Effective bioremediation strategy for rapid in situ cleanup of anoxic marine sediments in mesocosm oil spill simulation

Abstract

The purpose of present study was the simulation of an oil spill accompanied by burial of significant amount of petroleum hydrocarbons (PHs) in coastal sediments. Approximately 1000 kg of sediments collected in Messina harbor were spiked with Bunker C furnace fuel oil (6500 ppm). The rapid consumption of oxygen by aerobic heterotrophs created highly reduced conditions in the sediments with subsequent recession of biodegradation rates. As follows, after 3 months of ageing, the anaerobic sediments did not exhibit any significant levels of biodegradation and more than 80% of added Bunker C fuel oil remained buried. Anaerobic microbial community exhibited a strong enrichment in sulfate-reducing PHs-degrading and PHs-associated Deltaproteobacteria. As an effective bioremediation strategy to clean up these contaminated sediments, we applied a Modular Slurry System (MSS) allowing the containment of sediments and their physical-chemical treatment, e.g., aeration. Aeration for 3 months has increased the removal of main PHs contaminants up to 98%. As revealed by CARD-FISH, qPCR, and 16S rRNA gene clone library analyses, addition of Bunker C fuel oil initially affected the activity of autochthonous aerobic obligate marine hydrocarbonoclastic bacteria (OMHCB), and after 1 month more than the third of microbial population was represented by Alcanivorax-, Cycloclasticus-, and Marinobacter-related organisms. In the end of the experiment, the microbial community composition has returned to a status typically observed in pristine marine ecosystems with no detectable OMHCB present. Eco-toxicological bioassay revealed that the toxicity of sediments after treatment was substantially decreased. Thus, our studies demonstrated that petroleum-contaminated anaerobic marine sediments could efficiently be cleaned through an in situ oxygenation which stimulates their self-cleaning potential due to reawakening of allochtonous aerobic OMHCB.

Keywords: aerated slurry system; crude oil pollution; hydrocarbonoclastic bacteria; in situ bioremediation; marine anoxic sediments.

Figure 1

(A,B) Schematic representation (A) and detail (B) of “Modular Slurry System” used throughout this study. Abbreviation used: A, Modular Slurry System; B, temperature controller; C, external air pump; D, steel plate with needles to supply an oxygen into deep sediments; E, exhaust tube; F, seawater inlet; G, seawater outlet; H, contaminated sediments; I, overflow regulation system.

Figure 2

Dynamic of oxygen consumption (BOD values) measured in MSS external (untreated) superficial sediments (white bars) and internal sediments (gray bars). Error bar indicates the standard deviation of triplicate measurements.

Figure 3

Reduction potential (E_h) measured in external anaerobic sediments (AN) and in internal aerated sediments (OX) during 3 months of experimentation.

Figure 4

Mortality of Corophium orientale organisms in polluted (AN), treated (OX), and control (native) sediments. Error bar indicates the standard deviation of duplicate measurements.

Figure 5

Dendrogram representation of taxonomic analysis of 16S crDNA clones retrieved from five libraries. The numbers at the base of columns represent the percentage of clones in corresponding libraries. Abbreviations used: PB, Proteobacteria divisions; UC, Unaffiliated cluster. Bacterial groups involved in, or associated to, hydrocarbon degradation are marked in bold and red.

Figure 6

Phylogenetic affiliation of the Eubacteria clones retrieved in Messina harbor native sediments and during mesocosm experiment. Neighbor-joining analysis using 1000 bootstrap replicates was used to infer tree topology. The scale bar represents 10% of sequence divergence. Bootstrap values ≥50 are shown as open circles. Cenarcheum symbiosum was used as the outgroup. The scale bar represents the expected number of changes pet nucleotide position. Sequences from this study are indicated in bold.

Figure 7

Phylogenetic affiliation of the Gammaproteobacteria clones retrieved in Messina harbor sediments and in mesocosm experimentation. Neighbor-joining analysis using 1000 bootstrap replicates was used to infer tree topology. The scale bar represents 10% of sequence divergence Bootstrap values ≥50 are shown as open circles. Cenarcheum symbiosum was used as the out-group. Sequences obtained in this study are indicated in bold.

Figure 8

Phylogenetic affiliation of the Alphaproteobacteria clones retrieved in Messina harbor sediments and in mesocosm experimentation. Neighbor-joining analysis using 1000 bootstrap replicates was used to infer tree topology. The scale bar represents 10% of sequence divergence. Bootstrap values ≥50 are shown as open circles. Cenarcheum symbiosum was used as the outgroup. Sequences obtained in this study are indicated in bold.

Figure 9

Dendrogram of microbial biodiveristy and similarity analysis of the 16S rRNA transcripts detected at different sampling time and treatment. The UPGMA cluster analysis was obtained by using group average clustering from Euclidean distance on relative abundance matrix of OTUs detected in the analyzed libraries.