Bacterial Degradation of Cyanide and Its Metal Complexes under Alkaline Conditions

A bacterial strain able to use cyanide as the sole nitrogensource under alkaline conditions has been isolated. The bacteriumwas classified as Pseudomonas pseudoalcaligenes by comparisonof its 16S RNA gene sequence to those of existing strains anddeposited in the Colección Española de CultivosTipo (Spanish Type Culture Collection) as strain CECT5344. Cyanideconsumption is an assimilative process, since (i) bacterialgrowth was concomitant and proportional to cyanide degradationand (ii) the bacterium stoichiometrically converted cyanideinto ammonium in the presence of L-methionine-D,L-sulfoximine,a glutamine synthetase inhibitor. The bacterium was able togrow in alkaline media, up to an initial pH of 11.5, and toleratedfree cyanide in concentrations of up to 30 mM, which makes ita good candidate for the biological treatment of cyanide-contaminatedresidues. Both acetate and D,L-malate were suitable carbon sourcesfor cyanotrophic growth, but no growth was detected in mediawith cyanide as the sole carbon source. In addition to cyanide,P. pseudoalcaligenes CECT5344 used other nitrogen sources, namelyammonium, nitrate, cyanate, cyanoacetamide, nitroferricyanide(nitroprusside), and a variety of cyanide-metal complexes. Cyanideand ammonium were assimilated simultaneously, whereas cyanidestrongly inhibited nitrate and nitrite assimilation. Cyanaseactivity was induced during growth with cyanide or cyanate,but not with ammonium or nitrate as the nitrogen source. Thisresult suggests that cyanate could be an intermediate in thecyanide degradation pathway, but alternative routes cannot beexcluded.

INTRODUCTION

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Introduction

Cyanide is a natural compound produced by many organisms, includingbacteria, algae, fungi, and plants, and is bioproduced in somecases as a defensive metabolite (32) or with invasive purposes(19). Plants are the main source of cyanide in the biospherebecause they cogenerate cyanide with ethylene (34) in additionto generating cyanoglycosides and cyanolipids. Moreover, cyanidehas also been shown to be produced as part of active iron-cyanidecomplexes of catalytic proteins (39). Cyanide causes severeenvironmental problems when produced in high amounts by anthropogenicactivities, such as mining and the electroplating industry.As an example, the jewelry industry located in the city of Cordova,Spain, produces an alkaline residue (pH > 13) containingup to 20 g of free cyanide liter^–1 (around 0.77 M) togetherwith high amounts of heavy metals complexed with cyanide, thusmaking this effluent highly toxic and environmentally hazardous.An event showing the potential risk of cyanide occurred on 30January 2000, when almost 100,000 m³ of wastewater with a highconcentration of cyanide was discharged into the Tisza River,a Danube tributary, causing a massive poisoning of the ecosystem.

Cyanide is highly toxic for most living organisms because itforms very stable complexes with transition metals that areessential for protein function, i.e., iron in cytochrome oxidase.Consequently, organisms growing in the presence of cyanide musthave a cyanide-insensitive metabolism, such as the alternativeoxidase described for plants (7) or the cytochrome bd (or cyanide-insensitiveoxidase) in bacteria (24, 31, 40). The presence of cyanide inthe environment causes an additional problem, the formationof extremely stable metal-cyanide complexes that make essentialmetals unavailable to the organisms. Therefore, bacterial proliferationin the presence of cyanide requires specific metal uptake systems.The strategy for iron uptake consists of the production of organiccompounds, generically called siderophores, which strongly bindiron and are subsequently transported and assimilated (for reviews,see references 4 and 16). Finally, a cyanotrophic microorganismrequires an assimilatory pathway able to convert cyanide intoammonium (for reviews, see references 12 and 38 and the referencestherein). To summarize, the biological assimilation of cyanideneeds, at minimum, the concurrence of three separate processes,i.e., a cyanide resistance mechanism, a system for metal acquisition,and a cyanide assimilation pathway. Although all of these factorsin conjunction with one another have never been taken into account,a number of microorganisms that are able to degrade cyanideand its metal complexes have been described to date (6, 12,20, 22, 38).

