Introduction

Cadmium (Cd) is a nonessential element in macroalgae. It is highly mobile and toxic and can be enriched in animals and plants as it moves throughout the food chain. Cd can enter the human body when consumed and may cause kidney damage and cancer and impair lung function (Li et al. 2017; ATDR 2012). Therefore, the remediation of Cd pollution has become an important concern. Bioremediation is a promising technique for Cd remediation because of its eco-friendliness and cost-effectiveness (Zeraatkar et al. 2016). The green alga Cladophora, an important and ubiquitous component of freshwater environments, can be used for the phytoremediation of heavy metals (Cao et al. 2015a; Yang and Li 2015; Zeraatkar et al. 2016) and exists widely in natural water bodies of Anhui Province, China.

The subcellular distribution of heavy metals can provide information on the mechanism of heavy metal enrichment and tolerance (Hou et al. 2013). Cd exists in different chemical forms after assimilation by pokeweed (Fu et al. 2011). The toxicity and tolerance mechanisms for Cd are linked to its subcellular and chemical forms, greatly affecting its transport in plants (Meyer et al. 2015; Ma et al. 2015; Lavoie et al. 2009a). Similarly, the heavy metal tolerance and enrichment characteristics of hyperaccumulator plants are closely related to metal subcellular distribution and plant morphology (Lwalaba et al. 2017). Li et al. (2016) suggested that Arbuscular mycorrhizal fungi mainly enhance rice resistance to cadmium by altering the subcellular distribution and chemical forms of Cd. Mwamba et al. (2016) demonstrated that the difference between the subcellular distribution and chemical forms of Cd and Cu can improve the toxicity to Brassica napus plants. Therefore, investigating the subcellular and chemical forms of Cd in Cladophora rupestris is necessary to reveal the detoxification and absorption mechanisms of this alga.

Various absorption isotherms have been used to evaluate sorption characteristics (Kumar et al. 2016). A thermodynamic equation was used to determine the Cd biosorption capacity of living microalgae (Zhou et al. 2017). Xie et al. (2015) reported that the Freundlich model fitted biosorption data well and illustrated the complexation–biosorption properties of rice roots. Anastopoulos and Kyzas (2015) used isotherm models and kinetic equations to investigate the ability of algae to adsorb heavy metals and confirmed that micro- and macroalgae are promising biosorbents that can be used to remove heavy metals from wastewater. Cd2+ was affected on metal homeostasis in plants. The metal homeostasis of Fe, Mn, Ca, and Mg in Camellia sinensis was affected at Cd concentration of 1.0–15.0 mg/L (Cao et al. 2018).

The present investigation analyzed the subcellular distribution and chemical forms of Cd in C. rupestris under different levels of Cd stress. Langmuir and Freundlich, pseudo-first- and pseudo-second-order reaction, Elovich, and double-constant equations were used to analyze the Cd adsorption characteristics of C. rupestris. Moreover, the effects of Cd2+ on metal homeostasis in C. rupestris were analyzed. The findings of this study can provide references for the remediation of water polluted with heavy metals.

Materials and methods

Algae cultivation and preparation

Cladophora rupestris was collected from surface water of a pond in Hefei in Anhui Province, China (31°50′ N, 117°11′ E). The samples were grown in a sterilized medium at 25 °C and kept under an illumination intensity ranging from 3000 to 4000 lx (with a light:dark photoperiod of 12:12 h) in an incubator (SPX-250B-G). The cultures were kept at a pH of 7.0 ± 0.5 and grown without antibiotics (Cao et al. 2015a, 2015b).

