INTRODUCTION

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Thiocyanate (NC---S) is a C₁ sulfur species which can be produced both as a natural compound (mainly in biological cyanide detoxification processes) and as a waste product, largely by coke and metal plants (23, 46). Microorganisms can utilize thiocyanate as an energy, carbon, nitrogen, or sulfur source after it is hydrolyzed to sulfide, ammonia, and CO₂. Like degradation of other C₁ sulfur compounds, CNS degradation requires the primary action of a specific enzyme(s) to release the sulfan atom for further microbial oxidation (23, 35). Currently, two distinct pathways of microbial degradation of thiocyanate are recognized, and either H₂S or NH₃ is the first product. For the autotrophic thiocyanate-oxidizing bacterium Thiobacillus thioparus (formerly known as Thiobacillus thiocyanooxidans) it has been postulated that thiocyanate is degraded via cyanate (NC-O), which is converted to ammonia and CO₂ by the specific enzyme cyanase (13, 47). The liberated sulfide is utilized as an electron donor and energy source:

(1)

(2)

(3)

       [HCO₃]

Apparently, the first enzyme in this pathway should be able to break the C---S bond. Nothing is known yet about the identity of such an enzyme(s). Moreover, no direct proof of production of cyanate as an intermediate during bacterial thiocyanate degradation has been obtained for this autotrophic bacterium so far. To our knowledge, formation of cyanate from thiocyanate has been observed only once in a mixed bacterial population from thiocyanate-degrading sludge (14). Another strain of T. thioparus degrades thiocyanate via carbonyl sulfide (O==C==S) by using the specific enzyme thiocyanate hydrolase, which has substantial homology to nitrile hydratase (19, 21, 22). Such homology is hardly surprising, assuming that both enzymes break the nitrile bond (NC). The COS produced is hydrolyzed to sulfide and CO₂ (the enzymology of this reaction remains to be investigated), and sulfide is eventually oxidized to sulfate:

(4)

(5)

A similar two-stage hydrolysis via COS has been observed during carbon disulfide (S==C==S) degradation by T. thioparus TK-m, which is also able to oxidize thiocyanate (33). It seems likely that in this bacterium hydrolytic cleavage of CS₂ and CNS to sulfide proceeds through the same pathway (i.e., via COS).

Oxidation of thiocyanate to sulfate, ammonia, and CO₂ yields eight electrons. Among the neutrophilic sulfur-oxidizing bacteria, the ability to grow with thiocyanate as an electron donor for energy generation and CO₂ fixation is limited to a few strains of T. thioparus (7, 12, 13, 20, 32, 33, 47) and Thiobacillus denitrificans (7). The ability to utilize thiocyanate as an electron donor has recently been claimed for a newly described Paracoccus species, Paracoccus thiocyanatus (18), but it is difficult to analyze the evidence because no actual data for growth and oxidation kinetics were provided in the paper. The potential for active thiocyanate degradation has also been described for two bacterial consortia consisting of Pseudomonas and Acinetobacter species (3) and of Pseudomonas and Bacillus species (30). Both of these consortia were able to grow on thiocyanate mineral media at neutral pH values and produced sulfate, like the T. thioparus strains. However, no evidence concerning the ability of such consortia to grow autotrophically with other reduced sulfur compounds was presented. Although the possible existence of autotrophic thiocyanate specialists which utilize only thiocyanate as an energy source cannot be ruled out, so far all pure cultures of thiocyanate autotrophs are represented by sulfur bacteria able to grow on other reduced inorganic sulfur compounds. Therefore, whether the thiocyanate-oxidizing consortia may have contained a fraction of sulfur-oxidizing autotrophs morphologically indistinguishable from the heterotrophic components is an interesting question.

