Cr⁶⁺ bioremediation efficiency of Oscillatoria laete-virens (Crouan & Crouan) Gomont and Oscillatoria trichoides Szafer: kinetics and equilibrium study

Abstract

Two species of cyanobacteria, Oscillatoria laete-virens (Crouan & Crouan) Gomont and Oscillatoria trichoides Szafer, were isolated from a polluted environment and studied for their Cr⁶⁺ removal efficiency from aqueous solutions. The parameters studied included the solution pH, contact time, initial concentration of Cr⁶⁺ and culture density. Living biomass is more efficient than dead biomass in Cr⁶⁺ removal. Removal by living biomass involves bioreduction and biosorption. Below pH 3.1, bioreduction is favored and biosorption is dominant at higher pH. The highest removal through biosorption for living biomass was achieved between pH 5 and 5.9 and for dead biomass at pH 2. The maximum removal was on the tenth day of exposure for both the species. Cr⁶⁺ removal increased from 0.2 to 0.4 g L⁻¹ of culture biomass with a decrease with further increase in biomass. Increased Cr⁶⁺ concentration decreases growth of both the species over time. Both species tolerate a concentration as high as 30 mg L⁻¹ Cr⁶⁺. There was no evidence of bioreduction in the case of dead biomass. Living biomass of O. laete-virens followed both Langmuir and Freundlich models with maximum sorptive capacity (q _max) of 21.88 mg g⁻¹. The results of dead biomass were well fitted only to Langmuir isotherm. O. trichoides living biomass did not follow either of the isotherms, but removed the metal to a maximum extent of 38.7mg g⁻¹. The removal was better described by Freundlich isotherm in case of dead biomass. The pseudo-first-order model describes the kinetics better than the pseudo-second-order model in the case of living biomass. Participation of carboxylic, carbonyl, and amino groups in Cr⁶⁺ removal was confirmed by FTIR analysis. Both species seem to be promising biosorbents for Cr⁶⁺.

Introduction

Heavy metals as pollutants cause serious ecological problems since they are extremely toxic and tend to bioaccumulate throughout the food chain. Chromium is one such pollutant that is released from industries relating to alloys, film and photography, leather tanning, dyes, pigments, electroplating, textile, and wood preservation (Cervantes et al. 2001; Sarin and Pant 2006; Ozturk et al. 2009; Cheung and Gu 2007; Qaiser et al. 2009; Saha and Orvig 2010). Chromium is also an essential nutrient for plants (Prado et al. 2010).

Chromium exists in several oxidation states, but the most stable and common forms are Cr³⁺ and Cr⁶⁺ of which Cr⁶⁺ is more toxic (Cheung and Gu 2007; Kiran et al. 2007; Gupta and Rastogi 2008). Cr⁶⁺ occurs as chromate (CrO ²⁻₄ ) or di-chromate (Cr₂O ²⁻₇ ) ions (Sarin and Pant 2006; Ozturk et al. 2009) and Cr ³⁺ in the form of oxides, hydroxides, or sulfates and exists mostly bound to organic matter in soil in the aquatic environments. Cr⁶⁺ is a strong oxidizing agent and in the presence of organic matter it is reduced to Cr³⁺, a process which is fast in acidic environment. But at higher concentration, it may overcome the reducing capacity of the environment and persist as a pollutant (Cervantes et al. 2001).

Chromate is actively transported across biological membranes in microorganisms (Dreyfuss 1964) through membrane sulfate channels (Cheung and Gu 2007). Inside the cells, Cr⁶⁺ is reduced to Cr³⁺ via unstable Cr⁵⁺ and Cr⁴⁺ states (Arslan et al. 1987). Cr⁶⁺ exists mainly in the oxyanion form and can only be trapped by the cationic components of biosorbent surfaces in an acidic pH. The cell membrane is nearly impermeable to Cr³⁺ because of its insolubility, which facilitates its precipitation and removal (Alcedo and Wetterhahn 1990). The biotransformation of Cr⁶⁺ to Cr³⁺ is considered as an alternative process for treating Cr⁶⁺ contaminated waste (Cheung and Gu 2007; Cervantes et al. 2001).

Cr⁶⁺ has been classified as a human carcinogen. The permissible limit for chromium in drinking water is 0.05 mg L⁻¹ while for waste water it is 1 mg L⁻¹ of Cr⁶⁺ (WHO 2004; US EPA 1998) and therefore it is essential to reduce Cr⁶⁺ concentration in water or waste water to acceptable limits. Physicochemical methods of toxic metal removal are energy consuming and utilize a large amount of reagents (Ozturk et al. 2009), whereas biosorption is environmentally friendly and particularly efficient with low concentration of metals. Microorganisms with high level of tolerance to chromium can be candidates for bioremediation of chromium pollution. This tolerance may be due to biosorption, diminished accumulation, precipitation, and reduction of Cr⁶⁺ to Cr³⁺.

Both live and dead mass of microalgae are useful as biosorbents. Microalgae are preferred for their photosynthetic efficiency, simple nutritional requirements, ease of mass cultivation, and greater production of mucilage with high binding affinity (Khattar et al. 2007; Gupta and Rastogi 2008). Also, compared to fungi and bacteria, they have a greater capacity of metal uptake (Tüzün et al. 2005) due to the presence of functional groups like carboxyl, hydroxyl, sulfate, phosphate, and amines in their cell walls which can sequester heavy metal ions in a short period of time (Gong et al. 2005).

