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International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Potential of Live Spirulina platensis on Biosorption of Hexavalent Chromium and Its Conversion to Trivalent Chromium a
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Luciane Maria Colla , Clinei Dal’Magro , Andreia De rossi , AntôNio Thomé , Christian b
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Oliveira Reinehr , Telma Elita Bertolin & Jorge Alberto Vieira Costa
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University of Passo Fundo, Graduate Program in Civil and Environmental Engineering, Bairro São José, Passo Fundo/RS, Brazil b
University of Passo Fundo, Food Engineering Program, Building L1, Campus I, BR Bairro São José, Passo Fundo/RS, Brazil c
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Federal University of Rio Grande, Graduate Program in Food Engineering and Science, P.P. Box, Rio Grande, RS, Brazil Accepted author version posted online: 01 Dec 2014.
To cite this article: Luciane Maria Colla, Clinei Dal’Magro, Andreia De rossi, AntôNio Thomé, Christian Oliveira Reinehr, Telma Elita Bertolin & Jorge Alberto Vieira Costa (2015) Potential of Live Spirulina platensis on Biosorption of Hexavalent Chromium and Its Conversion to Trivalent Chromium, International Journal of Phytoremediation, 17:9, 861-868, DOI: 10.1080/15226514.2014.964846 To link to this article: http://dx.doi.org/10.1080/15226514.2014.964846
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International Journal of Phytoremediation, 17: 861–868, 2015 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2014.964846
Potential of Live Spirulina platensis on Biosorption of Hexavalent Chromium and Its Conversion to Trivalent Chromium ˆ LUCIANE MARIA COLLA1, CLINEI DAL’MAGRO1, ANDREIA DE ROSSI1, ANTONIO THOME´ 1, CHRISTIAN 2 2 OLIVEIRA REINEHR , TELMA ELITA BERTOLIN , and JORGE ALBERTO VIEIRA COSTA3 1
University of Passo Fundo, Graduate Program in Civil and Environmental Engineering, Bairro S˜ao Jos´e, Passo Fundo/RS, Brazil University of Passo Fundo, Food Engineering Program, Building L1, Campus I, BR Bairro S˜ao Jos´e, Passo Fundo/RS, Brazil 3 Federal University of Rio Grande, Graduate Program in Food Engineering and Science, P.P. Box, Rio Grande, RS, Brazil
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Microalga biomass has been described worldwide according their capacity to realize biosorption of toxic metals. Chromium is one of the most toxic metals that could contaminate superficial and underground water. Considering the importance of Spirulina biomass in production of supplements for humans and for animal feed we assessed the biosorption of hexavalent chromium by living Spirulina platensis and its capacity to convert hexavalent chromium to trivalent chromium, less toxic, through its metabolism during growth. The active biomass was grown in Zarrouk medium diluted to 50% with distilled water, keeping the experiments under controlled conditions of aeration, temperature of 30◦ C and lighting of 1,800 lux. Hexavalent chromium was added using a potassium dichromate solution in fed-batch mode with the aim of evaluate the effect of several additions contaminant in the kinetic parameters of the culture. Cell growth was affected by the presence of chromium added at the beginning of cultures, and the best growth rates were obtained at lower metal concentrations in the medium. The biomass removed until 65.2% of hexavalent chromium added to the media, being 90.4% converted into trivalent chromium in the media and 9.6% retained in the biomass as trivalent chromium (0.931 mg.g−1). Keywords: toxic metal, potassium dichromate, supplements
Introduction Contamination by toxic metals, because of their continuous accumulation in the food chain, is one of the most serious environmental problems and therefore is a matter of great concern (Doshi, Ray, and Khotari 2007) due to the risk to human and animal health (Merlino et al. 2010; Mudgal et al. 2006; Das, Vimala, and Karthika 2008). Numerous human activities can pollute soils and underground with metals, as the disposal of solid and industrial waste into poorly waterproofed landfills, contaminating the soil and water resources through leaching (Melo et al. 2010; Maus, Costa, and Righes 2009), leakage and spillage, industrial activities (galvanoplasty, mining, stell and cement production, tannery), application of fertilizers and agrochemicals, and disposal of agroindustrial waste and wastewater treatment plant sludge into the soil (Korf 2011; Canuto et al. 2007). Algae have been used to biosorption of toxic metal due to the need of new technologies for the treatment of wastewaters
Address correspondence to Luciane Maria Colla, University of Passo Fundo, Graduate Program in Civil and Environmental Engineering – Building G2, Campus I, BR 285, Bairro S˜ao Jos´e, Passo Fundo/RS, CEP: 99052-900. E-mail: [email protected]
contaminated by toxic metals. The use of algal biomass for the biosorption of toxic metals has some advantages such as low cost and good efficiency (Das et al. 2008), with the accumulation of metals in the outer walls of the algae by physical, chemical and biological mechanisms (Mane et al. 2011). Biosorption consists of the sorption of toxic metals by living or dead microorganisms (biomass) (Quintelas et al. 2008). According to Pietrobelli et al. (2009), biosorption is an efficient alternative to conventional wastewater treatments, as the microorganisms retain the metals, promoting wastewater selfregeneration and qualifying the process. Thus, biosorption is a thriving technology whose application in the treatment and final polishing of wastewaters containing toxic metals has cur´ rently expanded (Kratochvil and Volesky 1998; Modenes et al. 2009). Among toxic metals, chromium is one of the most dangerous and could contaminate water reservoirs, rivers, and sources of water for the population (Merlino et al. 2010; Canuto et al. 2007). The current technology for the removal of total chromium consists basically of two stages: conversion of hexavalent chromium (Cr VI) to trivalent chromium (Cr III) using reducing agents and the later precipitation of Cr III in the form of hydroxides. Conversion of Cr VI to Cr III is an essential stage in the process as the former one is quite mobile in nature, and is not easily adsorbed or precipitated. Given
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862 the wide array of sources of wastewaters containing Cr VI, the development of new technologies for its removal has been extensively investigated (Rutuolo and Gubulin 2003). Several studies are been conducted using Spirulina platensis biomass to remove metallic ions from aqueous solutions (Arunakumara and Xuecheng 2007; Doshi, Ray, and Kothari 2007). Spirulina platensis has been used for environmental purposes by several authors, such as Mezzomo et al. (2010) for the removal of COD (chemical oxygen demand) in swine wastewater. Aneja et al. (2010) assessed the biosorption of Pb+2 and Zn+2 from an aqueous solution containing different concentrations of these metals, using the dry biomass of Spirulina sp., with biosorption rates of 82% for Pb+2 and of 90% for Zn+2. In our research group, we already studied the active and passive biosorption of hexavalent chromium by Spirulina platensis, obtaining 61.97% of Cr VI removal using dead biomass (passive biosorption), in pH 3, with maximal adsorption capacity of 100.39 mg.g−1 (Dal Magro et al. 2013). In another work we show that Spirulina platensis Leb-52 strain can perform the active biosorption of Cr VI from wastewaters with removal rates of 45% to 60.92% for initial concentrations of Cr VI of 11.7 mg. L−1 and 3 mg.L−1, respectively (Dal’Magro et al. 2012). On the other hand, Spirulina is widely used as a nutritional supplement to humans and animals and has been produced by a great number of companies, due to the proof that the biomass could help in the treatment and prevention of many diseases (Ambrosi et al. 2008). Al-Dhabi (2013) and Hsu, Hwang, and Yeh (2001) studied the presence inorganic elements in Spirulina products and proved that the concentration of was not to exceed the present regulation levels. But if this microalgae is grown in contaminated culture media, it is important whether the growth will be maintained at high contaminant concentrations, as well as the contaminant is adsorbed by microalga biomass. Doshi, Ray and Khotari (2007) demonstrated that live and dead Spirulina sp. took up a significant amount of Cd+2 ions, but no studies were found using live biomass of Spirulina to biosorption of chromium species. Considering the described use of Spirulina biomass in biosorption of toxic metals, the potential of chromium contaminations in waters, and the widely use of Spirulina biomass as food, the highlight of this work was the study of the ability of this microalga to growth in media contaminated with hexavalent chromium and the bioconversion of Cr VI to Cr III in the culture. The addition of contaminant was accomplished in fed-batch mode in order to evaluate the effect of several contaminations of chromium over the growth of microalga.