Experimental design and C. rupestris cultivation

Three grams C. rupestris was exposed in 1000 mL BG11 medium (1.5 g NaNO3, 40 mg K2HPO4·3H2O, 75 mg MgSO4·7H2O, 36 mg CaCl2·2H2O, 6 mg citric acid, 6 mg ferric ammonium citrate, 1 mg Na2EDTA·2H2O, 20 mg Na2CO3, 2.86 mg H3BO3, 1.81 mg MnCl2·4H2O, 0.22 mg ZnSO4·7H2O, 0.39 mg Na2MoO4·2H2O, 0.079 mg CuSO4·5H2O, and 0.0494 mg CO(NO3)2·6H2O per liter) (Wang et al. 2017), and containing different concentrations of Cd (0.0, 0.5, 1.0, 2.5, 5.0, 7.5, or 10.0 mg/L) for 7 days. Cd (II) stock solutions (1000 mg/L) were prepared by dissolving Cd(NO3)2 in purified water. All solutions used in the experiments were obtained by means of stock solution dilution (Cao et al. 2015a). Each treatment was performed in three replicates.

Experiment and measurements

Before doing the subcellular fractionation and chemical form characterization of intracellular Cd, the carry-over Cd from the exposure solution and extracellular loosely bound Cd should be washed off from the algae samples with distilled water, and it is assumed not to affect cell physiology (Hassler et al. 2004; Lavoie et al. 2009b).

Subcellular partitioning procedure

After 7 days, the tissue fractionation of C. rupestris was on the basis of Cao et al. (2018): 1.0 g samples were frozen prior to the experiment. Cladophora rupestris tissues were homogenized in extraction buffer [50 mM HEPES, 1.0 mM DTT, 500 mM sucrose, 5.0 mM ascorbic acid, 1.0% (w/v) Polyclar ATPVPP, adjusted to pH 7.5]. Separation of subcellular fractions by differential centrifugation was the following process: the homogenate was centrifuged at 300×g for 30 s and the pellet was designated the cell wall fraction, consisting mainly of cell walls and cell wall debris (Lavoie et al. 2009a; Zhao et al. 2015). The filtrate was centrifuged at 20000×g for 45 min and the pellet designated as the organelle and the supernatant as the soluble fraction. The resultant pellets were resuspended in extraction buffer. All steps were performed at 4 °C.

Chemical form extraction

Following the method of Cao et al. (2018) and Zhao et al. (2015), frozen algal material (1.000 g) was mixed with 10 mL of extraction solution at 25 °C (24 h). The extraction solution was then separated, and the residual material was re-extracted with the same amount of extraction solution for 2 h. The twice-extracted solutions containing each chemical form of Cd were collected and separately wet digested. Different cadmium chemical forms were extracted successively by the following order: (1) 80% alcohol, extracting inorganic Cd, which included nitrate/nitrite, chloride, and aminophenol cadmium (FE); (2) d-H2O, extraction water-soluble fraction Cd of organic acid complexes and Cd(H2PO4)2 (FW); (3) 1MNaCl, extracting pectate and protein-integrated Cd (FNaCl); (4) 2% HAC, extraction insoluble fraction CdHPO4 with organic acids and Cd(H2PO4)2 and other Cd-phosphate complexes (FHAC); (5) 0.6 M HCl, extracting oxalate acid-bond Cd (FHCl); and (6) Cd in the residue (FR).

Determination of Cd and other element concentration

The samples were placed into 50-mL polytetrafluoroethylene tubes with 10 mL HNO3 for 12 h under the fume hood and then heated on the graphite digestion apparatus at 105 °C for 1.5 h (ZEROM ProD40, Changsha Zerom Instrument and Meter Co., Ltd., China). Afterwards, 2 mL HClO4 was added and heated again at 135 °C for 30 min (Qiu et al. 2011; Cao et al. 2015a), and diluted with water to 50 mL. Cd concentrations in the digestion were determined by M5 Thermo flame atomic absorption spectrometry, operated at 228.8 nm with a hollow cathode lamp under an air/acetylene flame while the fuel gas flow rate was 40.0 L/h, and the slit width was 0.2 nm. The content of Ca, Zn, Mn, Fe, Mg, and Cu were analyzed by M5 flame atomic absorption spectrometry, used hollow cathode lamp as light source, and set the wavelength at 422.7 nm (Ca), 213.9 nm (Zn), 279.5 nm (Mn), 248.3 nm (Fe), 285.2 nm (Mg), and 324.7 nm (Cu) respectively.