Cr⁶⁺ removal by non-viable cells of cyanobacteria has been demonstrated, using alginate-immobilized Lyngbya putealis (Kiran et al. 2007), Nostoc muscorum (Gupta and Rastogi 2008), and immobilized Nostoc calcicola and Chroococcus sp. (Anjana et al. 2007). However, only a few reports exist on the removal by viable cells such as Nostoc linckia (Bala et al. 2004), Anacystis nidulans (Khattar et al. 2007), and Synechocystis sp (Ozturk et al. 2009). This may be due to the low pH range required for Cr⁶⁺ removal. Living cells are advantageous as biosorbents because they can create a continuous supply of unsaturated metal removing biomass (Terry and Stone 2002), and some living systems can have higher metal removal efficiency than the dead cells (Rahmani and Sternberg 1999). However, the viability and metabolic activity of cells is a major limiting factor affecting the detoxification efficiency. Metal removal using cyanobacterial species isolated from polluted environments has been reported previously (Monteiro et al. 2009a, b; 2010).

To the best of our knowledge, no study has been carried out on the removal of Cr⁶⁺ using living biomass of Oscillatoria species which are very common in both fresh and polluted water habitats with acidic pH. Accordingly, the objectives of the study were to assess the Cr⁶⁺ removal capacity of both living and dead cells of two species of fresh water filamentous cyanobacteria, Oscillatoria laete-virens and Oscillatoria trichoides, which were isolated from an industrial polluted region in Mangalore, India. This study also tries to understand the adsorption and absorption capacities of living cells as well as to compare their biosorption capacities and compares the Cr⁶⁺ removal efficiency of living and dead cells, with respect to pH, contact time, initial metal ion concentration, adsorbent dose, and the impact of this metal on growth. Adsorption isotherms were applied to fit the experimental data and the kinetic models were also used to determine the sorption rates. Biomass–metal ion interactions were evaluated by FTIR analysis.

Materials and methods

Oscillatoria laete-virens and O. trichoides were isolated from the industrial waste waters near Mangalore, India, and identified referring to the monograph of Desikachary (1959). Pure cultures were obtained following standard isolation and culturing techniques on BG-11 medium (Stein 1973). Batch cultures were grown in sterilized optimal BG-11 medium without EDTA in 100-mL borosilicate culture flasks. Cultures were maintained at 25 ± 2°C under a 16:8 light–dark cycle and an irradiance of ∼30 μmol photons m⁻² s⁻¹ provided by cool white fluorescent lamps. Filaments from the exponential growth phase were used as inoculum in the subsequent experiments. Growth was assessed by estimating the Chl-a content (Jeffrey and Humphrey 1975).

A stock solution of Cr⁶⁺ ions was prepared by dissolving K₂Cr₂O₇ in deionized water. Desired concentrations of Cr⁶⁺ were obtained by diluting the stock solution. Glassware and other materials used to handle and grow the cyanobacteria were previously rinsed with dilute nitric acid and then with double distilled water to prevent interferences with analytical assays. Experiments were carried out in triplicate.

Cyanobacterial growth at different pH and Cr⁶⁺ concentrations

A pH range of 2–7 was chosen for the study. Then 0.4 g L⁻¹ of cells was added to BG-11 medium whose initial pH was adjusted using 0.1 N HCl or 0.1 N NaOH. Change in the pH was recorded once in 2 days. Moreover, 0.4 g L⁻¹ of biomass each was also grown in culture media having a suitable pH with 10, 20, 30, 40, and 50 mg L⁻¹ of Cr⁶⁺ in triplicates .The cells were grown for up to 12 days. Chl-a content was measured at regular intervals. There was a prior adaptation to 5–10 mg L⁻¹ Cr⁶⁺ during isolation of cyanobacterial species.

Metal removal by varying factors

The metal removal capacity of these cyanobacteria were studied by culturing 0.4 g L⁻¹ of the biomass in media with initial concentration of 10, 20, 30, 40, and 50 mg L⁻¹ Cr⁶⁺. The extent of Cr⁶⁺ removal was determined as a function of pH by exposing aliquots of 0.4 g L⁻¹ of living biomass to a medium containing Cr⁶⁺ at a pH previously adjusted between pH 2 and 7 using 0.1 N HCl or 0.1 N NaOH. Change in pH was recorded once in 2 days. To see the effect of culture density on Cr⁶⁺ removal, the experiments were carried out at 30 mg L⁻¹ of Cr⁶⁺ with a culture density of 0.2, 0.4, 0.8, and 1.2 g L⁻¹.

Each of these experiments was conducted in triplicate for 12 days. Blank controls were used to ensure that the supernatant Cr⁶⁺ concentration initially provided was not affected by any other factor. Flasks were agitated at 100 rpm in a shaker. Reaction mixtures were separated by centrifugation at 6,000 rpm for 15 min. The supernatants were collected for chromium analysis and the pellet was washed with 0.02 M EDTA for 15 min at 100 rpm to remove Cr⁶⁺ ions adsorbed onto the cell surface and then centrifuged at 10,000 rpm. The pellet was acid-digested and the biosorbed intracellular Cr⁶⁺ estimated.

