J.L. Gardea-Torresdey1 , J.L. Arenas2, R. Webb2, N.M.C. Francisco2, and K.J. Tiemann1

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, University of Texas at El Paso, El Paso Texas 79968


Synechococcus 7942 has the ability to grow in mass quantity under ideal conditions; such an ability provides usable biomass at a minimal effort. Using lyophilized biomass grown under normal conditions, Synechococcus was tested for its potential to bind metal ions from solution. Batch experiments have determined the optimum binding pH, time dependency, and metal binding capacities for copper(II), lead(II), nickel(II), cadmium(II), chromium(III), and chromium(VI), along with desorption of the metal bound. The biomass studied showed an affinity for five of the metal ions, with an optimum binding at pH 5. Time dependency studies showed that this cyanobacteria had rapid binding, while capacity experiments showed this cyanobacteria strain to bind 11.3 mg of copper(II) per gram of biomass, 30.4mg of lead(II) per gram of biomass, 3.2 mg of nickel(II) per gram of biomass, 7.2 mg of cadmium (II) per gram of biomass, and 5.4 mg of chromium (III) per gram of biomass. More than 90% of copper(II), lead(II), and nickel(II) metal ions were recovered, while over 50% of cadmium(II) and chromium(III) were recovered when treated with 0.1M HCl. The biomass was immobilized in a silica polymer and tested for its binding ability under flow conditions. Using 0.1mM concentrations of the previously indicated metals, individual experimental results showed an average of 143 ppm copper (II), 1456 ppm lead(II), 142 ppm nickel(II), and 529 ppm cadmium(II) were bound by the immobilized biomass. Treatment with 0.2M HCl resulted in nearly 100% recovery for both copper(II) and lead(II) from the column, 79% recovery of cadmium(II), while recovery for nickel(II) was 40%. Experiments were conducted to determine if many cycles of metal binding-stripping by the immobilized biomass were possible. Further, attempts were made to determine the presence of metallothioneins in various strains of cyanobacteria which may serve as defense mechanisms for stressed growth conditions. The presence of such proteins may serve to help develop engineered strains of Synechococcus sp. PCC 7942 with increased metal ion binding ability. Synechococcus can eventually be used as a source for a novel approach in using biosystems to remediate contaminants from solution and making those contaminants available to industry through an environmentally friendly biofiltration system.

Keywords: synechococcus, cyanobacteria, heavy metal binding, bioremediation, metal recovery


Environmental pollution and contamination have become a key focus of concern. However, changes in technology and manufacturing practices are providing relief to these problems. Yet, some of the present methods of environmental cleanup result in the production of harmful by-products. Thus, environmentally friendly processes need to be developed to clean up the environment without creating harmful waste products.

Cyanobacteria (also known as blue-green algae) are the largest and most diverse group of photosynthetic prokaryotes whose habitats vary from fresh and marine water to terrestrial environments. They are oxygen-evolving organisms that respond to stress conditions such as light deprivation (Borbely, et al.., 1990.). These cells have developed natural methods of responding to metals such as copper, lead, and cadmium through passive accumulation in cells and through surface binding to various functional groups. They have been found to remove harmful metals from the environment. For example, Spirulina platensis, a cyanobacterium, was determined to contain detectable levels of mercury and lead when grown under contaminated conditions (Slotton, et al..,1989), implying that this cyanobacterium was taking up the toxic metal ions from its environment. Further studies confirmed that these cyanobacteria both absorb and take up metal ions (Bender, et al., 1994). Other reports also indicate carboxyl groups on algal cell biomass as being responsible for binding to various ions (Gardea-Torresdey, et al., 1990). Live algae possess intracellular polyphosphates responsible for metal sequestration as well as algal extracellular polysaccharides serving to chelate or bind metal ions (Zhang and Majidi,1994; Kaplan, et al., 1987; Van Eykelenburg and Glucan, 1978). Strains of Synechococystis have been proven to develop a thickened calyx when exposed to copper-stressed growth conditions (Gardea-Torresdey, et al., 1996(a)). Synechococcus sp. PCC 7942 was found to posses a copper-transporting P-type ATPase in the thylakoid membrane (Bonilla, et al., 1995). Synechococcus cedrorum 1191 was proven to be tolerant to heavy metals and pesticides (Gothalwal and Bisen, 1993). Such findings show the possibility of manipulating or overexpressing existing resistance mechanisms and use of such organisms to remove harmful metals from the environment.

