Uptake of heavy metals byStylonychia mytilusand its possible use in ...

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The heavy metal resistant ciliate, Stylonychia mytilus, isolated from industrial wastewater has been shown to be potential bioremediator of contaminated ...

World J Microbiol Biotechnol (2008) 24:47–53 DOI 10.1007/s11274-007-9436-1

ORIGINAL PAPER

Uptake of heavy metals by Stylonychia mytilus and its possible use in decontamination of industrial wastewater A. Rehman Æ Farah R. Shakoori Æ A. R. Shakoori

Received: 29 January 2007 / Accepted: 10 May 2007 / Published online: 3 June 2007  Springer Science+Business Media B.V. 2007

Abstract The heavy metal resistant ciliate, Stylonychia mytilus, isolated from industrial wastewater has been shown to be potential bioremediator of contaminated wastewater. The ciliate showed tolerance against Zn2+ (30 lg/mL), Hg2+ (16 lg/mL) and Ni2+ (16 lg/mL). The metal ions slowed down the growth of the ciliate as compared with the culture grown without metal stress. The reduction in cell population was 46% for Cd2+, 38% for Hg2+, 23% for Zn2+, 39% for Cu2+ and 51% for Ni2+ after 8 days of metal stress. S. mytilus reduced 91% of Cd2+, 90% of Hg2+ and 98% of Zn2+ from the medium after 96 h of incubation in a culture medium containing 10 lg/mL of the respective metal ions. Besides this, the ciliate could also remove 88% of Cu2+ and 73% Ni2+ from the medium containing 5 lg/mL of each metal after 96 h. The ability of Stylonychia to take up variety of heavy metals from the medium could be exploited for metal detoxification and environmental clean-up operations. Keywords Heavy metal resistance  Heavy metal uptake  Bioremediation  Stylonychia mytilus  Heavy metal resistant ciliate

A. Rehman Department of Microbiology and Molecular Genetics, University of the Punjab, New Campus, Lahore 54590, Pakistan A. R. Shakoori (&) School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan e-mail: [email protected] F. R. Shakoori Department of Zoology, Government College University, Lahore, Pakistan

Introduction The presence of toxic heavy metal contaminants in aqueous streams, arising from the discharge of untreated metal containing effluents into water bodies, is one of the most important environmental issues. Metal pollutants can easily enter the food chain if heavy metal-contaminated soils are used for production of food crops. Farm productivity has been decreased in toxic metal polluted areas (Gosavi et al. 2004; Principi et al. 2006). Cadmium is one of the most dangerous heavy metals both to human health and aquatic ecosystems. Cd is carcinogenic, embryotoxic, teratogenic and mutagenic and may cause hyperglycemia, reduced immunopotency and anaemia, due to its interference with iron metabolism (Sanders 1986). The toxicity of Cd has also been well documented in selective types of almost all major phyla of eukaryotes (Unger and Roesijadi 1996; Coeurdassier et al. 2004). Copper is rarely found in natural water but is found in man-polluted environments (Udom et al. 2004). Any copper present normally originates from industrial effluents, seepage, water from refuse dumps, pesticides or corrosive water that has come into contact with fitting and pipes containing copper. Copper ions inhibit macromolecules synthesis and other enzymatic reactions (Company et al. 2004). Mercury is a unique element that has no essential biological function. Compounds of mercury such as mercuric chloride and organomercurials are toxic to both eukaryotic and prokaryotic cells. These compounds can pass through biological membranes (Gutknecht 1981) and bind with high affinity to thiol (SH) groups in proteins, thus causing damage to membranes and inactivating enzymes. Mercury is also genotoxic; inorganic Hg (II) is capable of strong reversible interactions with the nitrogens in purines and