Cr⁶⁺ removal by dead cells

Biomass harvested from an exponential growth phase was dried at 60°C for 24 h. The amount of 0.4 g L⁻¹ dry weight of dead biomass was then transferred to a metal solution with the required concentration of Cr⁶⁺. The Cr⁶⁺ removal capacity of dead cells was assessed against pH, equilibrium time, initial metal ion concentration, and biomass density. To determine Cr⁶⁺ removal as a function of pH, the aliquots of 0.4g L⁻¹ of biomass was added to a metal solution previously adjusted to pH 2 to 7 and containing an initial Cr⁶⁺ concentration of 10 mg L⁻¹. To determine the contact time required for maximum adsorption, 0.4 g L⁻¹ biomass was suspended in 10 mg L⁻¹ metal solution. To check the effect of initial metal ion concentration on removal of Cr⁶⁺, the biomass was suspended in Cr⁶⁺ solution having a concentration of 10, 20, 30, 40, and 50 mg L⁻¹. The system was kept on a rotary shaker at 100 rpm and the samples were removed at 0, 15, 30, 60, 90, 120, and 150 min and the supernatant assayed for residual Cr⁶⁺ concentration.

Analysis of metal ions

Cr⁶⁺ in the supernatant was measured spectrophotometrically at 540 nm with diphenyl carbazide as the complexing agent (Clesceri et al. 1998). The concentration range studied yields a linear plot indicating adherence to the Beer–Lambert’s law. A new calibration curve was prepared for each analysis. To measure the chromium incorporated into the cells, the pellets were subjected to FA-AAS (flame atomized atomic absorption spectrophotometer) in a GBC 932 plus AVANTA spectrophotometer with a lamp current of 6 mA at wavelength 359.3 nm having an optimum working range 0.1–20 mg L⁻¹.

The total Cr⁶⁺ removed by the cyanobacterial cells was calculated as

$$ q = \frac{{\left( {{C_{\text{i}}} - {C_{\text{f}}}} \right)v}}{m} $$

(1)

where q = metal removed (mg g⁻¹), C _i = initial Cr⁶⁺concentration in the supernatant (mg L⁻¹), C _f = final Cr⁶⁺concentration in the supernatant (mg L⁻¹), v = volume of medium (L), and m = amount of biomass taken (g).

The amount of Cr⁶⁺ adsorbed onto the cell surface was calculated as the difference between the total Cr⁶⁺ removed and the amount incorporated into the cells. The amount of reduced Cr⁶⁺ is the difference between final total chromium and final Cr⁶⁺ in the supernatant (Aoyama and Tsuda 2001; Park et al. 2006).

Adsorption isotherms

The sorption data obtained for chromium uptake at 25 ± 2°C were plotted using Langmuir equation (Langmuir 1918) (Eq. 2) and Freundlich equation (Freundlich 1907) (Eq. 3) given below.

$$ \frac{{1}}{{{q_{\text{eq}}}}} = \frac{{1}}{{{q_{{{ \max }}}}}} + \frac{{1}}{{b\,{q_{{{ \max }}}}\,{C_{\text{eq}}}}} $$

(2)

Where q _eq is the amount of metal adsorbed per unit weight of adsorbent at equilibrium (mg g⁻¹), q _max is the maximum metal uptake per unit mass of adsorbent (mg g⁻¹), b is the Langmuir constant (L mg⁻¹) related to energy of sorption which quantitatively reflects the affinity between the sorbent and sorbate, and C _eq is the equilibrium concentration of adsorbate (mg L⁻¹).

$$ { \log }\,{q_{\text{eq}}} = { \log }\,{K_{\text{f}}} + {1}/n\,{ \log }\,{C_{\text{eq}}} $$

(3)

Where K _f (mg g⁻¹) is the biosorptive uptake and n is the biosorption equilibrium constant indicative of the general shape of the isotherm.

The Langmuir equation suggests the monolayer sorption onto a surface containing definite number of identical sites. Freundlich equation is based on sorption on a heterogeneous surface. Parameters of both models were calculated from the intercepts and slopes of 1/q _e versus 1/C _e and log q _e versus log C _e plots, respectively.

Kinetic studies

Pseudo-first-order and pseudo-second-order kinetic models are used in this study (Ho and Mckay 1998). The pseudo-first-order model is based on sorbent capacity and considers that the rate of adsorption is proportional to the number of unoccupied sites. The pseudo-second-order equation is based on adsorption capacity of solid phase (Aksu and Tunc 2005). The pseudo-first-order model is given by the derivative expression:

$$ \frac{{{\text{d}}{q_t}}}{{{\text{d}}t}} = {k_{{1}}}\left( {{q_{\text{e}}} - {q_t}} \right) $$

(4)

On integration, Eq. (4) and using the conditions q _t  = 0 at t = 0, the linear form obtained is given as

$$ { \log }\left( {{q_{\text{e}}}-{q_t}} \right) = { \log \;}{q_{\text{e}}} - \frac{{{k_1}t}}{{2.303}} $$

(5)

Where k ₁ is the rate constant of pseudo-first-order adsorption (min⁻¹), q _t is the amount of Cr⁶⁺ adsorbed by the sorbent at time t (mg g⁻¹), and q _e is the amount adsorbed at equilibrium (mg g⁻¹). A plot of log(q _e − q _t ) against t is linear and k ₁ can be determined from the slope.

