| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Microbiology, University of Aarhus, Ny Munkegade Building 1540, DK-8000 Aarhus C, Denmark
Correspondence
Kjeld Ingvorsen
kjeld.ingvorsen{at}biology.au.dk
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
40 °C). These extreme conditions serve to minimize chemical corrosion and limit bacterial growth. However, oligotrophic conditions may not be sufficient to prevent biofilm formation in a flow system, since nutrients are constantly being replenished and tend to concentrate on submerged surfaces, thus promoting attached bacterial growth. Such biofilm formation can locally alter the surface chemistry, which in turn can result in (bio)corrosion of the surface (Hamilton, 1995
; Little et al., 1990). Sulfate-reducing bacteria are generally considered as the major causative agent of biocorrosion because of their ability to produce corrosive hydrogen sulfide and/or to consume molecular hydrogen (Lee et al., 1995; Hamilton, 1995). Biocorrosion is of particular importance in high-sulfate marine technical systems, especially those in the offshore oil industry, and can cause substantial economic losses (Marshall, 1992). However, biocorrosion can also occur in low-sulfate freshwater systems such as district heating networks, as was highlighted by recent surveys showing that several Danish district heating systems occasionally suffer from biocorrosion (Kjellerup et al., 2003, 2005). Attempts by those authors to link the occurrence of biocorrosion specifically to the presence of sulfate-reducing bacteria proved unsuccessful, suggesting that other types of micro-organisms might also be involved in biocorrosion processes. Here, we describe a novel alkalitolerant, sulfide-producing, anaerobic bacterium, strain sk.kt5T, isolated from a biofilm sample from a Danish district heating system that had previously suffered from failures due to biocorrosion.
Strain sk.kt5T was enriched from biofilms that developed on mild steel coupons during a 4 month incubation period in a corrosion-monitoring reactor connected to the return line (bulk water pH 9.5–10.0, 40 °C) of Skanderborg District Heating Plant in Denmark. The water-quality parameters of this system have been reported previously (Abildgaard et al., 2006). Enrichment cultures were initiated by inoculating biofilm material aseptically scraped off metal coupons and suspended in approximately 1 ml filter-sterilized (0.2 µm pore size) district heating water into a modified version of Postgate's medium B (Postgate, 1984) containing the following (l–1 MilliQ water): 0.5 g KH2PO4, 0.25 g NH4Cl, 1.26 g CaSO4.2H2O, 1.0 g MgSO4.7H2O, 0.1 g FeSO4.7H2O, 0.2 g yeast extract, 1.0 ml trace element mixture (Widdel & Bak, 1992), 1.0 ml selenite/tungstate mixture (Widdel & Bak, 1992) and 50 µl resazurin solution (2 %, w/v). After sterilization of the medium, the following solutions were added aseptically from sterile anoxic stocks (l–1 medium): 1.0 ml vitamin mixture [modified after Widdel & Bak (1992) by including 30 mg folic acid l–1], 1.0 ml vitamin B12 solution and 1.0 ml thiamine solution (Widdel & Bak, 1992). The following substrates were each added to a final concentration of 5 mM: propionate, acetate, lactate and ethanol. Before use, the pH of the medium was adjusted to 9.0 using sterile, anoxic NaOH (4 M) and the medium was finally reduced by adding a 2 % (w/v) sodium dithionite solution (3 ml l–1). Enrichment cultures were incubated at 40 °C in the dark. Strain sk.kt5T was isolated from one of the enrichment cultures by repeated (3x) application of the roll-tube technique (Hungate, 1976), using the same medium as described above but further supplemented with 10 mM thiosulfate and washed agar (2 %, w/v). The purity of the isolated culture was examined by phase-contrast microscopy after growth (at pH 7.0, 8.0 and 9.0) in the above-mentioned medium supplemented with 20 mM glucose, 20 mM lactate and 10 mM thiosulfate.