In addition to being oxidized for energy transduction purposes, CNS can be metabolized as a nitrogen source. Several neutrophilic heterotrophic bacteria (Arthrobacter sp., Pseudomonas spp., Methylobacterium thiocyanatum) able to utilize the nitrogen atom from thiocyanate were isolated from different sources which may have contained thiocyanate (2, 11, 28, 41, 42, 45). It has been suggested that such bacteria employ the same primary thiocyanate degradation pathways as autotrophs (e.g., either cyanate pathways or COS pathways), but again, no direct proof of accumulation of these intermediates has been presented. In these cases, ammonium produced from thiocyanate is utilized as the nitrogen source, while the reduced sulfur can be utilized as a sulfur source but not as the energy source.

The thiocyanate-oxidizing T. thioparus strains are likely to be able to utilize the nitrogen of thiocyanate as an N source during growth solely on CNS. However, there is no evidence concerning whether autotrophic sulfur bacteria or any other chemolithoautotrophs are able to assimilate thiocyanate nitrogen but are not able to use it as an electron donor, as is the case for the heterotrophic thiocyanate-utilizing bacteria.

CNS-containing wastewaters can be treated by acclimated bacterial sludge containing a high density of T. thioparus-like thiocyanate-oxidizing autotrophs (3-5, 15, 16, 32) or heterotrophs if an alternative carbon source is available (17). Such biosystems proved to be able to remove millimolar amounts of CNS at neutral or slightly alkaline pH values. The possibility of bioremoval of thiocyanate under highly alkaline conditions was not investigated.

This study demonstrated that thiocyanate can be used as the nitrogen source and as the energy source under highly alkaline conditions by alkaliphilic obligately organoheterotrophic and obligately lithoautotrophic sulfur-oxidizing bacteria, respectively, isolated from natural alkaline environments, such as those encountered in soda lakes.

	MATERIALS AND METHODS

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Samples. Four composite samples were used for enrichment of thiocyanate-degrading alkaliphiles. Two soil samples were composed of 8 to 10 subsamples of soda solonchak soils collected near soda lakes in Burjatia (southeast Siberia) and Kenya (East African Rift Valley). The other two samples were composed of five to eight sediment subsamples collected from soda lakes in Burjatia and Kenya. The pH values of the subsamples varied from 9.7 to 11.0, and the salt contents ranged from 0.05 to 20% (wt/vol).

Bacterial strains. Pure cultures of alkaliphilic heterotrophic and chemolithoautotrophic sulfur-oxidizing bacteria described previously (36-40) were tested for the ability to utilize CNS as a nitrogen or energy source. The heterotrophs used are members of the Halomonas-Deleya cluster in the gamma subdivision of the division Proteobacteria (gamma-Proteobacteria). The autotrophs belong to the new genera Thioalkalimicrobium and Thioalkalivibrio, also in the gamma-Proteobacteria. Some of the properties of the alkaliphilic autotrophs are shown in Table 1.

Media and culture conditions. Mineral base medium containing 0.6 M total Na⁺ as sodium carbonates and sodium chloride (pH 10) (38) was used in all growth experiments. It contained (per liter) 21 g of sodium carbonate, 9 g of sodium bicarbonate, 5 g of NaCl, 1 g of K₂HPO₄, and 0.5 g of KNO₃. A trace elements solution (31) (2 ml/liter) and Mg salts (0.5 mM) were added after sterilization. KCNS, sodium thiosulfate, and sodium acetate were also supplied after sterilization from filter-sterilized 2 M stock solutions. CNS was fairly stable under the alkaline conditions used; no chemical decomposition was observed during more than 1 month of incubation of uninoculated medium at pH 9.8 to 10.2. Media with higher salt contents (up to 4 M Na⁺; pH 10.0 to 10.1) were prepared by proportionally increasing the concentration of sodium carbonates.

