble surfaces, thereby increasing bubble production, resulting in higher recovery but low enrichment.

It is known that the pH of the surfactin solution influences the enrichment and recovery of heavy metal ions due to various factors. The effect of pH on the removal of mercury ions is shown in Figure 6, from which it can be seen that the mercury ion recovery was 47.5% and 47.8% at pH values of 8 and 9, respectively, and the mercury ion enrichment was about 2.43 and 2.33 at those pH values. However, mercury ion recovery was only 18.2% when the pH of the surfactin solution was decreased to 6, with an enrichment of 1.2. In addition, a lower pH of the surfactin solution may result in precipitation, thereby limiting the efficiency of the mercury removal process. It has also been reported that chelates of Hg²⁺ such as Cl⁻ and OH⁻ chelates are quite stable, leading to poor or no separation using sodium lauryl sulfate when the pH is reduced by HNO₃ or kept alkaline [24]. Hence, the low recovery and enrichment values of mercury under acidic conditions could be attributed to undissociated carboxyl groups to form complexation with surfactin molecules. However, surfactin resulted in better separation of metal ions with increased pH. These results show that the efficiency of separation is closely related to pH, as well as to the concentration of the positively-charged mercury-containing species.

In this study, surfactin was produced by wild-type Bacillus subtilis (BBK006) in culture media containing glucose as the sole carbon source. Production of surfactin using B. subtilis has been reported elsewhere [45,46]. Typically, a loop of bacteria grown on an LB agar plate was added to M9 medium (with 0.2% glucose) as a seed culture to make 30 mL of solution in a 50-mL tube. This was incubated for 24 h on an orbital shaker (200 rpm) at 37 °C in order to produce a seed culture. The exact composition of M9 medium was reported elsewhere [47]. Prior to sterilization, the medium pH was adjusted to 7.0 with 0.5 M NaOH. The medium was then sterilized at 121 °C for 20 min without glucose, which was filtersterilized (Millipore membrane PVDF, 0.22 μm filter unit; Millipore, Watford, UK). 300 mL M9 medium in a 500-mL Erlenmeyer flask was mixed with 0.2% glucose to produce surfactin. After 24 h of fermentation, the biomass was centrifuged (12,000 rpm, 10 min), followed by addition of 1 M HCl to adjust the pH of the supernatant solution to below 2.0. The precipitate was collected after centrifugation (12,000 g, 10 min) and the surfactin was purified as previously reported [40] by extraction with dichloromethane, dissolving in milli Q water to the required concentration, and stored at 4 °C. SDS and Tween-80 solutions were prepared at the time of experiment. The chemicals used in the growth media were purchased from Riedel-de Haën, GR, USA; the other surfactants, sodium dodecyl sulfate (98.5%) and Tween-80, were purchased from Sigma-Aldrich, Saint Louis, MO, USA.

All experiments were conducted at room temperature (25 °C). The foam fractionation system used for removal of mercury consists of a glass J-type column (L = 400 and 100 mm, ID = 22 mm) connected to 1-L Erlenmeyer flasks at both the ends with rubber corks. Required concentrations (0.5, 1, 2.5, 5, 10 and 30 times of CMC of different surfactants (SDS, Tween-80 and surfactin) solution were taken in one of the Erlenmeyer flasks (connected to long arm) and connected to air pump) and foam was collected in another flask. The airflow rate (0.5, 1, 1.5, 2 and 2.5 L min⁻¹) was controlled by a flow meter and the foam was collected in another flask to enable measurement of the concentration of mercury. The concentration of mercury in the artificially polluted water was varied from 2 to 100 mg L⁻¹. The foam was generated from fresh and overnight digestion solutions. The time of foam collection was varied from 3 to 7 min. Final fate of the mercury was in the form of solution after bubbles collapsed in the collector flask.

The mercury ion concentrations in the foam were analyzed using an atomic absorption spectrophotometer (AAS, Perkin Elmer Analyst 200, Shelton, CT, USA). The samples were digested in 3% HNO₃ prior to the analysis. Mercury solution was diluted to 2 to 20 μmL⁻¹ and the instrument was calibrated in this concentration range. All experiments were performed in triplicate, and the average of the results is presented. The metal removal efficiency was calculated by using the following formula, as reported elsewhere [48]:

where C_F and C_R indicate the mercury concentration in the foamate and remnant, respectively, and V_F and V_R indicate the foamate and remnant volume, respectively.