The pseudo-second-order equation (Ho and Mckay 1998) is given by the derivative expression:

$$ \frac{{{\text{d}}{q_t}}}{{{\text{d}}t}} = {k_{{2}}}{\left( {{q_{\text{e}}} - {q_t}} \right)^{{2}}} $$

(6)

On integration, Eq. (6) and using condition q _t  = 0 at t = 0 the linear form is obtained as

$$ \frac{t}{{{q_t}}} = \frac{{1}}{{{k_{{2}}}q_{\text{e}}^2}} + \frac{t}{{{q_{\text{e}}}}} $$

(7)

Where k ₂ is the rate constant of pseudo-second-order adsorption (g mg⁻¹ min⁻¹). A plot of t/q _t as a function of t is linear and the value of k ₂ is determined from the slope and intercept.

FTIR analysis

Biomass in the solid phase was subjected to IR analysis to assess the chemical groups present on the cell wall and analysis of control was compared with treated cultures. Infrared spectra were obtained with the help of FTIR spectrophotometer (IR Prestige-21, Shimadzu). Translucent sample disks were prepared by encapsulating 0.1 g of finely sized biomass in 1 g of KBr.

Statistical analysis

All experiments were performed in triplicate and the results were expressed as means ± SD. SPSS software was used for statistical analysis. A two-way ANOVA was used to compare the significant differences and multiple comparison was performed by Bonferroni test. The relationship between total metal removed, adsorbed, and absorbed was obtained by Karl Pearson’s correlation coefficient.

Results

Cyanobacterial growth

Growth of Oscillatoria sp. was assessed for the pH range of 2–7. At pH 2, death of algal cells was seen on the second day and at pH 3 chlorophyll-a content decreased with prolonged culture time (Fig. 1a, b). pH 4 showed delayed growth (growth rate = 0.004 mg L⁻¹ h⁻¹). In the pH range 5–7, there was an increase in growth with increase in pH. Change in pH that occurred during the course of the experiment is shown in Table 1. This change in pH was significant (p < 0.05) in the range 5–7. At the end of 12 days of cultivation, initial pH of 4, 5, 6, and 7 increased to about pH 5.91 ± 0.01, 6.33 ± 0.014, 7.55 ± 0.04, and 8.75 ± 0.05 for O. laete-virens and pH 5.9 ± 0.1, 6.26 ± 0.05, 7.56 ± 0.03, and 8.74 ± 0.05 for O. trichoides.

Fig. 1

Effect of pH on the growth of a O. laete-virens and b O. trichoides. Results are expressed as means; error bars represent standard deviation (n = 3)

Table 1 Variation in pH during the growth of Oscillatoria laete-virens and Oscillatoria trichoides at different pH values

Increased Cr⁶⁺ concentration showed a significant decrease (p < 0.01) in growth in both the species over time (Fig. 2a, b). A two-way ANOVA indicates a significant change in cell growth in response to Cr⁶⁺ concentration and incubation time for each organism. The F values associated with biomass vs. time vs. initial Cr⁶⁺ concentration vs. their interactions were 436.13 (p < 0.001), 123.65 (p < 0.001), and 17.17 (p < 0.001) for O. laete-virens. The values for O. trichoides were 504.40 (p < 0.001), 287.77 (p < 0.001), and 22.02 (p < 0.001). Further, Bonferroni test suggests O. trichoides to be significantly more tolerant to Cr⁶⁺ ions (p < 0.05) in all days of growth than O. laete-virens except on the fourth, sixth, and 12th days at 50 mg L⁻¹.

Fig. 2

a, b Growth of O. laete-virens and O. trichoides at various initial Cr⁶⁺ concentrations. Results are expressed as means; error bars represent standard deviation (n = 3)

Effect of initial concentration on metal removal

Cr⁶⁺ removal is both by adsorption onto the cell surface and intracellular uptake. The total amount of Cr⁶⁺ removed, adsorbed, and incorporated into the cells as a function of exposure time and initial Cr⁶⁺ concentration is shown in Table 2. The initial concentration of Cr⁶⁺ remarkably influenced the equilibrium metal uptake (Fig. 3). When C _i was increased from 10 to 30 mg L⁻¹, the metal removal capacity increased from to 5.4 ± 0.4 to 13.7 ± 1.7 mg g⁻¹ for O. laete-virens, and from 6.19 ± 0.36 to 38.7 ± 3.8 mg g⁻¹ for O. trichoides. Further increase in C _i does not show much effect on the removal. Figure 3 shows almost similar values for q at higher C _i. The highest percent removal (20%) of Cr⁶⁺ was at the initial concentration of 10 mg L⁻¹ for O. laete-virens and at 30 mg L⁻¹ (51.6%) for O. trichoides. Increased Cr⁶⁺ concentrations increase Cr⁶⁺ absorption for both the species (Table 2). Total Cr⁶⁺ removal showed an initial slow uptake with maximum removal for both the species on the tenth day of exposure, irrespective of the initial concentration.

Table 2 Total amounts of Cr⁶⁺ removed, adsorbed, and accumulated by Oscillatoria laete-virens and Oscillatoria trichoides at various initial concentrations of Cr⁶⁺

Fig. 3

Effect of initial concentration on the removal of Cr⁶⁺ at equilibrium by living cells

The two-way ANOVA shows a highly significant change in Cr⁶⁺ removal in response to incubation time and concentration for each organism. The F values for metal removal vs. time vs. initial concentration vs. their interactions for O. laete-virens were 126.23 (p < 0.001), 183.41 (p < 0.001), and 5.79 (p < 0.001), respectively, and for O. trichoides were 71.51 (p < 0.001), 236.44 (p < 0.001), and 5.90 (p < 0.001). Further, Bonferroni test for concentrations of 10–50 mg L⁻¹ of Cr⁶⁺ indicate higher Cr⁶⁺ removal by O. trichoides (p < 0.05) compared to O. laete-virens at all incubation times except on the second, fourth, sixth, and eighth days at 10 mg L⁻¹.

