Uptake of Metal Ions from Solution by Inactivated Cells of Cyanobacteria J.L
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UPTAKE OF METAL IONS FROM SOLUTION BY INACTIVATED CELLS OF CYANOBACTERIA J.L. Gardea-Torresdey1,*, J.L. Arenas2, R. Webb2, K.J. Tiemann1, and J.H. Gonzalez1, 1Department of Chemistry, The University of Texas at El Paso, El Paso, TX, 79968, Phone 915- 747-5359, FAX: 915-747-5748, and 2Department of Biological Sciences, The University of Texas at El Paso, El Paso, TX, 79968 ABSTRACT Some species of cyanobacteria have the ability to grow under stress conditions that would kill many other bacteria. Solutions of 0.25 mM Cu2+ were inoculated with the cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 with the idea that these cyanobacteria may develop certain defense mechanisms allowing their survival in such stressed environments. Transmission electron microscopy showed that Synechocystis sp. grown in 0.25 mM copper(II) developed a filamentous sheath around the cell wall, which could be responsible for binding copper ions on the cell surface. The presence of copper adsorbed by Synechocystis sp. was corroborated using environmental scanning electron microscopy equipped with an x-ray energy-dispersive spectrometer. Synechococcus has the ability to grow in mass quantity under ideal conditions, providing usable biomass at a minimal effort. Using lyophilized biomass grown under normal conditions, Synechococcus was tested for its potential to bind copper(II), lead(II), and nickel(II) ions from solution. Batch experiments were performed to determine the optimum binding pH, time dependency, and metal binding capacities for copper(II), lead(II), and nickel(II), along with desorption of the metal bound. The biomass studied showed a high affinity for all metal ions as the pH increased from two to six with optimum binding occurring at pH 5. Time dependency studies showed that this cyanobacterium had rapid binding to all three metals. Capacity experiments showed that this cyanobacterium bound 11.3 mg of copper(II) per gram of biomass, 30.4 mg of lead(II) per gram of biomass, and 3.2 mg of nickel(II) per gram of biomass. More than 98% of the copper(II), lead(II) and nickel(II) adsorbed by the biomass was recovered when treated with 0.1M HCl. Cyanobacterial biomass can eventually be used as the source for a novel approach in using biosystems to remediate contaminants from solution. KEYWORDS: Synechococcus, Synechocystis, cyanobacteria, heavy metal binding, bioremediation INTRODUCTION they had no nucleus. These cells, which first appeared millions of years ago, gained the Few people doubt that environmental energy necessary for their metabolism by contamination has become an important fermenting the limited supply of organic source of concern. There exists a general compounds then available. Simultaneously, belief that the major environmental threats inorganic molecules were uptaken into the faced by mankind can be corrected with biomass. Therefore, the biosphere was some changes in technology and “cleaned” or “detoxified” of compounds manufacturing practices. However, new such as hydrogen cyanide, carbon monoxide, environmentally-friendly processes need to and hydrogen sulfide-through the use of a be developed to clean the already natural process. Cells have also assisted in contaminated areas of our environment. The detoxification, using other natural methods. first cells on earth were prokaryotic, i.e., *To whom all correspondence should be addressed (email: [email protected]). Heavy metals such as cadmium, copper, and Bonilla, et al., studied the interaction of lead were removed by the biomass by boron and calcium with live cyanobacteria biomineralization and by passive species such as Anabaena and accumulation in cells through surface Synechococcus [24]. Kanamaru, et al., binding with chemical functional groups. found a copper-transporting P-type ATPase These chemical functional groups evolved in the thylakoid membrane of the most likely as a response mechanism to the cyanobacterium Synechococcus sp. metal toxicity. PCC7942 [25]. On the other hand, other investigators isolated a strain of Cyanobacteria (also known as blue-green Synechococcus cedrorum 1191 tolerant to algae) are oxygen evolving, photosynthetic heavy metals and pesticides [26]. Although prokaryotic organisms that respond to stress the uptake of some heavy metal ions to conditions such as light deprivation [1]. several live species of cyanobacteria has Spirulina platensis, a cyanobacterium, has been studied, no reports have yet appeared been found to adapt to grow in cobalt and to relate the uptake of heavy metals to iodine-enriched media [2], and it has been inactivated biomass of cyanobacteria, determined that this cyanobacterium may be specifically Synechococcus spp. In addition, resistant to ammonia at high pH values [3]. the possible morphological alterations that Slotton, et al., determined that cyanobacteria undergo, when cultured in commercially-grown Spirulina contains high concentrations of copper ions, have not detectable levels of mercury and lead [4]. been studied. Their