Material and Methods Microorganism and Inoculum Maintenance Spirulina platensis Leb-52 strain was used, and the inocula were kept in a non-sterile thermostat-controlled greenhouse under controlled aeration (diaphragm pumps), temperature (∼30◦ C) and lighting (1800 lux). We used the Zarrouk (1966) medium, which is standard for the cultivation of this microalga, diluted to 50% with sterile distilled water. Lighting
L. M. Colla et al. was provided by 20 W fluorescent lamps and the temperature was maintained by resistors whose heating is controlled by thermocouples and fans. Growth Conditions The experiments were performed in 2 L Erlenmeyer flasks with an initial medium volume of 1.3 L, with a 12-hour photoperiod, and constant aeration. Culture media received Cr VI through standardized potassium dichromate (K2 Cr2 O7 ) solutions at variable concentrations according to the experimental design (Table 1). Potassium dichromate solutions were added in fed batch mode after cultures reached a certain biomass concentration (0.5 g. L−1 or 1.0 g. L−1). After the first contamination, the other additions of Cr VI occurred at 5-day intervals, totaling five additions of Cr VI at the end of the growth period in all assays. Kinetic Growth Parameters The pH of the samples was assessed regularly by a pH meter (DIGIMED, DM-22). Microalgae growth was evaluated every two days by absorbance reading of cultures using a spectrophotometer (PG INTRUMENTS, T60) at 670 nm and the results were obtained from a previously built standard biomass curve. The maximum specific growth rate and the doubling time were calculated using Equations 1 and 2. X2 1 Ln t X1 ln 2 tg = μmax
μmax =
(1) (2)
Where: μmax = maximum specific growth rate (d−1) X1 = Concentration of cells in the early exponential growth stage (g.L−1) X2 = Concentration of cells in the late exponential growth stage (g.L−1) t = duration of the exponential growth stage (d) tg = doubling time (d) Determinations of Chromium in Biomass and Wastewater At the end of the growth period, the presence of Cr VI and Cr III in the cell-free medium and in the biomass was determined in order to assess their removal and the conversion of Cr VI to Cr III, which is less toxic, by the microalga. The wastewater biomass was separated by two consecutive filtrations, using a vacuum pump (MARCONI, MA 058) and a polyester filter with 180 filament yarns. Later on, the biomass was dried at 60◦ C until a constant weight was obtained and submitted to digestion for extraction of the metal from the intracellular medium by EPA method 3050B (USEPA 2012). The filtrated wastewater was submitted to digestion for oxidation of the existing organic matter by EPA method 3050A (USEPA 2012).
Bioconversion of Cr(VI) to Cr(III) by Live Spirulina platensis
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Table 1. Growth parameters of microalga S. platensis during the active biosorption of Cr VI Exp. 1 2 3 4 5 6 7
Cc (g.L−1)
[Cr VI]added (mg.L−1)
Cmax (g.L−1)
μmax (d−1)
Tg (d)
log (d)
R2
0.5 1 0.5 1 0.75 0.75 0.75
5 5 10 10 7.5 7.5 7.5
1.411 1.478 0.906 1.374 1.186 1.289 1.384
0.088 0.1045 0.1045 0.1196 0.0906 0.0960 0.0980
7.92 6.64 6.64 5.79 7.65 7.23 7.10
0-18 0-16 0-12 0-12 0-16 0-16 0-16
0.96 0.97 0.94 0.98 0.94 0.95 0.95
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Cc: Cell concentration (g.L−1) in the first addition of Cr (VI); [Cr VIadded ] : Cr VI concentration added in each new addition (mg.L−1); Cmax = maximum cell concentration (g.L−1); μmax = maximum specific growth rate (d−1); Tg = generation time (d); log = time values used in the exponential regression (d); R2 = determination coefficients of the regressions.
The total chromium concentration in the sample was determined by flame atomic absorption spectrophotometry (ANALYST 200-PERKIN ELMER). Hexavalent chromium was determined by the 1.5- diphenylcarbazide (APHA 2000) method while trivalent chromium was determined by the difference between total chromium and hexavalent chromium, as shown in Equation 3. This is due to the fact that Cr III complexes in aqueous solutions have relative kinetic inertia (GOVINDRAO 1980). Cr (total) = Cr I I I + Cr V I
%Cr (total)r ec =
Cr (total)r ec .100 Cr (total)i ni ti all y added
%Cr (I I I)bi omass =
(4) (5)
Based on total chromium recovery, we calculated the percentage of Cr VI removed from the wastewater and the percentage of Cr VI converted to Cr III, both in the wastewater and in the biomass. The removal of Cr VI from the wastewater was calculated by Equation 6. Cr (total)r ec − Cr V Imedi um Cr (total)r ec
%Cr I I Imedi um =
Where: Cr(total)rec = total chromium recovered (mg) by analytical determinations as a function of the initial amount of Cr VI added to each culture. Cr(total)biomass = total chromium (mg) in the biomass at the end of the growth period, considering the dry biomass weight. Cr(total)medium = total chromium (mg) in the medium the end of the growth period, considering the final volume of the culture. Crinitially added = total hexavalent chromium (mg) added in the experiment during cultivation. % Cr(total)rec = percentage of total chromium recovered at the end of the growth period.
RemovalCr (V I)medi um =
The percentages of Cr III in the wastewater and in the biomass at the end of the growth period were calculated by Equations 7 and 8 and the mass of Cr III retained per g of dry biomass was calculated by Equation 9.
(3)
The total chromium recovered by analytical determinations was calculated by the mass balance, according to Equations 4 and 5. Cr (total)r ec = Cr (total)bi omass + Cr (total)medi um
Where: Removal of Cr VImedium = hexavalent chromium removed from the medium (mg). Cr VImedium = total Cr (VI) which remained in the medium at the end of the growth period (mg).
(6)
q
mg g
=
Cr I I Imedi um Cr (total)r ec −Cr V Imedi um Cr I I Ibi omass Cr (total)r ec −Cr V Imedi um
.100
(7)
.100
(8)
Cr I I Ibi omass (mg) Cmax .V (g)
(9)
Where: % Cr IIImedium = percentage of Cr III in the medium at the end of the growth period. Cr IIImedium = Cr III which remained in the medium at the end of the growth period (mg). % Cr IIIbiomass = percentage of Cr III in the biomass at the end of the growth period. Cr IIIbiomass = Cr III which remained in the biomass at the end of the growth period (mg). Cmax = maximum biomass concentration (g/L) V = volume of cultures in the end of the experiments, that was 1.3 L
Results and Discussion Influence of Chromium Addition on Microalga Growth The pH of cultures ranged from 9.63 to 10.15, a range that is deemed appropriate for the cultivation of Spirulina platensis, offering the ideal conditions for its growth (Andrade and Costa 2008). Figure 1 shows the growth curves for the microalga as a function of time. In all experiments, there was exponential growth on the first days of culture (from 12 to 14 days). In experiment 3, from the 14th day of growth, the cell concentration
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Fig. 1. Microalgae growth versus culture time.