Data analysis

Isotherm adsorption model

The Langmuir isotherm model assumes monolayer adsorption on a uniform surface with a finite number of adsorption sites and once a site is filled (Islam et al. 2015). The linear form is expressed as:

$$ \frac{C_e}{Q_e}=\frac{1}{Q_{\mathrm{max}}b}+\frac{C_e}{Q_{\mathrm{max}}} $$
(1)

where Qe is the amount of Cd(II) adsorbed per unit mass of C. rupestris at equilibrium (mg/g), Ce is the concentration of Cd(II) remaining in solution during adsorption equilibrium (mg/L), Qmax is Langmuir constant related to the maximum adsorption capacity (mg/g), and b is Langmuir constant related to free energy of adsorption (L/mg). The values of Qmax and b can be calculated from the slope and intercept of the Ce / Qe − Ce.

The Freundlich isotherm model is an empirical equation based on non-ideal adsorption on a heterogeneous surface, describes the heterogeneity of the adsorbent surface or the interaction between the adsorbent surface and the adsorbate (Chakravarty and Banerjee 2012). The linear is expressed as:

$$ 1\mathrm{n}{Q}_e=1\mathrm{n}{K}_f+\frac{1}{n}1\mathrm{n}{C}_e $$
(2)

where Qe is the amount of Cd(II) adsorbed per unit mass of C. rupestris at equilibrium (mg/g), Ce is the concentration of Cd(II) remaining in solution during adsorption equilibrium (mg/L), Kf is the Freundlich constant and indicative of the relative adsorption capacity of the adsorbent [(mg/g)(kg/mg)1/n], and n is the Freundlich constant, indicating the intensity of adsorption (Farhan et al. 2013). LnQe is a function of lnCe, and n and Kf can be obtained from the slope and intercept.

Kinetics of adsorption of Cd by algae

The kinetic equations used to describe the metal ion biosorption process (Shen et al. 2017; Zhao et al. 2017; Zhang et al. 2017) are the following:

Pseudo-first-order adsorption rate equation,

$$ \ln \left({Q}_e-{Q}_t\right)=\ln \kern0.1em {Q_e}^{- kt} $$
(3)

Pseudo-second-order adsorption rate equation,

$$ t/{Q}_t=1/k\ {Q_e}^2+t/{Q}_e $$
(4)

Elovich equation,

$$ {Q}_t=a+b\kern0.1em \ln t $$
(5)

Double-constant equation,

$$ \ln {Q}_t=a+b\kern0.1em \ln t $$
(6)

where Qt is the amount of Cd(II) adsorbed at t days (mg/kg), Qe is the amount of Cd(II) adsorbed per unit mass of C. rupestris at equilibrium (mg/kg), K is apparent rate constant, a and b are defined as the rate constants of the Elovich and double-constant kinetics equations, and t was the time (days).

The data of this paper are expressed in Excel 2010 in the form of mean ± standard deviation (SD) with three repetitions. Pearson correlation coefficient and bilateral test method were used for correlation analysis; the conditions (normality and variance homogeneity) have been validated prior to correlation analysis. SPSS18.0 and Originpro8.5 software were used to analyze data and create diagrams.

