Optimization of copper bioremoval from hypersaline environments by the halophilic archaeon Halalkalicoccus sp. Dap5 via response surface methodology

Abstract

Copper pollution in hypersaline environments poses a significant challenge due to the inefficiency of conventional bioremediation strategies under high salinity and metal stress. Halophilic archaea represent a promising solution for heavy metal removal in saline environments due to their biocompatibility and cost-effectiveness. Here, we investigated the copper removal potential of a Halalkalicoccus sp. Dap5, a halophilic archaeon isolated from the Urmia Lake in Iran. This strain exhibited copper tolerance (MIC: 80 mg/L Cu²⁺) and tolerance to several other toxic metals, including cadmium (Cd²⁺), cobalt (Co²⁺), lead (Pb²⁺), zinc (Zn²⁺), and arsenite (As³⁺) under 15% (w/v) salinity. A Central Composite Design (CCD) was employed within the Response Surface Methodology (RSM) to optimize three key parameters: pH, initial copper concentration, and inoculum percentage, to maximize copper removal. The resulting model was statistically significant (R² = 0.9972, p < 0.0001) and attained a maximum copper removal efficacy of 90.8% at pH 8.1, 28.8 mg/L Cu²⁺, and 4.8% (v/v) inoculum. Microscopic and spectroscopic analyses revealed that copper removal occurred through both biosorption and bioaccumulation mechanisms, supported by increased extracellular polymeric substance (EPS) production and specific functional group interactions identified via FTIR. The results demonstrate that Halalkalicoccus sp. Dap5 exhibits marked tolerance to copper and efficiently removes copper ions from saline environments, making it a valuable candidate for sustainable bioremediation under extreme conditions. This is the first report on optimization of copper bioremoval in a Halalkalicoccus strain using RSM, underscoring its biotechnological significance for green environmental management.

Keywords: Halalkalicoccus sp. Dap5; Copper bioremediation; Extracellular polymeric substances (EPS); Halophilic archaeon; Response surface methodology (RSM).

Fig. 1

Morphological characterization of Halalkalicoccus sp. Dap5. (a) Colony morphology. (b) Microscopic view.

Fig. 2

Effects of copper and physicochemical parameters on the growth of Halalkalicoccus sp. Dap5. (a) Minimum inhibitory concentration (MIC) of copper: Strain Dap5 showed tolerance to copper up to 80 mg/L in SW-23 medium. Notably, growth was enhanced at copper concentrations up to 40 mg/L compared to the control. Although growth was significantly reduced at 80 mg/L, the strain maintained the ability to grow at this concentration. All differences were statistically significant (p < 0.05). (b) Influence of physicochemical factors in the absence of copper: The effects of (1) Salinity, (2) pH, and (3) Temperature on Dap5 growth were assessed without copper. Optimal growth was observed at 15% NaCl, 40 °C, and pH 8.0, with the highest biomass recorded on day 6. (c) Influence of physicochemical factors in the presence of copper. (1) Copper ion concentration. (2) Salinity. (3) pH, and (4) Temperature. Under 40 mg/L copper, the strain exhibited the same optimal growth conditions (15% NaCl, 40 °C, pH 8.0) as in the absence of copper, but maximum growth was achieved one day earlier, on day 5.

Fig. 3

Neighbor-joining phylogenetic tree of the 16 S rRNA gene sequence of strain Dap5 (PV052243). Strain Dap5 is closely related to species within the genus Halalkalicoccus. Methanospirillum hungatei CaP2L-L1A (MW498161) was used as the outgroup. The sequence had a length of 1046 bp with 70.8% completeness, and bootstrap values were calculated based on 500 replicates. The scale bar represents 0.02 substitutions per nucleotide site.

Fig. 4

Copper removal experiments and measurement of final copper concentration using anthocyanin colorimetry. (a) Experimental design and responses for copper bioremoval analyzed using RSM. (b) Standard curves prepared using anthocyanins extracted from (1) eggplant peel and (2) red cabbage.

Fig. 5

Analysis of residual graphs, 2-D and 3-D response surface plots, and desirability ramp for optimizing copper removal parameters. (a) Diagnostic residual plots: (1) Normal probability plot confirmed the normality assumption; (2) Residuals vs. predicted values showed a random scatter, indicating homoscedasticity and model adequacy; (3) Residuals vs. run order confirmed the independence of residuals over time; (4) Cook’s distance plot revealed no influential outliers; and (5) Box-Cox plot indicated that no data transformation was required. These diagnostics collectively confirm the adequacy and validity of the fitted model. (b) 2D and 3D response surface plots. In all plots, the red color indicates the highest response, while dark blue represents the lowest response. For each 3D plot, the third parameter is fixed at its central value. (1) The A-axis denotes pH and the B-axis shows inoculation percentage. (2) The A-axis indicates pH and the B-axis shows the initial copper concentration. (3) The A-axis represents inoculation percentage and the B-axis shows the copper concentration. (c) Desirability ramp charts for the optimization of initial solution pH, copper concentration, and inoculation percentage to maximize copper removal efficiency. The optimal values for each parameter and the predicted maximum removal percentage are indicated. This figure was created by using Design-Expert software (version 12.0.3.0, Stat-Ease Inc., Minneapolis, USA).

Fig. 6

Mechanisms of copper removal and interaction with surface functional groups in Halalkalicoccus sp. Dap5 using SEM, TEM, EDS, and FTIR analyses. (a) Control sample and (b) Copper-treated sample: (1) SEM images show an increase in EPS production in the copper-treated sample; (2) TEM images highlight bioaccumulated copper, indicated by the red arrow in the treated sample; (3) Both the EDS spectra and the corresponding tables of elemental weight percentages demonstrate copper biosorption on the cell surface. (c) FTIR spectra identify functional groups involved in copper binding to the cells, where the red graph represents copper-treated cells and the black graph represents control cells.

Fig. 7

Effects of arsenite, cobalt, mercury, lead, cadmium, and zinc ions on the growth of Halalkalicoccus sp. Dap5 and copper removal. (a) Growth of the strain in the presence of different metal ions. (b) Copper removal efficiency by Halalkalicoccus sp. Dap5 in the presence of these metal ions.