Bacterial degradation of cyanide and its metal complexes under alkaline conditions

Abstract

A bacterial strain able to use cyanide as the sole nitrogen source under alkaline conditions has been isolated. The bacterium was classified as Pseudomonas pseudoalcaligenes by comparison of its 16S RNA gene sequence to those of existing strains and deposited in the Coleccion Espanola de Cultivos Tipo (Spanish Type Culture Collection) as strain CECT5344. Cyanide consumption is an assimilative process, since (i) bacterial growth was concomitant and proportional to cyanide degradation and (ii) the bacterium stoichiometrically converted cyanide into ammonium in the presence of l-methionine-d,l-sulfoximine, a glutamine synthetase inhibitor. The bacterium was able to grow in alkaline media, up to an initial pH of 11.5, and tolerated free cyanide in concentrations of up to 30 mM, which makes it a good candidate for the biological treatment of cyanide-contaminated residues. Both acetate and d,l-malate were suitable carbon sources for cyanotrophic growth, but no growth was detected in media with cyanide as the sole carbon source. In addition to cyanide, P. pseudoalcaligenes CECT5344 used other nitrogen sources, namely ammonium, nitrate, cyanate, cyanoacetamide, nitroferricyanide (nitroprusside), and a variety of cyanide-metal complexes. Cyanide and ammonium were assimilated simultaneously, whereas cyanide strongly inhibited nitrate and nitrite assimilation. Cyanase activity was induced during growth with cyanide or cyanate, but not with ammonium or nitrate as the nitrogen source. This result suggests that cyanate could be an intermediate in the cyanide degradation pathway, but alternative routes cannot be excluded.

FIG. 1.

Utilization of cyanide by P. pseudoalcaligenes CECT5344. Cells were precultured with 2 mM ammonium to the stationary phase (A₆₀₀ ≈ 0.3). At this point (time zero), the culture was separated into two flasks and 1 mM cyanide was added to one of them, whereas the other remained as a control without any additions. At the indicated times, the increments of cell growth of the culture growing with cyanide with respect to the control culture (•) and the cyanide concentration in the culture supernatant (▴) were measured. The concentration of cyanide in a noninoculated flask containing culture medium (▵) was measured at the same times. Data are from a representative experiment.

FIG. 2.

Growth of P. pseudoalcaligenes CECT5344 with cyano-metal complexes as the sole nitrogen source. Cells were inoculated in media at pH 9.5 containing 1 mM potassium ferrocyanide (A) and 2 mM K₂Cu(CN)₄ (B) as the sole nitrogen sources. The cell growth was measured at the indicated times. No significant growth (less than 10%) was observed for cultures without cyanide. The experiment was repeated three times with similar results.

FIG. 3.

Induction of the cyanide removal system in P. pseudoalcaligenes CECT5344. The cells were grown with 5 mM ammonium (A) and 4 mM cyanide (B) and harvested by centrifugation. After washing with nitrogen-free minimal medium, cells were resuspended in minimal medium containing 1 mM cyanide up to an A₆₀₀ of 0.6 and placed in a rotatory shaker at 30°C. The cyanide concentrations in the culture supernatants were measured at the indicated times. In both cases, the open symbols and dashed lines correspond to the evolution of cyanide in noninoculated controls. The experiment was repeated three times with similar results.

FIG. 4.

Utilization of cyanide by P. pseudoalcaligenes CECT5344 in the presence of additional nitrogen sources. Cells were previously grown with a limiting amount of nitrogen (2 mM ammonium) with respect to the carbon source (70 and 50 mM acetate in panels A and B, respectively) in two separate flasks. After the complete ammonium consumption, cyanide was added to both of them, plus either ammonium (A) or nitrate (B). At the indicated times, aliquots of the cultures were centrifuged and the concentrations of the nitrogen sources was determined as indicated in Materials and Methods. The data are from a single experiment. Three independent experiments gave similar results.