Bioremediation potential of microalgae for copper ion from wastewater and its impact on growth and biochemical contents: equilibrium isotherm studies

The use of microalgae to remediate heavy metal-contaminated wastewater has attracted more and more interest. In this investigation, the green microalgae Chloroidium ellipsoideum and Desmodesmus subspicatus were used to study copper uptake from nutrient media and its effect on algal growth and metabolism. The growth of C. ellipsoideum and D. subspicatus generally decreased with increasing copper concentrations. There was a decrease in the carbohydrate content of C. ellipsoideum, but an increase was observed in D. subspicatus by treatment with various copper concentrations. Low concentrations of copper helped to increase the protein content of C. ellipsoideum, but a decline in protein content was reported for D. subspicatus. By increasing the copper concentrations, an increase in the free amino acids and a decrease in the total lipid content of C. ellipsoideum and D. subspicatus were recorded. At 0.1 mgl–1 copper concentration, pH of 6.8, and algal dose of 1 g L−1, the maximum biosorption capacity of C. ellipsoideum was 0.398 mg g−1, corresponding to the maximum reduction of 68.66% of Cu2+, and 0.396 mg/g for D. subspicatus, corresponding to the maximum reduction of 59.52%. The Langmuir, Freundlich, Temkin and Dubinin–Radushkevich models were applied to describe the isothermal biosorption of Cu2+ ions in studied algae. The Dubinin–Radushkevich model indicated that the copper biosorption mechanism was physical in nature. Cu2+ has a greater affinity for D. subspicatus than C. ellipsoideum, suggesting that C. ellipsoideum was relatively more resistant to Cu2+ toxicity than D. subspicatus. Moreover, FT-IR analysis revealed that carboxyl, amide, amino, carbonyl, hydroxyl, methyl and alkyl groups were the key groups responsible for the biosorption process. Therefore, D. subspicatus and C. ellipsoideum are efficient biosorbents for Cu2+ and can be used as biosorbents for heavy metals removal from wastewater.


Introduction
Owing to human activities (primarily from agriculture, industy and waste disposal), the concentrations of heavy metal in surface and groundwater have raised significantly in recent decades.Heavy metals can accumulate in organisms, and these metals can accumulate at higher nutritional levels through the food chain, thereby causing great harm to end users and even human health [1].Some heavy metals are needed by living organisms in metabolism, but high concentrations of metals may be harmful [2].
Among the harmful heavy metals, copper is the most common metal contaminant in the ecosystem [3].
Copper is essential element for immune system protection, respiration, and red blood cell formation [4].However, exceeding 1.0 mg L −1 in receiving water can cause toxicity, damage to the nervous system, failure of 2. Materials and methods

Experimental materials
Chloroidium ellipsoideum and Desmodesmus subspicatus algae used in this study were obtained from a local polluted agricultural drain in El-Minia Governorate.All chemical reagents such as sodium hydroxide (NaOH, 98% purity) and sulfuric acid (H 2 SO 4 , 98% purity) for pH adjustment, as well as copper sulfate pentahydrate (CuSO 4 .5H 2 O, 98% purity) and a total lipids kit, were purchased from El-Gomhouria Co, for chemicals & medical appliances, Egypt.To prepare a copper solution (1000 mgL −1 ), a specific amount of CuSO 4 .5H 2 O was dissolved in 1 L of deionized water, then the stock solution was diluted to achieve various concentrations and stored at 4 °C until use.

Algae cultivation and treatments
Green microalgae namely; Chloroidium ellipsoideum (Gerneck) Darienko & al. and Desmodesmus subspicatus (Chodat) E.Hegewald & A.W.F.Schmidt were isolated from Al-Moheet drain (a large agricultural drain at El-Minia Governorate, Egypt).Copper ions were separately added at various concentrations of 0.1, 0.2, 0.5, 1 and 2 mg L −1 to Bischoff and Bold [25] medium containing algal cells.Cultures were incubated at a temperature of 20 ± 2 °C under constant light conditions of 4000 lux and a pH of 6.8.Algal cultures were collected for determining growth and various metabolites at the end of the exponential phase.Three replicates were established for each treatment.

Determination of chlorophyll a
Chl. a was extracted in hot methanol (70 °C) and measured daily in algal cultures.The concentration of chl. a was determined as μg/ml algal suspension according to Metzner et al [26].

