Succession of internal sulfur cycles and sulfur-oxidizing bacterial communities in microaerophilic wastewater biofilms

Abstract

The succession of sulfur-oxidizing bacterial (SOB) community structure and the complex internal sulfur cycle occurring in wastewater biofilms growing under microaerophilic conditions was analyzed by using a polyphasic approach that employed 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization, microelectrode measurements, and standard batch and reactor experiments. A complete sulfur cycle was established via S(0) accumulation within 80 days in the biofilms in replicate. This development was generally split into two phases, (i) a sulfur-accumulating phase and (ii) a sulfate-producing phase. In the first phase (until about 40 days), since the sulfide production rate (sulfate-reducing activity) exceeded the maximum sulfide-oxidizing capacity of SOB in the biofilms, H(2)S was only partially oxidized to S(0) by mainly Thiomicrospira denitirificans with NO(3)(-) as an electron acceptor, leading to significant accumulation of S(0) in the biofilms. In the second phase, the SOB populations developed further and diversified with time. In particular, S(0) accumulation promoted the growth of a novel strain, strain SO07, which predominantly carried out the oxidation of S(0) to SO(4)(2-) under oxic conditions, and Thiothrix sp. strain CT3. In situ hybridization analysis revealed that the dense populations of Thiothrix (ca. 10(9) cells cm(-3)) and strain SO07 (ca. 10(8) cells cm(-3)) were found at the sulfur-rich surface (100 microm), while the population of Thiomicrospira denitirificans was distributed throughout the biofilms with a density of ca. 10(7) to 10(8) cells cm(-3). Microelectrode measurements revealed that active sulfide-oxidizing zones overlapped the spatial distributions of different phylogenetic SOB groups in the biofilms. As a consequence, the sulfide-oxidizing capacities of the biofilms became high enough to completely oxidize all H(2)S produced by SRB to SO(4)(2-) in the second phase, indicating establishment of the complete sulfur cycle in the biofilms.

FIG. 1.

Time courses for different sulfur compounds in the reactor influent (inf) and effluent (eff) during the experiments. (A) RDR1. (B) RDR2.

FIG. 2.

Accumulation of S⁰, AVS (H₂S, HS⁻, S²⁻, and FeS), and CRS (FeS₂) in the developing biofilms. (A) RDR1. (B) RDR2. The error bars indicate the standard deviations of the measurements for three biofilm specimens.

FIG. 3.

Potential sulfur and sulfide oxidation rates of 18-day-old and 65-day-old biofilms taken from RDR1 (A) and RDR2 (B). The rates were determined in batch experiments with O₂ or NO₃⁻ as the sole electron acceptor (e-acceptor). The error bars indicate the standard deviations for duplicate experiments.

FIG. 4.

(A) Steady-state concentration profiles for O₂, H₂S, NO₃⁻, and pH in a 14-day-old wastewater biofilm taken from RDR1. The biofilm was incubated in DO-controlled (DO concentration, approximately 60 μM) synthetic medium with about 250 μM NO₃⁻. The error bars indicate the standard deviations for three measurements at different positions. (B) Spatial distributions of the specific H₂S production and oxidation rates. The rates were calculated based on the corresponding microprofiles shown in panel A. The biofilm surface was at a depth of 0 μm.

FIG. 5.

(A) Steady-state concentration profiles for O₂, H₂S, NO₃⁻, and pH in a 60-day-old wastewater biofilm taken from RDR1. The biofilm was incubated in DO-controlled (DO concentration, approximately 80 μM) synthetic medium with no addition of NO₃⁻ (solid circles) and with 90 μM NO₃⁻ (open circles). The error bars indicate the standard deviations for three measurements at different positions. (B) Spatial distributions of the specific H₂S production and oxidation rates when NO₃⁻ was absent and present. The rates were calculated based on the corresponding microprofiles shown in panel A. (C) Vertical distributions of S⁰ in the biofilms obtained from RDR1 (gray bars) and RDR2 (open bars). The error bars indicate the standard deviations of the measurements for four biofilm specimens. The biofilm surface was at a depth of 0 μm.

FIG. 6.

Phylogenetic distance tree showing the affiliations of 16S rRNA gene clone sequences related to SOB shown in Table 1. The nearly full-length sequences of 16S rRNA genes (>1,380 bp) were retrieved from the RDR1 biofilm (Biofilm-) and RDR1 biofilms precultured with H₂S-O₂ (HSO-), H₂S-NO₃⁻ (HSN-), S⁰-O₂ (SO-), and S⁰-NO₃⁻ (SN-) as combinations of sole electron donor and sole electron acceptor. The bar represents 5% estimated divergence, and the numbers at the nodes are bootstrap values (100 replicates) with more than 50% bootstrap support. Aquifex pyrophilius served as an outgroup.

FIG.7.

In situ detection of sulfur-oxidizing bacteria in a thin vertical section (thickness, 20 μm) of a 70-day-old wastewater biofilm taken from RDR1. (A and B) FISH with fluorescein isothiocyanate-labeled probe G123T (specific for Thiothrix sp.) at the surface of the biofilm (0 to 100 μm): confocal scanning laser microscope projection image (A) and differential interference contrast (DIC) image (B). (C) FISH with TRITC-labeled probe for strain SO07 in the surface 100 μm of the biofilm. (D) DIC image of the field shown in the panel C. (E) FISH with TRITC-labeled probe for Thiomicrospira denitrificans at a depth of approximately 400 μm in the biofilm. (F) DIC image of the field shown in panel E. The yellowish signals were autofluorescence. Bars = 20 μm (A), 10 μm (B), and 5 μm (C to F).