Another resistance mechanism is the production of metallothioneins. Metallothioneins are low-molecular weight proteins or polypeptides (6,000-8,000 daltons) which bind metal ions in metal-thiolate clusters. These polypeptides are abundant in cysteine residues and often possess a characteristic pattern of sulfur containing amino acids (Turner, et al., 1995). They are commonly found in association with essential metal ions such as zinc and copper but have also been shown to bind toxic metals like cadmium, mercury, and lead. Their metal binding properties are mediated via the abundant cysteine residues and their characteristic organization into -Cys-Cys-, -Cys-X-Cys-, or -Cys-X-X-Cys- sequences. Synthesis of metallothioneins has been shown to be prompted by elevated concentrations of some metals (Turner, et al., 1995). Metallothioneins can be characterized into three specific classes: class I contains most animal metallothioneins; class II consists of metallothioneins whose cysteine locations in the polypeptide are distantly related to those found in equine renal metallothioneins; and class III metallothioneins are characterized by atypical non-translationally synthesized metal-thiolate polypeptides. Cyanobacterial metallothioneins belong to class II. They possess approximately 56 amino acids including nine cysteine residues. This amount of cysteine residues is somewhat less than the 20 cysteine residues out of 60 amino acids for the animal class I metallothioneins and 12 cysteine residues out of 75 amino acids for plant class I metallothioneins (Silver et al.,1994).

Until now, a complete study of the uptake of heavy metal ions by inactivated biomass of cyanobacteria (under equilibrium and flow conditions), specifically Synechococcus spp. has not been fully performed. In addition, metallothionein expression by various strains of cyanobacteria has yet to be investigated.

The purpose of our investigation was to study the ability of Synechococcus sp. PCC 7942 to uptake copper(II), lead(II), cadmium(II), nickel(II), chromium(III), and chromium(VI) under batch conditions. As well as the uptake of copper (II), lead (II), cadmium (II), and nickel (II) by silica immobilized cells of this strain of cyanobacteria under flow conditions. Experiments were conducted to find optimal binding pH, optimal time for binding, and maximal capacity for binding of each metal. Further, tests were performed to investigate the ability to recover bound metals. Synechococcus sp. PCC 7942 was immobilized in a silica polymer matrix, then tested for its ability to bind copper(II), lead(II), nickel(II), and cadmium(II). Additionally, genetic investigation was performed to identify genes for class II metallothioneins in this and seven other strains of cyanobacteria.


Cyanobacterial Collection

Synechococcus sp. PCC 7942 was cultured under normal conditions using liquid BG-11 media, which is commonly used for growing unicellular blue-green algae on plates (Allen, 1968). This media contains only trace amounts of metal ions, thus allowing for rich growth. The resulting biomasses were then washed with sterile distilled water, freeze-dried (in a Labconco freeze-dryer), and ground and sieved to pass a 100-mesh screen. All experiments were conducted using biomass collected in this manner.

pH Profile Studies for Metal Binding

Batch laboratory methods were used for the pH studies. 250-mg samples of inactivated cyanobacterial biomass were washed twice with 0.01 M HCl to remove any debris or soluble biomolecules that might interact with metal ions. Samples were centrifuged and washings were collected to account for any biomass weight loss. The remainder of the experiment for the pH profile for copper(II) binding was similar to that reported previously (Gardea-Torresdey, et al., 1996(b)). Comparable methods were used to study pH profiles for lead(II), nickel(II), cadmium(II), chromium(III), and chromium(VI). Concentrations of 0.1 mM were used for each metal tested.

