Diversity and dynamics of microbial community during bioremediation of polycyclic aromatic hydrocarbons in polluted marine sediments
Student thesis: Doctoral Thesis
The prevalence of polycyclic aromatic hydrocarbons (PAHs) in marine sediments and their adverse impacts on biota and humans have long been of great environmental concerns. Natural attenuation based on microbial degradation has been recognized as the major means for bioremediation processes; but little is known about the ecology and diversity of microbial communities in the contaminated environment and their dynamics during bioremediation processes. The present research aims: (i) to investigate the microbial diversity and community dynamics during the bioremediation of PAHs in heavily-polluted marine sediments; (ii) to identify the responsible PAH-degraders and assess their catalytic potential and (iii) to elucidate the interactions between the microbial community and the contaminated environment. Spatial and temporal changes of the microbial community structure as well as eight heavy metals and 16 US EPA PAHs at different stations around an old floating dock, Yiu Lian in Hong Kong were examined for a one-year period right after its relocation. The levels of pollution indicated the dock as a significant source of mixed pollutants, but no significant temporal trend in pollution levels was observed. Several hot spots of contamination were consistently detected near the old dock site over time. A shift in the PAH inputs, reflected by the ratios of individual PAH compounds (i.e., Phe/Ant and Fla/Pyr) and 16 PAH profiles, was observed over the course of one year. Accordingly, a marked shift in microbial ester-linked fatty acid methyl esters (EL-FAMEs) profiles was detected after the removal of the dock, with an increase in the diversity and richness indices of the microbial community. Redundancy analysis (RDA) further showed that the pollution level had a relatively minor effect on microbial community structure compared to the sampling time. Nevertheless, strong relationships were revealed between certain fatty acid biomarkers indicative of Gram-negative bacteria and the total PAH concentration, suggesting their special importance in PAH bioremediation processes. Two 16S rDNA based molecular techniques, terminal restriction fragment length polymorphism (T-RFLP) and Barcoded Illumina paired-end (PE) sequencing (BIPES), were employed to depict the genotypic diversity of microbial community in polluted marine sediments. T-RFLP analysis using two different restriction enzymes showed similar significant temporal patterns in the microbial community structure based on the presence/absence data of peaks. This was in good agreement with the result of EL-FAME analysis. However, different patterns were produced on the basis of the peak area data, where samples between certain impacted stations and the reference stations were clearly separated. Such discrepancy highlighted the importance of certain less frequent members in the microbial community, whose relative abundance varied greatly in response to the pollution stress. BIPES, a rapid high-throughput sequencing method, was employed to explore the metagenomics of the microbial community of eight representative sediment samples. The number of bacterial 16S rDNA sequences obtained from each site varied from 33,397 to 95,837. The most abundant bacterial groups across all the samples were the γ-Proteobacteria (37%), δ-Proteobacteria (26%) and Planctomycetes (7%). The community composition in one sample from an extremely polluted hot spot showed a distinct distribution pattern at the phylum level, with significantly high abundance of Actinobacteria, Firmicutes and JS1. The result of BIPES was in good consistency with the diversity of PAH-degrading microbial populations based on traditional culture-dependent approach. A total of 15 PAH degrading bacterial strains were isolated from marine sediments in the present study. The partial 16S rRNA gene sequences assigned them to three main bacterial phyla: Actinobacteria (Micrococcus sp., Gordonia sp., and Rhodococcus sp.); Firmicutes (Bacillus sp., Exiguobacterium sp. and Oceanobacillus sp.) and Proteobacteria (Agrobacterium sp., Ochrobactrum sp., Paracoccus sp., Pseudomonas sp. and Rhodobacter sp.). These were the most relevant members in the microbial community related to pollution. A microcosm study was carried out to investigate the microbial community dynamics and biodegradation processes of the polluted marine sediments artificially spiked with different levels of PAH contamination. Microbial community dynamics differed significantly with the magnitude of PAH contamination. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that a noted reduction in species diversity and a distinct community profile occurred only in the sediments receiving the highest level of PAH treatment, but such differences among different levels of PAH treatment were not shown by EL-FAME analysis during the first seven days. Afterwards, the temporal shifts in the microbial community structure were consistent between these two methods. In all levels of PAH treatment, increases in the number of PAH-degraders and the relative abundance of fatty acid biomarkers for Gram-negative bacteria were observed with a notably decrease in species diversity when PAH concentrations in the sediment significantly decreased, suggesting that the Gram-negative populations became predominant and might be the responsible members in PAH biodegradation. Both methods showed that the diversity indices increased later on as a sign of recovery after PAH exposure. The present study demonstrated the use of multiple techniques for microbial community analysis to clarify the complex interactions between microbial groups and pollutants during PAH bioremediation processes. The strong relationships between microbial community and the degree as well as the types of mixed pollutants were addressed during natural attenuation in the field study. The hot spot of contamination harbored a distinct microbial diversity, with significantly high abundances of Actinobacteria, Firmicutes and JS1, which have many bacterial genera that contain PAH-degrading species. An extremely high level of PAH contamination did alter the microbial community structure and the important role of Gram-negative bacteria in PAH biodegradation was confirmed in the microcosm study.
- Polycyclic aromatic hydrocarbons, Marine sediments, Bioremediation, Microbiology