Routine cultivation and all growth tests were performed in 16x125 mm Hungate anaerobic culture tubes (Bellco Glass) containing 10 ml basal medium buffered with CAPSO (3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid, pKa 9.6) prepared as described previously (Abildgaard et al., 2006). Unless noted otherwise, the pH of the basal medium was adjusted to 9.0 (measured at 40 °C), the medium was supplemented with betaine and thiosulfate (20 mM each) and all incubations were carried out in duplicate at 40 °C in the dark. Cell growth was quantified by measuring optical density at 600 nm (OD600). Because of the formation of inorganic precipitates in the basal medium at pH values greater than 9.0, OD600 measurements were sometimes supplemented with measurements of sulfide production (performed using the method of Cline, 1969) and with total counts of SYBR Gold-stained cells (performed according to Mogensen et al., 2005). Transmission electron microscopy was performed as described previously (Mogensen et al., 2005). The Gram-staining reaction was determined by using a standard procedure. The pH range for growth was determined at 11 different pH values ranging from 6.3 to 10.5, obtained by titrating the medium with sterile, anoxic 2 M HCl or NaOH solutions. The pH did not remain stable when titrated to values above 10.5, so growth of the strain was not evaluated at higher pH. The temperature range for growth was determined using a temperature-gradient block (Elsgaard et al., 1994) at 21 different temperatures ranging from 0.6 to 50.2 °C. The effect of NaCl on growth was determined at 0.1, 0.5, 1.0, 2.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 10, 15 and 20 % (w/v). The ability of strain sk.kt5T to utilize different electron donors was tested in separate incubations using both thiosulfate (20 mM) and sulfite (10 mM) as electron acceptors. The fermentative abilities of strain sk.kt5T were tested in medium devoid of external electron acceptors. The utilization of different electron acceptors was tested using betaine (20 mM) and yeast extract (0.5 g l–1) as electron donors. Cultures grown in medium with 20 mM betaine and 0.5 g l–1 yeast extract without any external electron acceptors were used as inocula (5 %, v/v) for the latter experiments. The reduction of Fe(III) citrate (20 mM) was examined in unreduced medium by measuring the Fe(II) concentration by means of the ferrozine method (Lovley & Phillips, 1986). Notably, strain sk.kt5T was capable of initiating growth in unreduced medium when the inoculum was from betaine-grown cultures. The reduction of nitrate (20 mM) and nitrite (2 mM) was tested (five replicate cultures each) by measuring the OD600 and by monitoring the concentrations of nitrate and nitrite using HPLC (Kjeldsen et al., 2004), as well as by assessing the ammonium concentration by using a colorimetric method (Bower & Holm-Hansen, 1980). Plating on agar plates (1.5 %, w/v) consisting of basal medium (pH 9.0) was used to test for aerobic growth.
The cells of strain sk.kt5T were motile straight rods varying in size from 0.4 to 0.6 µm in diameter and from 2.5 to 9.6 µm in length (Fig. 1). Strain sk.kt5T stained Gram-positive and formed round terminal endospores (Fig. 1). The temperature range for growth was 23–44 °C, with an optimum at 35–37 °C. After 120 days incubation, growth was detected at temperatures down to 14.5 °C. No growth was detected at 48 °C or at 
11 °C. Cultures of strain sk.kt5T survived pasteurization for 4 h at 60 °C. The pH range for growth was pH 7.6 to approximately pH 10.5 (strain sk.kt5T exhibited only weak growth at this pH; higher values were not tested, as mentioned above); optimum growth occurred at pH 8.0–9.5. Growth was not observed at or below pH 7.5. Strain sk.kt5T grew at NaCl concentrations ranging from 0 to 5 % (w/v), the optimum being observed at 0–0.5 % (w/v) NaCl.
|
Genomic DNA of strain sk.kt5T was extracted and the dsrAB [encoding the alpha and beta subunits of dissimilatory (bi)sulfite reductase] and 16S rRNA gene sequences were retrieved as described previously (Abildgaard et al., 2006). A custom-designed internal dsrAB sequencing primer (5'-GATGCATGCACTGTATTAACAAA-3', annealing to Desulfovibrio vulgaris dsrA positions 728–751) was used to complete the dsrAB sequence of strain sk.kt5T. The 16S rRNA gene sequence of strain sk.kt5T was aligned according to the ssu_jan04_corr_opt.arb ARB database (available at http://www.arb-home.de). Phylogenetic trees (based on datasets of sequences >1350 nt) were constructed using the neighbour-joining (with Jukes–Cantor distance correction), maximum-parsimony and maximum-likelihood algorithms of the ARB package (Ludwig et al., 2004). A 50 % conservation filter calculated for the taxa shown in Fig. 2(a) was applied to select sequence positions (1277 nt) for the analyses. The phylogenetic analysis of the dsrAB sequence of strain sk.kt5T was carried out as described previously (Abildgaard et al., 2006) and included 457 unambiguously aligned amino acid sequence positions. Bootstrap analyses were performed and phylogenetic consensus trees were constructed as described previously (Abildgaard et al., 2006).