Determination of carbohydrates
The carbohydrate fraction was estimated using the anthrone sulfuric acid method adopted by Fales [27].

Determination of proteins
Soluble, insoluble and total protein were measured according to Lowry et al [28].

Determination of free amino acids
Free amino acid contents were estimated according to Lee and Takahashi [29].

Determination of total lipids
The sulfophospho-vanillin method (SPV) described by Drevon and Schmitt [30] was used to determine the total lipid content.

Estimation of supernatant metal concentrations
The cultures were centrifuged at 4000 rpm and 4 °C for 15 min and then the supernatant was kept for copper ion analysis.The filtrate was analyzed for Cu 2+ ions after acid digestion using inductively coupled plasma optical emission spectrometry (ICAP 6200).Based on the difference in copper ion concentration before and after biosorption, the quantity of copper biosorbed at equilibrium was determined in accordance with the following equation: where q e : amount of copper ions biosorbed on algal biomass (mg/g), V: the volume of liquid sample (ml), C i : initial copper ion concentration (mg/L), C eq : final copper ion concentration (mg/L) and W: biosorbent mass (g).
The reduction percentage of copper ions was determined from equation (2): where q max (mg/g) is the maximum biosorption capacity for copper ions that correspond to the saturation capacity, and b (L/mg) is the Langmuir constant value, which is correlated to the affinity of the metal ion for the biosorbent binding sites.The values of b and q max can be determined by plotting C eq /q e against C eq from the slope and intercept, respectively.

Sorption isotherms
The Freundlich isotherm model is presented from equation (4): where: n and K f (L/mg) are constants correlated to the adsorption intensity and the magnitude of adsorption capacity at equilibrium, respectively.The values of 1/n and K f can be obtained by plotting the slope and intercept of lnq e versus lnC eq The Temkin model equation is described by the equations (5), ( 6): where A (L/mg): Temkin constant isotherm, b: Temkin constant related to the heat of sorption (J/mol), T: absolute temperature (298.15K) and R: the constant of gas (8.314J mol −1 K −1 ).B and A values can be obtained by plotting q e versus ln C eq from the slope (B) and intercept (B lnA), respectively.
The isotherm model of Dubinin-Radushkevich (D-R) can be determined from equation (7): where β: the constant of (D-R) associated with adsorption energy (mol 2 /J 2 ), q o (mgg −1 ): theoretical saturation capacity and ε: Polanyi potentialgiven by equation (8): The values of the constants q o (mg/g) and β (mol 2 /J 2 ) were calculated by the linear plot of lnq e versus ε 2 from the intercept and slope, respectively.
Moreover, equation (9) expresses the average sorption energy (E, KJ mol -1 ) relative to the energy transmitted from the target molecule to the surface of biosorbent.

FT-IR analysis
In this study, Fourier Transform Infrared Spectroscopy (FT-IR) analysis was done to characterize the key functional groups occur in biomass responsible for metal sorption.After 2 days, certain volume of cultures were centrifuged at 5000 rpm for 5 min, and the pellet was dried in oven at 60 °C to attain the metal precipitate in powder form, which was measured by FT-IR (Bio-Rad, FTS, 3000 MX) by KBr disk technique (Faculty of Science, Assiut University).

Statistical analysis
Experiments were performed in replicates and averages were analyzed by one-way analysis of variance (ANOVA) and compared by Duncan's multiple test (SPSS 16.0 statistics software).Differences were considered statistically significant for p 0.05.