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pyrimidines, and organic mercury compounds, e.g. methylmercury, also produce irreversible damage to nucleic acids (Sletten and Nerdal 1997). Nickel is a problematic heavy metal (Joho et al. 1995). Higher concentrations of nickel are toxic (Dalton et al. 1985). A significant concentration of nickel is present in industrial and municipal discharges (3.8 · 106 kg/year), particularly in steel mill and electroplating wastes. The nickel burden of the world’s fresh waters is about 3.4 · 107 kg, and rivers transport 1.35 · 109 kg/year of this metal (Nriagu 1980). Nickel compounds are found to be nephrotoxic, hepatotoxic, immunotoxic and teratogenic (Ross 1995). Nickel sub-sulphide, a component of nickel refinery dust, can cause lung, throat and nasal cancer (Liesegang et al. 1993). Zinc is a major inorganic pollutant of water, which has shown inhibitory and promotory effects on the growth along with accumulation in plants (Kumar 1989). Seedling growth and enzymes activities have been found inhibited by zinc in Phaseolus aureus cv. R-851 (Veer 1989). Some reports indicate that Zn may inhibit apoptosis (Zalewski et al. 1991), others suggest that Zn actually induces apoptotic cell death (Haase et al. 2001; Iitaka et al. 2001) and propose Zn as a potential cytotoxic agent in treatment of thyroid cancer (Iitaka et al. 2001). The heavy metals have also been reported to affect aquatic life inhabiting industrial wastewater, tolerating high concentrations of toxic metals in the field as well as in the laboratory conditions. Madoni et al. (1992) described the sensitivities of the seven ciliate species to cadmium. The 24-h LC50 values of Cd ranged from 180 lg/L (Paramecium caudatum) to 2,650 lg/L (Euplotes patella). Shakoori et al. (2004) found that the ciliary movements decreased in the presence of CdCl2 and the cell number was reduced from 1,085 to 690 cells/mL. The LC50 values of copper were 1.75 and 3.51 lg/L for Drepanomonas revolute and Spirostomum teres, respectively. Blepharisma americanum showed a higher sensitivity to this metal, with a LC50 of 1.45 lg/L. The order of toxicity of metals to the ciliate species tested was generally: Cu > Hg > Cd > Pb > Cr > Zn (Madoni et al. 1994). Shakoori et al. (2004) found that the movement of Vorticella microstoma completely stopped in the presence of high concentrations of CuSO4. Twagilimana et al. (1998) reported 24-h LC50 value of mercury as 0.004 mg/L for S. teres. Gray and Ventilla (1971) reported that 14.8 lg Hg/L administered as mercuric chloride was 100% toxic to the marine ciliate, Cristigera sp. The LC50s values of mercury for Drepanomonas revoluta and S. teres were 5.37 and 5.94 lg/L, respectively (Madoni et al. 1994). Ruthven and Cairns Jr (1973) found that 1.36 mg Zn/L administered as zinc sulphate was lethal for Tetrahymena pyriformis, while other freshwater ciliates were killed with 1.2–24 mg Zn/L. Nickel was least toxic to a hypotrich E. patella, with the

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concentration range between 6 and 10 mg/L (24-h LC50 7.70 mg/L), but showed highest toxicity to S. teres with minimum and maximum values of 0.13 and 0.25 mg/L (24-h LC50, 0.17 mg/L) (Twagilimana et al. 1998). Conventional wastewater treatments such as chemical precipitation, lime coagulation, solvent extraction, ion exchange and adsorption have several disadvantages including high-energy requirements, incomplete metal removal, high-capital investment and running costs, and generation of toxic sludges (Ciba et al. 1999). Recently, microbial bioremediation has emerged as an alternative technique to such traditional chemical treatments (Brierley 1990). Microbial metal bioremediation is an efficient strategy due to its low cost, high efficiency and ecofriendly nature. Microbiological detoxification of polluted water is economical, safe and sustainable (Eccles 1995). The objective of this study was to evaluate the survival of Stylonychia mytilus in media containing heavy metals such as Cd2+, Hg2+, Zn2+, Cu2+ and Ni2+ and to determine the efficiency of uptake of these metals by the ciliate. This information could later be used for remediation of heavy metal contaminated wastewater. S. mytilus has already been reported to thrive in heavy metal contaminated industrial wastewater (Madoni et al. 1992, 1994; Shakoori et al. 2004; Martin-Gonzalez et al. 2006). A number of authors have already emphasized the role of protozoa in wastewater treatment plants (Fernandez-Leborans et al. 1998; Haq et al. 2000; Shakoori et al. 2004; Rehman et al. 2005, 2006). The resistance and bioaccumulation ability has made this organism a candidate for this study to clean low-strength metal polluted wastewater.