The Pearson correlation coefficient for the total amount of Cr⁶⁺ removed from the solution and the amount adsorbed and accumulated intracellularly shows a strong positive correlation (p < 0.01) for both species. Therefore, adsorption is proportional to total removal. A significant correlation (p < 0.01) in both species was also found between the total Cr⁶⁺ removed and bioaccumulated.

Effect of pH on metal removal

Cyanobacterial presence in the medium affects the pH with an increase in initial pH during cultivation (Table 3). The shift was marginal in the pH range of 2–3 but prominent at other pH values. pH remains unaltered in the absence of cyanobacterial cells (Table 3). The amount of bioremoval of Cr⁶⁺ as a function of pH is shown in Table 4. The maximum removal of Cr⁶⁺ by O. laete-virens was 12.6, 13.7, 10.7, and 5.5 mg g⁻¹ at an initial pH of 4, 5, 6, and 7, respectively, on the tenth day of exposure at 30 mg L⁻¹ of initial concentration. The corresponding values were 28.75, 38.75, 25.36, and 6.18 mg g⁻¹ for O. trichoides. The cells of both the species did not survive beyond the second day (48 h) at pH 2, and the removal of Cr⁶⁺ was 71.23 mg g⁻¹ for O. laete-virens and 70.33 mg g⁻¹ for O. trichoides. The growth decreased with culture time at pH 3 and the Cr⁶⁺ removal was 57.23 mg g⁻¹ for O. laete-virens, and 50.16 mg g⁻¹ for O. trichoides within 72 h. The maximum removal was at pH 2 followed by pH 3 due to the reduction of Cr⁶⁺ to Cr³⁺. This bioreduction was more pronounced in the acidic pH range and stopped beyond pH 5. Therefore, the experiments were restricted to pH 4–7. The Cr⁶⁺ removal by cyanobacteria decreased with increasing pH from 5 to 7. The highest removal was observed at an initial pH 5 (Fig. 4).

Table 3 Variation in pH during the growth of Oscillatoria laete-virens and Oscillatoria trichoides at 30 mg L⁻¹ of Cr⁶⁺

Table 4 Removal of Cr⁶⁺ and total chromium by Oscillatoria trichoides and Oscillatoria laete-virens at different pH values

Fig. 4

Effect of pH on the removal of Cr⁶⁺ by living cells of two species of Oscillatoria at 30 mg L⁻¹

Effect of culture density

Culture density affects metal removal efficiency. The amount removed increased from 0.2 to 0.4 g L⁻¹, and there was a decrease with the further increase in culture density (Fig. 5).

Fig. 5

Effect of culture density on the removal of Cr⁶⁺ by the living cells of a O. laete-virens and b O. trichoides. Results are means ± standard deviation (n = 3). White bars—removed, gray bars—adsorbed, black bars—accumulated

Adsorption by dead cells

For dead cells, pH 2 favors maximum Cr⁶⁺ removal (Fig. 6a, b). For an initial concentration of 10 mg L⁻¹, equilibrium was attained at 60 min with a removal of 3 ± 0.5 mg g⁻¹ by O. laete-virens and at 90 min with a removal of 3.33 ± 0.28 mg g⁻¹ by O. trichoides. The increase in initial concentration enhanced Cr⁶⁺ removal in both species; the highest removal for both the species was at 50 mg L⁻¹. O. trichoides removed 6.25 ± 0.52 mg g⁻¹ and O. laete-virens removed 7.83 ± 1.04 mg g⁻¹. The equilibrium was attained at 90 min and 120 min, respectively (Table 5).

Fig. 6

Effect of pH on the removal of Cr⁶⁺ by the dead cells of a O. laete-virens and b O. trichoides at 10 mg L⁻¹. Results are expressed as means; error bars represent standard deviation (n = 3)

Table 5 Total amounts of Cr⁶⁺ removed by dead biomass of Oscillatoria laete-virens and Oscillatoria trichoides at various initial concentrations of Cr⁶⁺

The F values for O. laete-virens Cr⁶⁺ removal vs. time vs. initial concentration vs. their interactions were 45.43 (p < 0.001), 136.18 (p < 0.001), and 5.05 (p < 0.001), respectively, while the F values for O. trichoides were 90.27 (p < 0.001), 54.43 (p < 0.001), and 4.80 (p < 0.001), respectively. The Bonferroni test showed a significantly higher metal removal by living cells than by non-living cells in all the concentrations tested. Living cells of O. trichoides are nearly nine times (38.7 ± 3.8 mg g⁻¹) more efficient than non-living cells and O. laete-virens living cells are almost twice (13.7 ± 1.7 mg g⁻¹) more efficient than non-living cells.