Enrichment procedure and isolation of pure cultures. Heterotrophic alkaliphiles utilizing CNS as a sole nitrogen source were enriched on a mineral base medium (pH 10.0) supplemented with 20 mM acetate as the carbon and energy source and 5 mM KCNS. Chemolithoautotrophic alkaliphilic bacteria utilizing the nitrogen from KCNS were enriched on the same medium, except that the acetate was replaced by 40 mM thiosulfate. After complete disappearance of CNS, several subcultures (1:100 dilution) were made. The cultures exhibiting stable thiocyanate disappearance were plated onto solid medium having the same composition, and different colonies were then isolated and checked for the ability to use thiocyanate in liquid culture. Media without a nitrogen source were used as controls.

Oxygen uptake experiments. Cells of autotrophic thiocyanate-oxidizing alkaliphiles were obtained from the cultures grown at pH 10.0 with thiocyanate or thiosulfate as the electron donor. After centrifugation, the cells were washed and resuspended at a protein concentration of about 10 mg ml¹ in sodium carbonate buffer (pH 10.0) (see below). The respiration activity was tested at pH values of 6.0 to 11.5 in buffers containing 0.6 M total Na⁺ and 50 mM KCl. For pH 6 to 8, 0.1 M HEPES-NaOH-NaCl was used; for pH 8.2, freshly prepared NaHCO₃ was used; and for pH 9 to 11.5, a combination of Na₂CO₃ and NaHCO₃ was used. The carbonate dependence of respiration was examined by using 0.1 M Tris-HCl-0.6 M NaCl at pH 9 to 10. The respiration rates were measured in a 5-ml thermostat-equipped chamber mounted on a magnetic stirrer and fitted with a Clark type of dissolved oxygen probe (Yellow Spring Instruments Co., Yellow Springs, Ohio). Stock solutions of sodium sulfide, polysulfide (S₆²; prepared by autoclaving a 0.2 M sodium sulfide solution with a large excess of powdered sulfur), and sulfite were prepared anaerobically in 0.1 M Tris-HCl with 5 mM EDTA to prevent autooxidation and were introduced into the chamber at final concentrations of 25 to 50 µM. Elemental sulfur was added from a saturated solution in acetone at a final concentration of 70 µM. CS₂ was added from a concentrated ethanol solution at final concentrations of 0.05 to 2 mM. COS, methane thiole (CH₃SH), and dimethyl sulfide [(CH₃)₂S] were supplied as saturated water solutions at a final concentration of 100 µM. Thiosulfate and tetrathionate were added at final concentrations of 50 to 200 µM from freshly prepared concentrated stock solutions in water. Kinetic parameters (V_max and K_s) were calculated from V-[S] plots.

Experiments with washed cells. The kinetics of degradation of various substrates by washed cells obtained either from batch cultures or from chemostat cultures was studied by using 10-ml serum bottles containing 2 ml of suspension, in which the cell protein concentration ranged from 0.1 to 1 mg ml¹. Anaerobic experiments were conducted after removal of oxygen with evacuation and argon flushing (five cycles). When CS₂ (2 mM) and COS (2 mM) were used as substrates, gray butyl rubber stoppers were used instead of black stoppers.

Analysis. Thiocyanate was analyzed colorimetrically as ferric thiocyanate (34). The same method was employed to determine the elemental sulfur content after extraction with acetone and cyanolysis. Thiosulfate, tetrathionate, and trithionate contents were measured by cyanolysis (24). Sulfate content was measured by a turbidimetric method (6). Sulfide content was determined as described by Trüper and Schlegel (43) after precipitation as ZnS. NH₄⁺ content was measured by a phenol-hypochlorite colorimetric procedure described by Weatherburn (44). Cell protein content was analyzed by the Lowry method. When elemental sulfur was produced, it was removed by extraction with acetone prior to alkaline digestion of the cell pellet for the protein assay.

Electron microscopy. For total-cell preparations, washed cells were directly fixed with formaldehyde (final concentration, 2.5%) in liquid medium and then positively stained with 1% phosphotungstic acid. Samples used for ultrathin sectioning were centrifuged, washed and resuspended in fresh 0.6 M NaHCO₃ (pH 8), fixed with 1% (final concentration) OsO₄ for 12 h at 4°C, dehydrated, and embedded in resin. Thin sections were stained with uranyl acetate and lead citrate. To detect intracellular accumulation of elemental sulfur, cells were sedimented, stained with a solution containing 2% AgNO₃ and 2% glutaraldehyde for 10 h, and then fixed with OsO₄. Postsectional staining was omitted in this case.

