FEMS Microbiology Ecology 30 (1999) 113^123 Anaerobic oxidation of thiosulfate to tetrathionate by obligately heterotrophic bacteria, belonging to the Pseudomonas stutzeri group Dimitry Yu. Sorokin a, Andreas Teske b, Lesley A. Robertson c;*, J. Gijs Kuenen c a Institute of Microbiology, Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, 117811 Moscow, Russia b Max-Planck-Institut fu«r Marine Mikrobiologie, Celsiusstr. 1, 28359 Bremen, Germany c Kluyver Laboratory of Biotechnology, TU Delft, Julianalaan 67, 2628 BC Delft, The Netherlands Received 3 March 1999; revised 3 June 1999; accepted 3 June 1999 Abstract A number of strains of heterotrophic bacteria were isolated from various environments on the basis of their potential to oxidize inorganic sulfur compounds to tetrathionate. The isolates were screened for the ability to oxidize thiosulfate under denitrifying conditions. Many of them could grow anaerobically with acetate and nitrate, and eight strains could oxidize thiosulfate to tetrathionate under the same conditions. In batch cultures with acetate as carbon and energy source, most active anaerobic thiosulfate oxidation occurred with N2O as electron acceptor. The level of anaerobic thiosulfate-oxidizing activity in cultures and cell suspensions supplied with nitrate correlated with the activity of nitrite reductase in cell suspensions. Some strains converted thiosulfate to tetrathionate equally well with nitrite, nitrate and N2O as electron acceptors. Others functioned best with N2O during anaerobic thiosulfate oxidation. The latter strains appeared to have a lower level of nitrite reductase activity. Thiosulfate oxidation under anaerobic conditions was much slower than in the presence of oxygen, and was obviously controlled by the availability of organic electron donor. The strains had DNA-DNA similarity levels higher than 30%. Sequence analysis of the 16S rRNA gene of four selected isolates showed their affiliation to specific genomovars of Pseudomonas stutzeri and the proposed new species, Pseudomonas balearica. As shown by 16S rRNA sequence analysis and DNA-DNA hybridization, the previously misnamed `Flavobacterium lutescens' (ATCC 27951) is also a P. stutzeri strain which can oxidize thiosulfate to tetrathionate aerobically and anaerobically in the presence of N2O. The data suggest that tetrathionate-forming heterotrophic bacteria, in particular those belonging to the P. stutzeri `superspecies', can play a much more significant role in the biogeochemical cycles than was previously recognized. ß 1999 Federation of European Micro- biological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Denitri¢cation; Heterotrophic tetrathionate-producing bacterium; Pseudomonas stutzeri 1. Introduction * Corresponding author. Tel.: +31 (15) 782421; Heterotrophic bacteria able to oxidize thiosulfate Fax: +31 (15) 782355; E-mail: [email protected] to tetrathionate are widely distributed in soil and 0168-6496 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0168-6496(99)00045-8 FEMSEC 1056 23-9-99 114 D.Yu. Sorokin et al. / FEMS Microbiology Ecology 30 (1999) 113^123 natural water [1^5]. Such bacteria dominate among bacteria represent a compact phylogenetic group af- sulfur-oxidizing communities in strati¢ed bodies of ¢liated with Pseudomonas stutzeri. water where there are low sul¢de concentrations in the interface layer, such as the Black Sea. Large numbers of tetrathionate-forming bacteria have 2. Materials and methods been found in freshwater lakes and specialized envi- ronments such as soda lakes and sul¢de-oxidizing 2.1. Strains bioreactors ([6]; D. Sorokin, unpublished data). Most of the strains of tetrathionate-forming hetero- Forty-¢ve strains of obligately heterotrophic, tet- trophic bacteria thus far investigated were able to rathionate-forming bacteria isolated from sea and reduce tetrathionate to thiosulfate under anaerobic fresh water, and from a sul¢de-oxidizing bioreactor conditions at the expense of organic electron donors [4], were screened for the ability to grow anaerobi- [1,7,8]. Moreover, some marine isolates could grow cally with acetate and nitrate. Nineteen of the pos- anaerobically using tetrathionate as electron accept- itive strains were then examined for the ability to or [9]. oxidize thiosulfate while growing anaerobically with Despite numerous examples of the presence of acetate as carbon and energy source, and nitrate, such bacteria in various sul¢de-containing environ- nitrite or N2O as electron acceptors. Of these, the ments, the reason for tetrathionate production by eight strains shown in Table 1 were able to oxidize heterotrophic bacteria has, until recently, remained thiosulfate anaerobically, and were selected