3.1Thiosulfate and sulfide oxidation by cultures and cell suspensions
All selected tetrathionate-forming heterotrophic isolates (Table 1) could be grown as denitrifiers on media containing acetate (±thiosulfate) as well as nitrate, nitrite or N2O, resulting in vigorous production of nitrogen gas which was clearly visible in cultures grown in media including 0.5% agar. In the presence of nitrate or N2O, thiosulfate was oxidized exclusively to tetrathionate (Table 2). If nitrite was the electron acceptor, active growth was observed, but thiosulfate was not converted. When acetate or the electron acceptors were not provided, the bacteria did not grow or oxidize thiosulfate. Although tetrathionate was also the sole product of thiosulfate oxidation in aerobic cultures, the extent of thiosulfate oxidation was significantly higher than in the absence of oxygen (Table 2).
Table 2. Oxidation of thiosulfate in batch cultures of tetrathionate-forming heterotrophic bacteria grown with different electron acceptors
|Strain||Product formation (mM)|
|Electron donors: acetate 10 mM; thiosulfate: anaerobic 12 mM, aerobic 20 mM; incubation 48 h at 30°C. Column 1: NO3−, 10 mM; column 2: NO3−, 20 mM; column 3: NO2−, 10–20 mM; column 4, N2O; column 5, O2.|
The most active anaerobic oxidation of thiosulfate occurred with N2O as electron acceptor, with more than 90% of the thiosulfate being converted to tetrathionate. When nitrate was supplied in excess, more thiosulfate was oxidized, but nitrite accumulation prevented complete conversion. If the nitrate concentration was limiting, acetate was preferentially oxidized. The most active anaerobic thiosulfate oxidation with nitrate as electron acceptor was found with strain TG 3. The formation of tetrathionate proceeded at the same time as growth and nitrate reduction (Fig. 1), and was highest at a nitrate:acetate ratio of 2:1. Less nitrite accumulated in nitrate-reducing cultures containing thiosulfate and acetate, presumably because of the changed electron donor:acceptor ratio.
Figure 1. Growth and thiosulfate oxidation in anaerobic batch culture of strain TG 3 with acetate(10 mM) and nitrate (20 mM). 1: growth, mg protein l−1; 2: thiosulfate; 3: tetrathionate; 4: nitrate; 5: nitrite.
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As might be expected, there was a clear relationship between the amount of available electron acceptor and tetrathionate production in acetate-limited anaerobic continuous cultures of strain TG 3 (Table 3). These experiments also demonstrated that the culture could not gain sufficient energy from thiosulfate oxidation to increase the growth yield observed with acetate alone.
Table 3. Anaerobic growth and thiosulfate oxidation in acetate-limited continuous culture of strain TG 3 (D=0.1 h−1, temp. 30°C, pH 7.5)
|Influent concentration (mM)||Biomass (mg protein l−1)||Concentration in steady-state culture (mM)|
The experiments with washed cells aimed to eliminate any potential influence of organic electron donors, and to clarify the roles of the different stable nitrogen oxides in anaerobic thiosulfate oxidation. In general, the strains could be divided into two main groups on the basis of their response to nitrite. Group 1 includes strains ChG 4-1, ChG 5-2, ChG 5-3 and ChG 7-4, all of which had highly active cytochrome cd1 nitrite reductase (see below). Group 2 (strains ChG 5-1, ChG 6-1 and BG 2) is characterized by relatively lower nitrite reductase activity, and no detectable cytochrome cd1. The behavior of the two groups during experiments with washed cells was quite different.
Group 1 strains (an example is given in Table 4) did not accumulate nitrite during the anaerobic reduction of nitrate, but produced substantial amounts of N2O when incubated anaerobically with nitrite and thiosulfate. The most favorable electron acceptors for anaerobic thiosulfate oxidation by these strains were nitrite (washed cells) and N2O (batch cultures). Acetate inhibited anaerobic thiosulfate oxidation by washed cells from this group. Group 2 strains (Table 5) accumulated substantial amounts of nitrite when growing anaerobically with acetate and nitrate, but did not produce N2O when washed cells were incubated anaerobically with nitrite and thiosulfate. N2O was the best electron acceptor for anaerobic thiosulfate oxidation by this group, and acetate did not inhibit anaerobic thiosulfate oxidation by the washed cells. In some experiments it even stimulated the reaction. Strain TG 3 combined properties from both groups.