stabilized and decreased, and there might have been a toxic effect of the metal on microalgae growth as, in this experiment, Cr VI additions were performed at the highest concentration (10 mg.L−1), and the experiment showed a lower biomass concentration at the time of first addition of the metallic solution (0.5 g.L−1). For the other experiments, cell growth was similar, revealing growth up to the 20th day followed by a decrease in concentration, indicating the death of some of the cells in the medium. This may have occurred due to nutritional restriction as no nutrients were added during the experiment, only at the beginning of cultures. Table 1 shows the microalgae growth parameters obtained during cultivation. The experiments with higher maximum concentrations were those in which chromium was added at the lowest concentrations (Experiments 1 and 2), reaching the maximum cell concentrations (Cmax ) of 1.411 and 1.478 g.L−1, respectively, demonstrating that Cmax depends on the concentration of toxic compounds added to the medium. The lowest Cmax was obtained in experiment 3 (0.906 g.L−1), to which a larger amount of Cr VI was added (50 mg.L−1, five additions of 10 mg.L−1) at the lowest cell concentration in the first addition of the metal (0.5 g.L−1), showing that the toxicity of the metal interfered with cell growth. When Cr VI, even at high concentrations (10 mg.L−1), was added to cultures with larger cell concentration in the first addition (1.0 g.L−1), Cmax was 1.374 g.L−1 (experiment 4), indicating that the larger amount of cells in the medium led to better uptake of the metal; however, the exponential growth interval ( log) decreased in both experiments with the highest concentration of the metal (experiments 3 and 4, with 50 mg.L−1 of Cr VI) (12 days) in relation to the other experiments, which had a log of 16 to 18 days. Doubling times (time intervals necessary for biomass doubling) ranged from 5.79 to 7.92 days. The lowest doubling times were observed in the experiments with the highest Cr VI concentration (50 mg.L−1of Cr VI), as a function of the influence of the amount of added chromium on the exponential growth stage. In these experiments, although the doubling time is shorter, indicating higher growth, the microorganisms remained in the exponential growth stage for shorter time periods ( log = 12 days) than those observed in the other experiments (16 to 18 days). Moreover, the extension of the
L. M. Colla et al. exponential growth stage may be also related to the fact that, in some experiments, chromium was only added after the microorganism reached concentrations of 0.75 g.L−1 (experiments 5, 6 and 7) or 1.0 g.L−1 (experiments 2 or 4). Dal Magro et al. (2012) observed that the addition of Cr VI in S. platensis cultures only at the beginning of the experiments interfered with the kinetic growth parameters, compromising good cell development and indicating the toxicity of this metal to the microbial metabolism. The cell growth was affected by the addition of wastewater containing around 3 mg.L−1 of Cr VI. Toxic metals can cause adverse effects on biological systems, indirectly producing breaks in the DNA double strand by oxidative stress (Gastaldo et al. 2008). This stress occurs when there is an imbalance produced by the excess generation of oxidants or by the decrease in antioxidants (Guo, Yang and Xu 2008). According to Moreira and Moreira (2004), toxic metals may hinder all and any biological activity, causing different types of biological responses to these metals. In the present study, the cell growth was poorly affected when the metal concentration was 5 mg.L−1 and we have to consider that five additions of contaminant were made in the cultures. With cells in growth, novel additions of contaminant do not seems to affect greatly the growth. This happens because the effect of the addition of the contaminant might have on the cells is minimized by the appearance of new cells, which represent new binding sites for chromium VI or, as will be shown later, the biomass is able to convert chromium VI to chromium III, which seemingly has lower toxicity to the cells. This is in accordance with Romera et al. (2007), which showed that biomass concentration is an important variable during chromium uptake. By the way, it should be remarked that the larger the biomass growth, the larger the amount of dry matter to be treated or sent for proper disposal, as the metal removed from the medium will accumulate in the biomass. Therefore, in experiments for the removal of metals, biomass growth should not be excessive so as not to increase the treatment cost and the final disposal of this biomass, reducing the cost-effectiveness of biosorption (Dal Magro et al. 2012). In comparison with the cultures accomplished without addition of Chromium VI using only the medium Zarrouk diluted to 50% with distilled water, Dal Magro et al. (2012) obtained maximum cell concentration (Cmax ) of 2.76 g.L−1 and maximum specific growth rate (umax ) of 0.095 d−1 to Spirulina platensis Leb-52, higher than the obtained in all experiments obtained in the recent work. Table 2 shows the analysis of variance of the full factorial design variables on the maximum cell concentration (Cmax ) and the maximum specific growth rate (μmax ). Both variables, as well as the interaction between these variables (X1 .X2 ), had significant effects on Cmax (p < 0.05). The variable X1 (cell concentration before Cr VI addition) had a positive effect, indicating that the best values of Cmax were obtained when Cr VI addition began with larger cell concentration in the medium. The variable X2 (Cr VI concentration added to each new addition) had a negative effect (–0.304667), indicating that the best values of Cmax were obtained when smaller Cr VI concentrations were added to the cultures.
Bioconversion of Cr(VI) to Cr(III) by Live Spirulina platensis
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Table 2. Regression coefficients of the model, estimated effects and significance level of full factorial design variables on Cmax and μmax Maximum cell concentration - Cmax Source of variation Means X1 (Cc) X2 ([Cr VI added ] X1 .X2 Regression model analysis Source of variation
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Means X1 (Cc) X2 ([Cr VI added ] X1 .X2 Regression model analysis
Regression coefficients
Estimated effects
1.289857 1.289857 0.133667 0.267333 −0.152333 −0.304667 0.100333 0.200667 Fcalculated = 57.28 Ftabulated = 3.19 Maximum specific growth rate - μmax Regression coefficients
Estimated effects
0.100038 0.008033 0.008000 −0.000450 Fcalculated = 16.33638 Ftabulated
0.100038 0.016067 0.016000 −0.000900 = 3.19
Significance level (p) 0.000000 0.000001 0.000000 0.000021 R2 = 91%; R2 adjusted = 89% Significance level (p) 0.000000 0.000120 0.000125 0.784629 R2 = 74%; R2 adjusted = 70%
Cc: Cell concentration (g.L−1) in the first addition of Cr (VI); [Cr VIadded ] : Cr VI concentration added in each new addition (mg.L−1).
Both variables (X1 and X2 ) showed significant effects on μmax (p < 0.05). Variables X1 and X2 had a positive effect, indicating that the best values of μmax were obtained at the highest levels of the variables (higher cell concentration in the first addition of Cr VI and higher concentration of Cr VI added to the cultures). The interaction between variables (X1 .X2 ) did not have a significant effect on μmax (p = 0.784629). According to Rodrigues and Iemma (2012), the Fcalculated of the regression model should be greater than the Ftabulated for the rejection of the null hypothesis at the significance level studied, i.e., the variation caused by the model is significantly greater than the unexplained variation. In this case, the value of Fcalculated was higher than the Fcritical (3.19) for both of the analyzed responses (57.28 and 16.33, respectively for Cmax and μmax ). Besides the value of F, the coefficient of determination (R2) is also useful for checking the quality of the model (Kaushik et al. 2010), with R2 values of 0.91 and 0.74, respectively, for Cmax and μmax . Cr VI Removal and Bioconversion in Cr III by Live Biomass Table 3 shows the mass balance of chromium species during cultivation. In each experiment, the amounts of added Cr VI were calculated. From the amounts of metal recovered by the chemical analysis of the biomass and of the medium, we calculated the total chromium amount recovered by the analytical methods. Note that total chromium recovery was smaller than the total chromium addition to the cultures, which may have occurred due to the culture medium conditions, as the microalga needs a high pH for its maintenance, yielding a better performance at pH values around 9.0. pH is one of the most important variables in the removal of toxic metals by biomass, since the speciation of the metal in the solution is pH-dependent and the charge of active sites on the surface may change depending on this value (Pino and Torem 2011).
According to Barros (2006), at a high pH, many metals undergo micro precipitation and may insolubilize some of the metal in the solution, and trivalent chromium can precipitate as hydroxides at near-neutral pH (Pirete et al. 2008). For Possa and Santos (2003), the pH required precipitating most metals in water ranges from 6 to 9, but there are some exceptions, such as ferric hydroxide, which precipitates at acidic pH around 3.5, and aluminum hydroxide, at a pH around 5.5. However, given the importance in separating and removing metals dissolved in solutions, precipitation should be regarded as positive as, according to Kotrba et al. (2011), in general, the precipitation of the metal increases the overall biosorption rates, thus improving the efficiency of the process. Table 4 shows the removal and conversion rates of Cr VI in the biomass and in the medium, taking total chromium recovery into account. Cr VI was not detected in the biomass in any of the experiments, and all chromium retained by the cell was converted to Cr III, demonstrating the capacity of the microalga to turn this metal species into a less toxic one. The conversion of hexavalent chromium removed from the wastewater to trivalent chromium was high – greater than 90%, corroborating the assumption that the presence of the microalga helped convert hexavalent chromium to a less toxic state (trivalent chromium). According to Pirete et al. (2008), the enzymatic reduction of hexavalent chromium to trivalent chromium may be one of the defense mechanisms of microorganisms that live in environments contaminated with this type of metal. According to Hayashi (2001), conversion of hexavalent chromium to trivalent chromium can occur by several mechanisms, which basically depend on the nature of the reducing agent and on reaction conditions. The hexavalent chromium metabolism involves its cellular reduction by small molecules and enzyme systems, producing reactive intermediates and trivalent chromium.
