Effects of Cellular Metabolism and Viability on Metal Ion Accumulation by Cultured Biomass from a Bloom of the Cyanobacterium Microcystis aeruginosa

Abstract

The sorption of nickel, cadmium, and copper by cultured biomass from a naturally occurring bloom of Microcystis aeruginosa was demonstrated in two systems: cells suspended in culture medium and cells immobilized in alginate. Incubation in the absence of light, in the presence of metabolic inhibitors, and at 4°C did not substantially decrease the copper accumulation by cells in culture medium. Heat-killed, formaldehyde-treated, and air-dried biomass samples sorbed nearly as much (or in some cases slightly more) copper as did viable samples.

The recovery of valuable or toxic metals by biological methods, attractive alternatives to conventional physiochemical processes, is finding significant application in various remediation strategies (3, 6, 7, 13, 26, 27, 29). Metal sequestration by organisms is often termed biosorption, which includes both passive adsorption of metals to cell envelopes and metabolically mediated uptake (4, 8, 12, 22, 23, 29). The nature of the biosorption depends on the metal and the microorganism (3, 5, 9, 23).

The cyanobacteria Microcystis aeruginosa and Microcystis flos-aquae exist in multicellular aggregates which are surrounded by a colonial capsule or exopolymeric matrix that can adsorb substantial amounts of various metal ions (1, 17, 20, 21). Metal accumulation by Microcystis has been reported for field samples (1, 10, 11, 25), axenic cultures (17, 18, 20, 21), and purified exopolymers (2, 17, 20, 21).

Growths of M. aeruginosa in Laxmi Pond (25°20′N, 83°0′E), Varanasi, India, have been studied at frequent intervals for several years (19). M. aeruginosa was consistently the dominant phototroph in this pond (19), which typifies a prevalent kind of subtropical body of water. The sequestration of metals by Microcystis in these ponds has numerous ecological and public health implications. Furthermore, similar ponds (including ones created in alkaline and saline wastelands) are potentially suitable for the mass cultivation of cyanobacteria and the harvesting of their products.

A procedure for obtaining large amounts of biomass from cyanobacterial blooms was developed with the goal of investigating the metal binding potential of material from natural growths. Surface samples from dense blooms in which M. aeruginosa was the only detectable phototroph were collected at 2 m from the shore of Laxmi Pond (19). The pH range of the sampled water was 8.9 to 9.2. The Microcystis colonies were concentrated by filtration through 100% polyester fabric (Gloria weave; Bombay Dyeing Corp.), suspended in a 15-fold excess (vol/vol) of deionized water, held for 2 h at ambient temperature, and collected by filtration. This washing procedure was repeated twice. The washed cells were cultured under standard conditions, which were 4 days of growth in DP medium (19) at 29 ± 2°C with 2,100 lx of continuous irradiance. The pHs of the cultures at 4 days of growth ranged between 8.8 and 9.1. At the beginning of each metal uptake experiment, the 4-day-old culture was diluted appropriately in DP medium, adjusted to pH 9 or 9.1, assayed (14) for chlorophyll α (CHL), and supplemented with a designated metal chloride. Deionized (18 MΩ) water and acid-soaked, low-metal plastics (or occasionally acid-leached glassware) were used for all experiments.

Metal sorption by biomass in a liquid medium.

Four-day cultures in DP medium alone (no metal added) or in DP medium containing 7.87 or 31.5 μM CuCl₂ (0.5 or 2.0 μg of Cu per ml), 4.45 μM CdCl₂ (0.5 μg of Cd per ml), or 8.52 μM NiCl₂ (0.5 μg of Ni per ml) were incubated under the standard growth conditions described above. At timed intervals after the addition of the metal, duplicate 10-ml portions of each culture were collected on nylon filters (5-μm pore size; Micron Separations, Inc. Westboro, Mass.) and washed twice with 5 ml (each) of deionized water. The filtered cells were digested by the method described by Martin (16). The digested cells and corresponding culture medium samples were resuspended in 1% HNO₃ and analyzed for metals in a Perkin-Elmer 2380 atomic absorption spectrophotometer.

Steady-state biosorption of cadmium, nickel, and copper was achieved at 2 h of incubation (Fig. 1). Figure 2 summarizes the concentrations of sorbed and free copper that were observed at 2 h of incubation in reaction mixtures containing various concentrations of copper (7.87 to 31.5 μM CuCl₂) and M. aeruginosa biomass (0.68 to 1.8 mg of CHL per liter). The arrow in Fig. 2 indicates the concentrations of sorbed and free copper at 2 h in subsequent experiments in which the effects of metabolic inhibition on metal accumulation by M. aeruginosa were examined.

FIG. 1.

Time course of metal sorption by cultured cells from an M. aeruginosa bloom. (A) ○, 31.5 μM CuCl₂ (2.0 μg of Cu²⁺/ml); ▵, 7.87 μM CuCl₂ (0.5 μg of Cu²⁺/ml); cells at a density of 1.5 mg of CHL per liter. (B) ◊, 4.45 μM CdCl₂ (0.5 μg of Cd²⁺/ml); □, 8.52 μM NiCl₂ (0.5 μg of Ni²⁺/ml); cells at a density of 1.8 mg of CHL per liter. Error bars indicate the ranges of duplicate measurements.

