Variations in abundance and community composition of denitrifying bacteria during a cyanobacterial bloom in a eutrophic shallow lake in China

<p>Although cyanobacterial blooms can change microbial communities, it is still unclear what impact such harmful blooms will have on denitrifying bacteria, the drivers of the removal of excessive nitrogen from water. In order to clarify the impact, populations of denitrifying bacteria, with periodic proliferation and dominance of cyanobacteria in a eutrophic shallow lake located in southeast China, were investigated using quantitative real-time polymerase chain reaction (qPCR) and 454-pyrosequencing based on the copper-containing nitrite reductase (<i>nirK</i>) gene, cytochrome cd1-containing nitrite reductase (<i>nirS</i>) gene and nitrous oxide reductase (<i>nosZ</i>) gene. Samples were collected periodically during a three-month period when the cyanobacterial density gradually increased. In the qPCR analyses, abundances of <i>nirK</i>, <i>nirS</i> and <i>nosZ</i> were intensely positively correlated with the biomass of cyanobacteria. Moreover, 454-pyrosequencing revealed that the community composition of denitrifying bacteria shifted with the increase in cyanobacterial density. These results indicated that the shifts of the community composition of denitrifying bacteria might be related to cyanobacterial blooms, which could potentially lead to alterations of denitrification in eutrophic water.</p

The location (A, B) and structure (C-G) of CMC gut.

<p>The cross-sections of CMC intestines were analyzed with light microscope (D, F) and with transmission electron microscope (E, G). ES represents endothelial cell surface, SL represents secretary layer, and GC represents gut cavity. Scale bar = 1.0 cm (A, B), 2.0 cm (C), 100 μm (D, F), 0.25 μm (E), and 5.0 μm (G).</p

Crabs used in this study.

<p>¹ DMx and DHx mean the midgut and hindgut from one crab individual if x is of the same number.</p><p>Crabs used in this study.</p

OTU richness, coverage and diversity richness index of the 16S rRNA gene obtained from metagenomic sequencing.

<p>OTU richness, coverage and diversity richness index of the 16S rRNA gene obtained from metagenomic sequencing.</p

RDP Classifier (Confidence threshold: 95%) results of bacterial community in the CMC intestines based on DGGE.

<p>¹ M, midgut; H, hindgut.</p><p>RDP Classifier (Confidence threshold: 95%) results of bacterial community in the CMC intestines based on DGGE.</p

FISH analysis of the two dominant bacterial phylotypes of <i>Epsilonproteobacteria</i> and <i>Bacteroidetes</i> in the intestines of CMC.

<p>Every two epifluorescence photomicrographs (A and B, D and E) show the same microscopic field of CMC intestinal sample double-hybridized with oligonucleotide probes specific for <i>Bacteria</i> (Eub338) (A, D) and for <i>Epsilonproteobacteria</i> (EPSY549) (B) or <i>Bacteroidetes</i> (CF319a) (E). (C) Merged photo of A and B. (F) Merged photo of D and E. Scale bar = 10.0 μm.</p

DGGE analysis of the intestinal bacterial community of CMC.

<p>The seventeen bands excised for sequencing were marked with numbers. DMx and DHx indicate midgut and hindgut, respectively. The samples were obtained from the same crab when x is of the same number. CM-1 represents CMC <i>Mollicutes</i> group 1 and CM-2 represents CMC <i>Mollicutes</i> group 2.</p

Phylogenetic affiliations of dominant OTUs from 16S rRNA metagenomic analysis of CMC intestines.

<p>Phylogenetic affiliations of dominant OTUs that contain more than 50 reads in the bacterial 16S rRNA gene metagenomic data sets obtained from crab intestines. Sequences derived from the present study are shown in bold. The background colors indicate the bacterial phylotypes of different phyla.</p

Rarefaction curves (A) and Venn diagram (B) of the bacterial 16S rRNA gene metagenomic data sets from the intestine of CMCs at 97% similarity.

<p>The rarefaction curves for all the samples reached the near plateau phase, representing good sampling depth. Most of the abundant species were common to all the samples. DMx and DHx indicate midgut and hindgut, respectively. The samples were obtained from the same crab when x is of the same number.</p

Phylogenetic affiliations of the bacterial 16S rRNA genes cloned from crab intestines.

<p>The clones obtained from crab intestines are shown in bold. The clones obtained in this study and those from a previous study (Li et al, 2007) are indicated with black square and black dot, respectively. The number of clones is shown in parenthesis. The background colors indicate the bacterial phylotypes of different phyla.</p