- Title
- Biodegradation of polycyclic aromatic hydrocarbons and dimethylformamide by aerobic, anaerobic sulfate-reducing, and phototrophic purple nonsulfur bacteria
- Creator
- Dhar, Kartik
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2022
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Polycyclic aromatic hydrocarbons (PAHs) are recognized as a group of priority organic pollutants because of their toxic, mutagenic, and carcinogenic potentials. Contaminated soil, water, and sediment pose serious risks to living organisms, including humans. Many former manufactured gas plant (MGP) sites still bear the legacy of PAH contamination. Removal of highly recalcitrant PAHs from impacted MGP sites requires efficient remediation technology. Bioremediation has emerged as an effective and sustainable technology for removing PAHs. PAHs are also widespread in anoxic environments, such as subsurface soils and sediments. However, knowledge of anaerobic biodegradation of PAHs is limited compared to its aerobic counterpart. Dimethylformamide (DMF) is an extensively used industrial and laboratory organic solvent. Consequently, a high concentration of potentially toxic DMF is often released into the environment with industrial wastewaters. In addition, some laboratory wastewaters also contain both DMF and PAHs. Bioremediation of DMF has also been shown as an efficient and cost-effective treatment option. The research aimed to identify specialized bacteria to develop promising bioremediation technology for cleaning up DMF and PAHs from contaminated environments. The objectives of this study were to evaluate the efficacies of aerobic, anaerobic sulfate-reducing bacteria (SRB) and phototrophic purple nonsulfur bacteria (PNSB) in biodegradation PAHs and DMF. Selective enrichment on pyrene was established using a PAHs-contaminated MGP soil to obtain an effective aerobic bacterial inoculant for bioremediation. Surprisingly, the mixed enrichment culture, designated as MM34X, exhibited utilization of the supposedly inert carrier solvent, DMF, and the supplied PAH. Mesorhizobium tamadayense MM3441 was isolated from the mixed culture and has been the only member of the genus, Mesorhizobium, capable of degrading DMF. The methylotrophic strain removed 95% of 5000 mg L‒1 DMF within nine days of incubation. Biochemical investigations revealed that strain MM3441 was capable of complete mineralization of DMF following the typical dimethylamine forming pathway. This study demonstrated the potential use of the strain in bioremediation of the environments contaminated with DMF. Subsequent investigation with MM34X focused on screening efficient PAHs-degrading bacteria. Remarkably, the mixed enrichment culture was more efficient in PAHs biodegradation than the culturable pure strains. Surprisingly, analysis of 16S rRNA sequences revealed that the culture was dominated by a previously uncultured member of the family Rhizobiaceae. When provided as the sole carbon source or with DMF as a co-substrate in the liquid medium, the culture rapidly degraded phenanthrene, pyrene, and the carcinogenic benzo(a)pyrene (BaP). Inspired by the promising performance, the efficiency of the culture in the bioremediation of PAHs from the MGP soil (MGPS) and a laboratory waste soil (LWS) was evaluated in bench-scale slurry systems. After 28 days, 80% of Σ16 PAHs were efficiently removed from the inoculated MGPS. Likewise, almost all phenanthrene, pyrene, and 65% BaP were removed from the bioaugmented LWS. This study established the ability of the methylotrophic enrichment culture dominated by an uncultured bacterium in the biodegradation of PAHs and demonstrated its suitability for the efficient bioremediation of PAHs. Further investigation indicated that the PAHs-degrading mixed culture MM34X was a superior DMF degrader. The culture efficiently degraded 98% of 20000 mg L‒1 DMF within 120 h. An effort was made to translate the combined degradation features of the culture into a green and sustainable technology for the bioremediation of laboratory wastewater co-contaminated with PAHs and DMF. LWW bioremediation was performed in stirred bottle laboratory-scale bioreactor. After 35 days of incubation, ≥96% of initial concentrations of DMF, phenanthrene, pyrene, and BaP in the LWW were removed. Furthermore, analysis of post-bioremediation transformation metabolites indicated the absence of any known toxic intermediates. In addition, the efficacy of bioremediation was further subjected to validation from cyto-genotoxicity assays using Allium cepa. Compared to the untreated samples, the bioaugmented LWW exhibited a significantly improved mitotic index, less DNA damage, and lower oxidative stress potential. The findings indicated that the culture might prime the development of inexpensive and efficient large-scale bioremediation of DMF and PAHs co-contaminated LWW. Enrichment and characterization of PAHs-degrading anaerobic sulfate-reducing cultures is a tedious and challenging task. In this research, an effort was made to develop a comprehensive, reliable, and recipe-style protocol for establishing and characterizing PAHs-degrading enrichment cultures under sulfate-reducing conditions. Critical steps in the sample preparation, anoxic media preparation, culture