- Email: [email protected]
A new chlorophycean nickel hyperaccumulator
Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 3930–3934
Short Communication
A new chlorophycean nickel hyperaccumulator Harish a, S. Sundaramoorthy a b
a,*
, Devendra Kumar b, S.G. Vaijapurkar
b
Department of Botany, JNV University, Jodhpur 342 001, India Defence Research Laboratory, DRDO, Jodhpur 342 001, India
Received 4 May 2007; received in revised form 4 July 2007; accepted 4 July 2007 Available online 10 September 2007
Abstract Bioremediation of nickel by chlorophycean bioremediator, Chlorococcum hemicolum was investigated. The growth rates at various concentrations of Ni2+ were assessed in terms of protein level and 12 mg L 1 of the Ni2+ is the tolerance limit (46.76% level of growth kinetics). Absorption/adsorption kinetics was estimated after 240 h of Ni2+ treatments. Absorptions were higher than adsorption with maximum accumulation factor (AF) of 1.37. Ni2+ concentration and absorption were linearly related (r = 0.98; p > 0.01). Other biochemical parameters like total sugar, chlorophyll and carotenoids were also quantified to correlate the state of metabolism and these exhibited reduction due to heavy metal stress. 2007 Elsevier Ltd. All rights reserved. Keywords: Bioremediation; Chlorococcum; Hyperaccumulator; Nickel
1. Introduction Discharge of heavy metals from metal processing industries is known to have adverse effects on the environment (Ahluwalia and Goyal, 2007). Though, some of these metals (Mg, Fe, Mn, Zn, Mo etc.) are essential for various physiological functions and are critical in many of the enzymatic and metabolic reactions occurring within an organism, but many (Cd, Cr, Pb, Co, Ag, Se etc.) have no known biological function. The essentiality of nickel is now generally acknowledged, based on the numerous symptoms caused by nickel deficiency (mainly in terrestrial vertebrates) and its role in various enzymes in bacteria and plants. The information on optimal and deficient concentrations of nickel is limited; and it can only be confirmed for plants and cyanobacteria for its role in the urease and hydrogenase metabolism. Deficiency levels ranged from 10 12 M to 2 · 10 6 M Ni2+ (external; in water of in vitro studies) for different species (Muyssen et al., 2004). None-
*
Corresponding author. Tel.: +91 291 2722081; fax: +91 291 2649465. E-mail address: jnvusundar@rediffmail.com (S. Sundaramoorthy).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.07.043
theless, excess of heavy metal is toxic to most plants and it is now for more than a decade since the significance of Ni2+ as a very serious pollutant were recognized (Iljin, 1991). It is known to be highly toxic to a wide range of algae (Rai and Mallick, 1993). In recent times, a number of attempts have been made to explore the ability of the plant to accumulate the heavy metal (hyperaccumulation) for remediation of the polluted water. Brooks et al. (1977) first coined the term ‘hyperaccumulator’ to define plants with Ni2+ concentrations higher than 1000 lg g 1 DW (0.1%). This value was not chosen arbitrarily. Ni2+ is a plant micronutrient and is found in the vegetative organs of most plants in the range of 1–10 lg g 1 DW (Assunc¸a˜o et al., 2003). Toxicity occurs at concentrations higher than 10–50 lg g 1 DW (Marschner, 1995). The ability of algae to remove heavy metals from aqueous solution has been known for some decades. Chmielewska and Medved (2001) confirmed the high Ni2+ bioaccumulation ability of Cladophora glomerata. Likewise, bioremediation potentiality of Microspora was also assessed (Axtell et al., 2003). The algae of chronically metal-contaminated localities tend to accumulate heavy
Harish et al. / Bioresource Technology 99 (2008) 3930–3934
metals to a dangerous extent. The metal content of algae can be used to predict the level of metal pollution in a water body (Abdallah et al., 2006). The high accumulation capacity can even be used for the enrichment or recycling of valuable metals (Can et al., 2006). Their relative comparison is generally made with the help of accumulation factor (AF). Metal accumulation factor (AF) is defined as the ratio of metal concentration in plant cell (micrograms per gram) and the metal concentration in water (micrograms per milliliter) and also known as a bioconcentration ratio, concentration ratio and enrichment ratio. Isolation of algal ecotype from local stream, containing polluted industrial effluent, that can tolerate high metal alloy, is essential for efficient functioning of bioremediation system in that particular environment. Dye of fabrics and metal alloy production are the major industrial activities in Boranada industrial area, Jodhpur releasing effluent that flow into the local Jojri River. The study region (which comes under the area of Indian Thar Desert) is classified as semi arid. Consequently, the algal flora have considerable ecological importance, even if they occur in river micro-habitat. Hence, the aim of this study was to explore the biotic potential of local chlorophycean alga Chlorococcum hemicolum for Ni2+ bioremediation. Further changes in protein, sugar, chlorophyll and carotenoids were also assessed.