findings implied that the cyanobacteria was taking up the toxic heavy metal ions. The purpose of this work was to study the Indeed, other investigators have found that binding or uptake of copper(II), lead(II), and various species of algae take up or adsorb nickel(II) ions from solution by inactivated metal ions [5-15]. Other reports have biomass of the cyanobacterium indicated that carboxyl groups on algal cell Synechococcus sp. PCC 7942 cultured under walls may be responsible for a great portion normal conditions. Batch laboratory of metal binding to inactivated algal biomass experiments were carried out in order to [16]. In live algae, intracellular determine optimal metal binding pH. We polyphosphate has been found to be also also determined optimal metal binding times responsible for metal sequestration [17]. and the capacity of the Synechococcus Other investigators have shown that algal biomass to adsorb each metal ion. The extracellular polysaccharides also chelate or possibility of desorbing the metal ions bind metal ions [18, 19]. Gordon, et al., adsorbed by the biomass was studied. found that extracellular proteins are Additionally, transmission electron produced in copper-stressed cultures of microscopy (TEM) and environmental Vibrio alginolyticus [20]. Production of scanning electron microscopy (ESEM) were extracellular metal-binding proteins has also used to determine any morphological been proposed as a resistance mechanism in changes that could occur when the bacteria such as Pseudomonads [21, 22]. In cyanobacterium Synechocystis sp. PCC addition, ultrastructural changes in live 6803 was cultured under high copper(II) Dunaliella minuta have been observed levels. Also, we wished to investigate (using following acute and chronic exposure to ESEM) if copper(II) ions were being taken copper and cadmium [23]. up when the live cultures of cyanobacteria were grown under high copper levels. PROCEDURES ethanol was removed and 100% acetone was added for 15 min, twice. The samples were Cyanobacterial strains and culture embedded in Poly/Bed 812 resin plastic. conditions Samples were sectioned and examined in a Zeiss EM 10 transmission electron Synechococcus sp. PCC 7942 was cultured microscope operating at 60 kV. under normal conditions using liquid BG-11 Environmental scanning electron media, which is utilized for growing microscopy (ESEM) was used to try to unicellular blue-green algae on plates [27]. determine if the copper was being taken up This media contains only trace µ by Synechocystis sp. PCC 6803. The concentrations of copper(II) ions (0.3 M uncoated Synechocystis freeze-dried copper). Synechocystis sp. PCC 6803 was biomass was placed in the ESEM sample also cultured using the BG-11 media but also chamber and run in a water vapor in the presence of 0.25 mM copper(II). The atmosphere at three torr in an Electroscan resulting biomasses were washed with sterile ESEM model 2020, equipped with an EDAX distilled water, freeze-dried (in a Labconco power MX system. freeze-dryer), ground and sieved to pass a 100-mesh screen. pH profile for copper(II), lead(II), Electron microscopy and nickel(II) uptake Transmission electron microscopy (TEM) of A batch laboratory method was utilized for Synechocystis sp. PCC 6803 biomasses the pH profile studies of metal binding to grown under normal copper levels (control) inactivated cells of Synechococcus sp. PCC and grown under high copper levels (0.25 7942. The cyanobacterial biomass (250 mg) mM, experimental) were used to determine was washed twice with 0.01 M HCl. It was morphological changes in the cells. The then centrifuged and the washings were biomass pellets (control and experimental), collected, dried, and weighed to account for after centrifugation, were immobilized in biomass weight loss. The rest of the 10% agarose. Sections of immobilized experiment for the pH profile for copper(II) cyanobacteria were prepared by fixing them binding is similar to that reported before with 2% glutaraldehyde in 0.1 M sodium [28]. A comparable method was used for the cacodylate buffer (pH 7.2) for 40 min. pH profile for lead(II) and nickel(II) binding Glutaraldehyde was removed, and the to the Synechococcus inactivated biomass. samples were rinsed twice with 0.1 M Concentrations of 0.1 mM lead(II) (as lead sodium cacodylate buffer (pH 7.2) for 1 min. nitrate) and of 0.1 mM nickel(II) (as nickel nitrate) were used. Samples were incubated in 2% OsO4 in 0.1 M sodium cacodylate buffer for 1 hr at 4ºC. Time-dependence studies for metal After OsO4 was removed, the samples were rinsed with sodium cacodylate buffer for 5 ions binding min. The buffer was removed, and 0.5% A batch laboratory method was used for the aqueous uranyl acetate was added and time dependence studies of metal binding to incubated for 40 minutes at 4ºC. After inactivated cells of Synechococcus sp. PCC incubation the samples were rinsed with 7942. The cyanobacterial biomass (250 mg) distilled water for 5 min. The samples were was washed twice with 0.01 M HCl. It was dehydrated with 75%, 95%, and 100% then centrifuged and the washings were ethanol for five minutes each.