Genetic analysis. Isolation of DNA, determination of the G+C contents of DNA preparations, and DNA-DNA hybridization were performed as described by Marmur (27) and De Ley et al. (8).

Amplification and sequencing of 16S rRNA genes. For amplification and sequencing of 16S rRNA genes, DNA was obtained by standard phenol-chloroform extraction. The 16S rRNA genes were selectively amplified by using primers 5'-AGAGTTTGATCCTGGCTCAG-3' (forward) and 5'-TACGGTTACCTTGTTACGACTT-3' (reverse). PCR products were purified from low-melting-point agarose by using a Wizard PCR-Prep kit (Promega) according to the manufacturer's instructions. Almost complete sequencing (1,400 to 1,450 nucleotides) was performed by using a Silver Sequencing kit (Promega) according to the manufacturer's instructions, with minor modifications.

16S ribosomal DNA sequence analysis. The sequences were aligned manually with sequences obtained from the database consisting of small-subunit rRNAs collected from the EMBL international nucleotide sequence library. The sequences were compared with the sequences of members of the Proteobacteria. Regions that were not sequenced in one or more reference organisms were omitted from the analyses. Pairwise evolutionary distances (expressed in estimated number of changes per 100 nucleotides) were computed by using the method of Jukes and Cantor. A phylogenetic tree was constructed by the neighbor-joining method. Bootstrap analysis (100 replications) was used to validate the reproducibility of the branching pattern of trees.

Nucleotide sequence accession numbers. 16S ribosomal DNA sequence data for strains ARh 1 and ARh 2 have been deposited in the EMBL and GenBank databases under accession numbers AF151432 and AF302081, respectively.

	RESULTS

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CNS uptake in pure cultures of alkaliphilic sulfur-oxidizing bacteria. Twenty-five strains of heterotrophic tetrathionate-forming alkaliphilic bacteria and 30 strains of obligately autotrophic sulfur-oxidizing alkaliphilic bacteria isolated previously from alkaline environments (35-39) were tested to determine their abilities to use thiocyanate as a sole source of nitrogen while they were growing with acetate and with thiosulfate, respectively, as the energy source.

Enrichment and isolation of alkaliphilic bacteria utilizing CNS as the nitrogen source. (i) Heterotrophic alkaliphiles. Incubation of samples composed of subsamples of the Kenyan soda lake sediments and subsamples of the Kenyan and Siberian soda soils with 40 mM acetate and 5 mM thiocyanate at pH 10.0 resulted in complete disappearance of CNS within 2 weeks. No consumption of thiocyanate was detected in cultures inoculated with composite samples obtained from the Siberian soda lake sediments. Plating of the cultures obtained after several successive passages in liquid medium resulted in domination by one or two morphological colony types in all three enrichments. Finally, we obtained five pure cultures (strains AGSCN 1 through AGSCN 5) that were able to utilize CNS as a nitrogen source while growing with acetate at pH 10.

View this table: [in this window] [in a new window]   TABLE 2.   Heterotrophic alkaliphilic isolates that utilize CNS as a nitrogen source

(ii) Obligately autotrophic sulfur-oxidizing alkaliphiles using CNS as the N source. During incubation of the composite soda lake samples with thiocyanate as the sole source of energy and nitrogen at pH 10, two types of obligately lithoautotrophic sulfur-oxidizing alkaliphiles were enriched. One type was bacteria able to utilize thiocyanate as the N source during growth with thiosulfate as the energy source at pH 10. The other type was bacteria able to utilize thiocyanate as both the energy source and the nitrogen source (see below).

