Bacterial communities from shoreline environments (costa da morte, northwestern Spain) affected by the prestige oil spill

Abstract

The bacterial communities in two different shoreline matrices, rocks and sand, from the Costa da Morte, northwestern Spain, were investigated 12 months after being affected by the Prestige oil spill. Culture-based and culture-independent approaches were used to compare the bacterial diversity present in these environments with that at a nonoiled site. A long-term effect of fuel on the microbial communities in the oiled sand and rock was suggested by the higher proportion of alkane and polyaromatic hydrocarbon (PAH) degraders and the differences in denaturing gradient gel electrophoresis patterns compared with those of the reference site. Members of the classes Alphaproteobacteria and Actinobacteria were the prevailing groups of bacteria detected in both matrices, although the sand bacterial community exhibited higher species richness than the rock bacterial community did. Culture-dependent and -independent approaches suggested that the genus Rhodococcus could play a key role in the in situ degradation of the alkane fraction of the Prestige fuel together with other members of the suborder Corynebacterineae. Moreover, other members of this suborder, such as Mycobacterium spp., together with Sphingomonadaceae bacteria (mainly Lutibacterium anuloederans), were related as well to the degradation of the aromatic fraction of the Prestige fuel. The multiapproach methodology applied in the present study allowed us to assess the complexity of autochthonous microbial communities related to the degradation of heavy fuel from the Prestige and to isolate some of their components for a further physiological study. Since several Corynebacterineae members related to the degradation of alkanes and PAHs were frequently detected in this and other supralittoral environments affected by the Prestige oil spill along the northwestern Spanish coast, the addition of mycolic acids to bioremediation amendments is proposed to favor the presence of these degraders in long-term fuel pollution-affected areas with similar characteristics.

FIG. 1.

(A) Detailed map of the northwestern coast of Spain (Galicia) known as the Costa da Morte or the Costa de la Muerte (from the Muros and Noia Estuary to Malpica), which means Coast of Death, for its strong swell and harsh weather. Punta Ínsua is the main landmark next to the sampling site. (B) Closer image of the sampling site.

FIG. 2.

Flow chart diagram illustrating the protocols used in this study. For both sample types (OR and OS), chemical, microbiological, and molecular analyses were done. Templates for PCR-DGGE were DNA extracted directly from the environment (samples A/DNAs a, in boxes), from the trophic populations (pop.) grown in MPN analysis plates (samples B and C/DNAs b and c, in boxes), or from degrading strains. The DGGE profiles of the total DNA of each sample and of its trophic populations (heterotrophic and alkane and aromatic degrading) were compared to identify common bands between them. Degrading strains isolated with different media were individually screened by DGGE to detect those that comigrated with specific bands in the profiles. A nearly full-length 16S rRNA gene PCR fragment from the total DNA (a) of the samples was cloned. The clone library was screened by restriction fragment length polymorphism analysis, and different operational taxonomic units were sequenced. Sequences from the most interesting bands and strains were compared with those from the clone library to determine the quantitative proportions of the different species found.

FIG. 3.

MPN analysis of heterotrophic (HET), alkane-degrading (ALK), and PAH-degrading populations (PAHs) in polluted samples of rocks (OR) and sand (OS) compared with those in nonoiled sand (NOS). Standard deviations (n = 8) are represented by error bars. *, significantly different from the other two samples by the Student t test. Polluted samples have similar populations, except for aromatic degraders, while unpolluted samples always have significantly smaller degrading populations than polluted ones do.

FIG. 4.

DGGE profiles of PCR-amplified 16S rRNA genes of bacterial communities from oiled samples (OR and OS) compared with those of bacterial communities from NOS (A). The hydrocarbon (alkane [Hx] and aromatic compound [PAH])-degrading bacterial populations from polluted rock (B) and sand (C) are compared with their total profiles (OR, OS). R1 to R18 and S1 to S13 indicate excised and sequenced bands from the total OR and OS profiles, respectively (see Table S2 in the supplemental material). Bands of the alkane (OR-Hx and OS-Hx)- and aromatic (ORPx and OSPx)-degrading populations from rock and sand, respectively, were sequenced as well (see Table S3 in the supplemental material). Bacterial diversity (Table 3), PCA (see Fig. S4 in the supplemental material), and a neighbor-joining tree from Pearson correlation factor data (see Fig. 5) were obtained from the complete densitometric curves of the different DGGE profiles (i.e., OR, OS, NOS, OR-Hx, OR-PAH, OS-Hx, and OS-PAH) to consider possible shifts in the composition of the microbial populations.

FIG. 5.

Cluster analysis from a similarity matrix generated from DGGE profiles (Fig. 4) according to the Pearson product moment and the unweighted-pair group method using average linkages. The DGGE profile of OS samples was close to that of hydrocarbon degraders, whereas the OR DGGE profile was more similar to that of alkane degraders than to that of PAH degraders. NOS, nonoiled sand; OS plus a number, oiled sand replicates; OR plus a number, oiled rock replicates. OR-Hx, OR-PAH, OS-Hx, and OS-PAH, total DNA from bacteria growing at the highest dilutions in hexadecane (Hx) and aromatic (PAH) MPN analyses, respectively.

FIG. 6.

DGGE screening of isolated alkane-degrading strains from rocks (A) and sand (B) and isolates related to PAH degradation from both matrices (C). DGGE patterns of total communities (OR and OS) and their respective hydrocarbon (Hx and PAH)-degrading populations were used as references to detect those strains playing roles in the biodegradation of the Prestige crude oil in the environment (e.g., hexadecane-degrading strain PDR24, identical to bands R1 and RH1, indicates the presence of Rhodococcus and its role in the in situ degradation of the alkane fraction of the Prestige fuel). The OR, OS, OR-Hx, OS-Hx, OR-PAH, and OS-PAH profiles, marked with arrows and bold letters, were identical to those described in Fig. 4. Some of the sequenced bands are referenced again in the present figure to assist in the detection of strains. The term “mix” indicate those isolates which were mixtures of more than one strain (e.g., PDR23 was composed of two strains of Rhodococcus, where the upper two bands were a heteroduplex of the two Rhodococcus 16S rRNA gene sequences).