Time-dependency Studies for Metal Ion Binding

Methods for time dependency studies of metal binding were also under batch conditions. 250 mg samples of the inactivated cyanobacterial biomass were washed twice with 0.01 M HCl. Samples were centrifuged and washings were collected, dried, and weighed to account for any biomass weight loss. The remainder of the experiment is analogous to that reported previously (Gardea-Torresdey, et al., 1996(b)). Metal ion concentrations were 0.3 mM for each metal while maintaining a pH 5 in 0.01 M sodium acetate buffer. The time intervals tested were 5, 10, 15, 30, 60, 90, and 120 min.

Metal Binding Capacity Studies

Samples of 50 mg of Synechococcus sp. PCC 7942 were washed twice with 0.01 M HCl and washings were collected and weighed to determine biomass loss. The remainder of the experiment was similar to that reported before in regards to copper(II) binding by Mucor rouxii (Gardea-Torresdey, et al., 1996(c)). Studies for the other metals were performed using a comparable method, with concentrations of 0.3 mM for each metal studied.

Desorption of Adsorbed Metal

In attempts to recover bound metal ions, the biomasses laden to capacity were exposed three times to 2 ml of 0.1 M HCl , reacting by agitation for 5 min intervals and then centrifuging. After centrifugation, the supernatants were analyzed. All analyses for metals tested were performed by flame atomic absorption spectroscopy.

Immobilization of Cyanobacterial Biomass

The methodology of immobilization of Synechococcus sp. PCC 7942 within a polysilicate matrix was similar to that reported by Huei-Yang and Rayson (Huel-yang,1993). A 2.5 g sample of biomass (100 mesh) was washed twice by vortexing the sample with deionized water and then centrifuged for 5 min. at 3000 rpm to remove solubles and debris. Next, 75 ml of 5% sulfuric acid (H2SO4) was mixed with enough 6% sodium silicate (Na2SiO3) solution to raise the pH to 2.0. The remainder of the immobilization was performed following procedures previously reported (Gardea-Torresdey, et al., 1996(b)).

Column Experiments

Using 3 ml of immobilized Synechococcus sp. PCC 7942, a column was prepared having one bed volume equal to the volume of immobilized biomass inside the column (3ml). The column was washed with 24 bed volumes of 0.01 M sodium acetate buffer at pH 5.0 and effluent pH was monitored to ensure that the column was continuously at the optimal binding pH. A flow rate of 2 ml per minute was used to pass 240 bed volumes of 6.3 ppm copper(II), 5.8 ppm nickel(II), 20.7 ppm lead(II), 33.7 ppm cadmium(II), 15.6 ppm chromium(III), and 15.6 ppm chromium(VI). Each metal solution was in 0.01 M sodium acetate at pH 5.0 and was tested in triplicate. Each metal ion was tested individually.

Recovery of Metal from the Column

To remove the bound metal, 0.2 M HCl was passed through the column at a flow rate of 2 ml per minute. Each effluent bed volume was collected and analyzed by flame atomic absorption spectroscopy.

Analytical Procedure

All metal analyses were performed by flame atomic absorption spectroscopy (FAAS) using a Perkin Elmer model 3110 with deuterium background subtraction. Analytical wavelengths used for the various metals were as follows: copper, 327.4 nm; lead, 217 nm; nickel, 352.5 nm; cadmium, 229 nm; and chromium, 359.4 nm. An impact bead was used to improve the sensitivity and samples were read three times with the mean value computed. Calibrations were performed within the calibration range of each metal and the correlation coefficients for the calibration curves were 0.98 or better. Controls of each of the metal solutions were run to detect any possible metal precipitation or contamination. The difference between the initial metal concentration and the remaining concentration in supernatants and effluents was assumed to be taken up by the biomass.

Identification of Cyanobacterial Metalothionein Genes

Bacterial Strains and Culture Conditions

Spirulina sp. ATCC 53843, Microcystis aeruginosa ATCC 22663, Anabaena flos aquae ATCC 22664, Nostoc sp. ATCC 29105, Fischerella Gomont ATCC 27929, and Synechococcus sp. PCC 7942 were cultured in BG-11 for two weeks under constant light (50 micromole quanta m-2s-1) first on a shaker (140 rpm) and later with air bubbling at 27oC. Prochlorothrix hollandica was obained from Dr. George Bullerjahn (Bowling Green University, Bowling Green, Ohio) and was grown in BG-11 with constant light (40 micromole quanta m-2s-1) and air bubbling at 25oC for two weeks.