Results and discussion
3.1.Effect of various copper concentrations on the growth and metabolites of Chloroidium ellipsoideum and Desmodesmus subspicatus Heavy metals can affect algae via penetration of algal cells by chelating proteins either through endocytosis or active transport, affecting algal growth and various biochemical and physiological processes.In the current study, the growth of C. ellipsoideum expressed as chl.a was decreased with increasing the concentrations of copper ions, while a non-significant increase in the growth of D. subspicatus was observed (figures 1(a), (b); table 1).Furthermore, the dry weight of C. ellipsoideum and D. subspicatus decreased with increasing copper concentrations compared to controls.In this respect, Fawzy et al [13] reported that chl. a and dry weight of Synechocystis pevalekii and Scenedesmus bernardii decreased with increasing the concentration of cobalt ion.As well as, Fawzy [31] stated that various Cd 2+ and Cu 2+ concentrations resulted in reduced pigment content in Merismopedia tenuissima.Afkar and Fathi [32] also reported the inhibitory impact of Cu 2+ ions on the growth rate and pigment content of Chlorella vulgaris.
The toxic effect of copper at higher concentrations observed in this study may be attributable to copper oxidative potential, which decreases oxygen evolution levels, chlorophyll and ATP by enzymes inhibition (nitrate reductase and alkaline phosphatase), which are involved in cellular metabolism of nitrates and ammonia [33].Bajguz [34] explained that the decreasing in chlorophyll a content may be due to the inhibition of chlorophyll synthesis and the enzymes involved in this process, and certain metals may substitute the magnesium atom of chlorophyll, causing photosynthesis breakdown.
Soluble, insoluble and total carbohydrate contents of C. ellipsoideum were decreased by increasing the concentrations of copper (table 1).Brahmbhatt et al [35] found the same results in Spirogyra cultures in response to various concentrations of Cd 2+ ions.Tripathi and Gaur [36] reported that the carbohydrate content of Scenedesmus sp. was reduced by Cu 2+ and Zn 2+ ion treatment.The total amount of carbohydrate in Chlorella vulgaris was declined gradually depending on the concentration of cobalt, copper and zinc ions in the culture medium [32].On the other side, the treatment of D. subspicatus with Cu 2+ ions in this investigation caused an increment in all carbohydrate fractions.The high value of soluble carbohydrates (5.5 mg g −1 dry wt.) was recorded in D. subspicatus at copper conc. of 0.5 mgl −1 , while the highest value of insoluble and total carbohydrate (44.01 and 47.9 mg g −1 dry wt., respectively) was recorded at 0.1 mgl −1 of copper conc.compared to control culture (p < 0.05).Chia et al [37] found that increased stress with Cd 2+ ion induced higher development of carbohydrates in Chlorella vulgaris.Fawzy and Issa [14] observed increases in insoluble and total carbohydrates in Cyanosarcina fontana when treated with wastewater.This may be due to the existence of heavy metals in sewage, which changed algal distribution and carbohydrate accumulation.
In the present study, low concentration (0.1 mgl −1 ) of copper ions leads to an increase in the insoluble and total protein content of C. ellipsoideum (table 1).Chia et al [37] observed that Cd 2+ stress would lead to an increase in total protein in Chlorella vulgaris.Shanab et al [38] also reported that lower Pb 2+ ion concentrations significantly stimulated protein protein content in Scenedesmus and Pseudochlorococcum.Increased protein content can be used as a defensive mechanism by the formation of binding proteins such as phytochelatins and metallothioneins [38].On the other hand, the treatment of D. subspicatus with various copper concentrations significantly decreased total and insoluble protein contents (p < 0.05; table 1).
In the cultures of Spirogyra spp.and Oscillatoria spp., Brahmbhatt et al [35] noticed a reduction in protein and proline content with increasing Cd 2+ ions.Fawzy [31] stated that the content of total protein of Mersmopedia tenuissima reduced with increasing concentrations of copper and cadmium ions.The inhibition of protein by high concentrations of metal is due to metals toxicity to the enzymatic reactions of protein synthesis [36].In other words, decreased protein synthesis may be due to the blockage of certain enzymatic activities by binding to free amino acids or carboxylic groups [39].
In the current study, the highest contents of the free amino acids (13.8 and 78.6 mg g −1 dry wt.) were recorded at 0.5 and 1 mg L −1 of copper in C. ellipsoideum and D. subspicatus, respectively (table 1).Omar [40] showed that the low concentrations of zinc ion increased the total amino acid content of Scenedesmus obliquus and Scenedesmus quadricauda, while it decreased at high concentrations.Chia et al [37] found that amino acids   involved in metal chelation, such as histidine, glutamine and proline elevated significantly after Chlorella vulgaris susceptibility to Cd 2+ stress.Afkar and Fathi [32] noticed that the total free amino acids slowly increased in Chlorella vulgaris with elevated the concentrations of cobalt, copper and zinc ions.The production of amino acids as a result of metal stress may be encouraged by the biosynthesis of protein, or may be attributed to some suppressing chelating mechanism against the toxicity of heavy metals [41].
The lipid content of algae relies on many factors such as salinity, media availability of nitrogen, light intensity and a number of other possible stressors such as heavy metals [42].In the present investigation, total lipid contents were decreased by treating C. ellipsoideum and D. subspicatus with different concentrations of copper ions.The maximum total lipid content was recorded in control cultures of the tested algae (table 1).In this respect, Pham et al [43] noticed a dramatic decrease in total lipid production in Scenedesmus sp. with Pb 2+ ion addition.These findings are inconsistent with the results of Yang et al [44] who stated that the total lipid contents of Chlorella minutissima were significantly increased by adding cadmium and copper ions.Pham [45] also reported that lipid accumulation of Scenedesmus sp. was significantly increased by the treatment of Cu 2+ ions.Tripathi and Gaur [36] established that in Scenedesmus sp., lipid levels were almost constant under stress of metal because of enzyme tolerance of lipid metabolism or decreased (or increased) enzymes activity involved in the consumption and development of lipid.