2. Methods 2.1. Experimental organism, growth and experimental conditions C. hemicolum, a local alga, was collected from Jojri River (pH 6.2–6.5). This alga occurred at various locations within this stream. All the industrial effluent of nearby Boranada Industrial area, Jodhpur, flow into this stream. Axenic culture of this alga was multiplied in BG-11 medium (Rippka et al., 1979) and grown in culture room under continuous light, illuminated with cool fluorescent tubes (14.4 W m 2) at 24 ± 1 C. All the experiments were conducted in triplicate at same culture conditions. The cultures contains glass beads (five in number in each culture flask with 0.5 mm size each) to prevent clumping of cells in growing algal mass, and were shaken gently everyday. 2.2. Changes in growth rate One milliliter of algal cells with protein value 100 lg mL 1 were withdrawn from exponentially growing homogenous culture of alga and inoculated in 100 mL freshly prepared BG-11 medium containing different ranges (0– 20 mg L 1) of Ni2+. Source for Ni2+ in medium was stock solution prepared with NiSO4Æ6H2O. Changes in protein
3931
content were measured by the method of Lowry et al. (1951) as modified by Herbert et al. (1971) using lysozyme (Sigma) as the standard. Protein content was measured after every 48 h, up to 480 h, starting from 96 h (4th day) from these newly established cultures. 2.3. Absorption/adsorption kinetics One milliliter of algal cells with protein value 100 lg mL 1 was withdrawn from exponentially growing homogenous culture of alga and inoculated in 100 mL freshly prepared BG-11 medium containing different Ni2+ ranges (0, 2, 6, 10 and 12 mg L 1). After 240 h of inoculation, 10 mL of algal sample was harvested from homogenous cultures and centrifuged (4000g, 15 min) and supernatant media were separated. The algal samples in the sediment were mixed individually with 10 mL of EDTA (10 lM) solution and gently shaken. Samples were once again centrifuged (4000g, 15 min). Supernatant EDTA was taken out for measuring the adsorbed ionic concentration. All three parts i.e. media, EDTA and algal pellets from each sample were dried, digested with double acid [HNO3: HClO4 mixture (10:1, v/v)] in boiling water bath for 1 h. After cooling, the samples were diluted to 10 mL with triple glass distilled water and analyzed for Ni2+ level by Atomic Absorption Spectrophotometer using Perkin Elmer model 373 AAS. Adsorbed here refers to extra-cellular Ni2+ (i.e., nickel not transported into the algal cells) which methodologically here chelates to EDTA, and that absorbed refers to intra-cellular Ni2+ (i.e., nickel transported into the interior of the cell), represented in method as algal pellets. 2.4. Other biochemical parameters Estimation of sugar: The soluble and insoluble sugars were estimated using the principle of hydroxymethyl furfural reaction with anthrone to produce green color (Plummer, 1971). Aqueous alcoholic soluble sugar part was considered as soluble and hydrolyzed part as insoluble (Plummer, 1971; Sadasivam and Manickam, 1992). Estimation of chlorophyll and carotenoids: Pigments were extracted in methanol and their relative amounts were calculated using equation (Mackinney, 1941): 13.42 · A665 = lg chlorophyll mL 200 · A420 = lg carotenoids mL 1
1
Sugar and pigment were estimated 240 h after the different ranges of Ni2+ treatment. All the experiments were triplicated and the results were statistically analyzed for variance (ANOVA) and cause effect relationship (Snedecor and Cochran, 1967). Growth rate experiment involved two factors (concentration of Ni2+ and period of growth) and performed as per strip-plot design, whereas in rest of the experiments concentration of
3932
Harish et al. / Bioresource Technology 99 (2008) 3930–3934
Ni2+ is the only factor, accordingly they were carried out following randomized block design.
The Ni2+ concentrations affected growth rate (Fig. 1). The tolerance limit (sub-lethal concentration of Ni2+) for C. hemicolum was observed to be 12 mg L 1 (level of growth kinetics was 46.76%). The effect of sub-lethal concentrations of Ni2+ on cultures appears to delay the onset of exponential phase. Such marked increase in lag phase of the growth has been manifested earlier also (Anand et al., 2006). The results of factorial analysis of variance suggested that Ni2+ toxicity to growth rate is a consequence of Ni2+ dosage, duration of exposure and their strong interaction (p > 0.01). Such results have been reported with cultures of Scenedesmus and Ankistrodesmus (Jin et al., 1996; Lin and Jiang, 2000). The results indicated that algal growth becomes oppressive as Ni2+ concentration increases, and ultimately inhibited at higher concentration (>12 mg L 1). Resistant cells that survive the lag phase are then able to enter the exponential phase of growth. Alternatively, the cells may release secondary metabolites which chelate the toxic ions (Gardea-Torresday et al., 1990) or resistance may be genetic, rather than physiological (Rubinelli et al., 2002). Further, resistance to one heavy metal may confer resistance to multiple metal ions as well (Okamura and Aoyama, 1994). In our other study, C. hemicolum exhibits tolerance to Cd2+ (up to 12 mg L 1) and Cu2+ (up to 7 mg L 1) as well (Harish et al., Unpublished).