From a chemical point of view, the biological treatment of industrialeffluents contaminated with cyanide requires an alkaline pHin order to avoid the formation of the volatile HCN (pK_a = 9.2).In addition, since cyanide is known to react chemically withsome keto groups (the Kiliani reaction), the use of glucoseor similar C sources should be avoided. Previous works havereported microbial degradation of cyanide at neutral or acidicconditions (6, 38, 46). Strains adapted to survive in the presenceof cyanide at alkaline pH but unable to degrade it have alsobeen described (13). By contrast, references describing cyanidebiodegradation at alkaline pHs are scarce. One of them refersto the fungus Fusarium solani (13), which uses cyanide as thesole nitrogen source. The bacterium Burkholderia cepacia isable to consume cyanide optimally at pH 10, but it needs glucoseas a carbon source and is relatively sensitive to metal ions,such as iron and copper (1, 2). In this report, we report forthe first time a bacterial isolate (Pseudomonas pseudoalcaligenesCECT5344) that is able to grow by using cyanide as the solenitrogen source under alkaline conditions and acetate as theC source. The regulation of the process and the assimilationof alternative nitrogen sources are also described. In addition,this bacterium tolerates up to 30 mM of free cyanide and mayalso use several cyano-metal complexes, even in the presenceof ammonium or nitrate. Therefore, this strain offers new perspectivesin the bioremediation of industrial effluents contaminated withcyanide.

MATERIALS AND METHODS

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Materials and Methods

Bacterial isolation.
P. pseudoalcaligenes CECT5344 was isolated after an enrichmentcultivation procedure. Basically, the medium used was the M9minimal medium (30) prepared without ammonium and citrate, atpH 9.5, with 2 mM NaCN and 50 mM acetate as the sole added nitrogenand carbon sources, respectively. The medium was inoculatedwith sludge from the left margin of the Guadalquivir River downstreamfrom the city of Córdoba and incubated in an Erlenmeyerflask at 30°C in a rotatory shaker. Cyanide was completelydepleted after 2 weeks, and the process was repeated four timesby reinoculation in fresh medium with 1% (vol/vol) of the previouslygrown culture. Samples of the enriched culture were plated onLuria-Bertani (LB) medium solidified with 1.8% Bacto agar (Difco),and individual colonies were purified and tested for axenicgrowth in liquid cultures with cyanide as the sole nitrogensource. Only one type of colony was able to assimilate cyanide,thus producing an axenic culture from which a single colonywas selected, obtained in pure culture, and kept for furtheranalysis.

Culture media.
The bacterium was grown either in M9 minimal medium (withoutammonium and citrate) adjusted to pH 9.5 or in LB medium ona rotatory shaker at 230 rpm and 30°C. Unless otherwisestated, 50 mM acetate was used as the carbon source. The appropriatenitrogen sources were added from sterilized stocks at the indicatedconcentrations. When the residue generated in the jewelry electroplatingbaths (hereafter called residue) was used as the nitrogen source,only the free cyanide was taken into account, because the methodused routinely for cyanide determination detects only free cyanide.Moreover, the residue was used mainly to demonstrate the applicabilityof the biodegradation technology (see Chemicals section below).

For the detection of siderophores, the chrome azurol S agarmedium was prepared as previously described (42). The plateswith Prussian blue {Fe₄[Fe(CN)₆]₃} were prepared by adding 0.75mM soluble Prussian blue (Fluka) to M9 agar plates (pH 7) containing50 mM acetate and, where indicated, 6 mM ammonium. The Prussianblue was added either to the whole plate or as top agar dissolvedin 1% agar.

