Isolation, Characterization, and In Situ Detection of a Novel Chemolithoautotrophic Sulfur-Oxidizing Bacterium in Wastewater Biofilms Growing under Microaerophilic Conditions

Abstract

We successfully isolated a novel aerobic chemolithotrophic sulfur-oxidizing bacterium, designated strain SO07, from wastewater biofilms growing under microaerophilic conditions. For isolation, the use of elemental sulfur (S 0 ), which is the most abundant sulfur pool in the wastewater biofilms, as the electron donor was an effective measure to establish an enrichment culture of strain SO07 and further isolation. 16S rRNA gene sequence analysis revealed that newly isolated strain SO07 was affiliated with members of the genus Halothiobacillus, but it was only distantly related to previously isolated species (89% identity). Strain SO07 oxidized elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions. Strain SO07 could not grow on nitrate. Organic carbons, including acetate, propionate, and formate, could not serve as carbon and energy sources. Unlike other aerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 150 mM was negligible. In situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells hybridized with a probe specific for strain SO07 were mainly present in the oxic biofilm strata (ca. 0 to 100 m) and that they often coexisted with sulfate-reducing bacteria in this zone. These results demonstrated that strain SO07 was one of the important sulfur-oxidizing populations involved in the sulfur cycle occurring in the wastewater biofilm and was primarily responsible for the oxidation of H 2 S and S 0 to SO 4 2؊ under oxic conditions.The sulfur cycle, driven by sulfate reduction and sulfide oxidation, is an important cycle of minerals in various sulfurand sulfide-rich environments (e.g., hot spring microbial mats, lake sediments, marine sediments, and wastewater biofilms), and it is closely linked with other cycles (e.g., carbon, oxygen, and nitrogen). For instance, up to 50% of organic matter can be mineralized by sulfate reduction in marine sediments (18). Successional development of sulfate reduction and subsequent sulfide oxidation were precisely determined within wastewater biofilms with a typical thickness of approximately 1,000 m, which accounted for a substantial part of oxygen consumption and mineralization of organic matter (25, 33).The sulfur cycle in wastewater biofilms is mainly driven by the close association of sulfate-reducing bacteria and sulfuroxidizing bacteria, both of which comprise phylogenetically diverse groups of microorganisms adapted to wastewater conditions, e.g., neutral pH and low dissolved oxygen concentrations. The diversity and in situ ecophysiology of sulfate-reducing bacteria in the wastewater biofilms have been intensively investigated by a 16S rRNA gene approach and microelectrodes (16,17,32,33,35), while the diversity and ecological importance of sulfur-oxidizing bacteria involved in the sulfur cycle are still largely unknown. Recently, novel sulfur-oxidizing bacteria belonging to the ␥ and ε subclasses of the Proteobacteria have been discovered and is...

Succession of Internal Sulfur Cycles and Sulfur-Oxidizing Bacterial Communities in Microaerophilic Wastewater Biofilms

“…This result suggested that the novel SOB strain SO07 could be responsible for oxidation of S 0 to SO 4 2Ϫ under oxic conditions. Further isolation and characterization of strain SO07 revealed that strain SO07 was an aerobic chemolithoautotrophic sulfur-oxidizing bacterium capable of oxidizing S 0 and H 2 S to SO 4 2Ϫ (13). More details concerning the ecophysiological characteristics of strain SO07 have been reported previously (13).…”

Community Structure of Subsurface Biofilms in the Thermal Sulfidic Caves of Acquasanta Terme, Italy

“…Related isolates include the sulfur-oxidizing lithoautotrophs "Ca. Thiobacillus baregensis" (25,26), Thiofaba tepidiphila (42), and Thiovirga sulfuroxydans (29,30). Representatives of the clade defined by these isolates (Fig.…”

Thiovirga sulfuroxydans gen. nov., sp. nov., a chemolithoautotrophic sulfur-oxidizing bacterium isolated from a microaerobic waste-water biofilm

“…The use of S 0 as an electron donor was an effective measure to establish an enrichment culture of strain SO07 T and for further purification (Ito et al, 2004). The 16S rRNA genes of large numbers of uncultured bacterial clones that are closely related to strain SO07 T have been found in GenBank (Benson et al, 2003), suggesting the widespread distribution and ecological importance of these types of bacteria in the environment.…”

“…The composition of SOB medium (in g l 21 ) was KH 2 PO 4 (0?5), K 2 HPO 4 (0?5), NH 4 Cl (0?5), MgSO 4 .7H 2 O (0?1), CaCl 2 (0?05) and NaHCO 3 (1?0), plus 1 ml trace element solution l 21 (Kuenen et al, 1991). The procedures for enrichment and isolation have been described previously (Ito et al, 2004). Cells grown in liquid SOB medium with thiosulfate (6?5 mM) as a sole electron donor were used for studies on phenotypic properties and chemotaxonomic traits unless otherwise specified.…”

“…PCR amplification, purification of PCR products and 16S rRNA gene sequencing were carried out as described previously (Ito et al, 2004). Phylogenetic inferences were made with the 16S rRNA gene sequence database associated with the ARB software package (Ludwig et al, 2004).…”