Materials and methods Culturing of Stylonychia mytilus Stylonychia mytilus was originally isolated from ponds receiving effluents from tanneries and other industrial units in a small town Kasur, close to Lahore, Pakistan. These ciliates have been adapted to tolerating increasing concentrations of several metals in these ponds. These isolates were brought to the laboratory, where they are in culture for the last 6 years now. This axenic culture of S. mytilus was used for the present study. One hundred millilitres of Bold-basal salt medium [NaNO3 (0.25 g/L), CaCl2  H2O (0.025 g/L), MgSO4  7H2O (0.075 g/L), K2HPO4 (0.075 g/L), KH2PO4 (0.175 g/L), NaCl (0.0025 g/L), EDTA (0.05 g/L), KOH (0.031 g/L), FeSO4  7H2O (0.04 g/L), H2SO4 (0.001 L/L), H3BO3 (0.01142 g/L), ZnSO4  7H2O (0.00881 g/L), MnCl2  4H2O (0.00144 g/L), MoO3 (0.00071 g/L), CuSO4  5H2O (0.00157 g/L) and Co(NO3)  6H2O (0.00049 g/L)], diluted 1:1,000 with distilled water, in 250 mL conical flask, was inoculated under

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aseptic conditions with 10 lL of inoculum containing 40– 50 ciliates. Glucose as carbon source was only added as 1 g/L in Bold-basal salt medium (Shakoori et al. 2004; Rehman et al. 2005, 2006). The cultures were maintained in the laboratory at room temperature (25–27 C). The pH of the medium was adjusted at 7.5. The growth of Stylonychia was observed in the cultures by counting the number of ciliates at regular intervals. Determination of growth curves The effect of different metal ions on growth of the culture was checked by counting the number of protozoan cells in the medium. The cells were grown in the salt medium, to which Cd2+, Hg2+, Zn2+, Cu2+ and Ni2+ ions were added at a concentration of 1 lg/mL/day for 8 days. At least three counts were taken every day to get a mean of every reading. The growth was compared with that of the control culture, which contained no added metal ions. The activity, shape and size of the protozoans were also noted. The size was measured with an ocular micrometer after restricting the movement of the ciliates by putting the culture in methylcellulose and staining with 1% neutral red.

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treated one but without the ciliates. The cultures were incubated for 4 days and from each medium (control and treated) 5 mL culture was taken out under sterile conditions after 0, 48, 72 and 96 h, respectively. The cultures were spun down at 350·g for 15 min and the supernatants were used for the estimation of Cd2+, Hg2+, Zn2+, Cu2+ and Ni2+ by atomic absorption spectrophotometer (Varian, Palo Alto, CA, USA) at wavelengths 228.8, 253.7, 213.9, 324.7 and 232.0 nm, respectively. The amount of metals in the supernatants was determined using standard curves. The percentage reduction in the amount of Cd2+, Hg2+, Zn2+, Cu2+ and Ni2+ in the medium was calculated. Statistical analysis All values are averages of three readings and have been shown as Mean ± SEM. For determining significance of differences between the control and the experimental, Student’s t-test was applied.

