Removal of nitrate from groundwater by cyanobacteria: quantitative assessment of factors influencing nitrate uptake

Abstract

The feasibility of biologically removing nitrate from groundwater was tested by using cyanobacterial cultures in batch mode under laboratory conditions. Results demonstrated that nitrate-contaminated groundwater, when supplemented with phosphate and some trace elements, can be used as growth medium supporting vigorous growth of several strains of cyanobacteria. As cyanobacteria grew, nitrate was removed from the water. Of three species tested, Synechococcus sp. strain PCC 7942 displayed the highest nitrate uptake rate, but all species showed rapid removal of nitrate from groundwater. The nitrate uptake rate increased proportionally with increasing light intensity up to 100 micromol of photons m(-2) s(-1), which parallels photosynthetic activity. The nitrate uptake rate was affected by inoculum size (i.e., cell density), fixed-nitrogen level in the cells in the inoculum, and aeration rate, with vigorously aerated, nitrate-sufficient cells in mid-logarithmic phase having the highest long-term nitrate uptake rate. Average nitrate uptake rates up to 0.05 mM NO(3-) h(-1) could be achieved at a culture optical density at 730 nm of 0.5 to 1. 0 over a 2-day culture period. This result compares favorably with those reported for nitrate removal by other cyanobacteria and algae, and therefore effective nitrate removal from groundwater using this organism could be anticipated on large-scale operations.

FIG. 1

Influence of light intensity on growth of Synechococcus sp. strain PCC 7942 cultures (A) and nitrate removal (B). Light intensities tested were 6 (■), 12 (□), 50 (▵), 100 (○), and 180 (▿) μmol of photons m⁻² s⁻¹. Cultures were inoculated with cells precultured in BG-11 medium which were harvested during the exponential growth phase and washed twice with distilled water. Groundwater contained 1.5 mM nitrate, the aeration rate was ca. 0.7 vvm, and the culture temperature was 32°C (error bars indicate standard deviations; n = 4).

FIG. 2

Changes in cell density (A) and external NO₃⁻ (B) and external PO₄³⁻ (C) concentrations in the medium during the course of cultivation of Synechococcus sp. strain PCC 7942 in groundwater in the presence of various concentrations of phosphate. Cells had been previously deprived of PO₄³⁻ by propagating the cells in groundwater without addition of phosphate for a week. The initial PO₄³⁻ concentrations were 0.16 μM (■) (the phosphate concentration in the groundwater used), 6.6 μM (□), 14.7 μM (○), 53.7 μM (▿), and 105.3 μM (▵). The light intensity was 160 μmol m⁻² s⁻¹, the culture temperature was 32°C, and the aeration rate was ca. 0.7 vvm (error bars indicate standard deviations; n = 4).

FIG. 3

Effect of initial NO₃⁻ concentrations in tap water on growth of (A) and NO₃⁻ removal (B) by Synechococcus sp. strain PCC 7942. Nitrate-deprived cells to be used for inoculation were prepared by growing Synechococcus sp. strain PCC 7942 in modified BG-11 medium without fixed nitrogen for 40 h. The initial NO₃⁻ concentrations were 0.007 mM (○), 0.09 mM (□), 0.17 mM (■), 0.32 mM (▿), 0.82 mM (◊), 1.61 mM (▵), and 2.36 mM (●). The light intensity was 160 μmol m⁻² s⁻¹, the culture temperature was 32°C, and the aeration rate was ca. 0.7 vvm (error bars indicate standard deviations; n = 3).

FIG. 4

Effect of the fixed-nitrogen level of cells in an inoculum on nitrate uptake (A) and growth (B) of Synechococcus sp. strain PCC 7942. Nitrate-sufficient cells (□) were obtained from an inoculum that had been propagated in BG-11 medium for 24 h. Nitrate-depleted cells (○) originated from a culture that had been maintained in nitrate-free BG-11 growth medium for 48 h. The groundwater used for this experiment contained 2.3 mM NO₃⁻, and PO₄³⁻ had been added to a final concentration of 100 μM. The light intensity was 160 μmol m⁻² s⁻¹, the culture temperature was 32°C, and the aeration rate was ca. 0.7 vvm (error bars indicate standard deviations; n = 3).

FIG. 5

NO₃⁻ uptake and biomass yield as affected by culture aeration and initial cell density. NO₃⁻ uptake rates (A) and biomass yields (B) were the average values calculated from a cultivation period of 45 h. To prepare inocula with various initial cell concentrations, cells were precultured in BG-11 medium for 24 h, concentrated by centrifugation, and washed twice with deionized water. The x axis indicates the cell density right after inoculation. The aeration rate was set up at two levels, 0.3 (□) and 0.8 (▵) vvm. The light intensity was 160 μmol m⁻² s⁻¹, and the culture temperature was 32°C (error bars indicate standard deviations; n = 4).