accumulation factor (AF = 1.37) found at 6 mg L 1 Ni2+ treatment. Even at 10 mg L 1 Ni2+ treatment AF value was 1.32. AF curve shows parabolic path (AF = 0.587 + 0.524 X 0.084 X2; R2 = 0.94; X = Ni2+ concentration). The adsorptions (Y) were comparatively low and linearly related with Ni2+ concentration [Y = 0.04 + 0.1757 X; r2 = 0.89]. The cultures with higher Ni2+ concentrations (X) revealed a linear relationship of absorption [Y = 3.66 + 3.4823 X; r2 = 0.97; Fig. 2]. The variance analysis revealed that Ni2+ concentration significantly contributes to the levels of adsorption (F ratio = 169.01; p > 0.01) and absorption (F ratio = 1393.69; p > 0.01). However, the variations among the replicates were non significant. Exposure of algae to elevated concentrations of nickel may lead to intra-cellular accumulation of high concentrations of the metals (Suresh and Ravishankar, 2004). Comparatively higher level of absorption than adsorption in our study and its comparison with other such study suggests that different species may have different mechanism to deal with excess amount of heavy metals. Some may responds by absorption (intra-cellular) strategy (Axtell et al., 2003) while other may with adsorption (extra-cellular; Anand et al., 2006). Energetic advantage of both the mechanisms is questionable and needs further exploration, since absorption may have metabolic requirement for active intra-cellular transport of the heavy metal or even if it is passive, species may have energy requirement for its conjugation/detoxification (in active mechanism also; PeralesVela et al., 2006), while, on the hand, greater amount of adsorption of heavy metal may consume more energy for synthesis of higher amount of extra-cellular biomass that chelates to metals (Singh et al., 1999).
3.2. Absorption/adsorption kinetics
3.3. Other biochemical parameters
C. hemicolum is not only just a tolerant species but also found to be hyperaccumulator of Ni2+ with maximum
The different range of Ni2+ treatments decreased both insoluble and soluble sugar and variations were
3. Results and discussion
-1
Protein (μg ml )
3.1. Changes in growth rate as measured by protein value
450
Control
400
1 mg/l
350
2 mg/l
300
4 mg/l
250
6 mg/l 8 mg/l
200
10 mg/l
150
12 mg/l
100
14 mg/l
50
16 mg/l
0
18 mg/l 0
96
144
192
240
288
336
384
432
480
20 mg/l
Elapsed time ( h) Fig. 1. Effect of Ni2+ on growth rate as measured by protein content in Chlorococcum hemicolum.
Harish et al. / Bioresource Technology 99 (2008) 3930–3934
3933
Ni conc. (mg 1-1)
14 12
Medium
10
Adsorbed
8
Absorbed
6 4 2 0 control
2
6 Treatment (mg 1-1)
10
12
Fig. 2. Nickel absorption/adsorption kinetics of Chlorococcum hemicolum.
Table 1 Level of sugar and pigment in Chlorococcum hemicolum due to nickel treatment Ni2+ Concentrations (mg L 1)
Sugar (mg mL 1)
Pigment (lg mL 1)
Insoluble
Soluble
Chlorophyll
Carotenoids
0 (control) 2 6 10 12
0.067 ± 0.0009 0.042 ± 0.0014 0.022 ± 0.0011 0.009 ± 0.0007 0.0002 ± 0.0005
0.020 ± 0.0001 0.013 ± 0.0008 0.011 ± 0.0001 0.007 ± 0.0005 0.004 ± 0.0003
14.977 ± 1.07 13.192 ± 0.251 4.133 ± 0.071 1.396 ± 0.097 0.573 ± 0.129
191 ± 2.227 226.8 ± 12.573 67.533 ± 5.672 25.4 ± 0.346 15.267 ± 1.617
Critical difference (at 0.05 level)
7.22E 03
3.70E 03
significantly (F = 2368.83; p > 0.01 and 443.31; p > 0.01, respectively) contributed by Ni2+ treatment only (Table 1). Insoluble sugar was found to be more than soluble sugar. Our results are in conformity of earlier trend observed for such study (Fathi et al., 2005) and once again establish the fact that physiology of the species is significantly affected by higher Ni2+ concentrations. Chlorophyll and carotenoid content reduced significantly with increase in Ni2+ concentration (F = 650.1; p > 0.01 and 658.03; p > 0.01, respectively; Table 1). Concentration dependent reduction of pigment was similar to those obtained earlier (Manankina et al., 2003). 4. Conclusion Our results revealed greater tolerance of chlorophycean alga C. hemicolum to the nickel as compared to other known algae and/or cyanobacteria. The high hyperaccumulation capability of alga for Ni2+ opens henceforth unexplored domain for research to exploit the hard genome of desert area for bioremediation. Our results showed reduced level of total sugar, chlorophyll and carotenoids, confirming significant changes occurred in physiology of algae due to heavy metal stress. Acknowledgements We are grateful to the Professor and Head, Department of Botany and to Director, Defence laboratory, Jodhpur
6.44E 02
6.20E 02