Results and discussion

Biosorption effects and subcellular distribution of Cd in C. rupestris

The subcellular distribution of Cd is shown in Table 1, and Cd concentration in culture medium after different times is shown in Fig. 1, clearly indicating that C. rupestris accumulates Cd. The cumulative Cd capacity of C. rupestris ranges from 288.19 to 2735.3 mg/kg, and that bioconcentration factor (BCF) values reached up to 576. Brooks and Lee (1977) emphasized that the aboveground parts of a hyperaccumulator plant can accumulate up to 100 mg·kg−1 Cd. Thus, C. rupestris can be considered as a Cd hyperaccumulator plant (Cao et al. 2015a). Among all subcellular fractions, the cell wall exhibited the highest Cd concentration (Fig. 2). Cd concentrations in different subcellular fractions decreased in the following order: the cell wall and debris fraction (42.8–68.2%) > soluble fraction (22.1–38.4%) > organelle (11.6–30.5%). Moreover, Cd concentration markedly increased in different subcellular fractions with Cd supply (Fig. 2). When Cd concentration exceeded 5.0 mg/L, its proportion in cell organelles increased dramatically, indicating that high Cd concentration weakened the cell wall adsorption and is gradually transferred to cell organelles. These findings suggested that in C. rupestris, the cell wall plays a significant role in Cd retention at Cd concentration of ≤ 2.5 mg/L. By contrast, intracellular mechanisms mediate Cd retention at Cd concentration of > 2.5 mg/L Cd.

Table 1 Measured Cd contents in different parts of C. rupestris and BCF values
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Fig. 1
figure 1

Concentration of Cd in culture medium after different time

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Fig. 2
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The distribution of Cd in subcellular components

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Previous studies showed that the cell wall and vacuoles play important roles in the tolerance, detoxification, and accumulation of heavy metals in plants (Zhang et al. 2011). The cell wall provides negatively charged sites on their multipleated surfaces that bind Cd ions and restrict Cd ion transport across the cytomembrane (Brune et al. 2008). Salt et al. (2002) reported that complexation with strong ligands, such as the thiol groups of phytochelatins and glutathione, also participates in detoxification. Consistent with the observations of Kramer et al. (2000), we found that under intensifying Cd stress, Cd is gradually transferred to organelles and soluble fraction components. So the C. rupestris has bioremediation potential of Cd.

Chemical forms of Cd

After the extraction of different chemical forms of Cd, the extracts exhibited the following order of Cd concentration: FHAc > FNaCl > FW > FHCl > FE (Fig. 3). The residual form of Cd in C. rupestris was lower than 0.1 mg/kg. Among these extracts, the 2% HAc extract had the largest proportion of Cd (43.2–56.0%) in the form of insoluble CdHPO4 complexed with organic acids, Cd(H2PO4)2, and other Cd-phosphate complexes (FHAC), followed by 1 M NaCl extract containing pectate and protein-integrated Cd (FNaCl), accounting for 30.8–43.2% Cd. The different chemical forms of Cd in C. rupestris increased in a concentration-dependent manner after Cd exposure.

Fig. 3
figure 3

Proportion of Cd in the storage forms in C. rupestris

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Water-soluble inorganic and organic fractions of Cd (extracted with 80% ethanol and d-H2O) have higher migration capacity and are more deleterious to plant cells than undissolved Cd phosphate (extracted with 2% HAc) and Cd oxalate (extracted with 0.6 M HCl) (Bai et al. 2014). In the present study, Cd was mainly present in the forms of insoluble CdHPO4 complexed with organic acids in C. rupestris, Cd(H2PO4)2, and other Cd-phosphate complexes (extracted with 2% HAc). These forms are less actively toxic than other forms, suggesting that their transport across cells is limited. At the Cd concentration of 2.5 mg/L, the proportion of low-activity and low-toxicity Cd (extracted with 2% HAC or 0.6 M HCl) increased by 12.8% (P < 0.05). Bai et al. (2014) reported that Viola tricolor L. resists Cd toxicity by decreasing the proportion of soluble forms (extract by water and alcohol) of Cd. Similarly, the subcellular experiment revealed that at Cd concentration of < 5.0 mg/L, the Cd localization in the cell wall and soluble fraction attenuated organelle damage in C. rupestris. Other studies found similar results, and the chemical forms of Cd are related to its accumulation in the cell wall and in soluble fractions (Hernandez-Allica et al. 2006; Kupper et al. 2000). Krzesłowska (2011) reported that the complexation of Cd with mercapto and the side chains of protein or other organic compounds promote the complexation of Cd with pectic acid and protein. In C. rupestris, Cd enrichment in the cell walls decreases free intracellular Cd. This response ultimately attenuates Cd toxicity by increasing the amount of actively binding forms of Cd.