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Table 3. Total chromium added and recovered by analytical methods in the experiments of biosorption with S. platensis Exp.
Cc (g.L−1)
[Cr VIadded ] (mg.L−1)
Cr VIadded (mg)
Cr totalbiomass (mg)
Cr totalmedia (mg)
Cr totalrec (mg)
Cr totalrec (%)
0.5 1 0.5 1 0.75 0.75 0.75
5 5 10 10 7.5 7.5 7.5
32.5 32.5 65.0 65.0 48.75 48.75 48.75
1.71 1.46 2.11 2.13 1.53 1.68 1.54
25.62 24.74 59.09 47.64 36.85 33.11 34.87
27.32 26.20 61.19 49.77 38.38 34.79 36.40
(84.1%) (80.6%) (94.1%) (76.6%) (78.7%) (71.4%) (74.7%)
1 2 3 4 5 6 7
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Cc: Cell concentration (g.L−1) in the first addition of Cr (VI); [Cr VIadded ] : Cr VI concentration added in each new addition (mg.L−1); Cr VIadded = total Cr (VI) mass added considering the final culture volume of 1.3 L (mg); Cr total = total chromium mass measured at the end of the experiment in the biomass or in the media, considering the final culture volume of 1.3 L (mg); Cr totalrec : total chromium recovered in comparison with the added considering the determinations of chromium species in biomass and in the media in the end of the experiments (calculated by Equations 3 and 4).
Biological membranes are impermeable to trivalent chromium, but hexavalent chromium can permeate through them and be reduced in the mitochondrion, nucleus and cytoplasm to trivalent chromium, binding to proteins and interacting with nucleic acids. This process may cause damage to the cell structure, compromising microbial growth. In water, hexavalent chromium is quite stable due to the small amount of reducing agents, while trivalent chromium is associated with particulate matter, suggesting that organic particles can reduce and bind to the element (Hayashi 2001). According to Cˆamera (2011), hexavalent chromium reduction processes through some mechanisms, such as anion exchange and adsorption and/or absorption, can be used to decrease the concentration of these ions in the medium and, consequently, minimize the damage. In in natura treatments, aquatic macrophytes have been used, showing great potential for adsorption and removal, converting hexavalent chromium to trivalent chromium within their tissues (Martin 2008). However, as can be seen in Table 4, of the total hexavalent chromium removed, the percentage values of chromium retained by the biomass (trivalent chromium) ranged from 6.3% (experiment 3) to 9.9% (experiment 6), which were lower than the rates for conversion of hexavalent chromium to trivalent chromium observed in the medium (90.1% (experiment 6) to 93.7% (experiment 3)), demonstrating that chromium concentration in the wastewater was still high in the trivalent state.
Concerning the removal of hexavalent chromium from the medium, rates ranged from 48.9% (experiment 6) to 65.2% (experiment 1). The best hexavalent chromium removal rates were obtained in experiments with lower addition of this metal (experiments 1 and 2 with removal rates of 65.2% and 58.7%, respectively), whereas the lowest percentage values were obtained from central points, with an intermediate concentration of added hexavalent chromium, with removal rates of 49.8% (experiment 5), 48.9% (experiment 6) and 50.9% (experiment 7), while cultures with higher hexavalent chromium addition (experiments 3 and 4) showed removal rates of 54.2% and 55.2%, respectively. By comparison with other studies in this field, we note that the removal rates are similar. Dal Magro et al. (2012) used the active biomass of Spirulina patensis for hexavalent chromium removal and COD and obtained satisfactory results, as Spirulina revealed a hexavalent chromium biosorption of 40% to 60% and COD removal of 60 to 70%. It should be highlighted that, in both studies, the behavior of the metal in the solution was not assessed due to culture conditions. The findings of the present study are in line with other ones conducted by other authors, who demonstrated the extensive use of microorganisms for the removal and bioconversion of metals in wastewaters. In the studies mentioned in what follows, there are a larger number of citations on the use of bacteria (Conceic¸a˜ o et al. 2007; Panigatti et al. 2012; Camargo
Table 4. Percentage of Cr VI removal by biomass and conversion to Cr III in biomass and in the media in relation to the total chromium recovered at the end of the experiments Exp. 1 2 3 4 5 6 7
Cr total rec Cr VI removed from the media Cr VI removed that was converted to Cr III Cr VI removed that was converted to Cr III 4 (mg)1 (mg) and (%)2 (mg and%)3 in the media (mg and%) 27.32 26.20 61.19 49.77 38.38 34.79 36.40
1calculated
17.82 (65.2) 15.39 (58.5) 33.19 (54.2) 27.47 (55.2) 19.13 (49.8) 17.01 (48.9) 18.53 (50.9)
16.11 (90.4) 13.92 (90.5) 31.08 (93.7) 25.34 (92.2) 17.60 (92.0) 15.32 (90.1) 16.99 (91.7)
1.71 (9.6) 1.46 (9.5) 2.10 (6.3) 2.13 (7.7) 1.19 (8.0) 1.29 (9.9) 1.38 (8.3)
in biomass q
(mg.g−1)5
0.931 0.763 1.786 1.192 0.993 1.007 0.854
by Equation 3; 2calculated by Equation 6; 3calculated by Equation 7; 4calculated by Equation 8; 5q: mass of Cr III retained per g of dry biomass, calculated by Equation 9.
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Bioconversion of Cr(VI) to Cr(III) by Live Spirulina platensis et al. 2005) and macroalgae (Sargassum sp.) (Saravanan et al. 2009) for the conversion of metals and fewer studies on the use of microalgae. Gagrai, Das and Golder (2013) performed the study of passive biosorption of Cr VI and its conversion to Cr III by non-living biomass of Spirulina, founding that the optimal conditions were in pH 0.5 in initial Cr (VI) concentrations of 0.96 mM. The major functional groups of Spirulina biomass involved with biosorption and reduction of Cr VI to Cr III is carboxyl, phosphate and amine. pH did not affect the adsorption of Pb II in the range of 3 to 5.5, nor of Cd in the range of 4 to 7. For Cr VI, adsorption was observed only at a pH equal to 2 or lower. Doshi, Ray and Kothari (2009) realized the biosorption of arsenic (V) by live and dead Spirulina from water, but chromium was not studied. As can be seen, most studies performed using Spirulina were accomplished with dead biomass and in the case of bioconversion of Cr VI to Cr III by this microalgae using dead biomass, very low pH was need, which implies in the use of chemicals. We demonstrated that living biomass of Spirulina could convert these species of chromium, maintaining the cell growth until five additions of 10 mg.L−1 of Cr VI. The greater amount of Cr III retained in biomass was 1.78 mg.g−1 in the Experiment 3 (Table 4). Whereas the recommended daily intake for humans is 50 μg (Hsu, Hwang, and Yeh 2001) the consumption by a person of 1 g of biomass would be very dangerous. Considering the environmental aspects of the application of this study, it was found that living cells of Spirulina are capable of withstanding high concentrations of Cr VI seen in the culture medium, converting most in Cr III in the liquid medium.
Conclusion We demonstrate the potential of live cells of Spirulina platensis Leb-52 to realize the biosorption of Cr VI and its conversion to Cr III, less toxic. The microalgae was capable to remove until 65.2% of Cr VI added in the culture, being 90.42% of which converted to trivalent chromium in the medium and 9.6% retained by the biomass. Besides, we can infer that the quality control of water used for the cultivation of microalgae is very important, because the microalgae were able to grow even with consecutive additions of 5 to 10 mg.L−1 of Cr VI in the culture media, with bioaccumulation of Cr in the trivalent form in concentrations above those which can be consumed by humans.
Funding The authors would like to thank the Foundation for Research Support of Rio Grande do Sul, Brazil.