Results Effect of metal on the growth of ciliate

Determination of resistance to metal ions Resistance of Stylonychia to three metal ions, i.e. Hg2+, Zn2+ and Ni2+ was checked by addition of the respective metal salts viz., HgCl2, ZnSO4  7H2O and NiCl2 to Bold-basal salt medium. Metal ions were sterilized separately and added to the medium when the temperature of the salt medium was slightly Cu2+ > Cd2+ + > Ni2+ > Zn2+. The Stylonychia population was maximum on day 8 in control as well metal treated cultures, except for mercury containing medium, where it was achieved on day 7. The maximum number of cells in control culture were 2,008.33 ± 01.53 (n = 3) on day 8, whereas it was 1,079.66 ± 01.53 (n = 3; P < 0.001) in Cd2+, 1,380.00 ± 02.00 (n = 3; P < 0.01) in Hg2+, 1,541.66 ± 01.53 (n = 3; P < 0.01) in Zn2+, 1,229.66 ± 01.53 (n = 3; P < 0.01) in Cu2+ and 980.00 ± 01.00 (n = 3; P < 0.001) in Ni2+

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Fig. 1 Growth curves of Stylonychia mytilus in Cd , Hg , Zn , Cu2+ and Ni2+ containing medium. The cells were grown in the salt medium to which metal ions were added at a concentration of 1 lg/ mL/day for 8 days. Growth was assessed by counting number of cells every day using haemocytometer for 8 days. Mean of at least three readings were plotted at each time point. Control cultures did not contain any metal ions

containing media during this period. The growth rate of Stylonychia was slower in the presence of all metal ions as compared with the control.

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Figure 2 shows the removal of heavy metal ions from the medium by S. mytilus. The S. mytilus growing in medium containing cadmium (10 lg/mL) could reduce 75% of cadmium from the medium after 48 h, 84% after 72 h and 91% after 96 h. Likewise ciliate reduced 60% mercury (10 lg/mL) from the medium after 48 h, 80% after 72 h and 90% after 96 h. It could also decrease 90% of zinc (10 lg/mL) from the medium after 48 h, 94% after 72 h and 98% after 96 h. S. mytilus also removed 80% of copper

Conc entr ation ( ppm)

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Heavy metal tolerance Stylonychia mytilus was found to resist Zn2+ up to a concentration of 30 lg/mL. The Zn-resistant ciliate could also tolerate Hg2+and Ni2+ at the maximum concentrations of 16 lg/mL each, respectively. There was apparently no reduction in the size of S. mytilus cells. Movement, which is a vital sign of life, was taken as a parameter of effect on growth rate. The movements of the ciliate slowed down in the presence of ZnSO4 (30 lg/mL) but almost stopped in the HgCl2 (16 lg/mL) and NiCl2 (16 lg/mL).

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0

48 72 Time (Hours)

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Fig. 2 Uptake of Cd2+, Hg2+, Zn2+, Cu2+ and Ni2+ by Stylonychia mytilus growing in Cd2+, Hg2+, Zn2+, Cu2+ and Ni2+ containing medium. The cadmium, mercury and zinc ions were added at a concentration of 10 lg/mL in the medium containing ciliates, whereas copper and nickel ions were added at a concentration of 5 lg/mL. One hundred mL ciliate culture was inoculated with 10 lL inoculum containing 40–50 ciliates in a 250 mL conical flask. The control did not contain cells of the isolate. The cultures were incubated for 4 days, and 5 mL culture was taken out after 48, 72 and 96 h, spun down and heavy metal ion concentration estimated with Atomic Absorption Spectrophotometer

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(5 lg/mL) from the medium after 48 h, 84% after 72 h and 88% after 96 h. Likewise, ciliate decreased 49% nickel after 48 h, 61% after 72 h and 73% after 96 h from the medium containing Ni2+ at a concentration of 5 lg/mL (Fig. 2). Notwithstanding the original concentration of metal ions in the media, S. mytilus could remove 91% of Cd2+, 90% of Hg2+, 98% of Zn2+, 88% of Cu2+ and 73% of Ni2+ from the medium, 96 h after inoculation with the protozoans.

