In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms

Abstract

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 microm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH(4)(+) and NO(2)(-) consumption rates decreased from 0.68 and 0.64 micromol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 micromol cm(-2) h(-1) at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH(4)(+) and NO(2)(-) and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O(2) or organic compounds, which consequently established suitable microenvironments for anammox bacteria.

FIG. 1.

Schematic drawing of anaerobic fixed-bed glass column reactor with three ports (diameter, 1 cm) for liquid sampling and microelectrode measurements. P1, P2, P3, and P4 indicate the sampling points of anammox biofilms for FISH and phylogenetic analyses.

FIG. 2.

Phylogenetic trees showing the positions of clones retrieved from the detached biomass (OTUs HU1 to HU5) and biofilm (OTU Biofilm-PU13) in the reactor after 74 days of operation in relation to previously published anammox-like bacterial sequences (A) and betaproteobacterial sequences (B). The trees were generated by using 1,429 bp (A) and 1,462 bp (B) of the 16S rRNA gene and the neighbor-joining method. Bars = 2% sequence divergence. The values at the nodes are bootstrap values (1,000 resampling analyses). The GenBank/EMBL/DDBJ accession numbers are also indicated.

FIG. 3.

(A) CLSM image of a vertical section (30 μm thick) of the anammox biofilm at P1 after 74 days of operation after FISH with FITC-labeled EUB338mix probe (green) and TRITC-labeled Amx820 probe (red). Yellow signals result from binding of both probes to one cell, indicating anammox bacteria. Bar = 100 μm. (B) Phase-contrast image of the vertical section in panel A. The biofilm surface is at the top of the images.

FIG. 4.

Relative abundance of anammox bacteria at different depths in the anammox biofilm (A) and of nitrifying and unidentified bacteria in the upper part of the biofilm (>1,000 μm) (B). Relative abundance is shown as the percentage of each probe signal in a microscopic field compared with the EUB338mix probe signal. The number of unidentified bacteria was calculated as the difference between the EUB338mix signal and the total signal from each specific probe. P1, P2, P3, and P4 refer to the biofilm sampling points, as shown in Fig. 1. The error bars indicate standard deviations.

FIG. 5.

CLSM images showing the in situ spatial organization of anammox bacteria and coexisting bacteria (nitrifying and putative heterotrophic bacteria) in the anammox biofilm. All red signals show FISH with the TRITC-labeled probe Amx820 (A to D). Green signals show FISH with the FITC-labeled probe EUB338mix at P2 (A) and P3 (B), with probe NmV at P2 (C), and with probe Ntspa662 at P2 (D). Bars = 10 μm. Yellow signals result from binding of both probes to one cell, indicating anammox bacteria (A and B). The biofilm surface is at the top of the images.

FIG. 6.

Steady-state concentration profiles of O₂ (closed circles), NH₄⁺ (gray squares), NO₂⁻ (open circles), and NO₃⁻ (triangles) and pH profiles (crosses) in the anammox biofilms measured at P2 (A), P3 (B), and P4 (C), as shown in Fig. 1. Spatial distributions of the estimated specific consumption rates of NH₄⁺ (gray bars), NO₂⁻ (open circles), and NO₃⁻ (black bars) at P2, P3, and P4 are shown in panels D, E, and F, respectively. Negative values indicate production rates. The surface of the biofilm was at a depth of 0 μm. The error bars indicate standard deviations (n = 3).