Alkaliphilic chemolithoautotrophic thiocyanate-oxidizing sulfur bacteria. (i) Enrichment and isolation of pure cultures. Chemolithotrophic alkaliphilic bacteria able to grow solely on thiocyanate were enriched on mineral soda medium (pH 10.0) supplemented with 10 to 12 mM thiocyanate as the electron donor and source of nitrogen. At higher thiocyanate concentrations (20 to 40 mM) enrichments were negative. Positive enrichments were obtained with the sediments from Kenyan and Siberian soda lakes but not from the soil samples. The Kenyan culture developed more rapidly and consumed 11 mM thiocyanate within 10 days. The Siberian culture started to grow only after a long lag phase and consumed 10 mM thiocyanate within 18 days. After several 1:100 transfers, two stable enrichment cultures were obtained. Both the Kenyan and Siberian cultures included large nonmotile rod-shaped cells in which sulfur was deposited and two or three types of small, actively moving vibrios which were numerically dominant in subsequent serial dilutions on mineral medium with thiocyanate.

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Phylogenetic tree showing the positions of thiocyanate-oxidizing alkaliphilic autotrophic strains ARh 1 and ARh 2 among the sulfur-oxidizing species in the gamma-Proteobacteria. The numbers at the branching points indicate the bootstrap values. Reference sequences were obtained from the GenBank, EMBL, and Ribosomal Database Project databases. Scale bar, 5 base substitutions per 100 bases.

(ii) Characteristics of growth of strain ARh 1 on thiocyanate. Interestingly, strain ARh 1 grew faster with thiocyanate at pH 10.0 than with thiosulfate. A small amount of elemental sulfur, mostly intracellular, was produced during the active thiocyanate consumption phase. In the stationary phase, elemental sulfur disappeared. At this point about 90% of the thiocyanate sulfur was converted to sulfate. The bacterium was able to grow at initial thiocyanate concentrations of up to 30 mM but utilized no more than 10 to 15 mM. During growth on thiosulfate (with NH₃ as the N source), strain ARh 1 produced much more elemental sulfur during the initial growth phase than it produced with thiocyanate. When most of the thiosulfate was consumed, elemental sulfur began to disappear concomitant with a more rapid increase in biomass. The maximum specific growth rate and the growth yield obtained with thiosulfate were lower than the values obtained in thiocyanate-grown cultures (Table 5). Stable growth in continuous cultures at pH 10.1 was achieved only with low influent thiocyanate concentrations (5 to 6 mM). The reason for such behavior is discussed below. The maximum specific growth rate obtained with low thiocyanate concentrations in chemostats was twofold higher than the maximum specific growth rate observed in batch cultures (Table 5). With 6 mM thiocyanate and a dilution rate of 0.09 h¹, the cultures started to produce intracellular sulfur (2 to 3 mM) but still oxidized all of the thiocyanate. Washout began at dilution rates greater than 0.11 h¹.

2

1

1

1

1

(iii) Characteristics of growth of the vibrio-shaped strains on thiocyanate. Unlike rod-shaped strain ARh 1, the vibrio-shaped strains grew much more slowly with thiocyanate than with thiosulfate (Table 5). On the other hand, under certain conditions, the vibrio cultures utilized two to three times more thiocyanate than ARh 1 utilized. Maximum thiocyanate consumption was observed in cultures of strains ARh 2 and ARh 3 cultivated in the fed-batch mode. Neither of the vibrio strains produced elemental sulfur or other intermediate sulfur compounds during growth with thiocyanate. The sulfur from thiocyanate was almost quantitatively converted to sulfate. The growth efficiency of the alkaliphilic vibrios with thiocyanate was lower than that of strain ARh 1 (Table 5). Strain ARh 4 differed from the other ARh strains by its ability to grow fast on a thiosulfate-thiocyanate mixture. In thiocyanate-limited continuous cultures, stable growth of strain ARh 4 was achieved with 11 mM thiocyanate at pH 10.2. At a higher influent thiocyanate concentration (15 mM) the culture began to wash out at very low dilution rates (<0.02 h¹).