Discussion The heavy metals, in the present study, have significantly hampered the growth of the ciliate cells. When the cell populations of metal-treated cultures were compared with those of the corresponding control culture, it was observed that the cadmium-treated culture had 46% lesser cell population when compared with 8 days of control culture. In the presence of Hg2+ ions, this decrease was 38%, whereas for Zn2+ it was 23%, for Cu2+ it was 39% and for Ni2+, it was 51% as compared with control after 8 days of metal exposure. The order of resistance, in terms of reduction in the cellular population, was Zn2+ > Hg2+ > Cu2+ > Cd2+ > Ni2+. Metal resistant protozoa have been reported in wastewaters and metal-polluted environments (Shakoori et al. 2004; Rehman et al. 2005; Madoni and Romeo 2006). It is well recognized that microorganisms have a high affinity for metals and can accumulate both heavy and toxic metals by a variety of mechanisms (Harrison et al. 2006; Jeyasingh and Philip 2005; Pas et al. 2004). These have been used to remove metals from polluted industrial and domestic effluents on a large scale. Microorganisms have a high-surface area-to-volume ratio because of their small size and therefore provide a large contact area that can interact with metals in the surrounding environment (Ledin 2000). Shakoori et al. (2004) reported that V. microstoma showed remarkable ability to pick up heavy metal ions from the culture medium. The concentration of Zn2+ and Cr6+ was reduced 99 and 48% after 96 h, respectively. Mortuza et al. (2005) reported that Paramecium bursaria accumulated 1.72–15.5 pg Cr/cell in a time and concentration-dependent manner. In one of the investigations S. mytilus accumulated 5.4 pg Cr and 6.3 pg Pb/cell from the medium after 96 h of incubation (Unpublished data). These microorganisms actively contribute to the amelioration of the effluent quality, since the majority of them feed upon dispersed bacteria (Madoni 2000). Conventional methods consume high amounts of energy and large quantities of chemical reagents. It is well known that bioremediation of toxic pollutants has advantages over

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other techniques as it is cheap, non-destructive and contamination remains localized (Rise-Roberts 1998). Uptake of metals by living cells has become one of the most attractive means for bioremediation of industrial wastes and other metal polluted environments. Heavy metal uptake processes by biological cells are known under the general term of biosorption. These phenomena include both passive adsorption of heavy metals to the cell walls and metabolically mediated uptake by the cells (Gadd 1990). In one of the previous reports from this laboratory Euplotes mutabilis grown in the medium containing Cu2+ (5 lg/mL) has been reported to reduce 60% of copper from the medium after 48 h, 82% after 72 h and 95% after 96 h (Rehman et al. 2006). It could also reduce 67% Hg2+ after 48 h, 75% after 72 h and 82% after 96 h from the medium containing Hg2+ at a concentration of 10 lg/mL. Likewise, Stylonychia has also been reported to actively take up Pb2+ from the medium. The protozoan culture grown in medium containing lead (10 lg/mL) could reduce 80% of lead from the medium after 48 h, 82% after 72 h and 86% after 96 h, respectively (Rehman et al. 2005). In one of the study from this laboratory, the live S. mytilus could remove 88% (Pb2+) and 80% (Cr6+) from the medium after 96 h of incubation, whereas killed organisms could remove only negligible quantity of heavy metal from the medium (Unpublished data). In the present study S. mytilus could remove 91% (Cd2+), 90% (Hg2+), 98% (Zn2+), 88% (Cu2+) and 73% (Ni2+) from the medium after 96 h of incubation. This clearly indicates that the ciliates actively take up the heavy metals. Metal bioaccumulation has also been reported to be the main mechanism of resistance to heavy metals in ciliates by others (Martin-Gonzalez et al. 2006; Diaz et al. 2006). In recent years it has been reported from different laboratories that metallothionein (MT) in lower eukaryotes may play the role of general stress protein because of their potent metal-binding and redox capabilities. Different Mt-isoforms, cadmium-zinc MTs, Cu-ATPase pumps and Cu MT have been reported in a ciliate protozoan, Tetrahymena, which are important in heavy metal homoeostasis and detoxification (Boldrin et al. 2003; Piccini et al. 1994, 1999; Santovito et al. 2001; Shang et al. 2002; Nilsson 2003). In this study, we are reporting the multiple uptake potential of S. mytilus which is resistant to highly toxic metal ions and may be employed for metal detoxification operations.

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