Characterization Of Marine Obligate Hydrocarbonoclastic Bacteria Cycloclasticus Spp. And Biodegradation Of High-molecular Weight Polycyclic Aromatic Hydrocarbons By Defined Synergic Co-cultures

Strains of genus Cycloclasticus are typical representatives of the marine obligatehydrocarbonoclastic bacteria (OHCBs) in recent years, which are distributed indifferent marine environments worldwide and can utilize polycyclic aromatichydrocarbons (PAHs) as sole source of carbon and energy. They are proved to playpivotal role in the natural biodegradation process of marine oil spills. However, thesebacteria are difficult to cultivate because of their peculiar substrate profile, and thus,information on their pure cultures is lacking. In this study, three Cycloclasticus strains,NY93E, PY97M, and PY97N, isolated from the sediments of Yellow Sea, arecharacterized with respect to their PAHs-metabolizing traits. Interaction betweenCycloclasticus strains and other PAHs-degrading strains (Marinobacter spp.,Alcanivorax sp., and Porphyrobacter sp.) in the biodegradation of high-molecularweight polycyclic aromatic hydrocarbons (HMW PAHs) is also studied. First, morethan120strains of culturable bacteria were obtained from the sediments of differentmarine environments through enrichment with selective carbon sources and platingmethod. Phylogenetic analysis based on16S rRNA gene sequences shows that thecrude oil/PAHs-degrading consortia mainly comprise the strains ofalpha-proteobacteria and gamma-proteobacteria. There are about25strains of themarine OHCBs among all the culturalbl strains, which belongs to the generaCycloclasticus, Alcanivorax, and Marinobacter. Second, the detailed characterizationand comparison of the three Cycloclasticus strains were conducted by multiplemethods including morphological observation, phylogenetic analysis, determinationof PAH utilization profiles and PAH degradation rates, and homology analysis of thegenes encoding the ISP of PAH dioxygenase, which greatly enhance our knowledgeof the traits of genus Cycloclasticus. The three Cycloclasticus strains exhibited novelcharacteristics and better catalytic activity with respect to PAHs biodegradation whencompared with their counterparts of the same genus. When used as sole source of carbon and energy, pyrene and fluoranthene can be degraded by each of the threestrains, and the degradation rates of0.02g L-1pyrene and fluoranthene determined byGC–MS are in the range51.65%to63.43%and49.32%to65.21%after21d,respectively. The gene clusters encoding the iron–sulfur protein of the PAHdioxygenase were PCR amplified from these strains and their nucleotide sequencesexhibit more than98%nucleotide similarity with their counterpart in Cycloclasticussp. A5, and99%nucleotide similarity with their counterpart in Cycloclasticus sp. P1.Finally, more than46strains of culturable petroleum hydrocarbons/PAHs-degradingbacteria or biosurfactant-producing bacteria were obtained through biodegradabilitytest and surface tension test, which are selected as the candidates to construct thedefined co-cultures with Cycloclasticus spp.. Meanwhile, three Marinobacter strainsincluding Marinobacter sp. PY97S, Marinobacter nanhaiticus D15-8WT, andMarinobacter aromaticivorans D15-8PTare characterized as new species bypolyphasic taxonomic study.Then the defined co-cultures were constructed based on the above mentionedresults, combined with the analysis and previous knowledge of the structures ofPAHs-degrading bacterial consortia. First, the defined consortium of Cycloclasticus sp.PY97M and Marinobacter sp. D15-8W exhibites obvious synergic effects in HMWPAH biodegradation among all the defined batetrial associations composed of theCycloclasticus strains and the co-cultured bacteria. The degradation rates of pyreneand fluoranthene by the consortium PY97M+D15-8W was increased from47.10%to67.40%and42.50%to62.79%compared to the pure culture of strain PY97M,respectively (the initial concentration of the carbon sources is0.1g/L,25℃,21d).Second, the degradation rates of pyrene and fluoranthene by the consortiumPY97M+D15-8W are higher when the other HMW PAHs co-substrates are present.The consortium PY97M+D15-8W can degrade71.05%pyrene and67.36%fluoranthene in the two-HMW PAHs mixture under the same experiment conditions,including inuculum size, initial concentrations of carbon sources, incubationtemperature, and rotate speed (the initial concentration of the carbon sources is0.1g/L,25℃,14d). The toxicological assays based on the luminescent bacteria test alsoshow that the consortium PY97M+D15-8W can not only enhance the degradationrates of HMW PAHs, but also reduce the acute toxicity and genotoxicity of HMWPAHs metabolites compared with their parent compounds. Finally, biodegradation test by consortium PY97M+D15-8W under lower incubation temperature were alsoconducted (the initial concentration of the carbon sources is0.02g/L,20℃,21d). Thedegradation rates of0.02g L-1pyrene and fluoranthene by the consortiumPY97M+D15-8W increased from63.43%to75.50%and65.21%to83.03%compared with that of the pure culture of strain PY97M, respectively.In conclusion, these results of synergic effects between different bacterial strainsare significant for understanding the mechanism of HMW PAHs biodegradation inmarine environment. The consortium PY97M+D15-8W could degrade HMW PAHssynergically and is promising in the bioremediation of the coast line or tidal flat whichis polluted by persistent organic pollutants (POPs) such as HMW PAHs.