Kinetics of Cd in C. rupestris

Thermodynamic characteristics

The experimental results were analyzed using Langmuir and Freundlich adsorption models to describe the Cd biosorption characteristics of C. rupestris (Fig. 4). The R2 value of the Freundlich isotherm model was 0.933, which was higher than that of the Langmuir isotherm model (R2 = 0.656). This result indicated that the Freundlich model better fitted Cd biosorption by C. rupestris than the Langmuir model (Fig. 4a and b). These results are consistent with those obtained by Anastopoulos and Kyzas (2015), who stated that the Langmuir model fits well Cr(VI) adsorption by Chlorella vulgaris. Valuable information can also be observed from the model parameters (Areco et al. 2012). The Freundlich constant Kf indicates that the relative biosorption capacity of the biosorbent is related to bonding energy (Pillai et al. 2013). Kf = 0.638 reflects the low affinity and mobility of Cd to binding sites in C. rupestris. The Freundlich constant n indicates adsorption intensity. If the slope 1/n is less than 1, then a normal Freundlich isotherm is observed. Otherwise, cooperative adsorption can be observed (Fan et al. 2011). In the present study, the 1/n value (0.519) was less than 1, indicating that Cd adsorption by C. rupestris can be fitted by the normal Freundlich isotherm model. Most of the literatures suggested that the Freundlich model (Doshi et al. 2007; Mehta and Gaur 2001) fits well heavy metal absorption by living microalgae, which can remove heavy metals from water not only through cell-surface biosorption but also by cellular bioaccumulation. By contrast, the Langmuir isotherm model fits well the uptake process, which involves passive biosorption, of dead microbial cells or solid porous biosorbents (Huang et al. 2013; Ding et al. 2016). In the present study, the Freundlich isotherm model fitted well the Cd sorption by C. rupestris on the assumption that this process involves passive biosorption and initiative absorption.

Fig. 4
figure 4

Langmuir and Freundlich isotherm plot of Cd biosorption in C. rupestris (a), (b) as Langmuir and Freundlich isotherm plot of Cd biosorption in C. rupestris (c), (d) as Langmuir and Freundlich isotherm plot of Cd biosorption in cell wall of C. rupestris

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The Langmuir and Freundlich adsorption models were used to analyze Cd biosorption on the cell walls of C. rupestris (Fig. 4c, d). R2 of the Freundlich model was 0.875, indicating that the Freundlich model fitted well the biosorption data. However, the results of the Freundlich adsorption models better fitted the biosorption of the whole cell of C. rupestris than the cell wall only.

Kinetics characteristics of Cd adsorption

The kinetics data of Cd adsorption by C. rupestris at 25 °C were fitted with the pseudo-first-order and pseudo-second-order adsorption rate, Elovich, and double-constant rate equations (Fig. 5). The result indicated that the pseudo-second-order adsorption rate (R2 = 0.997) and Elovich (R2 = 0.972) equations have higher correlation coefficients than the other equations. The Elovich equation provides a description of the heterogeneous diffusion on the basis of the reaction rate and the diffusion factor (Zhao et al. 2017). The pseudo-second-order model assumes that the adsorption rate is controlled by the chemisorption mechanism (Kumar et al. 2016). Thus, complexation–biosorption properties and more than one mechanism would be involved in Cd biosorption by C. rupestris. Here, we found that Cd biosorption by C. rupestris involves active chemisorption and diffusion. This result was consistent with that reported by Talebi et al. (2013), who found that the second phase of biosorption by algal cells includes the uptake of heavy metal ions, wherein the first phase of biosorption consists of fast inactive adsorption on the cell surface.