for extending necessary facilities. The financial assistance from CSIR, New Delhi in form of fellowship (F.NO. 9/ 98(53)/2002-EMR-1) to Harish is acknowledged. References Abdallah, A.M.A., Abdallah, M.A., Beltagy, A., Siam, E., 2006. Contents of heavy metals in marine algae from Egyptian Red Sea coast. Toxicol. Environ. Chem. 88, 9–22. Ahluwalia, S.S., Goyal, D., 2007. Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresource Technol. 98, 2243–2257. Anand, P., Isar, J., Saran, S., Saxena, R.K., 2006. Bioaccumulation of copper by Trichoderma viride. Bioresource Technol. 97, 1018– 1025. Assunc¸a˜o, A.G.L., Schat, H., Aarts, M.G.M., 2003. Thlaspi caerulescens, an attractive model species to study heavy metal hyperaccumulation in plants. New Phytol. 159, 351–360. Axtell, N.R., Sternberg, S.P.K., Claussen, K., 2003. Lead and nickel removal using Microspora and Lemnaminor. Bioresource Technol. 89, 41–48. Brooks, R.R., Lee, J., Reeves, R.D., Jaffre´, T., 1977. Detection of nickeliferous rocks by analysis of herbarium specimens of indicator plants. J. Geochem. Exp. 7, 49–57. Can, M.V., Kaya, Y., Algur, O.F., 2006. Response surface optimization of the removal of nickel from aqueous solution by cone biomass of Pinus sylvestris. Bioresource Technol. 97, 1761–1765. Chmielewska, E., Medved, J., 2001. Bioaccumulation of heavy metals by green algae Cladophora glomerata in a refinery sewage lagoon. Croat. Chem. Acta 74, 135–145. Fathi, A.A., Zaki, F.T., Ibraheim, H.A., 2005. Response of tolerant and wild type strains of Chlorella vulgaris to copper with special reference to copper uptake system. Protistology. 4, 73–78.
3934
Harish et al. / Bioresource Technology 99 (2008) 3930–3934
Gardea-Torresday, J.L., Becker-Hapak, M.K., Hosea, J.M., Dranall, D.W., 1990. Effect of chemical modification of algal carboxyl groups on metal ion binding. Environ. Sci. Technol. 24, 1372– 1378. Herbert, D., Phipps, P.J., Strange, R.E., 1971. The chemical analysis of microbial cells. In: Norris, J.R., Ribbons, D.W. (Eds.), Methods in Microbiology. London Academic Press, London, pp. 209–344. Iljin, V., 1991. Heavy metals in the plant-soil system. Publishing House Nauka, Novosibirsk, Russia. Jin, X., Nalewajko, C., Kushner, D.J., 1996. Comparative study of nickel toxicity to growth and photosynthesis in nickel resistant and sensitive strains of Scenedesmus acutus f. alternans. (Chlorophyceae). Micro. Eco. 31, 103–114. Lin, B., Jiang, B., 2000. Adsorption and absorption of nickel by Ankistrodesmus sp. and effect of nickel on morphological structure of its cell. Chi. J. Environ. Biol. 6, 535–541. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin reagent. J. Biol. Chem. 193, 265–275. Mackinney, G., 1941. Absorption of light by chlorophyll solution. J. Biol. Chem. 140, 315–322. Manankina, E.E., Mel’nikov, S.S., Budakova, E.A., Shalygo, N.V., 2003. Effect of Ni2+ on early stages of chlorophyll biosynthesis and pheophytinization in Euglena gracilis. Russ. J. Plant Physiol. 50, 390–394. Marschner, H., 1995. Mineral nutrition of higher plants. Academic Press, London. Muyssen, B.T.A., Brix, K.V., DeForest, D.K., Janssen, C.R., 2004. Nickel essentiality and homeostasis in aquatic organisms. Environ. Rev. 12, 113–131.
Okamura, H., Aoyama, I., 1994. Interactive toxic effect and distribution of heavy metals in phytoplankton. Environ. Toxicol. Water Qual. 9, 7– 15. Perales-Vela, H.V., Penˇa-Castro, J.M., Canˇizares-Villanueva, R.O., 2006. Heavy metal detoxification in eukaryotic microalgae. Chemosphere 64, 1–10. Plummer, D.T., 1971. An introduction to practical biochemistry. Tata McGraw-Hill Publishing Co. Ltd., New Delhi. Rai, L.C., Mallick, N., 1993. Heavy metal toxicity to algae under synthetic microcosm. Ecotoxicology. 2, 231–242. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stainer, R., 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1–61. Rubinelli, P., Siripornadulsil, S., Gao-Rubinelli, F., Sayre, R.T., 2002. Cadmium and iron stress inducible gene expression in the green alga Chlamydomonas reinhardtii: evidence for H43 protein function in iron assimilation. Planta. 215, 1–13. Sadasivam, S., Manickam, A., 1992. Biochemical methods for agricultural sciences. Wiley Eastern Ltd, New Delhi. Singh, N., Asthana, R.K., Kayastha, A.M., Pandey, S., Chaudhary, A.K., Singh, S.P., 1999. Thiol and exopolysaccharide production in a cyanobacterium under heavy metal stress. Process Biochem. 35, 63–68. Snedecor, G.W., Cochran, W.G., 1967. Statistical methods. Oxford & IBH Publishing Co. Ltd., New Delhi. Suresh, B., Ravishankar, G.A., 2004. Phytoremediation – a novel and promising approach for environmental clean-up. Crit. Rev. Biotechnol. 24, 97–124.