Thiocyanate degradation pathway in alkaliphilic bacteria. (i) Formation of cyanate from thiocyanate. We indicate above that the alkaliphilic strains which utilized thiocyanate as an N source did not excrete any intermediate nitrogen compounds into the medium and that all of the thiocyanate nitrogen was apparently used for assimilation. In contrast, the N balance in cultures and cell suspensions of all ARh strains grown with thiocyanate as the electron donor was far from complete. A maximum of only about 20% of the converted thiocyanate could be accounted for by assimilation plus excreted ammonia. Part of the ammonia, of course, was lost by volatalization from the liquid at pH 10. However, special experiments with sterile media demonstrated that stripping of NH₃ could have resulted in no more than 10 to 15% of the nitrogen loss that was not accounted for. Therefore, production of an intermediate nitrogen compound during thiocyanate dissimilation by alkaliphilic autotrophs had to be assumed. The most probable candidate species is cyanate (CNO), which has been suggested as an intermediate in one of the microbial thiocyanate degradation pathways (see reaction 1 above). Cyanate is known to be reasonably stable at high pH values but decomposes rapidly under highly acidic conditions (see reaction 3 above). Indeed, acidification to pH 2 to 3 by HCl allowed almost complete recovery of nitrogen as ammonium in the supernatants after degradation of thiocyanate by the ARh strains. Pure cyanate added to a sterile carbonate buffer and to media reacted in a similar way, instantly decomposing to ammonium after acidification. A specific colorimetric reaction with anthranilic acid confirmed the identity of the intermediate N compound as cyanate in all samples of culture supernatants with substantial N disbalance (see above). The amounts of cyanate formed during utilization of thiocyanate by cultures and cell suspensions of ARh strains are shown in Table 7. Additional tests confirmed that in carbonate-based media at pH 10 to 10.5 spontaneous decomposition of cyanate to ammonia was relatively slow (5 to 10% with 10 mM cyanate at 30°C within 24 h).

(ii) Ammonia toxicity at pH 10. The clear evidence that cyanate rather than ammonia accumulates during thiocyanate dissimilation by autotrophic alkaliphiles, in contrast to neutrophilic species, should have some explanations. One of the explanations could be that NH₃, which is absolutely dominant over NH₄⁺ at pH 10, is toxic and therefore accumulation of NH₃ should somehow be avoided. For example, the sulfur-oxidizing alkaliphiles belonging to the genera Thioalkalimicrobium and Thioalkalivibrio were unable to grow at pH 10 in the presence of NH₃ at concentrations higher than 2 to 3 mM (39). Therefore, the toxicity of ammonia for growth and activity of thiocyanate-utilizing alkaliphiles was tested at pH 10. While there was no inhibition of respiratory activity by NH₃ or CNO at concentrations up to 20 mM, ammonia inhibited growth of the autotrophic strains at relatively low concentrations (2 to 3 mM). Strain ARh 1 was the most sensitive ARh strain. The thiocyanate-oxidizing ARh strains were slightly more sensitive to ammonia than the heterotrophic alkaliphile AGCNS 1 was.

(iii) Cyanase activity. The activities of cyanase (the enzyme which splits cyanate into ammonia and CO₂ [see reaction 3 above]) were measured in cell extracts prepared from cells of different thiocyanate-utilizing alkaliphilic strains grown under different conditions. Considerably cyanase activity was found in (i) heterotrophic strain AGCNS 1 grown with thiocyanate as the N source and (ii) autotrophic strains ALRh and ARh 4 grown with thiosulfate as the energy source and ammonia, nitrate, or thiocyanate as the N source. Although constitutive, the cyanase activity in strain ALRh markedly increased in the presence of thiocyanate. In contrast, cyanase activity was undetectable in thiocyanate-dissimilating strains ARh 1, ARh 2, and ARh 3 and was extremely low in ARh 4 grown with thiocyanate as the energy source (Table 8). The activity was maximal at pH 8 and was HCO₃ dependent (K_s = 2 mM). At pH 7 and 10 the activities were 40 and 88% of the maximal activity, respectively.

