Denaturing gradient gel electrophoretic analysis of ammonia-oxidizing bacterial community structure in the lower Seine River: impact of Paris wastewater effluents

Abstract

The Seine River is strongly affected by the effluents from the Acheres wastewater treatment plant (WWTP) downstream of the city of Paris. We have shown that the effluents introduce large amounts of ammonia and inoculate the receiving medium with nitrifying bacteria. The aim of the present study was to investigate the diversity of the ammonia-oxidizing bacterial population by identifying autochthonous bacteria from upstream and/or allochthonous ammonia-oxidizing bacteria from the WWTP effluents. Measurements of potential nitrifying activity, competitive PCR, and denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments specific to ammonia-oxidizing bacteria (AOB) were used to explore the succession and shifts of the ammonia-oxidizing community in the lower Seine River and to analyze the temporal and spatial functioning of the system at several different sampling dates. A major revelation was the stability of the patterns. The CTO primers used in this study (G. A. Kowalchuk, J. R. Stephen, W. D. Boer, J. I. Prosser, T. M. Embley, and J. W. Woldendorp, Appl. Environ. Microbiol. 63:1489-1497, 1997) were shown not to be completely specific to AOB of the beta subclass of Proteobacteria. We further demonstrated that when DGGE patterns are interpreted, all the different bands must be sequenced, as one major DGGE band proved to be affiliated with a group of non-AOB in the beta subclass of Proteobacteria. The majority of AOB (75 to 90%) present in the lower Seine river downstream of the effluent output belong to lineage 6a, represented by Nitrosomonas oligotropha- and Nitrosomonas ureae-like bacteria. This dominant lineage was represented by three bands on the DGGE gel. The major lineage-6a AOB species, introduced by the WWTP effluents, survived and might have grown in the receiving medium far downstream, in the estuary; it represented about 40% of the whole AOB population. The other two species belonging to lineage 6a seem to be autochthonous bacteria. One of them developed a few kilometers downstream of the WWTP effluent input in an ammonia-enriched environment, and the other appeared in the freshwater part of the estuary and was apparently more adapted to estuarine conditions, i.e., an increase in the amount of suspended matter, a low ammonia concentration, and high turnover of organic matter. The rest of the AOB population was represented in equal proportions by Nitrosospira- and Nitrosococcus mobilis-like species.

FIG. 1.

Description of the study site: the Seine River from Paris (km 0) to the mouth of the estuary (km 356). Numbers 1 to 18 represent the sampling stations, as follows: 1, Saint Maurice; 2, Maison Laffites; 3, Conflans; 4, Triel; 5, Porcheville; 6, Vernon; 7, Les Andelys; 8, Poses; 9, Pont Arche; 10, Elbeuf; 11, Oissel; 12, Bassin des Docks; 13, La Bouille; 14, Duclair; 15, Heurtauville; 16, Caudebec; 17, Tancarville; 18, Honfleur. Caudebec represents the limit of saline intrusion.

FIG. 2.

Variations along the Seine river from Paris (km 0) to the mouth of the estuary (km 356) in (a) ammonium (NH₄⁺), nitrite (NO₂⁻), and nitrate (NO₃⁻) levels, (b) potential ammonia oxidation rates (pAO) and potential nitrification activities (pNA), (c) SPM concentrations, and (d) amoA gene copy numbers determined by cPCR for three sampling dates (July 2002, September 2002, and September 2003).

FIG. 3.

DGGE profiles of ammonia-oxidizing bacteria of the β subclass of the Proteobacteria, obtained by CTO PCR on July 2002 samples. Lanes 1 to 18 correspond to the 18 different sampling stations; lanes “WW in” and “WW out” are samples from domestic wastewater before and after treatment, respectively. The principal bands are labeled b1 to b8 (bands that were cut and sequenced). The denaturing gradient is from 39 to 52%.

FIG. 4.

DGGE profiles of ammonia-oxidizing bacteria of the β subclass of the Proteobacteria, obtained by nested PCR on July 2002, September 2002, and September 2003 samples. Lanes 1 to 18 correspond to the 18 different sampling stations; lanes “WW in” and “WW out” represent samples from domestic wastewater treatment plants before and after treatment. Lanes WM, weight markers. The principal bands observed are labeled with capital letters (A to L). All these bands have been cut and sequenced to determine the phylogeny of each ammonia-oxidizing bacterium and to compare the three sampling dates. The denaturing gradient is the same for the three DGGE gels; only 36.5 to 51.5% is shown here.

FIG. 5.

Phylogenetic neighbor-joining tree based on a comparison of 404 bp of 16S rRNA gene sequences from AOB of the β subclass of the Proteobacteria and some other β-proteobacteria. The tree is rooted with two sequences of AOB belonging to the γ subclass of the Proteobacteria. Our DGGE sequences are boldfaced. Their designations are composed of C, for CTO PCR products; J2, S2, or S3, for a sampling date of July 2002, September 2002, or September 2003; b1 to b8, for the band as labeled in Fig. 3; and the name of the sampling station from which the sequence originated.

FIG. 6.

Phylogenetic neighbor-joining tree based on a comparison of 152 bp of 16S rRNA gene sequences from AOB of the β subclass of the Proteobacteria and some other β-proteobacteria. The tree is rooted with two sequences of AOB belonging to the γ subclass of Proteobacteria. Our DGGE sequences are boldfaced. Their designations are composed of N, for nested PCR products; J2, S2, or S3, for a sampling date of July 2002, September 2002, or September 2003; A to L, w1, or w2, for the band as labeled in Fig. 4; and, at the end, either the name of the sampling station from which the sequence originated or WWin or WWout (explained in the legend to Fig. 4).