Bioresource Technology 99 (2008) 3930–3934
Short Communication
A new chlorophycean nickel hyperaccumulator Harish a, S. Sundaramoorthy a b
a,*
, Devendra Kumar b, S.G. Vaijapurkar
b
Department of Botany, JNV University, Jodhpur 342 001, India Defence Research Laboratory, DRDO, Jodhpur 342 001, India
Received 4 May 2007; received in revised form 4 July 2007; accepted 4 July 2007 Available online 10 September 2007
Abstract Bioremediation of nickel by chlorophycean bioremediator, Chlorococcum hemicolum was investigated. The growth rates at various concentrations of Ni2+ were assessed in terms of protein level and 12 mg L 1 of the Ni2+ is the tolerance limit (46.76% level of growth kinetics). Absorption/adsorption kinetics was estimated after 240 h of Ni2+ treatments. Absorptions were higher than adsorption with maximum accumulation factor (AF) of 1.37. Ni2+ concentration and absorption were linearly related (r = 0.98; p > 0.01). Other biochemical parameters like total sugar, chlorophyll and carotenoids were also quantified to correlate the state of metabolism and these exhibited reduction due to heavy metal stress. 2007 Elsevier Ltd. All rights reserved. Keywords: Bioremediation; Chlorococcum; Hyperaccumulator; Nickel
1. Introduction Discharge of heavy metals from metal processing industries is known to have adverse effects on the environment (Ahluwalia and Goyal, 2007). Though, some of these metals (Mg, Fe, Mn, Zn, Mo etc.) are essential for various physiological functions and are critical in many of the enzymatic and metabolic reactions occurring within an organism, but many (Cd, Cr, Pb, Co, Ag, Se etc.) have no known biological function. The essentiality of nickel is now generally acknowledged, based on the numerous symptoms caused by nickel deficiency (mainly in terrestrial vertebrates) and its role in various enzymes in bacteria and plants. The information on optimal and deficient concentrations of nickel is limited; and it can only be confirmed for plants and cyanobacteria for its role in the urease and hydrogenase metabolism. Deficiency levels ranged from 10 12 M to 2 · 10 6 M Ni2+ (external; in water of in vitro studies) for different species (Muyssen et al., 2004). None-
*
Corresponding author. Tel.: +91 291 2722081; fax: +91 291 2649465. E-mail address: jnvusundar@rediffmail.com (S. Sundaramoorthy).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.07.043
theless, excess of heavy metal is toxic to most plants and it is now for more than a decade since the significance of Ni2+ as a very serious pollutant were recognized (Iljin, 1991). It is known to be highly toxic to a wide range of algae (Rai and Mallick, 1993). In recent times, a number of attempts have been made to explore the ability of the plant to accumulate the heavy metal (hyperaccumulation) for remediation of the polluted water. Brooks et al. (1977) first coined the term ‘hyperaccumulator’ to define plants with Ni2+ concentrations higher than 1000 lg g 1 DW (0.1%). This value was not chosen arbitrarily. Ni2+ is a plant micronutrient and is found in the vegetative organs of most plants in the range of 1–10 lg g 1 DW (Assunc¸a˜o et al., 2003). Toxicity occurs at concentrations higher than 10–50 lg g 1 DW (Marschner, 1995). The ability of algae to remove heavy metals from aqueous solution has been known for some decades. Chmielewska and Medved (2001) confirmed the high Ni2+ bioaccumulation ability of Cladophora glomerata. Likewise, bioremediation potentiality of Microspora was also assessed (Axtell et al., 2003). The algae of chronically metal-contaminated localities tend to accumulate heavy
Harish et al. / Bioresource Technology 99 (2008) 3930–3934
metals to a dangerous extent. The metal content of algae can be used to predict the level of metal pollution in a water body (Abdallah et al., 2006). The high accumulation capacity can even be used for the enrichment or recycling of valuable metals (Can et al., 2006). Their relative comparison is generally made with the help of accumulation factor (AF). Metal accumulation factor (AF) is defined as the ratio of metal concentration in plant cell (micrograms per gram) and the metal concentration in water (micrograms per milliliter) and also known as a bioconcentration ratio, concentration ratio and enrichment ratio. Isolation of algal ecotype from local stream, containing polluted industrial effluent, that can tolerate high metal alloy, is essential for efficient functioning of bioremediation system in that particular environment. Dye of fabrics and metal alloy production are the major industrial activities in Boranada industrial area, Jodhpur releasing effluent that flow into the local Jojri River. The study region (which comes under the area of Indian Thar Desert) is classified as semi arid. Consequently, the algal flora have considerable ecological importance, even if they occur in river micro-habitat. Hence, the aim of this study was to explore the biotic potential of local chlorophycean alga Chlorococcum hemicolum for Ni2+ bioremediation. Further changes in protein, sugar, chlorophyll and carotenoids were also assessed.