|
; GenBank accession numbers AF486695, AY540791, AY540795, AY540811, AY540821 and AY540824). Phylogenetic treeing consistently placed strain sk.kt5T within the class Clostridia of the phylum Firmicutes as a novel sister lineage to the genus Moorella (Fig. 2a
). Calculation of pairwise similarities from full-length alignment of all taxa included in the tree shown in Fig. 2(a), using the Firmicutes filter of the ssu_jan04_corr_opt.arb ARB database or a filter based on the Lane mask (which excludes all hypervariable sequence positions of the 16S rRNA gene; Lane, 1991), identified the two above-mentioned Moorella species and Moorella glycerini DSM 11254T as the closest relatives of strain sk.kt5T (91.5–91.8 % similarity). Similarity values that were 0.5–1.5 % lower were shared with several species belonging to different genera, including certain members of the sulfate-reducing genus Desulfotomaculum. Even lower levels of similarity (89.1–89.2 %) were shared with Moorella mulderi DSM 14980T, which, together with the three above-mentioned species, constitute the only members of the genus Moorella that have validly published names. Like strain sk.kt5T, the latter species are all anaerobic, Gram-positive, spore-forming rods capable of using thiosulfate as an electron acceptor (Fontaine et al., 1942; Wiegel et al., 1981; Ljungdahl, 1986; Slobodkin et al., 1997; Balk et al., 2003). However, apart from the above-mentioned similarities, a number of phenotypic and genotypic properties differentiate strain sk.kt5T from this genus. Most notably, the genomic G+C content of strain sk.kt5T (41.6 mol%; determined by HPLC analysis at the Identification Service of Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) differs by more than 12 mol% from the G+C contents of the four Moorella species, which suggests that strain sk.kt5T should be placed outside this genus (Vandamme et al., 1996). The genus Moorella belongs to the family Thermoanaerobiaceae; however, as can be seen from the consensus tree shown in Fig. 2(a), the phylogenetic analyses used failed to resolve the phylogenetic relationships among the families of the class Clostridia. Thus, strain sk.kt5T cannot be confidently assigned to a family.
According to phylogenetic analyses based on DsrAB amino acid sequences, strain sk.kt5T constitutes a novel lineage related to that of the syntrophic Gram-positive bacterium Pelotomaculum sp. MGP (Fig. 2b), which, like strain sk.kt5T, is unable to reduce sulfate (Imachi et al., 2006). Unfortunately, dsrAB sequence information is available only for a single Moorella species, namely M. thermoacetica. As can be seen from Fig. 2(b), this sequence clusters together with the xenologous dsrAB sequences of certain members of the class Clostridia, which probably acquired their dsrAB sequences by lateral gene transfer from a deltaproteobacterial donor related to Desulfobacterium anilini DSM 4660T (Zverlov et al., 2005). In contrast, strain sk.kt5T probably carries an orthologous dsrAB gene, as shown by the congruence between its dsrAB and 16S rRNA gene sequence-derived phylogenies (Fig. 2a, b).
The genotypic and phenotypic analyses clearly differentiate sk.kt5T from its 16S rRNA gene sequence-based phylogenetically closest relatives. Thus, we propose that strain sk.kt5T should be considered as representing a novel species within a novel genus, for which the name Desulfitibacter alkalitolerans gen. nov., sp. nov. is proposed. Desulfitibacter alkalitolerans was isolated from biofilms grown on mild steel coupons in a reactor connected to a district heating plant exhibiting problems with biofilm formation and corrosion. Strain sk.kt5T is capable of sulfide production through the reduction of elemental sulfur, thiosulfate or sulfite. The latter compounds could be formed transiently in the district heating system as a result of the chemical oxidation of biologically produced sulfide when oxygen is temporary introduced during maintenance/repair of the pipe system. As described in a separate publication (Abildgaard et al., 2006), a novel sulfate-reducing bacterium, Desulfovibrio alkalitolerans DSM 16529T, was recently isolated from a biofilm sample of the same district heating plant.