Fig. 5
figure 5

Adsorption dynamics equation fitting results: a Pseudo-first-order adsorption rate equation, b Pseudo-second-order adsorption rate equation, c Elovich equation, d Double-constant equation

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Correlation analysis between the subcellular distribution and chemical forms of Cd in C. rupestris

Correlation analysis revealed that in C. rupestris, the chemical forms of Cd present in soluble fractions and organelles are strictly correlated with Cd concentration (P < 0.01) (Table 2). The content of each chemical form of Cd in soluble fractions increased with Cd concentration and decreased in cell organelles with Cd concentration. Under intensifying Cd stress levels, Cd was gradually transferred to organelles and vacuolar components, and vacuolar compartmentalization transformed the chemical forms of Cd into low-toxicity forms to defend against toxicity. This response is consistent with the conclusion presented in Cao et al. (2018).

Table 2 Correlation analysis of subcellular distribution and chemical forms of Cd in C. rupestris
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Effects of Cd on metal homeostasis in C. rupestris

Cd stress caused mineral imbalance and disrupted the internal stability of mineral nutrition in C. rupestris. Cd treatment promoted the uptake of Ca and Zn (Fig. 6, RCa2 = 0.9194, P < 0.05; RZn2 = 0.899, P < 0.05). The absorption of Cu, Fe, Mn, and Mg was promoted by Cd concentrations, especially at Cd concentrations lower than 7.5 mg/L, but Mg uptake was inhibited by 10 mg/L Cd. The Mg concentration was lower than 25 mg/kg (Fig. 6). Cd can indirectly affect the growth and metabolism of plants by affecting the absorption of mineral nutrients, and nutrition status can greatly influence the capability of plants to accumulate heavy metals (Sarwar et al. 2010; Singh et al. 2010; Cao et al. 2018). Cd uptake and toxicity were observed in green alga (Chlamydomonas reinhardtii) after long-term exposure (60 h) to a range of environmentally realistic free Zn, Co, Fe, Mn, Ca, and Cu (Lavoie et al. 2012). Ca plays an important role in maintaining the stability of cell wall structure and thus enhances plant resistance (Cao et al. 2015b). Furthermore, enhancing cell wall stability by increasing Ca content is related to Cd enrichment in the cell wall. Zn is involved in plant transpiration and participates in auxin formation, photosynthesis, and protein synthesis. Increased Zn uptake may also be related to the coaccumulation relationship between Zn and Ca (Sarret et al. 2006). The decrease in Mg content inhibited the photosynthesis of C. rupestris because Mg is an important component of chlorophyll. Siedlecka and Krupa (1999) reported that the replacement of Mg, which is the central atom of chlorophyll, with Hg, Cu, Cd, or other heavy metal ions, disrupts photosynthesis. Importantly, Ren and Sheng (2017) proposed that the homeostatic balance between Ca2+ and Mg2+ is a critical feature of plant mineral nutrition for optimal growth and development under changes in soil nutrient status.

Fig. 6
figure 6

Nutrient contents in C. rupestris affected by Cd

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Conclusion

Our study demonstrated that C. rupestris could accumulate Cd. Cd mainly accumulates in the cell walls and soluble fractions in C. rupestris. Under Cd stress, a large quantity of Cd ions exists in C. rupestris in the forms of insoluble CdHPO4 complexed with organic acids, Cd(H2PO4)2, and other Cd-phosphate complexes. Cd complexation–biosorption involves passive biosorption and initiative absorption. Cell wall adsorption has an important role in the Cd adsorption ability and process of C. rupestris. Cell wall sequestration, vacuolar compartmentalization, and chemical morphological transformation are likely essential mechanisms for Cd detoxification and biosorption by C. rupestris. The adsorption data were well fitted by the Freundlich model (R2 = 0.933) and could be described by the pseudo-second-order reaction rate (R2 = 0.997) and Elovich (R2 = 0.972) equations. Related parameters indicated that Cd adsorption by C. rupestris is a heterogeneous diffusion. Cd promoted Ca and Zn uptake by C. rupestris. Cu, Fe, Mn, and Mg adsorption was promoted by low Cd concentrations and inhibited by high Cd concentrations. This study suggests that C. rupestris has bioremediation potential of Cd.