Isolation and Characterization of a New CO-Utilizing Strain, Thermoanaerobacter Thermohydrosulfuricus Subsp
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Extremophiles (2009) 13:885–894 DOI 10.1007/s00792-009-0276-9 ORIGINAL PAPER Isolation and characterization of a new CO-utilizing strain, Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans, isolated from a geothermal spring in Turkey Melike Balk Æ Hans G. H. J. Heilig Æ Miriam H. A. van Eekert Æ Alfons J. M. Stams Æ Irene C. Rijpstra Æ Jaap S. Sinninghe-Damste´ Æ Willem M. de Vos Æ Serve´ W. M. Kengen Received: 19 April 2009 / Accepted: 3 August 2009 / Published online: 23 August 2009 Ó The Author(s) 2009. This article is published with open access at Springerlink.com Abstract A novel anaerobic, thermophilic, Gram-posi- is most closely related to Thermoanaerobacter thermohy- tive, spore-forming, and sugar-fermenting bacterium (strain drosulfuricus and Thermoanaerobacter siderophilus (99% TLO) was isolated from a geothermal spring in Ayas¸, similarity for both). However, strain TLO differs from Turkey. The cells were straight to curved rods, 0.4–0.6 lm Thermoanaerobacter thermohydrosulfuricus in important in diameter and 3.5–10 lm in length. Spores were terminal aspects, such as CO-utilization and lipid composition. and round. The temperature range for growth was 40–80°C, These differences led us to propose that strain TLO repre- with an optimum at 70°C. The pH optimum was between sents a subspecies of Thermoanaerobacter thermohydro- 6.3 and 6.8. Strain TLO has the capability to ferment a wide sulfuricus, and we therefore name it Thermoanaerobacter variety of mono-, di-, and polysaccharides and proteina- thermohydrosulfuricus subsp. carboxydovorans. ceous substrates, producing mainly lactate, next to acetate, ethanol, alanine, H2, and CO2. Remarkably, the bacterium Keywords Thermoanaerobacter sp. Á Geothermal was able to grow in an atmosphere of up to 25% of CO as springs Á Thermophiles Á Bacteria Á Thermoanaerobacter sole electron donor. CO oxidation was coupled to H2 and CO2 formation. The G ? C content of the genomic DNA was 35.1 mol%. Based on 16S rRNA gene sequence anal- Introduction ysis and the DNA–DNA hybridization data, this bacterium Diverse thermophilic heterotrophic anaerobes have been isolated from a variety of habitats. Members of the genus Communicated by F. Robb. Thermoanaerobacter, in the order Thermoanaerobacteri- ales, are widely distributed in hydrothermal and oil- The GenBank accession number for the 16S rRNA sequence of Thermoanaerobacter sp. is AY 328919. producing vents, volcanic hot springs, non-volcanic geothermally heated subsurface aquifers, soil, sugar beet, M. Balk Á H. G. H. J. Heilig Á M. H. A. van Eekert Á and sugar cane extraction juices (Klaushofer and Parkkinen A. J. M. Stams Á W. M. de Vos Á S. W. M. Kengen 1965; Wiegel and Ljungdahl 1981; Schmid et al. 1986; Laboratory of Microbiology, Wageningen University, Cayol et al. 1995; Cook et al. 1996; Kozianowski et al. Dreijenplein 10, 6703 HB Wageningen, The Netherlands 1997; Larsen et al. 1997; Slobodkin et al. 1999; Fardeau I. C. Rijpstra Á J. S. Sinninghe-Damste´ et al. 2000; Kim et al. 2001; Onyenwoke et al. 2007; Department of Marine Organic Biogeochemistry, Wagner et al. 2008). Thermoanaerobacter species are NIOZ Royal Netherlands Institute for Sea Research, strictly anaerobic, thermophilic, rod-shaped bacteria, P.O. Box 59, 1790 AB Den Burg, Texel, The Netherlands growing between 55 and 75°C, and most of them form Present Address: round to oval terminal spores. Thermoanaerobacter species M. Balk (&) have been investigated for their sensitivity to different Netherlands Institute of Ecology (NIOO-KNAW), antibiotics but no differences have been found. A wide Centre for Limnology, Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands range of carbohydrates can be utilized by this group of e-mail: [email protected] organisms. Although the end products are mainly acetate, 123 886 Extremophiles (2009) 13:885–894 lactate, ethanol, H2, and CO2, the most abundant end cooled to room temperature under a stream of O2-free N2 product depends on the species and the growth conditions. gas. The medium was anaerobically dispensed into serum Generally, thiosulfate can be used as electron acceptor in bottles under a N2/CO2 (80/20, v/v) gas atmosphere. The anaerobic respiration. Here we describe a new anaerobic bottles were closed with butyl rubber stoppers sealed with thermophilic bacterium which belongs to the genus Ther- crimp seals. The medium was autoclaved for 20 min at moanaerobacter, and which differs from its closest rela- 121°C. Prior to inoculation, the medium was reduced with tives with respect to CO-utilization, lipid composition and sterile stock solutions of Na2S, 7–9 H2O and NaHCO3 to fermentation pattern. obtain final concentrations of 0.04 and 0.2%, respectively. The pure culture of strain TLO was either maintained by weekly transfer