Restriction Enzymes and DNA Markers

The following restriction enzymes were used in the restriction digestion analysis, HaeIII, Hinfl, MspI, RsaI, and TaqI. Restriction enzymes, Taq polymerase, oligonucleotide size markers, and agarose were purchased from New England BioLabs, Promega, and GIBCO-BRL.

Polymerase Chain Reaction

Metallothionein (SmtA) locus amplification was performed using oligonucleotides described by Robinson, et al., 1990, using standard protocols described in Ausubel, et al. 1987.

Sequence Alignment of the Class II Metallothioneins

PIMA sequence alignment was performed using the algorithms of Smith and Smith (1990, 1992).

Results and Discussion

Previous screening experiments with Synechococcus sp. PCC 7942 revealed the effect of pH on uptake of copper(II), lead(II), and nickel(II) (Gardea-Torresdey, et al., 1996(a)). Figure 1 shows the results of the pH profile for those three metals as well as for cadmium(II), chromium(III), and chromium(VI). It is evident that increased sorption of copper(II), nickel(II), cadmium(II), and chromium(III) was observed as the pH increased from 2 to 6. On the other hand, Synechococcus sp. PCC 7942 bound lead over a wide pH range in a rather pH- independent manner. The maximum binding observed was between pH 5.0 and 6.0. Previous studies showed that certain metal ions begin to precipitate out of solution once pH reaches 6.0 (Gardea-Torresdey, et al., 1996(b)). Therefore, maximum pH binding was taken as being pH 5. Further, the similar trends seen in copper(II), nickel(II), cadmium(II), and chromium(III) binding suggest that carboxyl groups play a role in the metal binding. The acid-dissociation constants (pKa's) for carboxyl groups have been reported to be around 3-4 (Hunt, 1986; Segel, 1976). As pH increases, these groups become deprotonated and attract the positively-charged metal ions.

Figure 2 represents the time dependency results. As can be seen, lead(II), cadmium(II), and nickel(II) are optimally bound within the time elapsed to mix the biomass and metal ions and to centrifuge. Copper(II) and chromium(III), on the other hand, required longer times in order to be adsorbed.

Experiments were carried out to determine the metal binding capacities of the Synechococcus biomass at optimum binding pH. Previous studies determined the capacity of Synechococcus sp. PCC 7942 to bind copper(II), lead(II), and nickel(II) (Gardea-Torresdey, et al., 1996(a)). Using similar methods as used to test those three metals, binding capacities for cadmium(II), chromium(III), and chromium(VI) were also determined. Table 1 shows the results of the tests. The higher binding capacities for copper(II) and lead(II) are clearly observed.

Experiments were also performed to determine the possibility of recovering metal ions bound by this particular strain of cyanobacteria. Previous studies showed that binding of copper(II) and nickel(II). and to a lesser extent lead(II), were favored at higher pH (Gardea-Torresdey, et al., 1996(a)). Our pH profile experiments also showed similar trends for cadmium(II) and chromium(III). This suggests that binding may be reversed by lowering pH. Therefore, as previously mentioned, 0.1 M HCl was used to strip the bound metal. Table 2 shows results of these experiments. While copper(II), lead(II), and nickel(II) had already shown efficient removal (Gardea-Torresdey, et al., 1996(a)), cadmium(II) and chromium(III) showed less removal percentage. This indicates the need for a stronger stripping agent, possibly EDTA. Currently, experiments are being performed to find better stripping agents for cadmium(II) as well as chromium(III).