Taxonomic position and analysis of 16S rRNA gene sequence of P. pseudoalcaligenes CECT5344.
Genomic DNA from the strain CECT5344 was obtained by using aWizard kit (Promega) following the instructions of the manufacturer.The gene encoding the small ribosome subunit (16S rRNA) wasentirely amplified by PCR with the universal eubacterial primersEUB-8F and EUB-1492R targeting positions 8 and 1492, respectively,of the Escherichia coli 16S rRNA numbering. PCR products werethen cleaned by using a QIAGEN kit according to the manufacturer'sprotocol and sequenced in both directions by the Big Dye (AppliedBiosystems) method.

Forward and reverse sequences thus obtained were aligned andmanually checked to produce a high quality consensus sequence.After a BLAST analysis of the sequence, the isolate was tentativelyidentified as P. pseudoalcaligenes (99% similarity).

Analytical determinations.
Bacterial growth was monitored by determining the absorbanceat 600 nm. Nitrate, nitrite, and ammonium concentrations weredetermined as previously described (9). The ammonium concentrationwas also determined by using an ammonium-sensitive electrode.The free cyanide concentration was determined colorimetrically(5). The formamide concentration was calculated by the methoddescribed by Powell et al. (35) and modified by Schygulla-Banek(43). The protein concentration was determined by using a modifiedversion of the Lowry method (44).

Cell dry weights were obtained by drying the pellets at 100°Cto a constant weight, after centrifugation of the cultures.

Enzymatic activities.
Nitrate reductase activity was determined as previously described(8).

Cyanase was assayed by the method described by Anderson (3).

Cyanide oxygenase was assayed according to the method of Fernándezet al. (17), by monitoring the cyanide-dependent (0.1 to 2 mM)oxidation of NADH (0.2 mM) by cell extracts (up to 1 mg ml^–1).In addition to NADH, NADPH or H₄biopterin were also used. Theactivity was tested from pH 7 to 9 at intervals of 0.5 unitsby using the appropriate buffers (phosphate and Tris at 50 mMconcentrations). Cyanide and ammonium were also assayed at theend of the experiments and compared to control test tubes containingboiled extracts.

Cyanidase (also called nitrilase and cyanide dihydratase) wasassayed by measuring the ammonium formed from cyanide. The assaymixture contained, in a total volume of 5 ml, a 50 mM concentrationof buffer (either phosphate or Tris, pH 7 to 9), 2 mM cyanide,and cell extract (up to 5 mg of protein). The same protocolwas used to detect cyanide hydratase activity, but in this caseformamide was measured instead of ammonium.

Formamide hydrolase was measured by monitoring the enzymaticconversion of formamide into ammonium as described above forcyanidase.

ß-Cyanoalanine synthase was assayed as described byDunnill and Fowden (14) with cysteine, serine, or O-acetyl-serineas the substrate.

Rhodanese activity was determined according to the method ofRay et al. (37).

Chemicals.
The residue from the electroplating industry was kindly suppliedby GEMASUR (Córdoba, Spain). The total cyanide concentrationvaries from 15 to 37 M, depending on the sample (47) whereasthe free cyanide varies from 25 mM to 1 M. The difference betweenfree and total cyanide is due to the presence of metals thatcomplex most of the cyanide. The analysis of the residue usedin this study revealed the presence of 0.76 M free cyanide (20g liter^–1). The concentrations of the most abundant metals,estimated by atomic absorption, were 3.5 mM for Fe, 0.6 mM forCu, 0.76 mM for Au, and 3.5 mM for V (B. Vallejo-Pecharrománand M. D. Luque de Castro, personal communication). The residueshowed a pH higher than 13. When used as the nitrogen source,the residue was diluted in the culture medium to give the desiredconcentration of free cyanide (usually 2 mM, which correspondsto 2.63 ml of residue per liter of medium).

A 12.5 mM stock solution of [Cu(CN)₄]^–2 was prepared bymixing equal volumes of 100 mM KCN (sterilized by filtration)and 25 mM CuSO₄ (sterilized by autoclaving). When used as thenitrogen source, this solution was diluted in the culture mediumto give the desired concentration.