for fur- obscure. However, research on the physiology of ther investigation. Table 1 also shows the depth and some marine isolates has shown that tetrathionate- sul¢de concentration in the water from which the producing heterotrophs can bene¢t from thiosulfate isolates were taken. oxidation when grown under organic carbon limita- `Flavobacterium lutescens' LMD 95.190 was ob- tion [10]. Moreover, biological tetrathionate produc- tained from the Culture Collection of the Kluyver tion also can drive chemical sul¢de oxidation [10]. Laboratory for Biotechnology, Delft University of Heterotrophic tetrathionate-producers may thus be Technology, Delft, The Netherlands. important in the natural sulfur cycle in some envi- ronments. 2.2. Growth experiments Denitri¢cation among the known colorless sulfur bacteria is not common. Thiosulfate- or sul¢de-de- For batch cultivation, all media contained the fol- 31 pendent denitri¢cation to N2 is best known in the lowing (g l ): NH4Cl 0.5, KH2PO4 2, K2HPO4 5, obligately autotrophic Thiobacillus denitri¢cans and MgSO4W7H2O 0.4, CaCl2W2H2O 0.1, NaCl (for ma- Thiomicrospira denitri¢cans [11], although it has been rine strains) 15, trace elements solution [15] 1 ml, reported for the facultatively autotrophic Paracoccus yeast extract 0.1. Unless otherwise mentioned, 10^ pantotrophus (formerly Thiosphaera pantotropha 20 mM acetate, 10 mM thiosulfate and 10^20 mM [12,13]). Other colorless sulfur bacteria such as Thi- of nitrite or nitrate were used. Anaerobiosis was obacillus thioparus can carry out partial denitri¢ca- achieved by £ushing the medium with sterile argon tion, often to nitrite, while oxidizing thiosulfate [11]. for 10^15 min. When N2O was the electron acceptor, Preliminary indications that some tetrathionate- the gas phase in the £ask was ¢rst displaced with forming heterotrophs were able to denitrify while argon before the liquid was £ushed with sterile oxidizing thiosulfate were of obvious interest. In par- N2O for 2 min. Autotrophic growth was tested ticular, heterotrophic bacteria from marine sul¢de- using the mineral medium without organic addi- containing environments formed gas when grown tions. The pH of the medium was adjusted to 7.8 anaerobically in sea water medium with thiosulfate with NaHCO3. The results presented are averages and nitrate [2,3,14]. of at least ¢ve sets of data, with a variation of 5^ This paper describes thiosulfate-dependent denitri- 10%. ¢cation by strains of tetrathionate-forming hetero- Anaerobic continuous cultivation of strain TG 3 trophic bacteria from di¡erent environments. These was performed in a laboratory fermenter with a pH FEMSEC 1056 23-9-99 D.Yu. Sorokin et al. / FEMS Microbiology Ecology 30 (1999) 113^123 115 and dO2 Biocontroller (Applicon, The Netherlands) 2.4. Cytochrome spectra and a 1.5-l working volume. The mineral base used for continuous culture was the same as for batch Extracts for cytochrome spectra were prepared cultivation, except that the magnesium and calcium from cells grown aerobically in batch culture with concentrations were halved. The pH was maintained acetate and thiosulfate, or anaerobically with ace- at 7.5 by auto-titration with 1 M HCl. The medium tate, thiosulfate and nitrate. Cells were centrifuged, bottles and the gas phase of the fermenter were kept washed and resuspended in 0.05 M potassium phos- under a gentle argon £ow. Measurements began after phate bu¡er (pH 7.5) with 15 g l31 NaCl and dis- 5^6 volume changes when a steady state had become rupted by sonication. Unbroken cells and cells debris established, and there was less than 1% di¡erence were removed by additional centrifugation at between measurements made on di¡erent days. At 15 000Ug for 10 min. Solid dithionite was used for least two sets of measurements were made per steady the complete reduction of cytochromes, and bub- state, with an interval of at least 1 volume change bling with air for their oxidation. CO di¡erence spec- between measurements. The results are presented as tra were made by comparing the results from di- averages of the two measurements. thionite-reduced preparations with those obtained after pure CO had been bubbled through the prepa- 2.3. Experiments with washed cells rations in the cuvettes. The di¡erence cytochrome spectra were recorded with a Pye-Unicam 1800 Cells were collected by centrifuging anaerobic cul- (UK) spectrophotometer. tures grown to stationary phase with acetate, thio- sulfate and either nitrate, nitrite or nitrous oxide, as 2.5. Chemical analysis indicated in the text. Cells were washed with anaer- obic 0.05 M potassium phosphate bu¡er, pH 7.5, Nitrite was assayed with N-(1-naphthyl) ethylene containing 15 g l31 NaCl, and resuspended