Synechococcus showed good metal ion binding under batch conditions, but in biofiltration Synechococcus sp. PCC 7942 would be most useful if capable of binding metal ions under flow conditions. By immobilizing the biomass in a silica polymer, the biomass could be packed into a column through which high flow rates could be achieved. Previous studies showed that optimal flow can be maintained if a polysilicate matrix support material was used to immobilize the biomass (Gardea-Torresdey, et al., 1996(b)). Using this matrix, column experiments were conducted to study the effects of metal binding by the algal biomass under flow conditions. Figure 3 represents the breakthrough curve of copper passed through the column. The curve shows the amount of metal remaining after solutions at pH 5 were passed through the column. Only after 100 bed volumes were trace amounts of metal detected in the effluent. Further, after 140 bed volumes, the column was nowhere near saturation. After elution with 0.2 M HCl, the same bound metal ion was recovered with near immediate effects seen in bed volumes 2-6. Figure 4 represents the curve showing this recovery.

Table 3 represents the average results of binding by the immobilized biomass to the various metals tested, as well as the average amount of recovery of each metal by using 0.2 M HCl. Excellent binding capacity was obtained for lead(II) ions. Further, copper(II) and lead(II) were almost completely stripped after treatment with 0.2M HCl, while lower recoveries were obtained for cadmium(II) and nickel(II).

Further, Synechococcus could provide a recyclable system for filtration. Thus, using lead(II), the column was recycled for six cycles to test the column's ability to continue to remove lead(II) ions. Figure 5 shows the results of this experiment. As can be seen, the amount of lead(II) bound never dropped below 1000 ppm. The fluctuating pattern seen in the graph is likely due to channel formation in the column. Thus, such data suggests the column containing immobilized Synechococcus sp. PCC 7942 could be recyclable at least for lead(II) removal.

Polymerase chain reaction was used to demonstrate the presence of previously undescribed class II metallothionein genes in seven species of cyanobacteria. The one established example of this gene from Synechococcus sp. PCC 7942 yielded a PCR product of 187 base pairs. Products of this size were also obtained from chromosomal DNA's of five other cyanobacterial species, Synechocystis (2 species), Spirulina, Prochlorothriz, and Nostoc. Slightly larger products were obtained (220 base pairs) from the chromosomal DNA's of Anabaena and Fischerella species. Restriction analysis, using four restriction enzymes which restrict DNA frequently, demonstrated that the amplified products were unique. These results, provided in Table 4, suggest that class II metallothionein genes are widespread among the cyanobacteria.

Class II metallothioneins may participate in the sequestration of toxic metal ions by the cyanobacteria. Previously, genes for these proteins were shown to be present only in three species of cyanobacteria, all of the genus Synechococcus. We have demonstrated that genes for these proteins are widespread among the various genera and species of cyanobacteria. Our goal is to overexpress these proteins and survey such engineered cyanobacteria for increased metal tolerance and for improved ability to sequester toxic metal ions. Derived amino acid sequences resulting from the further characterization of these genes will also drive experimentation aimed at elucidating the structure and function of these proteins. For example, as shown in Figure 6, the class II metallothioneins contain a lower fraction of sulfur containing amino acids (cysteine = C) than the metallothioneins of class I but their positions in the polypeptides are very well conserved. Multiple sequence alignment of some of the known class II proteins also reveals a high level of conservation of other amino acid residues such as those containing carboxylates (aspartic acid = D, glutamic acid = E) and hydroxyls (serine = S, threonine = T, tyrosine = Y) which may also participate in metal ion binding by these proteins.


Batch laboratory experiments have shown that inactivated cells of Synechococcus sp. PCC 7942 are able to bind copper(II), lead(II), nickel(II), cadmium(II), and chromium(III) ions. This ability provided preliminary data showing the potential for the silica immobilized biomass to be used as a biosorption resin for removal and recovery of metal ions from contaminated waters. Every metal was sorbed and desorbed three times and lead(II) as many as six times. After one adsorption/desorption cycle, the column containing silica-immobilized biomass was capable of binding the metal ions. Future experiments will attempt to identify the metal ion binding sites.

Further, metallothionein genes were shown to be present not only in Synechococcus sp. PCC 7942, but in seven other cyanobacterial strains. These proteins may be a sort of defense employed by the organism. Future experiments will attempt to overexpress these genes in engineered strains of Synechococcus sp. PCC 7942 with the idea that these strains will be better capable of binding metal ions from solution.