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International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Potential of Live Spirulina platensis on Biosorption of Hexavalent Chromium and Its Conversion to Trivalent Chromium a
a
a
a
Luciane Maria Colla , Clinei Dal’Magro , Andreia De rossi , AntôNio Thomé , Christian b
b
Oliveira Reinehr , Telma Elita Bertolin & Jorge Alberto Vieira Costa
c
a
University of Passo Fundo, Graduate Program in Civil and Environmental Engineering, Bairro São José, Passo Fundo/RS, Brazil b
University of Passo Fundo, Food Engineering Program, Building L1, Campus I, BR Bairro São José, Passo Fundo/RS, Brazil c
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Federal University of Rio Grande, Graduate Program in Food Engineering and Science, P.P. Box, Rio Grande, RS, Brazil Accepted author version posted online: 01 Dec 2014.
To cite this article: Luciane Maria Colla, Clinei Dal’Magro, Andreia De rossi, AntôNio Thomé, Christian Oliveira Reinehr, Telma Elita Bertolin & Jorge Alberto Vieira Costa (2015) Potential of Live Spirulina platensis on Biosorption of Hexavalent Chromium and Its Conversion to Trivalent Chromium, International Journal of Phytoremediation, 17:9, 861-868, DOI: 10.1080/15226514.2014.964846 To link to this article: http://dx.doi.org/10.1080/15226514.2014.964846
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Potential of Live Spirulina platensis on Biosorption of Hexavalent Chromium and Its Conversion to Trivalent Chromium ˆ LUCIANE MARIA COLLA1, CLINEI DAL’MAGRO1, ANDREIA DE ROSSI1, ANTONIO THOME´ 1, CHRISTIAN 2 2 OLIVEIRA REINEHR , TELMA ELITA BERTOLIN , and JORGE ALBERTO VIEIRA COSTA3 1
University of Passo Fundo, Graduate Program in Civil and Environmental Engineering, Bairro S˜ao Jos´e, Passo Fundo/RS, Brazil University of Passo Fundo, Food Engineering Program, Building L1, Campus I, BR Bairro S˜ao Jos´e, Passo Fundo/RS, Brazil 3 Federal University of Rio Grande, Graduate Program in Food Engineering and Science, P.P. Box, Rio Grande, RS, Brazil
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2
Microalga biomass has been described worldwide according their capacity to realize biosorption of toxic metals. Chromium is one of the most toxic metals that could contaminate superficial and underground water. Considering the importance of Spirulina biomass in production of supplements for humans and for animal feed we assessed the biosorption of hexavalent chromium by living Spirulina platensis and its capacity to convert hexavalent chromium to trivalent chromium, less toxic, through its metabolism during growth. The active biomass was grown in Zarrouk medium diluted to 50% with distilled water, keeping the experiments under controlled conditions of aeration, temperature of 30◦ C and lighting of 1,800 lux. Hexavalent chromium was added using a potassium dichromate solution in fed-batch mode with the aim of evaluate the effect of several additions contaminant in the kinetic parameters of the culture. Cell growth was affected by the presence of chromium added at the beginning of cultures, and the best growth rates were obtained at lower metal concentrations in the medium. The biomass removed until 65.2% of hexavalent chromium added to the media, being 90.4% converted into trivalent chromium in the media and 9.6% retained in the biomass as trivalent chromium (0.931 mg.g−1). Keywords: toxic metal, potassium dichromate, supplements
Introduction Contamination by toxic metals, because of their continuous accumulation in the food chain, is one of the most serious environmental problems and therefore is a matter of great concern (Doshi, Ray, and Khotari 2007) due to the risk to human and animal health (Merlino et al. 2010; Mudgal et al. 2006; Das, Vimala, and Karthika 2008). Numerous human activities can pollute soils and underground with metals, as the disposal of solid and industrial waste into poorly waterproofed landfills, contaminating the soil and water resources through leaching (Melo et al. 2010; Maus, Costa, and Righes 2009), leakage and spillage, industrial activities (galvanoplasty, mining, stell and cement production, tannery), application of fertilizers and agrochemicals, and disposal of agroindustrial waste and wastewater treatment plant sludge into the soil (Korf 2011; Canuto et al. 2007). Algae have been used to biosorption of toxic metal due to the need of new technologies for the treatment of wastewaters
Address correspondence to Luciane Maria Colla, University of Passo Fundo, Graduate Program in Civil and Environmental Engineering – Building G2, Campus I, BR 285, Bairro S˜ao Jos´e, Passo Fundo/RS, CEP: 99052-900. E-mail: [email protected]
contaminated by toxic metals. The use of algal biomass for the biosorption of toxic metals has some advantages such as low cost and good efficiency (Das et al. 2008), with the accumulation of metals in the outer walls of the algae by physical, chemical and biological mechanisms (Mane et al. 2011). Biosorption consists of the sorption of toxic metals by living or dead microorganisms (biomass) (Quintelas et al. 2008). According to Pietrobelli et al. (2009), biosorption is an efficient alternative to conventional wastewater treatments, as the microorganisms retain the metals, promoting wastewater selfregeneration and qualifying the process. Thus, biosorption is a thriving technology whose application in the treatment and final polishing of wastewaters containing toxic metals has cur´ rently expanded (Kratochvil and Volesky 1998; Modenes et al. 2009). Among toxic metals, chromium is one of the most dangerous and could contaminate water reservoirs, rivers, and sources of water for the population (Merlino et al. 2010; Canuto et al. 2007). The current technology for the removal of total chromium consists basically of two stages: conversion of hexavalent chromium (Cr VI) to trivalent chromium (Cr III) using reducing agents and the later precipitation of Cr III in the form of hydroxides. Conversion of Cr VI to Cr III is an essential stage in the process as the former one is quite mobile in nature, and is not easily adsorbed or precipitated. Given
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862 the wide array of sources of wastewaters containing Cr VI, the development of new technologies for its removal has been extensively investigated (Rutuolo and Gubulin 2003). Several studies are been conducted using Spirulina platensis biomass to remove metallic ions from aqueous solutions (Arunakumara and Xuecheng 2007; Doshi, Ray, and Kothari 2007). Spirulina platensis has been used for environmental purposes by several authors, such as Mezzomo et al. (2010) for the removal of COD (chemical oxygen demand) in swine wastewater. Aneja et al. (2010) assessed the biosorption of Pb+2 and Zn+2 from an aqueous solution containing different concentrations of these metals, using the dry biomass of Spirulina sp., with biosorption rates of 82% for Pb+2 and of 90% for Zn+2. In our research group, we already studied the active and passive biosorption of hexavalent chromium by Spirulina platensis, obtaining 61.97% of Cr VI removal using dead biomass (passive biosorption), in pH 3, with maximal adsorption capacity of 100.39 mg.g−1 (Dal Magro et al. 2013). In another work we show that Spirulina platensis Leb-52 strain can perform the active biosorption of Cr VI from wastewaters with removal rates of 45% to 60.92% for initial concentrations of Cr VI of 11.7 mg. L−1 and 3 mg.L−1, respectively (Dal’Magro et al. 2012). On the other hand, Spirulina is widely used as a nutritional supplement to humans and animals and has been produced by a great number of companies, due to the proof that the biomass could help in the treatment and prevention of many diseases (Ambrosi et al. 2008). Al-Dhabi (2013) and Hsu, Hwang, and Yeh (2001) studied the presence inorganic elements in Spirulina products and proved that the concentration of was not to exceed the present regulation levels. But if this microalgae is grown in contaminated culture media, it is important whether the growth will be maintained at high contaminant concentrations, as well as the contaminant is adsorbed by microalga biomass. Doshi, Ray and Khotari (2007) demonstrated that live and dead Spirulina sp. took up a significant amount of Cd+2 ions, but no studies were found using live biomass of Spirulina to biosorption of chromium species. Considering the described use of Spirulina biomass in biosorption of toxic metals, the potential of chromium contaminations in waters, and the widely use of Spirulina biomass as food, the highlight of this work was the study of the ability of this microalga to growth in media contaminated with hexavalent chromium and the bioconversion of Cr VI to Cr III in the culture. The addition of contaminant was accomplished in fed-batch mode in order to evaluate the effect of several contaminations of chromium over the growth of microalga.
