| Summary: | 博士 === 國立成功大學 === 環境工程學系 === 103 === Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), collectively called “dioxins”, comprise 210 congeners and are by-products of combustion and industrial manufacturing processes. Because of their strong hydrophobicity and xenobiotic nature, a high concentration of PCDD/Fs has accumulated in the soil environment. Octachloro DD/Fs are among the most abundant congeners distributed. Through bioaccumulation in the food chain, it already poses a serious ecological threat and health risk. Soil and compost are inhabited by diverse microbial populations that reportedly can decompose chlorinated dioxins. Potentially, these microorganisms with relevant genes and metabolic activities can be used to develop an efficient, cost-effective, and environmentally friendly bioremediation method for reducing the concentration of PCDD/Fs in soil. However, understanding of the microbial population quantity, distribution, and role in PCDD/F biodegradation remains limited. The aim of this thesis was to examine the soil microbiome (i.e., microbial community structure and functionality) associated with PCDD/F contamination and develop a biological treatment system for removing PCDD/F toxicants from a contaminated site.
To study the feasibility of bioremediation, closed microcosms were constructed with PCDD/Fs-contaminated soil in oxygen-stimulating conditions. After incubation for 6 weeks, octachlorodibenzofuran (OCDF) decreased by 330 mg/kg, whereas heptachlorodibenzofuran (HpCDF) concentration decreased from 88.8 to 0.24 μmol/kg. To further validate the degradation of OCDFs, microbial consortia from the first-batch experiment were transferred to serum bottles containing fresh minimal medium and 50 mg of OCDF powder. The results showed that the OCDF decreased from 5096 to 913 μmol/kg after 3 weeks, which corresponded to an efficiency of 82.1% and a degradation rate of 199 μmol/kg/day. Clone library analysis of a polymerase chain reaction-amplified 16S rRNA gene from the OCDF-degrading consortia determined that 98.3% of the detected sequences were affiliated with Proteobacteria. Hierarchical oligonucleotide primer extension analysis revealed that Micrococcus, Rhizobium, Pseudoxanthomonas, and Brevudimonas-related populations increased sharply when high concentrations of OCDF decreased. Furthermore, the obtained strains with putative aromatic dioxygenase genes were closely related to the members of Actinobacteria, Firmicutes, and Proteobacteria. Among the members of Actinobacteria, Firmicutes, and Proteobacteria, certain Rhodococcus, Micrococcus, Mesorhizobium, and Bacillus strains degrade OCDF with efficiencies of 26% to 43% within 21 days. This study determined that the contaminated soil evolved diverse bacterial populations able to degrade highly chlorinated dioxins.
Next-generation high-throughput sequencing technology was used to determine the microbial composition and population dynamics transited from the original soil to the OCDF microcosms. The results of 16S rRNA gene analysis showed complex microbial diversity in the PCDD/F-contaminated soil and revealed that the community composition changed considerably, becoming concomitant with the OCDF degradation; thus, the distinctive population structure developed rapidly in the OCDF microcosm. Anaerobic Sedimentibacter (within Firmicutes) emerged first, and several aerobic participants, such as Brevudimonas (within Alphaproteobacteria), Pseudoxanthomonas, and Lysobacter (within Gammaproteobacteria) increased considerably within the timeframe. The results revealed a temporal population dynamic and collaborative contributions to OCDF degradation under hypoxic conditions.
Rhodococcus erythropolis effectively degraded PCDD/Fs and aromatic compounds. To study microbial functionality, strain B11 was isolated from the PCDD/F-contaminated soil and decoded. Genome sequencing yielded a draft genome of 6 838 862 bp, with a guanine-cytosine content of 62.5%. The draft genome contained 6748 genes and 6332 protein-coding sequences (CDSs; 94% of the entire genome). These CDSs exhibited a distinct distribution pattern in the functional categories of the Clusters of Orthologous Groups database, reflecting the unique niche of Rhodococcus erythropolis in the contaminated environment. The annotation identified various genetic features that contribute to its lifestyle and the degradation of xenobiotics, indicating the potential of using ring-hydroxylation oxygenase (RHOs) systems to metabolise these xenobiotics. The B11 genome comprises several gene segments that can produce trehalose as a biosurfactant via 4 biosynthetic pathways (i.e., TreS, OtsA-OtsB, TreP, and TreY-TreZ), elucidating its ability to enhance substrate availability for microbial degradation in soil. In addition, merA and merR genes were identified in the B11 genome, suggesting that B11 may regulate mercury resistance and reduce Hg (II) to Hg (0). Phylogenetic analysis revealed a horizontal gene transfer from Janibacter hoylei and genomic analysis showed that the lifestyle of the B11 strain has valuable applications in removing organic and inorganic environmental pollutants.
Lastly, a novel landfill reactor system with compost amendment was developed in this study. Intermittent aeration and leachate recirculation were applied in the reactor to establish hypoxic conditions. After 280 days, the average removal efficiencies of total dioxin toxicity in the R6 (10% sewage sludge compost) and R7 (5% sewage sludge compost and 5% cow manure compost) bioreactors were 53% and 79%, respectively, both higher than that recorded by the control bioreactor (27%). This result suggests that adding compost effectively enhanced the degradation of PCDD/Fs in the landfill reactor operated under hypoxic conditions. In such conditions, the microbial community inside the reactors was dominated by the groups associated with the classes Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, and Clostridia, changing markedly. In particular, the removal of PCDD/Fs strongly correlated with an increase in the population of anaerobic Clostridium. The compost amendment increased the complexity of microbial composition and aided in stably reducing the toxicity equivalence of PCDD/Fs. The stimulating landfill reactor developed in this thesis has field applications, such as the bioremediation of An-shun, the site of a former chemical plant in Southern Taiwan. In conclusion, this thesis study affords insight into the association of the soil microbiome with PCDD/Fs-contaminated soil, microcosms, and landfill reactors developed, and indicates a new direction for future studies on the bioremediation of PCDD/Fs.
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