Table 4. Changes in the concentrations (mM) of tetrathionate and nitrogen oxides during anaerobic incubation of washed cells of strain ChG 5-2 (group 1) grown with different electron acceptors, and with either thiosulfate or a mixture of thiosulfate and acetate as electron donors
|Electron donor/acceptor||Cells grown with NO3−||Cells grown with NO2−||Cells grown with N2O|
|Experimental conditions: buffer, 0.05 M potassium phosphate+15 g l−1 NaCl, pH 7.5; biomass, 0.25 mg protein ml−1; incubation time, 2 h (with O2 1 h); S2O32−, 10.5 mM; NO3−, 11 mM; NO2−, 11.5 mM; N2O, 20 mM (saturation); acetate, 10 mM. nd=not detected; −=consumed; +=produced.|
Table 5. Anaerobic thiosulfate oxidation by washed cells of strain BG 2 (group 2) grown anaerobically with acetate, thiosulfate and nitrate
|Electron||Changes in tetrathionate and nitrogen oxide concentration (mM) during incubation of cell acceptor suspension|
| ||no acetate||+10 mM acetate|
|Experimental conditions as indicated in Table 4. nd=not detected; −=consumed; +=produced.|
If cultures were grown aerobically, they could not denitrify with thiosulfate as the electron donor, even though low levels of the nitrate and nitrite reductases could be detected if acetate was supplied. Cells grown under denitrifying conditions with acetate and thiosulfate were capable of active aerobic thiosulfate oxidation. 10 mM nitrite inhibited aerobic thiosulfate oxidation.
Diethyldithiocarbamate (DDC), an inhibitor of Cu-containing NO2− reductase, did not influence anaerobic thiosulfate oxidation by washed cells from either group in the presence of nitrate or nitrite. Acetylene (an inhibitor of N2O reductase; 5% v/v in the gas phase) did not influence anaerobic thiosulfate oxidation by washed cells with nitrate, slightly inhibited the process in the presence of nitrite, and completely stopped thiosulfate oxidation with N2O as electron acceptor.
Washed cells of strain ChG 5-2, grown anaerobically with acetate, thiosulfate and nitrate, were able to oxidize sulfide aerobically to tetrathionate. Under anaerobic conditions, the rate of sulfide oxidation was very low. The highest rate (10 nmol mg protein−1 min−1) was observed in the presence of nitrite, when the distinctive yellow color associated with the accumulation of polysulfide was transiently evident.
Difference spectra of cell-free extracts prepared from strains from group 1 showed the presence of cytochrome cd1. The absorption maxima typical of this nitrite reductase (γ 460–470 nm; α 610–615 and 660–670 nm) were not observed in spectra made on similar protein samples from the group 2 isolates. While this might indicate the presence of a copper-containing nitrite reductase, rather than cytochrome cd1, this seems unlikely in view of the failure of DDC to inhibit nitrite reduction. It is possible that the cytochrome cd1 in such strains was simply below the detection limit of the method.
Cytochromes c and b were detectable in cell-free extracts of all eight isolates when they were grown aerobically with acetate and thiosulfate. Cytochrome cd1 (nitrite reductase) was not expressed under aerobic conditions in any of the isolates from either group. Cytochrome b was present at relatively low concentrations, compared to the level of cytochrome c. CO difference spectra of dithionite-reduced extracts had the maxima and minima in the γ region (415 and 432 nm respectively), and minima in the α region (556–558 nm) typical of cytochrome oxidase type o.
All of the isolates were Gram-negative, oxidase- and catalase-positive, curved, motile rods with polar flagella when grown anaerobically in liquid culture. Two colony types were formed. R-type colonies were yellowish or pinkish, spreading, skin-like and with a very complex surface. S-types were small, smooth, soft, slimy and colorless. The R-type dominated when plated on dry agar surfaces and when incubated under aerobic conditions. The S-type predominated on wet surfaces and during anaerobic incubation. When sub-cultured, the R-type was stable, but the S-type produced both types under appropriate conditions. The morphology of the cells within the R- and S-type colonies was sufficiently different to suggest two different species. However, DNA-DNA hybridization on R and S clones from each of three of the strains (TG 3, BG 2 and ChG 5-2) confirmed that in each case, the clones were the same organism (hybridization level 99–100%). Cells from S-colonies and anaerobic liquid cultures were usually motile, curved rods, while cells from R-colonies were mostly coccoid and non-motile, being embedded in a slime matrix that was hard to homogenize. Motile cells were only found at the very edge of such colonies.