Description of the genus Desulfitibacter gen. nov.
Desulfitibacter (De.sul.fi.ti.bac'ter. L. pref. de from, off, away; N.L. n. sulfis -itis sulfite; N.L. masc. n. bacter a rod; N.L. masc. n. Desulfitibacter rod-shaped bacterium that reduces sulfite).
Motile, Gram-positive rods. Form terminal endospores. Anaerobic, growing chemoheterotrophically by fermentation or by reduction of sulfite or thiosulfate. The type species is Desulfitibacter alkalitolerans.
Description of Desulfitibacter alkalitolerans sp. nov.
Desulfitibacter alkalitolerans (N.L. n. alkali alkali; L. pres. part. tolerans tolerating; N.L. part. adj. alkalitolerans alkali-tolerating).
Displays the following properties in addition to those given in the genus description. Cells are 0.4–0.6x2.5–9.6 µm in size, alkalitolerant and mesophilic. The temperature range for growth is 23–44 °C, with an optimum at 35–37 °C. Extended incubation (130 days) facilitates growth at temperatures down to 14.5 °C. The pH range for growth at 40 °C is 7.6–10.5, with an optimum at pH 8.0–9.5. NaCl concentrations up to 5 % (w/v) are tolerated, but the optimal concentration for growth is 0–0.5 % (w/v). Yeast extract is required for growth. The following substrates are utilized as carbon and energy sources in the presence of thiosulfate: betaine, formate, lactate, methanol and pyruvate. In the presence of sulfite, choline is also utilized, but formate is not. The following substrates do not support growth: H2/CO2, acetate, acetone, D(–)-arabinose, benzoate, 2-butanol, butyrate, Casamino acids, choline chloride, ethanol, D(–)-fructose, fumarate, D(+)-galactose, D(+)-glucose, glycerol, glycine, DL-malate, D(+)-mannose, 1-pentanol, 2-propanol, propionate, L(+)-rhamnose, succinate, sucrose and D(+)-xylose. Elemental sulfur, sulfite, thiosulfate, nitrate and nitrite (at low concentrations) are utilized as electron acceptors. Sulfate and Fe(III) citrate are not reduced. The DNA G+C content is 41.6 mol%.
The type strain, sk.kt5T (=DSM 16504T=JCM 12761T), was isolated from a biofilm growing in a high-pH district heating system in Denmark.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Baker, B. J., Moser, D. P., MacGregor, B. J. & 13 other authors (2003). Related assemblages of sulphate-reducing bacteria associated with ultradeep gold mines of South Africa and deep basalt aquifers of Washington State. Environ Microbiol 5, 267–277.[CrossRef][Medline]
Balk, M., Weijma, J., Friedrich, M. W. & Stams, A. J. M. (2003). Methanol utilization by a novel thermophilic homoacetogenic bacterium, Moorella mulderi sp. nov., isolated from a bioreactor. Arch Microbiol 179, 315–320.[Medline]
Bower, C. E. & Holm-Hansen, T. (1980). A salicylate-hypochlorite method for determining ammonia in sea water. Can J Fish Aquat Sci 37, 794–798.
Cline, J. D. (1969). Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol Oceanogr 14, 454–458.
Elsgaard, L., Isaksen, M. F., Jørgensen, B. B., Alayse, A. M. & Jannasch, H. W. (1994). Microbial sulfate reduction in deep-sea sediments at the Guaymas Basin hydrothermal vent area: influence of temperature and substrates. Geochim Cosmochim Acta 58, 3335–3343.[CrossRef]
Fontaine, F. E., Peterson, W. H., McCoy, E., Johnson, M. J. & Ritter, G. J. (1942). A new type of glucose fermentation by Clostridium thermoaceticum. J Bacteriol 43, 701–706.
