Polycyclic Aromatic Hydrocarbon-Induced Changes in Bacterial Community Structure under Anoxic Nitrate Reducing Conditions

Abstract

Although bacterial anaerobic degradation of mono-aromatic compounds has been characterized in depth, the degradation of polycyclic aromatic hydrocarbons (PAHs) such as naphthalene has only started to be understood in sulfate reducing bacteria, and little is known about the anaerobic degradation of PAHs in nitrate reducing bacteria. Starting from a series of environments which had suffered different degrees of hydrocarbon pollution, we used most probable number (MPN) enumeration to detect and quantify the presence of bacterial communities able to degrade several PAHs using nitrate as electron acceptor. We detected the presence of a substantial nitrate reducing community able to degrade naphthalene, 2-methylnaphthalene (2MN), and anthracene in some of the sites. With the aim of isolating strains able to degrade PAHs under denitrifying conditions, we set up a series of enrichment cultures with nitrate as terminal electron acceptor and PAHs as the only carbon source and followed the changes in the bacterial communities throughout the process. Results evidenced changes attributable to the imposed nitrate respiration regime, which in several samples were exacerbated in the presence of the PAHs. The presence of naphthalene or 2MN enriched the community in groups of uncultured and poorly characterized organisms, and notably in the Acidobacteria uncultured group iii1-8, which in some cases was only a minor component of the initial samples. Other phylotypes selected by PAHs in these conditions included Bacilli, which were enriched in naphthalene enrichments. Several nitrate reducing strains showing the capacity to grow on PAHs could be isolated on solid media, although the phenotype could not be reproduced in liquid cultures. Analysis of known PAH anaerobic degradation genes in the original samples and enrichment cultures did not reveal the presence of PAH-related nmsA-like sequences but confirmed the presence of bssA-like genes related to anaerobic toluene degradation. Altogether, our results suggest that PAH degradation by nitrate reducing bacteria may require the contribution of different strains, under culture conditions that still need to be defined.

Keywords: 2-methylnaphthalene; Acidobacteria; PAHs; anaerobic naphthalene biodegradation; compost pile; iii1-8; nitrate reducing bacteria.

Figure 1

MPN enumeration in the environmental samples of nitrate reducing bacteria able to grow on naphthalene (NAP), 2-methyl-naphthalene (2MN), 2-naphtoic acid (2NA), anthracene (ANT), acetate (Ace), and with no added carbon source (na). Counts were made in triplicate. Numerical values (95% C.I.) can be found in supplementary Table S4. Dotted lines indicate the basal growth with no added carbon source. (A) Rice paddy samples (B) Oil refinery and sediment samples.

Figure 2

Cumulative plot of bacterial phyla detected in the initial environmental samples. Samples were analyzed in duplicate (labeled Ia and Ib) except for RPW, for which one of the samples was lost. RPS, rice-paddy soil; RPW, rice-paddy water, RPCal, rice-paddy Calasparra; AS, activated sludge; CP, compost pile; MS, marine sediment, FdP, Fuente de Piedra athalassohaline lagoon. Numerical values can be found in Table S5.

Figure 3

Cumulative plot of bacterial phyla detected in the different enrichments. The average values of the duplicate initial samples (Figure 2) are included for comparison, labeled as I. All other labels are as in Figure 2. Numerical values can be found in Table S5.

Figure 4

(A) Principal coordinate analysis (PCoA) of distances between OTUs present in the initial environmental samples and the enrichments obtained from them. The percentage of the variation between the samples by principal coordinates is indicated on the axes. (B) Unweighted pair group method with arithmetic average (UPGMA) cluster analysis of bacterial community structure in the compost pile (CP) and activated sludge (AS) environmental sample and enrichments from them using pyrosequencing analysis data based on Pearson correlation using OTUs (>97% sequence similarity) as the species data.

Figure 5

Effect of PAHs on the relative abundance of the most relevant groups in the compost pile (CP) sample. (A) Differences in the relative abundance of the most abundant groups between control (HMN), naphthalene (N), and 2-methyl-naphthalene (2MN) enrichments from the compost pile sample. Asterisks (^*) indicate significant differences between rarefied samples according to the Mann-Whitney test (p < 0.01). (B) Evolutionary relationships of iii1-8 DS18 sequences retrieved from the compost pile initial sample and enrichments. The optimal Neighbour-Joining tree is shown. The bootstrap (1000 replicates) is shown next to the branches. The evolutionary distances are in the units of the number of base substitutions per site. Numbers in brackets indicate the relative abundance (%) of each sequence in the corresponding sample. The sequences of previously described Acidobacteria clones are included (Hugenholtz et al., 1998).