2. Methods 2.1. Experimental organism, growth and experimental conditions C. hemicolum, a local alga, was collected from Jojri River (pH 6.2–6.5). This alga occurred at various locations within this stream. All the industrial effluent of nearby Boranada Industrial area, Jodhpur, flow into this stream. Axenic culture of this alga was multiplied in BG-11 medium (Rippka et al., 1979) and grown in culture room under continuous light, illuminated with cool fluorescent tubes (14.4 W m 2) at 24 ± 1 C. All the experiments were conducted in triplicate at same culture conditions. The cultures contains glass beads (five in number in each culture flask with 0.5 mm size each) to prevent clumping of cells in growing algal mass, and were shaken gently everyday. 2.2. Changes in growth rate One milliliter of algal cells with protein value 100 lg mL 1 were withdrawn from exponentially growing homogenous culture of alga and inoculated in 100 mL freshly prepared BG-11 medium containing different ranges (0– 20 mg L 1) of Ni2+. Source for Ni2+ in medium was stock solution prepared with NiSO4Æ6H2O. Changes in protein
3931
content were measured by the method of Lowry et al. (1951) as modified by Herbert et al. (1971) using lysozyme (Sigma) as the standard. Protein content was measured after every 48 h, up to 480 h, starting from 96 h (4th day) from these newly established cultures. 2.3. Absorption/adsorption kinetics One milliliter of algal cells with protein value 100 lg mL 1 was withdrawn from exponentially growing homogenous culture of alga and inoculated in 100 mL freshly prepared BG-11 medium containing different Ni2+ ranges (0, 2, 6, 10 and 12 mg L 1). After 240 h of inoculation, 10 mL of algal sample was harvested from homogenous cultures and centrifuged (4000g, 15 min) and supernatant media were separated. The algal samples in the sediment were mixed individually with 10 mL of EDTA (10 lM) solution and gently shaken. Samples were once again centrifuged (4000g, 15 min). Supernatant EDTA was taken out for measuring the adsorbed ionic concentration. All three parts i.e. media, EDTA and algal pellets from each sample were dried, digested with double acid [HNO3: HClO4 mixture (10:1, v/v)] in boiling water bath for 1 h. After cooling, the samples were diluted to 10 mL with triple glass distilled water and analyzed for Ni2+ level by Atomic Absorption Spectrophotometer using Perkin Elmer model 373 AAS. Adsorbed here refers to extra-cellular Ni2+ (i.e., nickel not transported into the algal cells) which methodologically here chelates to EDTA, and that absorbed refers to intra-cellular Ni2+ (i.e., nickel transported into the interior of the cell), represented in method as algal pellets. 2.4. Other biochemical parameters Estimation of sugar: The soluble and insoluble sugars were estimated using the principle of hydroxymethyl furfural reaction with anthrone to produce green color (Plummer, 1971). Aqueous alcoholic soluble sugar part was considered as soluble and hydrolyzed part as insoluble (Plummer, 1971; Sadasivam and Manickam, 1992). Estimation of chlorophyll and carotenoids: Pigments were extracted in methanol and their relative amounts were calculated using equation (Mackinney, 1941): 13.42 · A665 = lg chlorophyll mL 200 · A420 = lg carotenoids mL 1
1
Sugar and pigment were estimated 240 h after the different ranges of Ni2+ treatment. All the experiments were triplicated and the results were statistically analyzed for variance (ANOVA) and cause effect relationship (Snedecor and Cochran, 1967). Growth rate experiment involved two factors (concentration of Ni2+ and period of growth) and performed as per strip-plot design, whereas in rest of the experiments concentration of
3932
Harish et al. / Bioresource Technology 99 (2008) 3930–3934
Ni2+ is the only factor, accordingly they were carried out following randomized block design.
The Ni2+ concentrations affected growth rate (Fig. 1). The tolerance limit (sub-lethal concentration of Ni2+) for C. hemicolum was observed to be 12 mg L 1 (level of growth kinetics was 46.76%). The effect of sub-lethal concentrations of Ni2+ on cultures appears to delay the onset of exponential phase. Such marked increase in lag phase of the growth has been manifested earlier also (Anand et al., 2006). The results of factorial analysis of variance suggested that Ni2+ toxicity to growth rate is a consequence of Ni2+ dosage, duration of exposure and their strong interaction (p > 0.01). Such results have been reported with cultures of Scenedesmus and Ankistrodesmus (Jin et al., 1996; Lin and Jiang, 2000). The results indicated that algal growth becomes oppressive as Ni2+ concentration increases, and ultimately inhibited at higher concentration (>12 mg L 1). Resistant cells that survive the lag phase are then able to enter the exponential phase of growth. Alternatively, the cells may release secondary metabolites which chelate the toxic ions (Gardea-Torresday et al., 1990) or resistance may be genetic, rather than physiological (Rubinelli et al., 2002). Further, resistance to one heavy metal may confer resistance to multiple metal ions as well (Okamura and Aoyama, 1994). In our other study, C. hemicolum exhibits tolerance to Cd2+ (up to 12 mg L 1) and Cu2+ (up to 7 mg L 1) as well (Harish et al., Unpublished).