Dr. Gardea-Torresdey acknowledges the financial support from the NIH (grant # GM08012-25). Javier Arenas acknowledges the support of the Howard Hughes Medical Institute. Dr. Webb acknowledges the support of NIH(Grant # 3 G12 RR08124-0451).


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Table 1. Metal ion binding capacities of inactivated cells of Synechococcus sp. PCC 7942 biomass.
COPPER (II) 11.3
LEAD (II) 30.4

Note: Each data represents the mean of three replicates.

Table 2. Percent of metal ion recovery from inactivated cells of Synechococcus sp. PCC 7942 biomass after treatment with 0.1 M HCL.
LEAD (II) 99

Note: Data represents the mean of three replicates.

Table 3. Average amount of metal ion bound by silica immobilized biomass as well as the percent recovery after treatment with 0.2 M HCl.
COPPER (II) 143 97
LEAD (II) 1456 99
NICKEL (II) 142 42
CADMIUM (II) 529 79

Note: Data represents results of three cycles performed. One cycle consisted of 240 bed volumes collected (3 ml bed volume) with 2ml per min. flow rate.

Table 4. Polymerase chain reaction (PCR) product and restriction digestion product sizes obtained for smtA gene (metallothioneine gene) amplification and restriction from DNA isolated from filamentous and unicellular cyanobacterial species.

Cyanobacterial Species

smtA PCR products





Anabaena ATCC 22664








Fischerella ATCC 27929








Synechocystis ATCC 22663












Nostoc ATCC 29105












Prochlorothrix hollandica












Spirulina ATCC 53843












Synechocystis PCC 6714










Synechococcus PCC 7942










Note: PCR products were digested with HaeIII, MspI, RsaI, and TaqI. PCR products and restriction digestion fragments are given in base pairs. U=Fragment sizes which could not be resolved and ND= no digestion.

Figure 1. Percent Copper (¨), Lead(D), Nickel(D), Cadmium(n), Chromium (III)(Ñ), and Chromium (VI)(l) removed from solution as a function of pH by inactivated cells of Synechococcus sp. PCC 7942. Biomass (5 mg/ml) was reacted for 1 hour at the appropriate pH with 0.1 mM copper(II). The same procedure was repeated with 0.1 mM lead(II), nickel(II), cadmium(II), chromium(III), and chromium(VI).

Figure 2. Percent Copper (¨), Lead(D), Nickel(D), Cadmium(n), Chromium (III)(Ñ), and Chromium (VI)(l) removed from solution at different reaction times by inactivated cells of Synechococcus sp. PCC 7942. Biomass (5 mg/ml) was reacted for appropriate times with 0.3 mM copper(II) in 0.01 M sodium acetate buffer at pH 5.0. The same procedure was repeated with 0.3 mM lead(II), nickel(II), cadmium(II), chromium(III), and chromium(VI).

Figure 3. Breakthrough curve for copper binding under flow conditions. A solution of 6.3 ppm copper(II) in 0.01 M sodium acetate at pH 5 was passed through a column containing immobilized biomass (3 ml bed volume). The flow rate was at 2 ml per min.

Figure 4. Desorption curve for Copper(II) after binding under flow conditions. Using the same column used to gather data for Figure 4, ten bed volumes of 0.2 M HCl were passed at a flow rate of 2 ml per min.

Figure 5. Testing of Lead(II) recyclability under flow conditions. A solution of 22.7 ppm lead (II) in 0.01 M sodium acetate at pH 5 was passed through a column containing immobilized biomass (3ml bed volume). The flow rate was 2 ml per min. and six cycles were ran collecting 240 bed volumes per cycle.

Figure 6. PIMA-alignment of representative Class II metallothionein protein sequences from Saccharomyces Cerevisiae (SC1 and SC 2) and Synechococcus sp. (7942 and 6803). Alignment shows conserved cysteine residues and equivalent replacement of electronegatvie amino acid residues.