Adsorption isotherms

The experimental results were plotted using the linearized Langmuir and Freundlich adsorption isotherms over a concentration range of 10–40 mg L⁻¹ (Figs. 7a, b and 8a, b). The obtained parameters are given in Table 6. The data indicate that the experimental results for O. laete-virens living biomass are consistent with both the isotherms with an R ² of 0.8408 and 0.8462 with a q _max of 21.88 mg g⁻¹. However, the non-living biomass of O. laete-virens followed only the Langmuir isotherm model with an R ² of 0.732 and a q _max of 7.58 mg g⁻¹. Living biomass of O. trichoides does not follow either of the models, but removes the metal to a maximum extent of 38.7 mg g⁻¹ at equilibrium. The removal by nonliving biomass was well described by the Freundlich model with a R ² of 0.7642. The adsorption capacity (q _max) in terms of monolayer was 5.67 mg g⁻¹.

Fig. 7

Langmuir isotherm for the removal of Cr ⁶⁺ by a living biomass of O. laete-virens y = 1.1567x + 0.0457 R ² = 0.8408, O. trichoides y = 1.4273x − 0.043 R ² = 0.8473 and b dead biomass of O. laete-virens y = 2.0086x + 0.1318 R ² = 0.732, O. trichoides y =1.3214x + 0.1761 R ² = 0.5715. Filled diamonds—O. laete-virens, filled squares—O. trichoides

Fig. 8

Freundlich isotherm for the removal of Cr ⁶⁺ by a living biomass of O. laete-virens y = 0.6848x + 0.2139 R ² = 0.8462, O. trichoides y = 1.199x − 0.2362 R ² = 0.6348 and b dead biomass of O. laete-virens y = 0.5674x − 0.121 R ² = 0.8388, O. trichoides y = 0.3733x + 0.1127 R ² = 0.7642. Filled diamonds—O. laete-virens, filled squares—O. trichoides

Table 6 Langmuir and Freundlich isotherm parameters for the removal of Cr⁶⁺ by Oscillatoria laete-virens and Oscillatoria trichoides

Kinetic studies

For pseudo-first-order kinetics, the straight line plots of log (q _e − q _t ) against t were made for both living and dead biomass (Fig. 9a, b) of both cyanobacterial species. The linear plots of t/q _t against t for pseudo-second-order model (Fig. 10a, b) were made for living and dead mass of both species. From the linear regression analysis, R ² values were determined (Table 7a, 7b).

Fig. 9

The pseudo-first-order plot of kinetic study of Cr⁶⁺ biosorption by a O. laete-virens y = −0.0061x + 1.3909 R ² = 0.9359, O. trichoides y = −0.0058x + 1.7887 R ² = 0.8267 at a concentration of 30 mg L⁻¹ and 28°C for living biomass and b O. laete-virens y = −0.0083x + 0.8803 R ² = 0.8366, O. trichoides y = −0.0129x + 0.8993 R ² = 0.6716 at a concentration of 50 mg L⁻¹ and a temperature of 28°C for dead biomass. Filled diamonds—O. laete-virens, filled squares—O. trichoides

Fig. 10

The pseudo-second-order plot of kinetic study of Cr⁶⁺ biosorption by a O. laete-virens y = 0.0318x + 11 R ² = 0.7443, O. trichoides y = 0.0137x + 3.4911 R ² = 0.8562 at a concentration of 30 mg L⁻¹ and 28°C for living biomass and b O. laete-virens y = 0.1123x + 3.0634 R ² = 0.945, O. trichoides y = 0.0971x + 8.319 R ² = 0.8354 at a concentration of 50 mg L⁻¹ and 28°C for dead biomass. Filled diamonds—O. laete-virens, filled squares—O. trichoides

Table 7 Comparison of pseudo-first-order and second-order adsorption rate constants and the calculated and experimental q _e values

FTIR analysis

The spectral studies reveal biosorbent heterogenesity shown by different characteristic peaks with the possible presence of amino, carboxylic, hydroxyl, and carbonyl groups. The IR adsorption bands and corresponding groups that are capable of interacting with metal ions are presented in Table 8 and Fig. 11a–d. It was observed that there were slight changes in the absorption peak frequencies in both species after chromium adsorption which is suggestive of metal binding process taking place on the biomass surface.

Table 8 Frequencies and assignments of FTIR bands of free and Cr⁶⁺ bound living cells of Oscillatoria laete-virens and Oscillatoria trichoides

Fig. 11

FTIR analysis for the biomass of O. laete-virens (a, b) and O. trichoides (c, d)—free and Cr⁶⁺ bound living cells at 30 mg L⁻¹ concentration

Discussion

The ability to grow at low pH is a desirable feature of cyanobacteria as waters polluted with heavy metals generally have low pH (Rai et al. 1981). pH below 4 was found to be toxic to both cyanobacteria. A reduced cation uptake (NH ⁺₄ , Na⁺, K⁺, Ca²⁺, Cu²⁺, and Ni²⁺) and an accelerated uptake of anions (NO ⁻₃ and PO ³⁻₄ ) builds a positive charge on the membranes preventing the entry of cations and also helps to maintain a near neutral cytoplasmic pH (Rai et al. 1996). These mechanisms may be involved to protect the cells from acidity in the present study.

There is a significant increase in pH of the culture medium (p < 0.05) due to CO₂ uptake for photosynthesis. This leads to a decrease in CO₂ partial pressure, when CO₂ replacement is slower than the utilization (Chen and Durbin 1994; Dubinsky and Rotem 1974).