Material and Methods Microorganism and Inoculum Maintenance Spirulina platensis Leb-52 strain was used, and the inocula were kept in a non-sterile thermostat-controlled greenhouse under controlled aeration (diaphragm pumps), temperature (∼30◦ C) and lighting (1800 lux). We used the Zarrouk (1966) medium, which is standard for the cultivation of this microalga, diluted to 50% with sterile distilled water. Lighting
L. M. Colla et al. was provided by 20 W fluorescent lamps and the temperature was maintained by resistors whose heating is controlled by thermocouples and fans. Growth Conditions The experiments were performed in 2 L Erlenmeyer flasks with an initial medium volume of 1.3 L, with a 12-hour photoperiod, and constant aeration. Culture media received Cr VI through standardized potassium dichromate (K2 Cr2 O7 ) solutions at variable concentrations according to the experimental design (Table 1). Potassium dichromate solutions were added in fed batch mode after cultures reached a certain biomass concentration (0.5 g. L−1 or 1.0 g. L−1). After the first contamination, the other additions of Cr VI occurred at 5-day intervals, totaling five additions of Cr VI at the end of the growth period in all assays. Kinetic Growth Parameters The pH of the samples was assessed regularly by a pH meter (DIGIMED, DM-22). Microalgae growth was evaluated every two days by absorbance reading of cultures using a spectrophotometer (PG INTRUMENTS, T60) at 670 nm and the results were obtained from a previously built standard biomass curve. The maximum specific growth rate and the doubling time were calculated using Equations 1 and 2. X2 1 Ln t X1 ln 2 tg = μmax
μmax =
(1) (2)
Where: μmax = maximum specific growth rate (d−1) X1 = Concentration of cells in the early exponential growth stage (g.L−1) X2 = Concentration of cells in the late exponential growth stage (g.L−1) t = duration of the exponential growth stage (d) tg = doubling time (d) Determinations of Chromium in Biomass and Wastewater At the end of the growth period, the presence of Cr VI and Cr III in the cell-free medium and in the biomass was determined in order to assess their removal and the conversion of Cr VI to Cr III, which is less toxic, by the microalga. The wastewater biomass was separated by two consecutive filtrations, using a vacuum pump (MARCONI, MA 058) and a polyester filter with 180 filament yarns. Later on, the biomass was dried at 60◦ C until a constant weight was obtained and submitted to digestion for extraction of the metal from the intracellular medium by EPA method 3050B (USEPA 2012). The filtrated wastewater was submitted to digestion for oxidation of the existing organic matter by EPA method 3050A (USEPA 2012).
Bioconversion of Cr(VI) to Cr(III) by Live Spirulina platensis
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Table 1. Growth parameters of microalga S. platensis during the active biosorption of Cr VI Exp. 1 2 3 4 5 6 7
Cc (g.L−1)
[Cr VI]added (mg.L−1)
Cmax (g.L−1)
μmax (d−1)
Tg (d)
log (d)
R2
0.5 1 0.5 1 0.75 0.75 0.75
5 5 10 10 7.5 7.5 7.5
1.411 1.478 0.906 1.374 1.186 1.289 1.384
0.088 0.1045 0.1045 0.1196 0.0906 0.0960 0.0980
7.92 6.64 6.64 5.79 7.65 7.23 7.10
0-18 0-16 0-12 0-12 0-16 0-16 0-16
0.96 0.97 0.94 0.98 0.94 0.95 0.95
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Cc: Cell concentration (g.L−1) in the first addition of Cr (VI); [Cr VIadded ] : Cr VI concentration added in each new addition (mg.L−1); Cmax = maximum cell concentration (g.L−1); μmax = maximum specific growth rate (d−1); Tg = generation time (d); log = time values used in the exponential regression (d); R2 = determination coefficients of the regressions.
The total chromium concentration in the sample was determined by flame atomic absorption spectrophotometry (ANALYST 200-PERKIN ELMER). Hexavalent chromium was determined by the 1.5- diphenylcarbazide (APHA 2000) method while trivalent chromium was determined by the difference between total chromium and hexavalent chromium, as shown in Equation 3. This is due to the fact that Cr III complexes in aqueous solutions have relative kinetic inertia (GOVINDRAO 1980). Cr (total) = Cr I I I + Cr V I
%Cr (total)r ec =
Cr (total)r ec .100 Cr (total)i ni ti all y added
%Cr (I I I)bi omass =
(4) (5)
Based on total chromium recovery, we calculated the percentage of Cr VI removed from the wastewater and the percentage of Cr VI converted to Cr III, both in the wastewater and in the biomass. The removal of Cr VI from the wastewater was calculated by Equation 6. Cr (total)r ec − Cr V Imedi um Cr (total)r ec
%Cr I I Imedi um =
Where: Cr(total)rec = total chromium recovered (mg) by analytical determinations as a function of the initial amount of Cr VI added to each culture. Cr(total)biomass = total chromium (mg) in the biomass at the end of the growth period, considering the dry biomass weight. Cr(total)medium = total chromium (mg) in the medium the end of the growth period, considering the final volume of the culture. Crinitially added = total hexavalent chromium (mg) added in the experiment during cultivation. % Cr(total)rec = percentage of total chromium recovered at the end of the growth period.
RemovalCr (V I)medi um =
The percentages of Cr III in the wastewater and in the biomass at the end of the growth period were calculated by Equations 7 and 8 and the mass of Cr III retained per g of dry biomass was calculated by Equation 9.
(3)
The total chromium recovered by analytical determinations was calculated by the mass balance, according to Equations 4 and 5. Cr (total)r ec = Cr (total)bi omass + Cr (total)medi um
Where: Removal of Cr VImedium = hexavalent chromium removed from the medium (mg). Cr VImedium = total Cr (VI) which remained in the medium at the end of the growth period (mg).
(6)
q
mg g
=
Cr I I Imedi um Cr (total)r ec −Cr V Imedi um Cr I I Ibi omass Cr (total)r ec −Cr V Imedi um
.100
(7)
.100
(8)
Cr I I Ibi omass (mg) Cmax .V (g)
(9)
Where: % Cr IIImedium = percentage of Cr III in the medium at the end of the growth period. Cr IIImedium = Cr III which remained in the medium at the end of the growth period (mg). % Cr IIIbiomass = percentage of Cr III in the biomass at the end of the growth period. Cr IIIbiomass = Cr III which remained in the biomass at the end of the growth period (mg). Cmax = maximum biomass concentration (g/L) V = volume of cultures in the end of the experiments, that was 1.3 L
Results and Discussion Influence of Chromium Addition on Microalga Growth The pH of cultures ranged from 9.63 to 10.15, a range that is deemed appropriate for the cultivation of Spirulina platensis, offering the ideal conditions for its growth (Andrade and Costa 2008). Figure 1 shows the growth curves for the microalga as a function of time. In all experiments, there was exponential growth on the first days of culture (from 12 to 14 days). In experiment 3, from the 14th day of growth, the cell concentration
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Fig. 1. Microalgae growth versus culture time.
