Microbial diversity of a heavily polluted microbial mat and its community changes following degradation of petroleum compounds

Abstract

We studied the microbial diversity of benthic cyanobacterial mats inhabiting a heavily polluted site in a coastal stream (Wadi Gaza) and monitored the microbial community response induced by exposure to and degradation of four model petroleum compounds in the laboratory. Phormidium- and Oscillatoria-like cyanobacterial morphotypes were dominant in the field. Bacteria belonging to different groups, mainly the Cytophaga-Flavobacterium-Bacteriodes group, the gamma and beta subclasses of the class Proteobacteria, and the green nonsulfur bacteria, were also detected. In slurry experiments, these communities efficiently degraded phenanthrene and dibenzothiophene completely in 7 days both in the light and in the dark. n-Octadecane and pristane were degraded to 25 and 34% of their original levels, respectively, within 7 days, but there was no further degradation until 40 days. Both cyanobacterial and bacterial communities exhibited noticeable changes concomitant with degradation of the compounds. The populations enriched by exposure to petroleum compounds included a cyanobacterium affiliated phylogenetically with Halomicronema. Bacteria enriched both in the light and in the dark, but not bacteria enriched in any of the controls, belonged to the newly described Holophaga-Geothrix-Acidobacterium phylum. In addition, another bacterial population, found to be a member of green nonsulfur bacteria, was detected only in the bacteria treated in the light. All or some of the populations may play a significant role in metabolizing the petroleum compounds. We concluded that the microbial mats from Wadi Gaza are rich in microorganisms with high biodegradative potential.

FIG. 1.

DGGE band patterns of PCR-amplified 16S rRNA fragments obtained by using cyanobacterium-specific primers and the Wadi Gaza field microbial mats (lane WG) and mats collected at the end of the experiments from the microbiological controls (lanes G and GC) and from preparations incubated in the light (lane L) and in the dark (lane D). The labeled bands were excised, reamplified, and sequenced.

FIG. 2.

Phylogenetic tree (maximum likelihood) for cyanobacteria and plastids based on publicly available, almost complete 16S rRNA genes from members of the cyanobacterial line. E. coli and B. subtilis were used as outgroups. The numbers next to the collapsed clusters indicate the number of sequences in each cluster. The 400-bp 16S rRNA sequences from our excised DGGE bands were placed phylogenetically by using parsimony criteria without changing the topology of the preestablished tree. The bar indicates 10% sequence divergence. The identities of strains correspond to the identities given in the databases, and we do not imply that they are necessarily taxonomically correct.

FIG. 3.

DGGE profiles of PCR-amplified 16S rRNA fragments obtained by using universal bacterial primers from the Wadi Gaza field microbial mats (lane WG) and mats collected at the end of the experiments from the microbiological controls (lanes G and GC) and from preparations incubated in the light (lane L) and in the dark (lane D). The labeled fragments were excised, reamplified, and sequenced. The unlabeled bands include those containing cyanobacterial sequences and bacterial bands with sequences similar to the sequences of the labeled bands.

FIG. 4.

Unrooted phylogenetic tree showing affiliations based on partial bacterial 16S rRNA sequences and selected sequences from members of different bacterial clusters, including the γ and β subclasses of the Proteobacteria, the Cytophaga-Flavobacterium-Bacteriodes group (CF), the newly described Holophaga-Geothrix-Acidobacterium phylum (HGA), and the green nonsulfur bacteria (GNSB). The tree was simplified for clarity by omitting all sequences between clusters. The numbers in parentheses indicate the references in which the clustered sequences were described. The scale bar indicates 10% estimated sequence divergence.

FIG. 5.

Plots of quantities of model compounds in slurry experiments with Wadi Gaza microbial mats versus time. (a) Twelve hours of light and 12 h of darkness; (b) 24 h of darkness; (c) control with autoclaved biomass, 12 h of light and 12 h of darkness; (d) control without biomass, 24 h of darkness. The data are expressed as percentages relative to the amounts of the compounds initially introduced into the system (i.e., 3.33 mg of each model compound adsorbed on 100 mg of organo-clay complex particles). Solid bars, pristane; bars with diamonds, dibenzothiophene; open bars, phenanthrene; cross-hatched bars, n-octadecane.