The significant decrease (p < 0.01) in growth shown by both the species over time in increased Cr⁶⁺ concentrations may be due to the oxidative stress occurring in the chloroplasts and other cell parts via a Fenton-type mechanism which has the capacity to reduce the activity of some antioxidant enzymes (Shi and Dalal 1990; Panda and Choudhary 2005). Khattar et al. (2007) have reported decreasing growth over time in Anacystis nidulans and Ozturk et al. (2009) in Synechocystis sp. Rehman et al. (2007) reported 71% growth after 192 h (8 days) of Cr⁶⁺ stress in Distigma proteus.

Both the cyanobacterial species studied by us tolerate a Cr⁶⁺ concentration as high as 30 mg L⁻¹, which has not so far been reported for any other algae. Exopolymeric substances (EPS) are common in many cyanobacteria. These are rich in uronic acids (De Philippis and Vincenzini 1998) which bind to metal ions conferring on these cyanobacteria an increased tolerance to metal ions. Uronic acids occurring in the EPS produced by these species (results not shown here) may bind to Cr⁶⁺ resulting in an increased tolerance to the metal. The ability of cyanobacteria to reduce toxic Cr⁶⁺ to less toxic Cr³⁺ and to accumulate this also results in an increased resistance of these algae to Cr⁶⁺ (Garcia et al. 2009).

Increased initial concentration increases the number of ions competing for the available binding sites in the biomass leading to saturation of functional groups at equilibrium. This also helps to overcome all mass transfer resistance of metal ions between the aqueous and solid phases. Hence, as initial concentration increases, it will increase the biosorption rate until it reaches an equilibrium state leading to saturation of functional groups (Arief et al. 2008). The greater biosorption capacity of O. trichoides over O. laete-virens could be explained by the fact that metal binding groups differ in their affinity and specificity for metal uptake (Crist et al. 1981). Concentration-dependent removal of Cr⁶⁺ has been reported by Dönmez and Aksu (2002) in Dunaliella sp. Bala et al. (2004) have reported a maximum uptake of 3.3 mg g⁻¹ at 20 mg L⁻¹ in Nostoc linckia. Energy-dependent Cr⁶⁺ uptake in the cyanobacterium Anabaena doliolum also showed concentration dependence (Rai et al. 1992).

Both the reduction and adsorption of Cr⁶⁺ are highly dependent on the solution pH. In most cases, Cr⁶⁺ is reduced to Cr³⁺ at pH 2–3 along with anionic adsorption at a lower pH (Aoyama et al. 2005; Aoyama and Tsuda 2001; Park et al. 2005; 2006; Sarin and Pant 2006; Sanghi et al. 2009; Hasan et al. 2008; Cui et al. 2011; Qaiser et al. 2009). Kratochvil et al. (1998), using the Nernst equation, showed a greater redox potential of chromate at lower pH. This supports the results of living biomass obtained in this study. Also, at these pH values, the sorption of Cr³⁺ is also not favored because positively charged H⁺ ions will compete with metal ions for the anions on the cell wall. In addition, there is repulsion of positively charged Cr³⁺ by positively charged ions of biomass surface at acidic pH (Tan et al. 2003).

The increase in the biosorption of Cr⁶⁺ at lower pH (anionic adsorption) suggests that these bind through electrostatic attraction to the positively charged functional groups on the biomass surface (Gupta et al. 2001) since at lower pH, the overall surface charge on the biomass is positive. This has also been reported in a variety of biosorbents (Tunali et al. 2005; Niu and Volesky 2003). At higher pH, the concentration of OH⁻ ions increases and overall negative charge on the biomass surface causes a hindrance to the biosorption of negatively charged ions such as Cr₂O ²⁻₇ , CrO ²⁻₄ resulting in a decrease of biosorption of Cr⁶⁺ (Tewari et al. 2005; Niu and Volesky 2003; Saha and Orvig 2010; Anjana et al. 2007).

In our study using living biomass, the reduction rate of Cr⁶⁺ increased at lower solution pH (2–3) and the contact time required for the removal of Cr⁶⁺ (anionic adsorption along with accumulation) increased from hours to days at pH 5. Since there was no Cr³⁺ left in the medium, reduction of Cr⁶⁺ was not observed and Cr⁶⁺ was removed mainly through adsorption at the cell surfaces at pH 5, as in the mechanism of anionic adsorption (Sarin and Pant 2006; Mungasavalli et al. 2007; Anjana et al. 2007). In the present study, there is a removal of Cr⁶⁺ at pH >3.0. But the percentage is less since at higher pH an electrostatic force of repulsion occurs. At the same time, some amount of removal could be due to the presence of other mechanism such as physical adsorption on the surface of biosorbent (Gupta et al. 2001).

pH influences both cell surface metal binding sites and metal chemistry in water. Hexavalent chromium exists in different forms in aqueous solution and the stability of these forms is pH dependent. HCrO ⁻₄ of Cr⁶⁺ form is more stable in aqueous solution up to pH 7. The H₂CrO₄ (chromic acid) form is stable in the low pH range, whose concentration decreases sharply with increasing pH. Similarly, the CrO₄ ²⁻ form is stable at higher pH range (above pH 6). At pH 2, it is the HCrO ⁻₄ form of Cr⁶⁺ that binds to the surfaces (Gardea-Torresdey et al. 2000; Cimino et al. 2000).

