2.1. Algae Culture and Pretreatment
A strain of M. aeruginosa (FACHB-1322) from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences, was selected for this study. This strain is a widely prevalent algae species in freshwater lakes in China. Before initiating the experiments, algal cells of M. aeruginosa (FACHB-1322) with the optical cell density (OD680) of 0.10 were centrifuged for 15 min at 5000 r·min−1. Subsequently, the algal cells were washed twice with the same volume of sterile ultrapure water in order to minimize any residual As and P attached to cells. The washed M. aeruginosa cells were then transferred to a modified sterilized M11 growth medium [MgSO4·7H2O (11.25 mg), K2HPO4 (1.5 mg), NaNO3 (15 mg), CaCl2·2H2O (6 mg), Na2CO3 (3 mg), Na2·EDTA·2H2O (0.15 mg), all reagents were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China] at 25 °C under a photon flux density of 40 μmol/m2/s, provided by cool white, fluorescent irradiation, with a 12 h:12 h light/dark interval orbital incubation (125 rpm). To ensure adequate gas exchange, the test flasks were manually rotated and shaken three times during each light cycle. Prior to use, all stock vessels and culture flasks were meticulously cleaned by being soaked in 10% HCl for at least 24 h, followed by rinsing with Milli-Q water, oven drying, autoclaving, and handling under sterile conditions.
2.2. Experimental Design
The algal cells of
M. aeruginosa (FACHB-1322) under phosphorus-starved conditions were centrifuged and washed twice with sterile deionized water. Subsequently, they were divided equally into 36 portions (3 P treatments, each with 12 parallels, as shown in
Table 1 [
19]), and incubated in modified M11 media with KH
2PO
4 (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) as the phosphorus source for 96 h. The media contained 0.02, 0.1, and 0.5 mg/L of P in the form of PO
43− to simulate As metabolism in freshwater algae, subject to the influence of varying P levels corresponding to different eutrophication statuses (low, medium, and high).
The As
V solution was prepared by dissolving Na
3AsO
4·12H
2O (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) in deionized water. Subsequently,
M. aeruginosa was exposed to 0.4, 0.6, 0.8, and 1 mg/L iAs
V at specific As addition time points during each growth period [
19] (lag, exponential, and stationary phases, as outlined in
Table 2 [
19]). The exposure took place in 250 mL Erlenmeyer flasks, containing 100 mL M11 medium with three different P levels, and each treatment was replicated three times (
Table 1). After a 2-day exposure to As, 30 mL algal cells from each replicate were harvested via centrifugation (7000×
g). Subsequently, the cells were cleaned three times with ice-cold 0.1 mol/L phosphate buffer (pH 7.0, 3.5% salinity to maintain the osmotic balance and cell integrity) for 2 min in order to eliminate any As bound onto the cell surface [
15]. Duplicate samples were then freeze-dried, and stored in a desiccator before As speciation analyses.
2.3. Speciation and Content Analysis of Extracellular and Intracellular As
To explore the impact of different PO
43− additions (0.02, 0.1 and 0.5 mg/L) on the metabolism of iAs
V in
M. aeruginosa across the lag, exponential, and stationary growth phases, we collected all algal solutions at the end of the experiments for the iAs
V treatment groups (0.4, 0.6, 0.8, and 1 mg/L). The intra- and extracellular total As and As speciation were then assessed in both the media and algal cells. Approximately 0.005 g of freeze-dried algal samples underwent digestion with 2 mL HNO
3 (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) at 120 °C [
15]. Total As concentrations were determined using a hydride generation atomic fluorescence photometer (AFS-8230, Beijing Titan Co., Beijing, China). The actual concentration of the As stock solution was determined and verified using As quality control samples in drinking water, which was sourced from the China National Center for Standard Reference Materials. Any remaining As-contained wastes were collected in waste liquid barrels to be disposed of as hazardous waste by a qualified organization.
Arsenic speciation analysis in algal samples followed previously described methods [
20]. In summary, freeze-dried cells were extracted with 1.75% HNO
3 at 90 °C in three successive cycles. The supernatants were combined and filtered through 0.22 µm nylon syringe filters for As species determination (i.e., iAs
III, DMA, MMA, and iAs
V), using high performance liquid chromatography coupled with HG–AFS (HPLC–HG–AFS, SA20, Beijing Titan Co., Beijing, China). The extraction method has demonstrated reliability, maintaining As speciation in
M. aeruginosa within such treatments [
21,
22]. The recovery of As species ranged between 87% and 110% using 1.75% HNO
3 extraction, indicating no significant loss or gain for iAs
III and iAs
V during the extraction process.
2.4. The Principles and Computation Methods for Various Factors
The algal cell densities varied under different growth conditions. Therefore, in this study, unit cell As concentrations were employed to assess both intra- and extracellular As concentrations [
20]. The intracellular As content was computed using Equation (1), while the extracellular As content was determined using Equation (2) [
23].
where
Asin (fg /cell) denotes the intracellular As content per unit cell;
CAs−in (fg/L) signifies the measured intracellular As content in the cells;
Vfix (mL) indicates the final fixed volume;
Vsep (mL) indicates the volume of separated algae;
Asex (fg /cell) indicates the extracellular As content per unit cell;
CAs−ex (f/L) is the concentration of dissolved As in the growing media;
Xi+2 (cells/mL) signifies the algal cell density after 2 days of interaction with iAsV.
However, the intra- and extracellular As concentrations are insufficient for evaluating the biological productivity of As through the use of algal cells [
20]. This is because, after part of the exposed As was absorbed by the algae as iAs
V, a portion of it might undergo conversion, primarily to iAs
III, and also, to a lesser extent, to DMA and MMA [
24]. Therefore, the present study was to further calculate the bioproductivities of As absorption (As
ab), bioaccumulation (BCF), transformation (K
t), and release (K
r) from algae through the use of the following equations:
Asab represents the As absorption per cell (fg/cell), encompassing both intra- and extracellular As, but excluding the iAsV form;
Kt denotes the transformation rate;
Kr denotes the releasing rate;
Asin-0, Asin-1, Asin-2, Asin-3, and Asin-4 denote the intracellular total As, iAsV, iAsIII, DMA, and MMA per unit cell concentration, respectively;
Asex-1, Asex-2, Asex-3, and Asex-4 represent the extracellular dissolved iAsV, iAsIII, DMA, and MMA per unit cell concentration, respectively.
Notably, the extracellular iAsV, designated as Asex-1, is not considered in Equation (5) since it can be viewed as the remaining unchanged As initially added to the growth media.