stabilized and decreased, and there might have been a toxic effect of the metal on microalgae growth as, in this experiment, Cr VI additions were performed at the highest concentration (10 mg.L−1), and the experiment showed a lower biomass concentration at the time of first addition of the metallic solution (0.5 g.L−1). For the other experiments, cell growth was similar, revealing growth up to the 20th day followed by a decrease in concentration, indicating the death of some of the cells in the medium. This may have occurred due to nutritional restriction as no nutrients were added during the experiment, only at the beginning of cultures. Table 1 shows the microalgae growth parameters obtained during cultivation. The experiments with higher maximum concentrations were those in which chromium was added at the lowest concentrations (Experiments 1 and 2), reaching the maximum cell concentrations (Cmax ) of 1.411 and 1.478 g.L−1, respectively, demonstrating that Cmax depends on the concentration of toxic compounds added to the medium. The lowest Cmax was obtained in experiment 3 (0.906 g.L−1), to which a larger amount of Cr VI was added (50 mg.L−1, five additions of 10 mg.L−1) at the lowest cell concentration in the first addition of the metal (0.5 g.L−1), showing that the toxicity of the metal interfered with cell growth. When Cr VI, even at high concentrations (10 mg.L−1), was added to cultures with larger cell concentration in the first addition (1.0 g.L−1), Cmax was 1.374 g.L−1 (experiment 4), indicating that the larger amount of cells in the medium led to better uptake of the metal; however, the exponential growth interval ( log) decreased in both experiments with the highest concentration of the metal (experiments 3 and 4, with 50 mg.L−1 of Cr VI) (12 days) in relation to the other experiments, which had a log of 16 to 18 days. Doubling times (time intervals necessary for biomass doubling) ranged from 5.79 to 7.92 days. The lowest doubling times were observed in the experiments with the highest Cr VI concentration (50 mg.L−1of Cr VI), as a function of the influence of the amount of added chromium on the exponential growth stage. In these experiments, although the doubling time is shorter, indicating higher growth, the microorganisms remained in the exponential growth stage for shorter time periods ( log = 12 days) than those observed in the other experiments (16 to 18 days). Moreover, the extension of the
L. M. Colla et al. exponential growth stage may be also related to the fact that, in some experiments, chromium was only added after the microorganism reached concentrations of 0.75 g.L−1 (experiments 5, 6 and 7) or 1.0 g.L−1 (experiments 2 or 4). Dal Magro et al. (2012) observed that the addition of Cr VI in S. platensis cultures only at the beginning of the experiments interfered with the kinetic growth parameters, compromising good cell development and indicating the toxicity of this metal to the microbial metabolism. The cell growth was affected by the addition of wastewater containing around 3 mg.L−1 of Cr VI. Toxic metals can cause adverse effects on biological systems, indirectly producing breaks in the DNA double strand by oxidative stress (Gastaldo et al. 2008). This stress occurs when there is an imbalance produced by the excess generation of oxidants or by the decrease in antioxidants (Guo, Yang and Xu 2008). According to Moreira and Moreira (2004), toxic metals may hinder all and any biological activity, causing different types of biological responses to these metals. In the present study, the cell growth was poorly affected when the metal concentration was 5 mg.L−1 and we have to consider that five additions of contaminant were made in the cultures. With cells in growth, novel additions of contaminant do not seems to affect greatly the growth. This happens because the effect of the addition of the contaminant might have on the cells is minimized by the appearance of new cells, which represent new binding sites for chromium VI or, as will be shown later, the biomass is able to convert chromium VI to chromium III, which seemingly has lower toxicity to the cells. This is in accordance with Romera et al. (2007), which showed that biomass concentration is an important variable during chromium uptake. By the way, it should be remarked that the larger the biomass growth, the larger the amount of dry matter to be treated or sent for proper disposal, as the metal removed from the medium will accumulate in the biomass. Therefore, in experiments for the removal of metals, biomass growth should not be excessive so as not to increase the treatment cost and the final disposal of this biomass, reducing the cost-effectiveness of biosorption (Dal Magro et al. 2012). In comparison with the cultures accomplished without addition of Chromium VI using only the medium Zarrouk diluted to 50% with distilled water, Dal Magro et al. (2012) obtained maximum cell concentration (Cmax ) of 2.76 g.L−1 and maximum specific growth rate (umax ) of 0.095 d−1 to Spirulina platensis Leb-52, higher than the obtained in all experiments obtained in the recent work. Table 2 shows the analysis of variance of the full factorial design variables on the maximum cell concentration (Cmax ) and the maximum specific growth rate (μmax ). Both variables, as well as the interaction between these variables (X1 .X2 ), had significant effects on Cmax (p < 0.05). The variable X1 (cell concentration before Cr VI addition) had a positive effect, indicating that the best values of Cmax were obtained when Cr VI addition began with larger cell concentration in the medium. The variable X2 (Cr VI concentration added to each new addition) had a negative effect (–0.304667), indicating that the best values of Cmax were obtained when smaller Cr VI concentrations were added to the cultures.
Bioconversion of Cr(VI) to Cr(III) by Live Spirulina platensis
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Table 2. Regression coefficients of the model, estimated effects and significance level of full factorial design variables on Cmax and μmax Maximum cell concentration - Cmax Source of variation Means X1 (Cc) X2 ([Cr VI added ] X1 .X2 Regression model analysis Source of variation
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Means X1 (Cc) X2 ([Cr VI added ] X1 .X2 Regression model analysis
Regression coefficients
Estimated effects
1.289857 1.289857 0.133667 0.267333 −0.152333 −0.304667 0.100333 0.200667 Fcalculated = 57.28 Ftabulated = 3.19 Maximum specific growth rate - μmax Regression coefficients
Estimated effects
0.100038 0.008033 0.008000 −0.000450 Fcalculated = 16.33638 Ftabulated
0.100038 0.016067 0.016000 −0.000900 = 3.19
Significance level (p) 0.000000 0.000001 0.000000 0.000021 R2 = 91%; R2 adjusted = 89% Significance level (p) 0.000000 0.000120 0.000125 0.784629 R2 = 74%; R2 adjusted = 70%
Cc: Cell concentration (g.L−1) in the first addition of Cr (VI); [Cr VIadded ] : Cr VI concentration added in each new addition (mg.L−1).
Both variables (X1 and X2 ) showed significant effects on μmax (p < 0.05). Variables X1 and X2 had a positive effect, indicating that the best values of μmax were obtained at the highest levels of the variables (higher cell concentration in the first addition of Cr VI and higher concentration of Cr VI added to the cultures). The interaction between variables (X1 .X2 ) did not have a significant effect on μmax (p = 0.784629). According to Rodrigues and Iemma (2012), the Fcalculated of the regression model should be greater than the Ftabulated for the rejection of the null hypothesis at the significance level studied, i.e., the variation caused by the model is significantly greater than the unexplained variation. In this case, the value of Fcalculated was higher than the Fcritical (3.19) for both of the analyzed responses (57.28 and 16.33, respectively for Cmax and μmax ). Besides the value of F, the coefficient of determination (R2) is also useful for checking the quality of the model (Kaushik et al. 2010), with R2 values of 0.91 and 0.74, respectively, for Cmax and μmax . Cr VI Removal and Bioconversion in Cr III by Live Biomass Table 3 shows the mass balance of chromium species during cultivation. In each experiment, the amounts of added Cr VI were calculated. From the amounts of metal recovered by the chemical analysis of the biomass and of the medium, we calculated the total chromium amount recovered by the analytical methods. Note that total chromium recovery was smaller than the total chromium addition to the cultures, which may have occurred due to the culture medium conditions, as the microalga needs a high pH for its maintenance, yielding a better performance at pH values around 9.0. pH is one of the most important variables in the removal of toxic metals by biomass, since the speciation of the metal in the solution is pH-dependent and the charge of active sites on the surface may change depending on this value (Pino and Torem 2011).
According to Barros (2006), at a high pH, many metals undergo micro precipitation and may insolubilize some of the metal in the solution, and trivalent chromium can precipitate as hydroxides at near-neutral pH (Pirete et al. 2008). For Possa and Santos (2003), the pH required precipitating most metals in water ranges from 6 to 9, but there are some exceptions, such as ferric hydroxide, which precipitates at acidic pH around 3.5, and aluminum hydroxide, at a pH around 5.5. However, given the importance in separating and removing metals dissolved in solutions, precipitation should be regarded as positive as, according to Kotrba et al. (2011), in general, the precipitation of the metal increases the overall biosorption rates, thus improving the efficiency of the process. Table 4 shows the removal and conversion rates of Cr VI in the biomass and in the medium, taking total chromium recovery into account. Cr VI was not detected in the biomass in any of the experiments, and all chromium retained by the cell was converted to Cr III, demonstrating the capacity of the microalga to turn this metal species into a less toxic one. The conversion of hexavalent chromium removed from the wastewater to trivalent chromium was high – greater than 90%, corroborating the assumption that the presence of the microalga helped convert hexavalent chromium to a less toxic state (trivalent chromium). According to Pirete et al. (2008), the enzymatic reduction of hexavalent chromium to trivalent chromium may be one of the defense mechanisms of microorganisms that live in environments contaminated with this type of metal. According to Hayashi (2001), conversion of hexavalent chromium to trivalent chromium can occur by several mechanisms, which basically depend on the nature of the reducing agent and on reaction conditions. The hexavalent chromium metabolism involves its cellular reduction by small molecules and enzyme systems, producing reactive intermediates and trivalent chromium.