The rest of the reagents were all of the maximal purity commerciallyavailable.

Nucleotide sequence accession number.
The nucleotide sequence reported in this paper, correspondingto the 16S RNA gene of P. pseudoalcaligenes CECT5344, has beenannotated and deposited in the EMBL, DDBJ, and GenBank nucleotidesequence databases under the accession number AJ628163.

RESULTS AND DISCUSSION

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Results and Discussion

Although biological removal of cyanide from industrial wastesis an attractive alternative to its chemical destruction, ithas been applied only in a few cases, e.g., the Homestake mine(49). The oxidation numbers of C and N in cyanide are C(+II)and N(–III), that is, equivalent to CO (or formic acid)and NH₃, respectively. According to the current knowledge, allof the microorganisms able to assimilate cyanide can use itonly as a nitrogen source, but not as the sole carbon source.By contrast, under anaerobic conditions, cyanide may be usedas an alternative electron acceptor producing methane and ammonia,either in mixed (45) or axenic (25) cultures.

The main factor accounting for cyanide elimination in biodegradationprocesses is the evaporation of HCN due to the neutral or acidicconditions of the medium (36). In addition, the formulationof the medium must be taken into account because cyanide canreact with aldehydes and ketones (i.e., glucose), masking thebiological degradation (Kiliani reaction). In this work, wereport for the first time the degradation of cyanide by a bacteriumin minimal medium without glucose and under alkaline conditions.The strain was isolated by enrichment cultivation in selectivemedia at pH 9.5 with 2 mM NaCN as the sole nitrogen source asdescribed in Materials and Methods. The bacterium was slightlycurved, rod-shaped (0.4 by 1.5 µm), and gram negative.It was able to use ammonium, nitrate, nitrite, cyanoacetamide,nitroferricyanide, several amino acids (Ser, Thr, Ala, Met,Glu, and Gln), and some cyanide-metal complexes as nitrogensources, in addition to cyanide. For carbon sources, the bacteriumwas able to use acetate, D,L-malate, and glucose. In all cases,metabolism was strictly aerobic. The bacterium does not containdetectable plasmids. In LB agar plates, the strain was resistantto the following antibiotics: tetracycline (10 µg ml^–1),ampicillin (100 µg ml^–1), chloramphenicol (10 µgml^–1), and spectinomycin (200 µg ml^–1). Bycontrast, it was sensitive to kanamycin (25 µg ml^–1),streptomycin (200 µg ml^–1), and gentamicin (20 µgml^–1). The optimum pH for growth, as deduced from thehighest growth rate and shortest lag phase, was 9, with 50 mMacetate and 5 mM ammonium as the respective carbon and nitrogensources. The bacterium has been classified as P. pseudoalcaligenesby comparison of the 16S RNA gene sequence with existing sequencesand deposited in the Colección Española de CultivosTipo (Spanish Type Culture Collection) as strain CECT5344.

Cyanide degradation by P. pseudoalcaligenes CECT5344 seems tobe an assimilative process since cyanide removal coincided withthe exponential growth phase as well as with the maximal rateof oxygen consumption. This finding was further corroboratedby using L-methionine-D,L-sulfoximine, which irreversibly inhibitsammonium assimilation at the level of the glutamine synthetase(10, 33). In the presence of L-methionine-D,L-sulfoximine, restingcells grown with cyanide catalyzed the stoichiometric conversionof cyanide into ammonium, which was accumulated in the culturemedium (data not shown). By contrast, ammonium was not detectedin the growth medium of untreated cultures.

