A doubling of microphytobenthos biomass coincides with a tenfold increase in denitrifier and total bacterial abundances in intertidal sediments of a temperate estuary.

Helen Decleyre,Filip Van Nieuwerburgh,Carl Van Colen,Kim Heylen,Anne Willems,Dieter Deforce,Bjorn Tytgat,Koen Sabbe,Hauke Smidt

doi:10.1371/journal.pone.0126583

Abstract

Surface sediments are important systems for the removal of anthropogenically derived inorganic nitrogen in estuaries. They are often characterized by the presence of a microphytobenthos (MPB) biofilm, which can impact bacterial communities in underlying sediments for example by secretion of extracellular polymeric substances (EPS) and competition for nutrients (including nitrogen). Pyrosequencing and qPCR was performed on two intertidal surface sediments of the Westerschelde estuary characterized by a two-fold difference in MPB biomass but no difference in MPB composition. Doubling of MPB biomass was accompanied by a disproportionately (ten-fold) increase in total bacterial abundances while, unexpectedly, no difference in general community structure was observed, despite significantly lower bacterial richness and distinct community membership, mostly for non-abundant taxa. Denitrifier abundances corresponded likewise while community structure, both for nirS and nirK denitrifiers, remained unchanged, suggesting that competition with diatoms for nitrate is negligible at concentrations in the investigated sediments (appr. 1 mg/l NO3 -). This study indicates that MPB biomass increase has a general, significantly positive effect on total bacterial and denitrifier abundances, with stimulation or inhibition of specific bacterial groups that however do not result in a re-structured community.

Highlights

The rate of terrestrial nitrogen input has more than doubled in the past century, mostly through fossil fuel combustion and increased use of agricultural fertilizers [1, 2]
The only detected differences between HBM and LBM sediments were related to the presence and activity of MPB: chlorophyll a, a proxy for MPB biomass, and extracellular polymeric substances (EPS) were significantly higher in the HBM sediments, while PO43- and Si were significantly lower (p < 0.05, Table 1)
The 16S rRNA gene rarefaction curves of a replicate per sample type (HBM2 and LBM3) flattened very quickly in comparison with the other replicates (S2 Fig). As no such discrepancies were observed when using the same DNA material for nir gene sequencing and Quantitative real-time PCR (qPCR), we suspect that the limited numbers of operational taxonomic units (OTUs) observed for HBM2 and LBM3 resulted from inconsistent emulsion PCR and excluded both samples from further analyses

Summary

Introduction

The rate of terrestrial nitrogen input has more than doubled in the past century, mostly through fossil fuel combustion and increased use of agricultural fertilizers [1, 2]. Denitrifying communities are commonly characterized using the two genes encoding cytochrome cd and copper-containing nitrite reductases (nirS and nirK respectively) as a proxy [16] These key enzymes convert fixed nitrogen to a gaseous form, as such distinguishing dissimilatory nitrate-reducing bacteria that produce nitrite as end-product from true denitrifiers. Fixed carbon by MPB is transferred to heterotrophic bacteria within hours, resulting in a quick use of labile biofilm DOC and hydrolysed EPS [28,29,30,31,32], while MPB can produce cytotoxins that can inhibit bacterial growth [33]. Responses of nirK and nirS denitrifier communities To investigate these hypotheses, we sampled estuarine sediments at the Paulina polder tidal flat (Westerschelde estuary, SW Netherlands), characterized by the presence of MPB biofilms stabilizing sediment surfaces [37, 41]. Abundant and non-abundant fractions of all three bacterial communities (i.e. all bacteria, nirK, nirS) were examined separately to assess influences of the low-abundant fraction of the bacterial community on diversity parameters

Experimental procedures

Results

Discussion