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Table 3. Total chromium added and recovered by analytical methods in the experiments of biosorption with S. platensis Exp.
Cc (g.L−1)
[Cr VIadded ] (mg.L−1)
Cr VIadded (mg)
Cr totalbiomass (mg)
Cr totalmedia (mg)
Cr totalrec (mg)
Cr totalrec (%)
0.5 1 0.5 1 0.75 0.75 0.75
5 5 10 10 7.5 7.5 7.5
32.5 32.5 65.0 65.0 48.75 48.75 48.75
1.71 1.46 2.11 2.13 1.53 1.68 1.54
25.62 24.74 59.09 47.64 36.85 33.11 34.87
27.32 26.20 61.19 49.77 38.38 34.79 36.40
(84.1%) (80.6%) (94.1%) (76.6%) (78.7%) (71.4%) (74.7%)
1 2 3 4 5 6 7
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Cc: Cell concentration (g.L−1) in the first addition of Cr (VI); [Cr VIadded ] : Cr VI concentration added in each new addition (mg.L−1); Cr VIadded = total Cr (VI) mass added considering the final culture volume of 1.3 L (mg); Cr total = total chromium mass measured at the end of the experiment in the biomass or in the media, considering the final culture volume of 1.3 L (mg); Cr totalrec : total chromium recovered in comparison with the added considering the determinations of chromium species in biomass and in the media in the end of the experiments (calculated by Equations 3 and 4).
Biological membranes are impermeable to trivalent chromium, but hexavalent chromium can permeate through them and be reduced in the mitochondrion, nucleus and cytoplasm to trivalent chromium, binding to proteins and interacting with nucleic acids. This process may cause damage to the cell structure, compromising microbial growth. In water, hexavalent chromium is quite stable due to the small amount of reducing agents, while trivalent chromium is associated with particulate matter, suggesting that organic particles can reduce and bind to the element (Hayashi 2001). According to Cˆamera (2011), hexavalent chromium reduction processes through some mechanisms, such as anion exchange and adsorption and/or absorption, can be used to decrease the concentration of these ions in the medium and, consequently, minimize the damage. In in natura treatments, aquatic macrophytes have been used, showing great potential for adsorption and removal, converting hexavalent chromium to trivalent chromium within their tissues (Martin 2008). However, as can be seen in Table 4, of the total hexavalent chromium removed, the percentage values of chromium retained by the biomass (trivalent chromium) ranged from 6.3% (experiment 3) to 9.9% (experiment 6), which were lower than the rates for conversion of hexavalent chromium to trivalent chromium observed in the medium (90.1% (experiment 6) to 93.7% (experiment 3)), demonstrating that chromium concentration in the wastewater was still high in the trivalent state.
Concerning the removal of hexavalent chromium from the medium, rates ranged from 48.9% (experiment 6) to 65.2% (experiment 1). The best hexavalent chromium removal rates were obtained in experiments with lower addition of this metal (experiments 1 and 2 with removal rates of 65.2% and 58.7%, respectively), whereas the lowest percentage values were obtained from central points, with an intermediate concentration of added hexavalent chromium, with removal rates of 49.8% (experiment 5), 48.9% (experiment 6) and 50.9% (experiment 7), while cultures with higher hexavalent chromium addition (experiments 3 and 4) showed removal rates of 54.2% and 55.2%, respectively. By comparison with other studies in this field, we note that the removal rates are similar. Dal Magro et al. (2012) used the active biomass of Spirulina patensis for hexavalent chromium removal and COD and obtained satisfactory results, as Spirulina revealed a hexavalent chromium biosorption of 40% to 60% and COD removal of 60 to 70%. It should be highlighted that, in both studies, the behavior of the metal in the solution was not assessed due to culture conditions. The findings of the present study are in line with other ones conducted by other authors, who demonstrated the extensive use of microorganisms for the removal and bioconversion of metals in wastewaters. In the studies mentioned in what follows, there are a larger number of citations on the use of bacteria (Conceic¸a˜ o et al. 2007; Panigatti et al. 2012; Camargo
Table 4. Percentage of Cr VI removal by biomass and conversion to Cr III in biomass and in the media in relation to the total chromium recovered at the end of the experiments Exp. 1 2 3 4 5 6 7
Cr total rec Cr VI removed from the media Cr VI removed that was converted to Cr III Cr VI removed that was converted to Cr III 4 (mg)1 (mg) and (%)2 (mg and%)3 in the media (mg and%) 27.32 26.20 61.19 49.77 38.38 34.79 36.40
1calculated
17.82 (65.2) 15.39 (58.5) 33.19 (54.2) 27.47 (55.2) 19.13 (49.8) 17.01 (48.9) 18.53 (50.9)
16.11 (90.4) 13.92 (90.5) 31.08 (93.7) 25.34 (92.2) 17.60 (92.0) 15.32 (90.1) 16.99 (91.7)
1.71 (9.6) 1.46 (9.5) 2.10 (6.3) 2.13 (7.7) 1.19 (8.0) 1.29 (9.9) 1.38 (8.3)
in biomass q
(mg.g−1)5
0.931 0.763 1.786 1.192 0.993 1.007 0.854
by Equation 3; 2calculated by Equation 6; 3calculated by Equation 7; 4calculated by Equation 8; 5q: mass of Cr III retained per g of dry biomass, calculated by Equation 9.
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Bioconversion of Cr(VI) to Cr(III) by Live Spirulina platensis et al. 2005) and macroalgae (Sargassum sp.) (Saravanan et al. 2009) for the conversion of metals and fewer studies on the use of microalgae. Gagrai, Das and Golder (2013) performed the study of passive biosorption of Cr VI and its conversion to Cr III by non-living biomass of Spirulina, founding that the optimal conditions were in pH 0.5 in initial Cr (VI) concentrations of 0.96 mM. The major functional groups of Spirulina biomass involved with biosorption and reduction of Cr VI to Cr III is carboxyl, phosphate and amine. pH did not affect the adsorption of Pb II in the range of 3 to 5.5, nor of Cd in the range of 4 to 7. For Cr VI, adsorption was observed only at a pH equal to 2 or lower. Doshi, Ray and Kothari (2009) realized the biosorption of arsenic (V) by live and dead Spirulina from water, but chromium was not studied. As can be seen, most studies performed using Spirulina were accomplished with dead biomass and in the case of bioconversion of Cr VI to Cr III by this microalgae using dead biomass, very low pH was need, which implies in the use of chemicals. We demonstrated that living biomass of Spirulina could convert these species of chromium, maintaining the cell growth until five additions of 10 mg.L−1 of Cr VI. The greater amount of Cr III retained in biomass was 1.78 mg.g−1 in the Experiment 3 (Table 4). Whereas the recommended daily intake for humans is 50 μg (Hsu, Hwang, and Yeh 2001) the consumption by a person of 1 g of biomass would be very dangerous. Considering the environmental aspects of the application of this study, it was found that living cells of Spirulina are capable of withstanding high concentrations of Cr VI seen in the culture medium, converting most in Cr III in the liquid medium.
Conclusion We demonstrate the potential of live cells of Spirulina platensis Leb-52 to realize the biosorption of Cr VI and its conversion to Cr III, less toxic. The microalgae was capable to remove until 65.2% of Cr VI added in the culture, being 90.42% of which converted to trivalent chromium in the medium and 9.6% retained by the biomass. Besides, we can infer that the quality control of water used for the cultivation of microalgae is very important, because the microalgae were able to grow even with consecutive additions of 5 to 10 mg.L−1 of Cr VI in the culture media, with bioaccumulation of Cr in the trivalent form in concentrations above those which can be consumed by humans.
Funding The authors would like to thank the Foundation for Research Support of Rio Grande do Sul, Brazil.
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