of a 1% (v/v) inoculum to fresh medium or Materials and methods inoculated from frozen glycerol stocks. Bottles were incubated in the dark without shaking. To investigate and Collection of the sample optimize lactate production, some experiments were per- formed with phosphate-buffered medium 640 of the DSMZ Strain TLO was enriched from a mud sample obtained (PB medium), which enabled a lower pH between 4.0 and from a geothermal spring in Ayas¸ in Turkey. The geo- 6.7. The reference strains used in this study were Ther- graphical coordinates of the sampling site were 40.2° lat- moanaerobacter siderophilus (DSM 12299) (Slobodkin itude, 32.4° longitude. The temperature and the pH of the et al. 1999) and T. thermohydrosulfuricus (DSM 567) sampling site was 75°C and 7.5 (at 75°C), respectively. (Klaushofer and Parkkinen 1965). Growth on different substrates was measured as the Isolation optical density at 600 nm (OD600). Uninoculated medium served as a reference. The results The mud sample (5 ml) was directly transferred to 50-ml represent duplicate experiments. The optimal growth tem- sterilized bicarbonate-buffered medium in a 117-ml serum perature was determined by following growth at various vial sealed with a butyl rubber stopper under a gas phase of temperatures between 35° and 85°C, with 5-degree N2/CO2 (80/20, v/v). Cellobiose (20 mM) was used as intervals. electron donor. The enrichment culture was grown at 68°C and after growth, contained predominantly spore-forming Preparation of cell-free extract and enzyme rods. Isolation of the dominant bacterium was achieved by measurement the soft-agar dilution method. The colonies, visible after three days of incubation, were uniformly round, 0.5– Cell extracts used for enzyme assays were obtained from 1.0 mm in diameter and white. They were picked from the cells grown in the PB medium supplemented with 25% of highest dilution (10-8) with a sterile needle and subcul- CO as electron donor. The preparation of cell extracts was tured in liquid medium containing 20 mM of cellobiose as performed under anoxic conditions in an anaerobic glove a substrate. Serial agar and liquid dilutions in cellobiose- box. Cells were collected by centrifugation at 10,000g for containing media were repeated until a pure culture was 10 min at 4°C. The cell pellet was suspended (1:2 [w/v]) in obtained. The pure culture was designated strain TLO 15 mM potassium/sodium phosphate buffer, pH 7.2. The (=DSM15750, =ATCC BAA-892). cells were disrupted by ultrasonic disintegration (Sonics & Materials Inc., Danbury, Conn.) at 40 Kc/s for 30 s fol- Media and cultivation lowed by cooling for 30 s on ice. The cycle was repeated four times. Cell debris and whole cells were removed by The composition of the BM medium used for routine centrifugation at 13,000g for 10 min at 4°C. The super- growth and substrate utilization experiments were based on natant was used for enzyme assays. Carbon monoxide medium 120 of the DSMZ (http://www.dsmz.de), with the dehydrogenase (CODH) activity was assayed at the tem- following modifications: casitone, methanol and cysteine- peratures between 60 and 80°C by following the CO- HCl were omitted and the amount of yeast extract was dependent reduction of oxidized methyl viologen (MV) as either lowered to 0.25 g l-1 or not added for growth described by Svetlitchnyi et al. (2001). One unit of CO experiments. Routinely, cellobiose (20 mM) was used as oxidation activity was defined as the amount of enzyme the carbon and energy source. The pH of the medium that catalyzes the reduction of 2 lmol of MV min-1, (standard pH was 6.7) was adjusted using temperature which is equivalent to 1 lmol of CO oxidized min-1. The corrected pH standards and by injecting calculated amounts protein content of the cell extracts was determined of sterile Na2CO3/NaHCO3 or HCl from the sterile, according to the method of Bradford (1976) with bovine anaerobic stock solutions. The medium was boiled and serum albumin as a standard. 123 Extremophiles (2009) 13:885–894 887 Substrates and electron acceptors utilization tests Phylogeny, DNA base composition and DNA reassociation The ability of strain TLO to metabolize substrates was tested in the bicarbonate-buffered medium (BM). Sub- DNA was extracted and purified using the UltraClean Soil strates were added from sterile, anoxic concentrated stock DNA kit (MoBio). PCR was performed with the bacterial solutions to final concentrations of 20 mM, unless other- primers 7f and 1510r (Lane 1991) by using the Taq DNA wise indicated. To test the use of potential electron polymerase kit (Life Technologies) to amplify the bacterial acceptors with glucose as electron donor, thiosulfate 16S rRNA gene. The PCR products were purified with the (20 mM), elemental sulfur (2%, w/v), sodium sulfite Qiaquick PCR purification kit (Qiagen, Hilden, Germany) (5 mM), FeCl3 (10 mM), Fe(III)-NTA (10 mM), Fe(III)- according to the manufacturer’s instructions.