Sorption isotherms of Cu 2+ ions by Chloroidium ellipsoideum and Desmodesmus subspicatus
The relationship between biosorbed metal ions concentration and the metal in solution at a given temperature is the biosorption isotherm.Sorption isotherms of Cu 2+ ions in solutions of C. ellipsoideum and D. subspicatus are seen in figure 2.
In this study, the maximum reduction of Cu 2+ by C. ellipsoideum and D. subspicatus was recorded at 0.1 mgl -

1
, pH of 6.8, and algal dose of 1 g/l (68.66% and 59.52%, respectively; figure 3).In general, the data in this study show that metal biosorption decreases with increasing metal ion concentration.The decreased percentage of biosorption may be due to the absence of adequate free metal biosorption sites.All copper ions are exist at lower concentrations, associated with binding sites in aqueous solution, and thus have a higher percent biosorption than higher Cu 2+ concentrations.In addition, lower biosorption may occur at higher concentrations because of the saturation of adsorption sites.The biosorption potential increases and reaches a saturation value as the concentration of metal ions in the aqueous medium increases [46].The efficacy of the removal of ion also differs according to microorganisms used and the types of heavy metal used [14,31].In this resopect, Li et al [47] reported that Scenedesmus spp.and Chlorella spp.are capable of removing up to 89% of Pb 2+ from aqueous solutions.To determine the potential biosorption capacity of metal ions per unit biomass weight, the Langmuir model was chosen to form a complete monolayer coverage on the surface of biomass at high equilibrium concentrations [48].The biosorption capacity values (q max ) and the Langmuir constant b were determined using linear regression.Langmuir constant q max (mgg -1 ) of C. ellipsoideum and D. subspicatus for Cu 2+ was 0.398 and 0.396 mgg -1 , respectively (figure 4(a); table 2).The results revealed that the biosorption of Cu 2+ by C. ellipsoideum and D. subspicatus was almost similar.The data of Fawzy [31], who stated that Merismopedia tenuissima had a maximum biosorption capacity of 0.47 mgg -1 , are in close agreement with our findings.The removal rate of heavy metals by algae depends on the initial metal concentration, exposure time, target species, algal surface  composition, and the tendency of surface functional groups for specific metals [47].Langmuir constant value (b) is usually used to relate ion-metal affinity of biosorbents [49].In the current study, the Langmuir constants b of C. ellipsoideum and D. subspicatus were 2.89 and 4.55 Lmg -1 , respectively (table 2).From these data, it was clear that, Cu 2+ affinity for D. subspicatus was higher than C. ellipsoideum.These results indicated that C. ellipsoideum was more tolerant to the Cu 2+ toxicity than D. subspicatus.Roy et al [50] stated that Nostoc muscorum has a high capacity for Cd 2+ , Pb 2+ , Zn 2+ and Cu 2+ bioremoval processes and a broad range biosorption capacity for these ions.High determination coefficient (R 2 ) values (0.954 and 0.985; table 2) for C. ellipsoideum and D. subspicatus, respectively confirmed the fit of the experimental data to the Langmuir model.The suit of Langmuir model on particular sites is compatible with the strong monolayer sorption.Therefore, the effect of Cu 2+ on the biomass of C. ellipsoideum and D. subspicatus is well described by Langmuir model.
Freundlich model was selected to determine the biosorbate's adsorption intensity on algal biomass.The fast uptake of Cu 2+ from aqueous media was shown by the magnitude of the biosorption capability (K f ) and biosorption intensity (n) (figure 4(b); table 2).The maximum K f values for Cu 2+ from C. ellipsoideum and D.  2).The n value is used for the evaluation of a good adsorbent, with values greater than 1 indicating favorable adsorption behavior [12].Higher n values (2.05 and 2.17) for C. ellipsoideum and D. subspicatus, respectively suggested better biosorption and the formation of strong bonds between the metal ions and the adsorbent (table 2).From these data, it can be seen that, the biosorption intensity of D. subspicatus for Cu 2+ was better than that of C. ellipsoideum.High determination coefficient (R 2 = 0.935) for C. ellipsoideum suggested that Freundlich model fitted well with the experimental results than D. Subspicatus (R 2 = 0.838; table 2).Temkin model is based on the assumption that sorption heat decreases linearly rather than logarithmically with coverage for all molecules, but ignores high and very low concentrations.Moreover, for the bounding energy, it assumes a uniform distribution up to a certain maximum binding energy [61].The higher values of Temkin isotherm constant (A; 37.5 and 52.01 L mg −1 ; table 2) for C. ellipsoideum and D. subspicatus, respectively proposed a higher potential for sorbent-metal ion.In addition, the higher values of Temkin constant (b; 30.97 and 29.8 J/mol) for C. ellipsoideum and D. subspicatus, respectively suggested the presence of high sorbatesorbent interactions [62].The results of determination coefficient suggest that the Temkin model was not sufficiently suited for Cu 2+ biosorption by C. ellipsoideum and D. subspicatus (figure 4(c); table 2).
The data showed that the models of Langmuir, and Freundlich were well suited to characterize the Cu 2+ biosorption isotherm for C. ellipsoideum.On the other hand, the experimental results of adsorption isotherm of D. subspicatus were well represented by Langmuir, and D-R models over the concentration range studied.