P. pseudoalcaligenes CECT5344 tolerated an initial pH of upto 11.5 and a NaCN concentration of up to 30 mM. Nevertheless,due to the trapping of atmospheric CO₂, the pH of the mediumbecomes acidified during the experiments and some cyanide evaporates.To avoid this problem, the cyanide was added to cells preculturedwith a limiting amount of ammonium (2 mM). The addition of cyanideto ammonium-limited cultures has been used previously to induceenzymatic activities in Pseudomonas fluorescens (27, 29, 41).Under these conditions, the strain CECT5344 consumed 1 mM NaCNin 5 h (Fig. 1). Moreover, the cell growth was nearly the samewith NaCN as with NH₄Cl, while it was higher with the residue(data not shown). Under these experimental conditions, neithercyanide (Fig. 1) nor ammonium (data not shown) evaporated fromnoninoculated control flasks. These results clearly show thatcyanide can be fully degraded by this strain and suggest thatthe strain may also use cyano-metal complexes, according tothe high quantity of heavy metal-cyanide complexes present inthe residue from the jewelry industry, in addition to free cyanide.A further indication for the utilization of complexes was theappearance of a reddish precipitate at the end of the exponentialgrowth phase, which probably corresponds to ferric iron. Therefore,the degradation of several metal complexes by P. pseudoalcaligenesCECT5344 was investigated. The strain was able to use as thesole nitrogen source the cyanocomplexes of Fe(II) and Cu(II)at pH 9.5 (Fig. 2) and also at pH 7.5 in the presence of 200µM 2,2'-bipyridyl, a strong iron chelator (data not shown).By contrast, the Zn(II) complex was poorly used at both pH values.P. pseudoalcaligenes CECT5344 was also able to use nitroprussideas the sole nitrogen source (data not shown). The productionof siderophores was checked with plates with chrome azurol Sagar (42), where the colonies formed a colorless halo due tothe elimination of iron from the green complex. In addition,the bacteria grew on agar plates containing Prussian blue asthe sole added iron source. The first microbial isolate foundto grow on a metal-cyanide complex was P. fluorescens with Ni(CN)₄^2–(41). The authors demonstrated that Cu(CN)₄^2– is alsoa suitable nitrogen source for P. fluorescens, but to a lesserextent than the Ni complex. P. pseudoalcaligenes CECT5344 alsoused K₂Cu(CN)₄ as a nitrogen source (Fig. 2) but, as in thereference cited above, it was a poorer substrate than ammoniaor free cyanide. The cyanide-metal complexes can be dividedinto weak acid-dissociable (WAD) and strong complexes. In WADcomplexes, cyanide is readily released from the complexes whenthe pH is lowered to 4.5 to 6. Therefore, WAD refers to anyfree cyanide already present and cyanide released from nickel,zinc, copper, and cadmium complexes (but not from iron or cobaltcomplexes). WAD cyanide is generally considered to be the bestcurrent measure for assessing human and animal toxicity. Theiron complexes are very stable and for this reason less toxicand more recalcitrant. In this sense, it was not surprisingthat K₃Fe(CN)₆ was the worst nitrogen source used by the CECT5344strain. The stability of the complexes depends on the pH; theyare, in general, more stable as the pH increases. Strikingly,P. pseudoalcaligenes CECT5344 degraded ferricyanide faster atpH 9.5 than at pH 7, in contrast to F. solani (6) and P. fluorescens(15) which use ferrocyanide only at pH 5 but not at alkalinepHs. Probably, the optimum pH for degrading metal-cyanide complexesis the result of a balance between the stability of the complexand the optimum pH for growth. Interestingly, an almost lineargrowth with cyano-metal complexes (Fig. 2) was observed, whichsuggests that some medium component is limiting or that somecellular component can't be synthesized under these circumstances.In any case, this bacterium is able to use both free cyanideand its metal complexes at alkaline pHs, thus providing a clearadvantage since both cyanide volatilization and precipitationof its metal complexes are prevented. In addition, the toleranceof the bacterium to heavy metals is also a clear improvementover other strains that can degrade cyanide under alkaline conditionsbut are very sensitive to the presence of heavy metals (2).

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FIG. 1. Utilization of cyanide by P. pseudoalcaligenes CECT5344. Cells were precultured with 2 mM ammonium to the stationary phase (A₆₀₀ ${approx}$ 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 ( ${blacktriangleup}$ ) were measured. The concentration of cyanide in a noninoculated flask containing culture medium ( ${triangleup}$ ) was measured at the same times. Data are from a representative experiment.