In experiments using living biomass which were conducted at pH 5, the percentage of Cr⁶⁺ removal is less compared to acidic pH of 2 and 3. At pH 4, since the growth was less compared to pH 5 (Fig. 1a, b), the removal capacity might have been affected even though this acidic pH is favorable for Cr⁶⁺ adsorption (anionic adsorption).

Decreased biosorption at higher culture density is due to the limited availability of metal electrostatic interactions (Khattar et al. 2007) or by the attainment of equilibrium between adsorbate and adsorbent (Rai and Kumar 1999).

Metal removal by dead cyanobacterial cells is exclusively through adsorption on to the cell wall surface (Chu and Hashim 2004). The Cr⁶⁺ removal by dead cells in this study seems to be by anionic adsorption and not by reduction at low pH since there was no Cr³⁺ left out in the medium. The process of adsorption reaches equilibrium after a few minutes (90–120 min) of contact in this study. Similar results were reported by Ozer et al. (1999).

The lesser amount of metal removal by dead cells over living cells may be due to the unavailability of functional groups such as carboxyl, hydroxyl, or phosphoryl present on the cell surface following heating in terms of stereochemically adequate positioning and chemical integrity (Monteiro et al. 2009a). Information on the comparative removal of Cr⁶⁺ by living and dead cells of cyanobacteria is limited. Cr⁶⁺ removal from polluted waters through dead biomass can be more economical because constrained environmental conditions for the growth and maintenance of the cells is not needed. They withstand highly toxic environments, and metals contained in them can be recovered when used in industrial applications (Aksu and Dönmez 2006).

The Freundlich and Langmuir isotherms are widely used in biosorption studies (Martins et al. 2006; Volesky 2007) to predict the adsorption capacity of a biosorbent. Langmuir model gives a good description of experimental behavior in a wide range of operating conditions (Yun et al. 2001) which helps us in evaluating q _max—maximum possible quantity of metal ions adsorbed per gram of adsorbent and the constant b which is related to the affinity of binding sites for the metal ions. Lower value of b, i.e., 0.03 for living biomass of O. laete-virens and 0.06 and 0.13 for dead biomass of O. laete-virens and O. trichoides, is indicative of high metal removal capacity of these cyanobacteria (Langmuir 1918). Similar observations were also proposed by Kratochvil and Volesky (1998). Hence, these species seem to be good biosorbents for Cr⁶⁺as they have a high q _max and a low b.

Living biomass of O. trichoides does not follow either of the models and may involve some other mechanism. Studies by Kratochvil and Volesky (1998) and Volesky and Holan (1995) suggest that these sorption isotherms do not necessarily reflect the adsorption mechanisms involved.

Even values of K _f and n (Table 6) for O. laete-virens living biomass and O. trichoides dead biomass suggest their higher biosorption capacity for Cr⁶⁺. Such observations were made in many previous studies (Gokhale et al. 2008; Gabr et al. 2009). Higher value of K _f and n and the lower values of b indicate better affinity of the biomass towards metal ions (Saravanane et al. 2002; Aksu et al. 1991).

The sorption of Cr⁶⁺ follows relatively well with pseudo-first-order kinetic model with higher values of R ² in living biomass of both species. Besides, the proximity of values of predicted q _e and experimental q _e shows predictive relevance of the model. The pseudo-second-order kinetic model fits better with the dead biomass of O. laete-virens with predicted q _e nearer to experimental q _e and a higher rate constant indicates increased uptake of adsorbate ions with higher values of R ². For the dead biomass of O. trichoides, pseudo-first-order model is relevant since predicted q _e is nearer to experimental q _e, and a higher rate constant.

The FTIR results obtained indicate the presence of carboxylic, amino, and carbonyl groups on the algal cell surfaces and is also indicative of the mechanism of adsorption. The extent of band shifting also gives an indication of degree of interaction of functional groups with Cr⁶⁺ ions. Studies on Cr⁶⁺ adsorption in Oedogonium hatei had similar results (Gupta and Rastogi 2009). FTIR and XPS spectra suggest that carboxylate and carboxyl groups on the surface of biomass are responsible for Cr⁶⁺ binding and reduction, whereas amide and other groups play a minor role in the Cr⁶⁺ removal process (Cui et al. 2011).

In conclusion, we found that the removal of toxic hexavalent chromium from solutions was possible using these two species. Both bioreduction and biosorption contribute to the bioremoval of Cr⁶⁺ during the growth of the cyanobacterial species. A comparison of the bioreduction and biosorption process in living cells reveals that bioreduction contributes to a greater extent to the overall bioremoval below pH 3.1, while biosorption is more dominant beyond that pH. The highest removal through biosorption for living biomass was achieved between pH 5 and 5.9 and for dead biomass at pH 2. Of the two species, living cells of O. trichoides were most effective for which removal was 38.7 mg g⁻¹ and reached 51.6% of the total Cr⁶⁺ at 30 mg L⁻¹ at pH 5–5.9. But the results of this species did not follow either of the models. Most of the Cr⁶⁺ removal occurred through adsorption on to the cell surface. Living cells of both species outperform dead cells significantly. Kinetics of living biomass was well described by pseudo-first-order model. FTIR analysis indicated the participation of carboxylic, carbonyl, and amino groups in Cr⁶⁺ removal. Thus O. laete-virens and O. trichoides can be good candidates for Cr⁶⁺ sorption from polluted environments. However, further research is needed to establish the process with specific attention to use in the industrial effluents by immobilization of cyanobacteria and regeneration of the sorbed metal.