accumulation factor (AF = 1.37) found at 6 mg L 1 Ni2+ treatment. Even at 10 mg L 1 Ni2+ treatment AF value was 1.32. AF curve shows parabolic path (AF = 0.587 + 0.524 X 0.084 X2; R2 = 0.94; X = Ni2+ concentration). The adsorptions (Y) were comparatively low and linearly related with Ni2+ concentration [Y = 0.04 + 0.1757 X; r2 = 0.89]. The cultures with higher Ni2+ concentrations (X) revealed a linear relationship of absorption [Y = 3.66 + 3.4823 X; r2 = 0.97; Fig. 2]. The variance analysis revealed that Ni2+ concentration significantly contributes to the levels of adsorption (F ratio = 169.01; p > 0.01) and absorption (F ratio = 1393.69; p > 0.01). However, the variations among the replicates were non significant. Exposure of algae to elevated concentrations of nickel may lead to intra-cellular accumulation of high concentrations of the metals (Suresh and Ravishankar, 2004). Comparatively higher level of absorption than adsorption in our study and its comparison with other such study suggests that different species may have different mechanism to deal with excess amount of heavy metals. Some may responds by absorption (intra-cellular) strategy (Axtell et al., 2003) while other may with adsorption (extra-cellular; Anand et al., 2006). Energetic advantage of both the mechanisms is questionable and needs further exploration, since absorption may have metabolic requirement for active intra-cellular transport of the heavy metal or even if it is passive, species may have energy requirement for its conjugation/detoxification (in active mechanism also; PeralesVela et al., 2006), while, on the hand, greater amount of adsorption of heavy metal may consume more energy for synthesis of higher amount of extra-cellular biomass that chelates to metals (Singh et al., 1999).
3.2. Absorption/adsorption kinetics
3.3. Other biochemical parameters
C. hemicolum is not only just a tolerant species but also found to be hyperaccumulator of Ni2+ with maximum
The different range of Ni2+ treatments decreased both insoluble and soluble sugar and variations were
3. Results and discussion
-1
Protein (μg ml )
3.1. Changes in growth rate as measured by protein value
450
Control
400
1 mg/l
350
2 mg/l
300
4 mg/l
250
6 mg/l 8 mg/l
200
10 mg/l
150
12 mg/l
100
14 mg/l
50
16 mg/l
0
18 mg/l 0
96
144
192
240
288
336
384
432
480
20 mg/l
Elapsed time ( h) Fig. 1. Effect of Ni2+ on growth rate as measured by protein content in Chlorococcum hemicolum.
Harish et al. / Bioresource Technology 99 (2008) 3930–3934
3933
Ni conc. (mg 1-1)
14 12
Medium
10
Adsorbed
8
Absorbed
6 4 2 0 control
2
6 Treatment (mg 1-1)
10
12
Fig. 2. Nickel absorption/adsorption kinetics of Chlorococcum hemicolum.
Table 1 Level of sugar and pigment in Chlorococcum hemicolum due to nickel treatment Ni2+ Concentrations (mg L 1)
Sugar (mg mL 1)
Pigment (lg mL 1)
Insoluble
Soluble
Chlorophyll
Carotenoids
0 (control) 2 6 10 12
0.067 ± 0.0009 0.042 ± 0.0014 0.022 ± 0.0011 0.009 ± 0.0007 0.0002 ± 0.0005
0.020 ± 0.0001 0.013 ± 0.0008 0.011 ± 0.0001 0.007 ± 0.0005 0.004 ± 0.0003
14.977 ± 1.07 13.192 ± 0.251 4.133 ± 0.071 1.396 ± 0.097 0.573 ± 0.129
191 ± 2.227 226.8 ± 12.573 67.533 ± 5.672 25.4 ± 0.346 15.267 ± 1.617
Critical difference (at 0.05 level)
7.22E 03
3.70E 03
significantly (F = 2368.83; p > 0.01 and 443.31; p > 0.01, respectively) contributed by Ni2+ treatment only (Table 1). Insoluble sugar was found to be more than soluble sugar. Our results are in conformity of earlier trend observed for such study (Fathi et al., 2005) and once again establish the fact that physiology of the species is significantly affected by higher Ni2+ concentrations. Chlorophyll and carotenoid content reduced significantly with increase in Ni2+ concentration (F = 650.1; p > 0.01 and 658.03; p > 0.01, respectively; Table 1). Concentration dependent reduction of pigment was similar to those obtained earlier (Manankina et al., 2003). 4. Conclusion Our results revealed greater tolerance of chlorophycean alga C. hemicolum to the nickel as compared to other known algae and/or cyanobacteria. The high hyperaccumulation capability of alga for Ni2+ opens henceforth unexplored domain for research to exploit the hard genome of desert area for bioremediation. Our results showed reduced level of total sugar, chlorophyll and carotenoids, confirming significant changes occurred in physiology of algae due to heavy metal stress. Acknowledgements We are grateful to the Professor and Head, Department of Botany and to Director, Defence laboratory, Jodhpur
6.44E 02
6.20E 02