FTIR analysis
Metal ions biosorption by microorganisms is due to different types of chemical groups present in the cell wall, like carboxyl, carbonyl, hydroxyl, thioether, sulfhydrl, phosphonate, sulfonate, phosphodiester, imidazole, amide and amine groups [64].
In this research, FT-IR was made to provide a preliminary and qualitative analysis of the chief functional groups present on C. ellipsoideum and D. subspicatus cell wall, that may be responsible for the biosorption of Cu 2+ ions.FTIR spectra of untreated and treated algal biomass by Cu 2+ ions are shown in figure (S1, S2).In this study, the bands located at 3378.74 cm -1 and 3385.70 cm -1 shifted to 3385.11 cm -1 and 3354.74cm -1 after treatment of C. ellipsoideum and D. subspicatus with Cu 2+ ions, respectively were correlated by O-H and N-H groups [65].Bands around 2920 cm −1 and 2850 cm -1 observed on the surface of control cultures and treated biomass of C. ellipsoideum and D. subspicatus were assigned to CHO and alkyl groups [66].The peak at 1653.72 cm -1 transferred to 1652.82 cm -1 after copper biosorption by C. ellipsoideum, as well as, the band at 1651.63 cm -1 shifted to 1658.93 cm -1 after biosorption of Cu 2+ ion on D. subspicatus biomass, suggested that the uptake of Cu 2+ ions involved carbonyl and amide groups [67].Dmytryk et al [68] proved the involvement of amide group in the sorption of Cu-loaded Spirulina maxima.A new stretching peak at 1744.82 cm -1 was found only on Cu 2+ ion-treated C. ellipsoideum and D. subspicatus biomass.
Oves et al [69] observed a band at 1740 cm -1 due to the presence of COO− anion.The bands present in the range of 1400 and 1500 cm -1 on the treated and untreated algal biomass reflect the presence of stretched C-C group aromatics.The presence of an absorption band at 1378.35 cm -1 was identified by Cu 2+ on the treated C. ellipsoideum surface.Bands around 1248.58 cm -1 (before biosorption) and 1246.73 cm -1 (after the biosorption of Cu 2+ ) in C. ellipsoideum, respectively; in addition, the peak at 1245.68 cm -1 that was present only in the control culture of D. subspicatus can be assigned to -CH 3 group [70].The bands from 1020-1250 cm -1 are due to C-N stretch aliphatic amines [71], within this range the peak at 1153 cm -1 was only present in the treated culture of C. ellipsoideum and the control culture of D. subspicatus; as well as, absorption bands at 1076.97 cm -1 , 1078.16 cm -1 and 1026.44 cm -1 , 1026.98, cm -1 were observed in the control and the treated culture of C. ellipsoideum and D. subspicatus, respectively.
After Cu 2+ biosorption, the band at 861.67 cm -1 only appeared in C. ellipsoideum ; while, the band at 778.13 cm -1 was only found on the control culture surface.Saleh [71] stated that the absorption bands between 550 and 850 cm -1 were attributed to the C-Cl stretching of alkyl halides.The peak located at 575.97 cm -1 was found on the surface culture of C. ellipsoideum treated with Cu 2+ ions.On the other side, absorption bands around 575.67 cm -1 (control) and 525.08 cm -1 (after treatment with Cu 2+ ) were observed in D. subspicatus, indicating the presence of alkyl halides (C-Br) [72].Microalgae possess variety of chemical groups capable of binding metals ions in their biomass.The cell wall of algae consists of lipids, polysaccharides and structural proteins that offer functional binding groups for metal ions like amine, hydroxyl, carboxylate, sulphate and phosphate [73].Therefore, the analysis of FT-IR suggested that carboxyl, amide, amino, carbonyl, hydroxyl, methyl and alkyl groups are the key groups responsible for the biosorption process.