All of the isolates were obligately heterotrophic. They did not grow in mineral medium with only thiosulfate or H2 as the sole electron donor. Among the organic compounds utilized as carbon and energy sources were: organic acids including acetate, succinate, malate, α-ketoglutarate, citrate, fumarate, lactate, pyruvate, propionate, butyrate, gluconate, glyoxylate; alcohols including ethanol, propanol, glycerol; hexoses –D-glucose, D-fructose and D-maltose; amino acids and amines including L-alanine, L-glutamate, L-glutamine, L-aspartate, L-asparagine, L-proline and L-leucine. Strains ChG 6-1 and ChG 5-3 differed from the others by their ability to utilize D-galacturonic and D-glucuronic acids. Sugars were not fermented.
The bacteria described here have a relatively narrow range of GC mol% values (60–63.5). Direct DNA-DNA hybridization between all isolates was therefore possible. As can be seen in Fig. 2, there was DNA homology of more than 30% among the assorted isolates. The levels of hybridization with a non-specific control (Escherichia coli) were very low (0.2%). Within the group were four clusters. Strain TG 3, from the Pacific Ocean, was sufficiently different from the Black Sea isolates to be considered a different species by current standards for DNA-DNA-hybridization data .
Figure 2. Dendrogram of the DNA-DNA homology among strains capable of thiosulfate-dependent denitrification. DNA from E. coli gave less than 0.1% homology with any of these samples.
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16S rRNA sequence analysis showed that isolates ChG 5-2, ChG 5-3, BG 2 and TG 3 were new Pseudomonas stutzeri strains. P. stutzeri has been subdivided into eight different genomic groups, genomovars, defined by DNA-DNA hybridization, G+C% values and 16S rRNA sequences [30–33]. This taxonomic category allows phenotypically variable strains of broadly defined species to be sorted into genetically distinct and stable groups . On the basis of 16S rRNA sequence distances, isolates ChG 5-2, ChG 5-3, BG 2 and TG 3 were affiliated with genomovars 5, 3, 4, and 6 (Fig. 3). The isolates also matched all P. stutzeri genomovar signature nucleotides , with the exception of an adenosine replaced by a guanosine in E. coli position 1036 of strain ChG 5-3 and in E. coli position 278 of strain BG 2. As can be seen from Fig. 3, the only other organism in the clump with ChG5.2 and P. stutzeri was ‘Flavobacterium lutescens’, and its taxonomic position was therefore given some attention.
Figure 3. Phylogenetic tree based on 16S rRNA sequence positions 34–1461 for P. stutzeri and related strains, including the new thiosulfate-oxidizing isolates described in this study. The numbers 1–8 indicate genomovars [32,33]. The tree was rooted with P. aeruginosa strain as outgroup. The scale bar corresponds to 5 substitutions per 1000 nucleotides.
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Strain TG 3 was separated from the P. stutzeri isolates by lower DNA-DNA hybridization and 16S rRNA similarity (30% and 97%, respectively). These molecular differences exclude strain TG 3 from the species . Strain TG 3 fell into genomovar 6, which has recently been proposed as a separate species, Pseudomonas balearica.
According to 16S rRNA analysis, ‘Flavobacterium lutescens’ appeared to be related to P. stutzeri, especially to genomovars 1 and 5, and to the isolate ChG 5-2 (Fig. 3). By DNA-DNA hybridization, ‘F. lutescens’ showed 60–70% homology with strains ChG 5-2 and BG 2, and is therefore placed within the species P. stutzeri. The physiological properties of the strain confirm this classification. ‘Flavobacterium lutescens’ was able to grow anaerobically with acetate and nitrate, nitrite or N2O as electron acceptors. It oxidized thiosulfate to tetrathionate in batch cultures in the presence of acetate with oxygen or N2O, but not with nitrate or nitrite as electron acceptors. Washed cells of ‘F. lutescens’ grown anaerobically with acetate, thiosulfate and either nitrate or N2O could oxidize thiosulfate anaerobically, but were totally inactive with nitrite as electron acceptor. Cytochrome cd1 nitrite reductase was not spectroscopically detected in cell-free extracts prepared from cells grown anaerobically with acetate and nitrate. ‘F. lutescens’ also formed two colony types on dry agar. However, the R-type was smoother, practically colorless, and grew down into the agar.