for extending necessary facilities. The financial assistance from CSIR, New Delhi in form of fellowship (F.NO. 9/ 98(53)/2002-EMR-1) to Harish is acknowledged. References Abdallah, A.M.A., Abdallah, M.A., Beltagy, A., Siam, E., 2006. Contents of heavy metals in marine algae from Egyptian Red Sea coast. Toxicol. Environ. Chem. 88, 9–22. Ahluwalia, S.S., Goyal, D., 2007. Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresource Technol. 98, 2243–2257. Anand, P., Isar, J., Saran, S., Saxena, R.K., 2006. Bioaccumulation of copper by Trichoderma viride. Bioresource Technol. 97, 1018– 1025. Assunc¸a˜o, A.G.L., Schat, H., Aarts, M.G.M., 2003. Thlaspi caerulescens, an attractive model species to study heavy metal hyperaccumulation in plants. New Phytol. 159, 351–360. Axtell, N.R., Sternberg, S.P.K., Claussen, K., 2003. Lead and nickel removal using Microspora and Lemnaminor. Bioresource Technol. 89, 41–48. Brooks, R.R., Lee, J., Reeves, R.D., Jaffre´, T., 1977. Detection of nickeliferous rocks by analysis of herbarium specimens of indicator plants. J. Geochem. Exp. 7, 49–57. Can, M.V., Kaya, Y., Algur, O.F., 2006. Response surface optimization of the removal of nickel from aqueous solution by cone biomass of Pinus sylvestris. Bioresource Technol. 97, 1761–1765. Chmielewska, E., Medved, J., 2001. Bioaccumulation of heavy metals by green algae Cladophora glomerata in a refinery sewage lagoon. Croat. Chem. Acta 74, 135–145. Fathi, A.A., Zaki, F.T., Ibraheim, H.A., 2005. Response of tolerant and wild type strains of Chlorella vulgaris to copper with special reference to copper uptake system. Protistology. 4, 73–78.
3934
Harish et al. / Bioresource Technology 99 (2008) 3930–3934
Gardea-Torresday, J.L., Becker-Hapak, M.K., Hosea, J.M., Dranall, D.W., 1990. Effect of chemical modification of algal carboxyl groups on metal ion binding. Environ. Sci. Technol. 24, 1372– 1378. Herbert, D., Phipps, P.J., Strange, R.E., 1971. The chemical analysis of microbial cells. In: Norris, J.R., Ribbons, D.W. (Eds.), Methods in Microbiology. London Academic Press, London, pp. 209–344. Iljin, V., 1991. Heavy metals in the plant-soil system. Publishing House Nauka, Novosibirsk, Russia. Jin, X., Nalewajko, C., Kushner, D.J., 1996. Comparative study of nickel toxicity to growth and photosynthesis in nickel resistant and sensitive strains of Scenedesmus acutus f. alternans. (Chlorophyceae). Micro. Eco. 31, 103–114. Lin, B., Jiang, B., 2000. Adsorption and absorption of nickel by Ankistrodesmus sp. and effect of nickel on morphological structure of its cell. Chi. J. Environ. Biol. 6, 535–541. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin reagent. J. Biol. Chem. 193, 265–275. Mackinney, G., 1941. Absorption of light by chlorophyll solution. J. Biol. Chem. 140, 315–322. Manankina, E.E., Mel’nikov, S.S., Budakova, E.A., Shalygo, N.V., 2003. Effect of Ni2+ on early stages of chlorophyll biosynthesis and pheophytinization in Euglena gracilis. Russ. J. Plant Physiol. 50, 390–394. Marschner, H., 1995. Mineral nutrition of higher plants. Academic Press, London. Muyssen, B.T.A., Brix, K.V., DeForest, D.K., Janssen, C.R., 2004. Nickel essentiality and homeostasis in aquatic organisms. Environ. Rev. 12, 113–131.
Okamura, H., Aoyama, I., 1994. Interactive toxic effect and distribution of heavy metals in phytoplankton. Environ. Toxicol. Water Qual. 9, 7– 15. Perales-Vela, H.V., Penˇa-Castro, J.M., Canˇizares-Villanueva, R.O., 2006. Heavy metal detoxification in eukaryotic microalgae. Chemosphere 64, 1–10. Plummer, D.T., 1971. An introduction to practical biochemistry. Tata McGraw-Hill Publishing Co. Ltd., New Delhi. Rai, L.C., Mallick, N., 1993. Heavy metal toxicity to algae under synthetic microcosm. Ecotoxicology. 2, 231–242. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stainer, R., 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1–61. Rubinelli, P., Siripornadulsil, S., Gao-Rubinelli, F., Sayre, R.T., 2002. Cadmium and iron stress inducible gene expression in the green alga Chlamydomonas reinhardtii: evidence for H43 protein function in iron assimilation. Planta. 215, 1–13. Sadasivam, S., Manickam, A., 1992. Biochemical methods for agricultural sciences. Wiley Eastern Ltd, New Delhi. Singh, N., Asthana, R.K., Kayastha, A.M., Pandey, S., Chaudhary, A.K., Singh, S.P., 1999. Thiol and exopolysaccharide production in a cyanobacterium under heavy metal stress. Process Biochem. 35, 63–68. Snedecor, G.W., Cochran, W.G., 1967. Statistical methods. Oxford & IBH Publishing Co. Ltd., New Delhi. Suresh, B., Ravishankar, G.A., 2004. Phytoremediation – a novel and promising approach for environmental clean-up. Crit. Rev. Biotechnol. 24, 97–124.