FIG. 2.

Relationship between bound and free copper at various concentrations of added copper (7.87 to 31.5 μM) and various biomass concentrations (0.68 to 1.8 mg of CHL/liter), for the steady-state conditions observed after 2 h of incubation at 29°C in culture medium. The arrow indicates the conditions of bound and free copper in subsequent studies of metabolic inhibition.

Effects of metabolic inhibition.

A 4-day culture was adjusted to 0.86 mg of CHL per liter of DP medium (pH 9.1). Culture aliquots were pretreated by incubation for 2 h as follows: (i) at 29°C and 2,100 lx; (ii) at 29°C in the dark; (iii) at 4°C in the dark; (iv) with 10 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at 29°C and 2,100 lx; (v) with 0.1% formaldehyde at 29°C and 2,100 lx; and (vi) with 2.0% formaldehyde at 29°C and 2,100 lx. Another aliquot was heated at 80°C for 3 h and then cooled to 29°C. Each suspension was brought to 7.87 μM CuCl₂ and then maintained under the pretreatment conditions, except that the 80°C-heated sample was held at 29°C and 2,100 lx. At 2 h, duplicate 10-ml aliquots from each culture were filtered, washed, and assayed for biosorbed metal as described above.

In all cases, the sorption of copper by the variously inhibited or killed M. aeruginosa cells was at least 84% of that observed for metabolically active cells (Table 1). This observation is noteworthy because nonviable material may be safer and more convenient than viable material in various biotechnological applications.

TABLE 1.

Effect of metabolic inhibition on copper accumulation by M. aeruginosa in DP liquid medium

Type of cells and additive	Culture conditions	μmol of Cu accumulated/mg of CHL (% of control)a
Viable
None (control)	29°C, light	4.84 (100)
None	29°C, dark	4.59 (95)
None	4°C, dark	4.19 (87)
DCMU (10 μM)	29°C, light	4.06 (84)
HCHO
0.1%	29°C, light	4.72 (98)
2.0%	29°C, light	5.59 (116)
Heat-killed	29°C, light	4.19 (87)

^aSamples containing 0.86 mg of CHL per liter were incubated for 2 h with 7.87 μM CuCl₂. Values are means of duplicate samples that did not differ by more than 3% of the means. 

The largest decrease in copper sorption occurred with relatively gentle metabolic inhibition, such as incubation with the photosynthesis inhibitor DCMU (Table 1). In contrast, cells exposed to the most harsh conditions, i.e., treatment with 2% formaldehyde, actually sequestered more copper than did viable cells (Table 1), supporting the reports of other investigators (28) who suggest that formaldehyde increases metal adsorption by modifying cellular structures or exposing intracellular binding sites.

Metal sorption by alginate-immobilized biomass.

A portion of a 4-day culture was collected on 5-μm-pore-size filters and dried for 36 h at 45°C with blowing air. A parallel portion of moist cells was maintained in DP medium under standard growth conditions. The dried biomass was suspended in DP medium to a concentration of 2 mg/ml (3.5 μg of CHL per ml), as was the moist biomass. Duplicate 2.5-ml portions of each sample were mixed with 2.5 ml of 10% (wt/vol) alginate and added dropwise from a syringe into 0.2 M CaCl₂. The alginate beads were washed with DP medium. Each 5-ml aliquot of beads was agitated for 2 h in 50 ml of DP medium containing 7.87 μM CuCl₂. Digestion of the beads and metal analysis by atomic absorption spectrophotometry have been described previously (15, 24). Background metal uptake by the alginate alone was less than 25% of that of any biomass-containing sample and was subtracted from the data.

For immobilized moist biomass, the initial amounts of metal sorption during the first 2 h of the reaction were, per mg of CHL per h, 3.56 μmol of copper, 1.88 μmol of cadmium, and 4.94 μmol of nickel. For immobilized dried biomass, these values were, per mg of CHL per h, 2.28 μmol of copper, 1.36 μmol of cadmium, and 3.58 μmol of nickel. The efficacies of Cu, Cd, and Ni sorption by the dried biomass were, respectively, 65, 72, and 73% of those of moist biomass. The activity of the dried biomass may have been influenced by both its clumping and its presumably altered metabolic activity.

In summary, this study suggests that copper accumulation by M. aeruginosa-dominated biomass is largely independent of the metabolic state of the cells. Furthermore, heat-killed, HCHO-treated, and air-dried biomass samples exhibit substantial sorption of copper, cadmium, and nickel and thus may be promising materials for metal reclamation technologies. Since naturally occurring M. aeruginosa can exhibit various metabolic states, the surprisingly minor effects on copper sorption that we observed for diverse inhibitory treatments of field-collected M. aeruginosa biomass are of interest for environmental modeling.