Biosorption performance of C. ellipsoideum and D. subspicatus for copper ions in real wastewater
Copper ion removal from real wastewater was also studied at three different levels (0.5, 1 and 2 mg l −1 ).Real wastewater sample was collected from the sewage wastewater treatment plant at Wady Al-Arj, Taif, Saudi Arabia (21°19′ N and 40°32′ E, and altitude of 1591 m).The pH, temperature, electrical conductivity (EC), and total dissolved solids (TDS) of the effluent were determined to be 8.35, 21.9 °C, 1684 μS cm −1 , and 1077.8 mg L −1 , respectively.Figure 5 depicts the efficient removal of Cu 2+ by C. ellipsoideum and D. subspicatus biomass, indicating that the algal biomass could efficiently eliminate copper ions from wastewater.

Conclusions
Copper toxicity was determined in this work on the green microalgae C. ellipsoideum and D. subspicatus.According to the experimental results, exposure to high concentrations of copper inhibited growth and reduced chlorophyll content in the studied microalgae.In general, treatment with high concentrations of copper enhanced the content of free amino acids in C. ellipsoideum and D. subspicatus.However, when the concentration of copper increased, the total lipid content of the investigated algae declined compared to control culture.At 0.1 mg l −1 copper content, pH of 6.8, and algal doses of 1 g l −1 , C. ellipsoideum and D. subspicatus showed the maximum reduction of Cu 2+ at 68.66% and 59.52%, respectively.The Langmuir and Freundlich models better described copper biosorption on C. ellipsoideum biomass, although the Langmuir and D-R models best fit the experimental data of D. subspicatus adsorption isotherm.A real wastewater sample was also treated with the studied algae, which were effective in eliminating copper from wastewater.The FTIR spectra indicated that O-H, N-H, COO−, and C-C groups played a key role in copper biosorption.As a result, D. subspicatus and C. ellipsoideum can be considered promising sustainable biosorbents for heavy metal removal from wastewater.
Sorption isotherm is explained by Langmuir, Freundlich, Dubinin-Radushkevich (D-R) and Temkin isotherm models.The Langmuir equation is expressed as the following equation (3):
Control, Chl.a = Chlorophyll a, D.W = Dry weight, S.C. = Soluble carbohydrates, Ins. C. = Insoluble carbohydrates and T.C. = Total carbohydrates, S.P. = Soluble proteins, Ins.P. = Insoluble proteins, T.P. = Total proteins.The data are given as averages of three replicates ± standard error.At p < 0.05, the values followed by the various letters differ significantly.

3. 3 .
Biosorption isotherms models Different models of isotherm were used in this study to define biosorption isotherm, such as Langmuir, Freundlich, Temkin and Dubinin-Radushkevich. Model parameters estimation allowed us to compare the properties of C. ellipsoideum and D. subspicatus metal binding.

Table 1 .
Influence of various copper concentrations (mgL −1 ) on the growth and metabolites of C. ellipsoideum and D. subspicatus.