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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.

In order to investigate the regulation and induction of thecyanide assimilation process in P. pseudoalcaligenes CECT5344,cyanide removal was checked in resting cell experiments withcells grown with several nitrogen sources. Cyanide-grown cellsbegan to consume cyanide around 60 min before ammonium-growncells (Fig. 3). Nitrate- and nitrite-grown cells behaved similarlyto ammonium-grown cells (data not shown). These results suggestthat the cyanide assimilation process is inducible. Curiously,the cyanide-grown cells needed a lag period to start cyanideconsumption, unless they were resuspended in the same supernatantmedium after centrifugation (data not shown). This result suggeststhat an extracellular metabolite may be necessary for cyanideremoval. In this sense, both ${alpha}$ -keto acids and siderophores havebeen described as putative components for cyanide assimilationby P. fluorescens NCIMB11764 (11, 27). Whether this is the casefor P. pseudoalcaligenes CECT5344 deserves further investigation.Preliminary results show the accumulation of ketoacids, mainly2-ketoglutarate, at the beginning of the exponential growthphase with cyanide as the sole nitrogen source.

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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.

Both the enhancement of biodegradation by second substratesand the inhibition of the process by alternative nutrients havebeen described widely in the literature. These effects are importantnot only regarding the applicability of the process, but alsofrom a regulatory point of view. Therefore, the effect of alternativenitrogen sources on cyanide removal was investigated. To avoidcyanide volatilization, cells grown with a limiting amount ofammonium (2 mM) were used as mentioned above. Cyanide was consumedin the presence of either ammonium (Fig. 4A), nitrate (Fig.4B), or nitrite (data not shown). Nevertheless, cyanide wasnot consumed by the bacterium in LB medium (data not shown).Ammonium is usually the preferred nitrogen source for bacteria.Consequently, ammonium (or some metabolite produced from it)usually exerts a negative regulatory effect on the assimilationof several nitrogen sources (9, 10). In this sense, the inhibitionof cyanide assimilation by ammonium has been described in theliterature and seems to be a general fact for cyanotrophic organisms(21, 22, 46). However, this is not the case for the CECT5344strain, because the bacterium was able to assimilate both compoundssimultaneously (Fig. 4A). Accordingly, the lag phase observedafter the addition of cyanide to ammonium-growing cells hasto be attributed to the need for the induction of other processes,i.e., the mechanism of resistance to cyanide, the acquisitionof metals, or both. The inhibition of nitrate assimilation bycyanide has not been reported to date, whereas the in vitroinhibition of the assimilatory nitrate reductases by cyanideis well known. The nitrate reductase of this strain was alsoinhibited by cyanide in vitro (data not shown), giving an explanationfor the inhibitory effect of cyanide on nitrate assimilation(Fig. 4B). Once more, and from the applicability point of view,P. pseudoalcaligenes CECT5344 seems to be superior to othercyanotrophic strains so far described because it can assimilatecyanide in the presence of ammonium or nitrate.

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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.

The enzymatic reactions for cyanide degradation can be categorizedinto four types: substitution/addition, hydrolysis, oxidation,and reduction (38). The hydrolytic pathways can be of two types;in one, ammonium forms directly by the addition of two moleculesof water, whereas in the second, water is added sequentiallywith formamide as the intermediate. The generation of formamidefrom cyanide, catalyzed by cyanide hydratase activity, was firstdescribed for pathogenic fungi of cyanogenic crops (18). Theaddition of a second molecule of water catalyzed by an amidaseleads to the formation of ammonium that may be used as the nitrogensource. This pathway is exclusive for fungi, whereas cyanidedihydratases have been described for both fungi and bacteria(23). P. pseudoalcaligenes CECT5344 was unable to use formamideas a nitrogen source. In addition, enzymatic activities catalyzingeither the formation of formamide from cyanide, the hydrolysisof formamide into ammonium, or the direct conversion of cyanideinto ammonium were not detected in cell extracts from cellsgrown with cyanide. Therefore, despite the fact that the bacteriumwas able to use some amides, such as cyanoacetamide, as a nitrogensource (data not shown), the existence of a hydrolytic pathwayfor the assimilation of cyanide in this strain seems to be unlikely.