(iv) Thiocyanate dissimilation. Previous experiments demonstrated that the primary reaction in thiocyanate dissimilation by the alkaliphilic autotrophs should be hydrolysis to cyanate and HS. In the neutrophilic T. thioparus strain, which may use the same thiocyanate degradation pathway, a substantial rate of sulfide production was observed when the cells were incubated with thiocyanate under anaerobic conditions. However, in our experiments performed with washed cells and cell extracts of the alkaliphilic ARh strains at pH 8.0 to 10.5, anaerobic thiocyanate degradation could not be detected. When ARh 1 and ARh 4 cells were crushed, the thiocyanate degradation activity decreased significantly. Nevertheless, it was still detectable after prolonged incubation (100 to 160 nmol mg of protein¹ h¹); 80 to 90% of this activity was recovered in the soluble fractions of the extracts after removal of the membranes by ultracentrifugation at 180,000 × g for 1 h. Thiocyanate was quantitatively converted to cyanate and elemental sulfur. As in whole-cell experiments, no thiocyanate degradation was observed under anaerobic conditions.

	DISCUSSION

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The results obtained in this study demonstrated for the first time that active thiocyanate biodegradation may occur under highly alkaline conditions. Thiocyanate can be used by heterotrophic and autotrophic alkaliphilic bacteria either as a nitrogen source or as an electron donor and energy source. Thiocyanate utilization either by pure bacterial cultures or by mixed populations in activated sludge has never been observed at pH values above 8.5. In fact, pH values higher than 8.0 negatively influenced thiocyanate degradation and growth of the neutrophilic bacteria (14, 25, 29), probably because of increased formation of undissociated NH₃ instead of NH₄⁺.

A substantial number of the previously isolated pure cultures of alkaliphilic sulfur-oxidizing autotrophic bacteria were able to utilize thiocyanate as a nitrogen source. While for heterotrophic bacteria this ability has been demonstrated previously more than once, no chemolithoautotrophs were known to grow with thiocyanate as a nitrogen source except for the known neutrophilic thiocyanate-oxidizing sulfur bacteria, which use thiocyanate as an electron donor and as a nitrogen source simultaneously. This is logical because in both assimilation and dissimilation pathways the thiocyanate molecule should first be split into sulfide and ammonium. In contrast, it is difficult to explain why many strains of alkaliphilic sulfur bacteria, which are able to utilize the nitrogen moiety, cannot grow solely with thiocyanate. The only way to obtain nitrogen from CNS is to hydrolyze it and release ammonia. This, in turn, means that sulfur is released eventually as sulfide, which is a natural electron donor for the alkaliphilic sulfur autotrophs. Perhaps CNS is transported inside the cells, where it cleaved to sulfide and ammonia. Then, if the sulfide-oxidizing system is located outside the cell membrane, difficulties with substrate oxidation might to be expected, whereas external thiosulfate or sulfide can be oxidized easily. Strong induction of the cyanase activity in heterotrophic (strain AGCNS 1) and autotrophic (strain ALRh) alkaliphiles during growth with thiocyanate as an N source could imply that they use a cyanate pathway for thiocyanate degradation, although the absence of any observed cyanate accumulation does not allow us to substantiate such a conclusion.