establishment and maintenance, viability evaluation, sulfate reduction activity measurement, and PAH extraction technique were optimized. The protocol enabled obtaining several highly enriched PAHs-degrading sulfate-reducing enrichment cultures from soil and sediment samples. The MGP soil was considered a probable source of PAHs-degrading SRB, assuming that soil micropores could provide niches to the anaerobic bacteria. Selective enrichment of the soil resulted in the development of highly enriched sulfate-reducing cultures capable of degrading naphthalene and four-ringed PAH pyrene. Bacterial community composition analysis revealed that a naphthalene-degrading enrichment culture, MMNap, was dominated (84.90%) by a Gram-positive endospore-forming member of the genus Desulfotomaculum with minor contribution (8.60%) from a member of Clostridium. The pyrene-degrading enrichment, MMPyr, was dominated (97.40%) by a species of Desulfotomaculum. The involvement of the Desulfotomaculum sp. in the PAHs biodegradation was confirmed based on concomitant consumption of sulfate and accumulation of sulfide. Furthermore, the addition of sulfate reduction inhibitor (20 mM molybdate) in the cultures resulted in complete cessation of the PAHs utilization and sulfate reduction, clearly indicating the major role of the Desulfotomaculum in the biodegradation of the two PAHs. This study has been the first report on anaerobic pyrene degradation by a matrix-free, strictly anaerobic, and sulfate-reducing enrichment culture. Later, the research utilized contaminated freshwater sediments for investigating anaerobic PAHs degradation under strictly anaerobic sulfate-reducing conditions. Selective enrichment under strictly anaerobic sulfate-reducing conditions resulted in the development of highly enriched cultures capable of degrading phenanthrene and pyrene. Degradation of phenanthrene and pyrene by the enrichments was tightly coupled to sulfate reduction. The phenanthrene-degrading enrichment was dominated (98%) by a member of the genus Desulfovibrio. While pyrene-degrading culture was also dominated (79%) by a Desulfovibrio sp., an anoxygenic purple nonsulfur bacterium, Rhodopseudomonas sp., constituted a significant fraction (18%) of the total microbial community. In addition, phenanthrene-2-carboxylic acid was detected in the culture extract, suggesting that carboxylation could be a widespread initial step of phenanthrene activation under sulfate-reducing conditions. Thus, the current study is one of the rare instances that describes the active involvement of Desulfovibrio sp. in the degradation of PAHs. Overall, this study demonstrates the ability of freshwater sediments containing sulfate-reducing bacteria (SRB) in the anaerobic degradation of PAHs, suggesting that SRB might play a crucial role in the natural attenuation of PAHs in anoxic freshwater sediments. The final investigation of the research focused on evaluating PAH degradation by phototrophic PNSB. Enrichment of PAHs-contaminated sediment under anaerobic light conditions yielded a phototrophic phenanthrene-degrading mixed enrichment culture, PMK23X. The culture was dominated (92.2±3.5%) by a PNSB of the genus Rhodopseudomonas. Subsequently, the dominant organism was isolated and identified as Rhodopseudomonas palustris strain PMM231. The mixed enrichment culture degraded 48 ±3.4 % of the added phenanthrene in six weeks under photoheterotrophic conditions. At the same time, the pure strain R. palustris PMM231 degraded 41.8±3.9 % phenanthrene. Interestingly, the omission of HCO3– from the growth medium caused a >20% decrease in phenanthrene biodegradation, suggesting that CO2 fixation facilitated the photoheterotrophic growth on the highly reduced phenanthrene. Both the cultures were also capable of fermentative growth on phenanthrene under anaerobic-dark conditions in the presence of dimethyl sulfoxide. This study reveals PAH degradation in photosynthetic PNSB for the first time. Overall, the research demonstrated biodegradation of PAHs by the aerobic, the strictly anaerobic sulfate-reducing, and phototrophic PNSB cultures. In addition, the aerobic enrichment culture was established as a promising tool for bioremediation of PAHs-contaminated soils and laboratory wastewater co-contaminated with DMF and PAHs. Although efficient degradation of high concentrations of DMF was achieved with the enrichment culture and M. tamadayense MM3441, degradation under anaerobic sulfate-reducing and phototrophic conditions could not be established. The research encourages future appraisals for the mechanistic understanding of PAHs and DMF degradation, especially under anaerobic sulfate-reducing and phototrophic conditions.
- Subject
- polycyclic aromatic hydrocarbons (PAHs); dimethylformamide (DMF); phenanthrene; pyrene; benzo(a)pyrene (BaP); manufactured gas plant; freshwater sediment; mesorhizobium tamadayense MM3441; biodegradation; bioremediation; methylotrophy; sulfate-reducing bacteria (SRB); purple nonsulfur bacteria (PNSB); anaerobic biodegradation; rhodopseudomonas palustris; naphthalene
- Identifier
- http://hdl.handle.net/1959.13/1512095
- Identifier
- uon:56586
- Rights
- Copyright 2022 Kartik Dhar
- Language
- eng
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