Regarding to the substitution/addition pathways, the catalyzedreaction of cyanide with cysteine, serine, O-acetyl-serine,or thiosulfate was undetectable.

An oxidative pathway was first proposed by Harris and Knowles(21), and later the incorporation of oxygen into the substratewas clearly demonstrated (48). Theoretically, the action ofa monooxygenase can produce cyanate, which in turn is the substrateof the cyanase that catalyzes the formation of ammonium andCO₂ from cyanate and bicarbonate. Therefore, a pathway composedof two enzymes was originally proposed. The cyanide oxygenase,measured either by oxygen or NADH consumption, was hardly detectedin cell extract of P. pseudoalcaligenes CECT5344 grown withcyanide. Only cell extracts obtained from cells treated withsuccessive additions of 1 mM cyanide showed some cyanide-stimulatedconsumption of NADH (data not shown). Nevertheless, ammoniawas never detected as a reaction product, as described for otherbacteria (17, 21, 29), even in the presence of H₄biopterin ascofactor. By contrast, high levels of cyanase activity weremeasured with cells grown with cyanide or cyano-metal complexesas nitrogen sources (more than 400 U g^–1 of protein).As this activity was not present in cells grown with ammoniumor nitrate (data not shown), a pathway including cyanate asthe intermediate can be proposed for the CECT5344 strain. Aswith P. fluorescens NCIMB11764 (26), the cyanase activity ofCECT5344 was also induced by cyanate (data not shown). In contrast,the cyanase of P. fluorescens is repressed by ammonium and isnot induced by cyanide. Further experiments demonstrated thatcyanase activity of P. fluorescens is not essential for cyanotrophicgrowth (29). Unfortunately, the induction of the cyanase activityof P. pseudoalcaligenes CECT5344 was not exclusive for mediacontaining cyanide, since the activity was also detected incells grown with urea and even in nitrogen-free medium, althoughat lower levels (around 50 U g^–1 of protein). Therefore,other routes for the assimilation of cyanide cannot be ruledout, as proposed for P. fluorescens NCIMB11764, which assimilatescyanide through several alternative routes (28, 29). Experimentsare in progress to generate both random cyanide assimilationmutants and a site-directed mutant of the cyanase gene in orderto elucidate the pathway used by this bacterium.

Finally, we note that P. pseudoalcaligenes CECT5344 is the firstbacterium described to date that is able to degrade, in minimal-mineralmedium without glucose, cyanide and cyano-metallic complexesunder alkaline conditions. This capability, in combination withthe ability to use cyanide in the presence of heavy metals,ammonium, or nitrate and the unusual resistance to cyanide,make this strain a very good candidate for the biotreatmentof cyanurated residues.

ACKNOWLEDGMENTS

This work was funded by the Ministerio de Ciencia y Tecnología(BMC2002-04126-C03-01 and -03) and the FEDER (1FD97-0653). V.M.L.-A.was the recipient of a fellowship from the MEC (Spanish Ministryof Education), and M.D.R. holds a postdoctoral fellowship fromthe Junta de Andalucía (Andalusia, Spain).

We thank M. Dolores Luque de Castro and B. Vallejo-Pecharrománfor help with analytical determinations and for discussionsof the results. We also thank GEMASUR for providing the residuefrom the jewelry electroplating industry and for its fruitfulcollaboration.

FOOTNOTES

* Corresponding author. Mailing address: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Avenida de la Universidad SN, E-10071 Cáceres, Spain. Phone: 34 927257160, ext. 1339. Fax: 34 927257161. E-mail: rblasco{at}unex.es.

REFERENCES

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