Hamilton, W. A. (1995). Biofilms and microbially influenced corrosion. In Microbial Biofilms, pp. 171–182. Edited by H. M. Lappin-Scott & J. W. Costerton. Cambridge: Cambridge University Press.
Hungate, R. E. (1976). A roll tube method for cultivation of strict anaerobes. Methods Microbiol 3B, 117–132.
Imachi, H., Sekiguchi, Y., Kamagata, Y. & 7 other authors (2006). Non-sulfate-reducing, syntrophic bacteria affiliated with Desulfotomaculum cluster I are widely distributed in methanogenic environments. Appl Environ Microbiol 72, 2080–2091.
Kjeldsen, K. U., Joulian, C. & Ingvorsen, K. (2004). Oxygen tolerance of sulfate-reducing bacteria in activated sludge. Environ Sci Technol 38, 2038–2043.[Medline]
Kjellerup, B. V., Olesen, B. H., Nielsen, J. L., Frølund, B., Ødum, S. & Nielsen, P. H. (2003). Monitoring and characterisation of bacteria in corroding district heating systems using fluorescence in situ hybridisation and microautoradiography. Water Sci Technol 47 (5), 117–122.[Medline]
Kjellerup, B. V., Thomsen, T. R., Nielsen, J. L., Olesen, B. H., Frølund, B. & Nielsen, P. H. (2005). Microbial diversity in biofilms from corroding heating systems. Biofouling 21, 19–29.[CrossRef][Medline]
Lane, D. J. (1991). 16/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 113–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
Lee, W., Lewandowski, Z., Nielsen, P. H. & Hamilton, W. A. (1995). Role of sulfate-reducing bacteria in corrosion of mild steel – a review. Biofouling 8, 165–193.
Little, B. J., Wagner, P. A., Characklis, W. G. & Lee, W. (1990). Microbial corrosion. In Biofilms, pp. 635–670. Edited by W. G. Characklis & K. C. Marshall. New York: Wiley.
Ljungdahl, L. G. (1986). The autotrophic pathway of acetate synthesis in acetogenic bacteria. Annu Rev Microbiol 40, 415–450.[Medline]
Lovley, D. R. & Phillips, E. J. P. (1986). Organic matter mineralization with reduction of ferric iron in anaerobic sediments. Appl Environ Microbiol 51, 683–689.
Ludwig, W., Strunk, O., Westram, R. & 29 other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.
Marshall, K. C. (1992). Biofilms: an overview of bacterial adhesion, activity, and control at surfaces. ASM News 58, 202–207.
Mogensen, G. L., Kjeldsen, K. U. & Ingvorsen, K. (2005). Desulfovibrio aerotolerans sp. nov., an oxygen tolerant sulfate-reducing bacterium isolated from activated sludge. Anaerobe 11, 339–349.[CrossRef][Medline]
Postgate, J. R. (1984). The Sulfate-reducing Bacteria, 2nd edn. Cambridge: Cambridge University Press.
Slobodkin, A., Reysenbach, A., Mayer, F. & Wiegel, J. (1997). Isolation and characterization of the homoacetogenic thermophilic bacterium Moorella glycerini sp. nov. Int J Syst Bacteriol 47, 969–974.
Vandamme, P., Pot, B., Gillis, M., De Vos, P., Kersters, K. & Swings, J. (1996). Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60, 407–438.
Widdel, F. & Bak, F. (1992). Gram-negative mesophilic sulphate-reducing bacteria. In The Prokaryotes, 2nd edn, pp. 3352–3378. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer.
Wiegel, J., Braun, M. & Gottschalk, G. (1981). Clostridium thermoautotrophicum sp. nov., a thermophile producing acetate from molecular hydrogen and carbon dioxide. Curr Microbiol 5, 255–260.
Zverlov, V., Klein, M., Lücker, S., Friedrich, M. W., Kellermann, J., Stahl, D. A., Loy, A. & Wagner, M. (2005). Lateral gene transfer of dissimilatory (bi)sulfite reductase revisited. J Bacteriol 187, 2203–2208.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
, also including Sporotomaculum hydroxybenzoicum DSM 5475T (Y14845); 
, a partial dsrAB sequence of Ammonifex degensii DSM 10501T was obtained through the NCBI homepage (