The thiocyanate-oxidizing alkaliphilic autotrophs can be enriched only when thiocyanate is used as the sole growth substrate. The presence of thiosulfate in addition to thiocyanate invariably resulted in enrichment of the sulfur-oxidizing alkaliphiles that were unable to grow with thiocyanate as an electron donor and grew faster than the thiocyanate specialists. All four alkaliphilic thiocyanate-oxidizing strains isolated were typical sulfur chemolithoautotrophs and were related to the other sulfur alkaliphiles belonging to the genus Thioalkalivibrio, which are unable to grow with thiocyanate (39). This supports the conclusion that the true electron donor in such bacteria is sulfide and, therefore, thiocyanate-oxidizing autotrophs are also sulfur-oxidizing autotrophs. Most of the previously described thiocyanate-degrading bacteria were isolated from thiocyanate-containing waste systems. The presence of thiocyanate-assimilating and thiocyanate-oxidizing bacteria in natural soda environments implies that there is a thiocyanate influx. Shallow soda lake sediments are usually rich in decaying organic material and reduced sulfur compounds. Perhaps thiocyanate can be formed from CN and reduced sulfur, like polysulfide, in a well-known cyanolytic reaction or with thiosulfate by the action of the enzyme rhodanese (10, 46). Alkaliphilic representatives of the thiocyanate-oxidizing autotrophs described in this paper differed from the neutrophilic T. thioparus strains by their ability to grow and to oxidize thiocyanate and other sulfur compounds under highly alkaline conditions (optimum pH, around 10) in combination with high salt concentrations. Both previously described neutrophilic species of thiocyanate-oxidizing sulfur autotrophs (T. thioparus and T. denitrificans) belong to the beta-Proteobacteria, while the alkaliphilic isolates belong to the gamma-Proteobacteria.

In contrast to strains that utilize thiocyanate as the N source, thiocyanate-oxidizing alkaliphilic autotrophs accumulated a large amount of cyanate during thiocyanate dissimilation under alkaline conditions. In fact, cyanate was the major nitrogen species in cultures of thiocyanate-grown ARh strains. This finding correlated well with the absence (ARh 1, ARh 2, ARh 3) or suppression (strain ARh 4) of cyanase activity when these bacteria grew with thiocyanate as the electron donor. The cyanate accumulation observed can be taken as the first direct proof of the involvement of the cyanate pathway biodegradation of thiocyanate by pure bacterial cultures. Thus, it seems quite sensible for alkaliphiles to use this pathway in combination with the absence of cyanase activity to prevent ammonia toxicity at highly alkaline pH values. Nevertheless, even production of cyanate as an N buffer would not save these bacteria from intoxication when high concentrations of cyanate (8 to 10 mM) accumulate, as in the case of chemostat cultures grown at pH 10 at low dilution rates with 12 to 15 mM thiocyanate. In this case the residence time is sufficiently long to allow slow, spontaneous cyanate decomposition, which releases more toxic ammonia than the culture needs for assimilation. This was probably a major reason for the observed instability of the continuous cultures compared to batch cultures of the alkaliphilic thiocyanate-oxidizing ARh strains. Stable growth was achieved only with low influent thiocyanate concentrations (<10 mM), which allowed us to decrease the liquid residence time. In this case the steady-state NH₃ concentration was kept below the toxicity level (0.1 to 1.3 mM).

Two specific enzyme activities and corresponding proteins associated with growth with thiocyanate were detected in alkaliphilic bacteria. The cyanase activity, detected in both thiocyanate-assimilating and dissimilating strains, resembled the enzyme activity of neutrophiles in the pH optimum (pH 8.0) and bicarbonate dependency (1). However, it was much more alkalitolerant. The catalysis of the breaking of the C---S bond of thiocyanate may be related to a protein with subunit mass of about 50 kDa which was heavily expressed only in ARh strains grown with thiocyanate as the energy source. It seems likely that this protein may be different from its analogue in neutrophilic T. thioparus strains in that it needs oxygen for activity. As nothing is known yet about this type of enzymes, it would be very interesting to purify this protein from the alkaliphilic bacteria.

The ability of alkaliphilic bacteria to degrade thiocyanate under highly alkaline conditions might also be important in improving bioremoval of thiocyanate from alkaline wastewater. Such wastewater, for example, can result from gold cyanidation, in which alkaline cyanide can react with polysulfide or reactive sulfur to form a less toxic alkaline thiocyanate-containing waste, which subsequently might be treated with the alkaliphiles.