Final Report Summary - BASIS (PAH Anaerobic Biodegradation Assessment by Stable Isotope Technologies)
The main goals of the current project were to prove and quantify the use of PAHs in polluted marine and freshwater systems by methanogenic and sulphate-reducing microbiota, and characterize the key microorganisms. My research has included other compounds (for example heteroatomic PAH, like dibenzothiophene, carbazole and dibenzofuran) and which were not considered in my initial plans, but have similar characteristics: they are abundant in oil and oily products, they are relatively water soluble, they have been only poorly studied, and some possess toxic and carcinogenic properties as well. The samples to work with were mainly collected from a contaminated site in Gölzau (Germany) and kindly provided by Dr. Yao Jun, our cooperating partner from the University of Science and Technology of Beijing, China. These samples were taken from an oil reservoir in China. In addition, thanks to Dr. Srikanth Mutnuri from the Birla Institute of Technology and Science (BITS-Pilani, Goa, India), I could obtain samples from a hydrocarbon-polluted harbour in Goa. Finally, a collaboration with the group of Dr. Anna Maria Solanas, from the Department of Microbiology of the University of Barcelona, offered me the opportunity to work in an alkylbenzenes-contaminated aquifer in Spain.
From all these polluted sites, different cultivation experiments were set up and the results are summarized below:
• Microcosms under sulphate-reducing conditions using groundwater from Gölzau, containing 2-methyl-phenanthrene, heteroatomic PAHs (carbazol, dibenzothiophene, dibenzofuran) and dissolved in a heptamethylnonane phase were set up. Growth in carbazol and dibenzothiophene as sole carbon sources was detected and future experiments performed in the Department should determine the activation mechanisms.
• A cometabolism experiment with ten different PAHs was performed in cooperation with a PhD student from my department, and potential cometabolic degradation of several of them (including carbazol, dibenzothiophene, methylphenanthrene and decylbenzene) has been detected. Several microcosms were extracted and the metabolites have to be identified. A publication is expected from this work.
• Thanks to the collaboration with Dr. Srikanth Mutmuri from the Birla Institute of Technology and Science (BITS-Pilani, Goa, India), Enrichment cultures under sulphate-reducing and methanogenic conditions were set up using contaminated sediments from Goa, using oil as substrate to avoid the loss of metabolic potential. Some of the microcosms exhibited degradation potential, but the biomass is still low to conduct further experiments.
• An experiment using linear alkylbenzenes (LAB) as carbon sources and groundwater from a LAB-contaminated aquifer was set up in collaboration with people from the Microbiology Department of the University of Barcelona. The method for Compound Specific Isotope Analysis (CSIA) by GC-IRMS of these compounds was wet up. However, after several months of incubation no fractionation (C or H) on the different compounds was observed. Nevertheless, the cultures are being maintained in the lab for future investigations.
• Finally, the fruitful collaboration with Prof Jun Yao, from the University of Science and technology of Beijing, gave me the chance to obtain samples from an oil reservoir. This system can be used as model for hydrocarbon anaerobic (in this case methanogenic) polluted sites, like groundwaters and marine sediments, as it is being constantly supplied water. I studied it in collaboration with the Chinese research group and the group of the Department of Geomicrobiology, Federal Institute for Geosciences and Natural Resources (BGR).
Isotopic measurements of reservoir fluids and GC-analysis of the oil confirmed that the reservoir is highly methanogenic and that PAH degradation is taking place in situ. In addition to the field measurements, microcosms containing 13C-labelled aromatic hydrocarbons (methylnaphthalene, ethylbenzene and m-xylene) were set up and we detected production of labelled methane in some of them, which confirms that methanogenic degradation of aromatic hydrocarbons (including PAH) is a feasible process in oil reservoirs. Microcosms amended with oil exhibited a high rate of methane production and the consortia could extensively degrade oil under these conditions. Moreover, the results of the isotopic measurements of the enrichment cultures at different temperatures and some additional experiments with 13C-labelled acetate suggest that the metabolic pathway for the mineralization of PAH is depending, among other factors, on the temperature. Finally, the microbial communities from the reservoir were characterized. High amounts of methanogenic archaea and putative syntrophic bacteria were detected, indicating syntrophic oxidation of hydrocarbons or fermentation products coupled to hydrogenotrophic methanogenesis is likely occurring. As a result of this work, a manuscript has been published (Jiménez et al., 2012. Org Geochem 2: 44-54), another one is to be submitted and some more are under preparation.
Regarding the use of BACTRAPs®, preliminary exposure experiments have been done in Gölzau using activated charcoal as carrying material. The performance (regarding the sorption and desorption properties) of this material had been previously tested in vitro and it had proven to be suitable to be used in situ. In the first step, naphthalene and phenanthrene have been selected for the experiments in situ, because their metabolism has already been studied.
From all these polluted sites, different cultivation experiments were set up and the results are summarized below:
• Microcosms under sulphate-reducing conditions using groundwater from Gölzau, containing 2-methyl-phenanthrene, heteroatomic PAHs (carbazol, dibenzothiophene, dibenzofuran) and dissolved in a heptamethylnonane phase were set up. Growth in carbazol and dibenzothiophene as sole carbon sources was detected and future experiments performed in the Department should determine the activation mechanisms.
• A cometabolism experiment with ten different PAHs was performed in cooperation with a PhD student from my department, and potential cometabolic degradation of several of them (including carbazol, dibenzothiophene, methylphenanthrene and decylbenzene) has been detected. Several microcosms were extracted and the metabolites have to be identified. A publication is expected from this work.
• Thanks to the collaboration with Dr. Srikanth Mutmuri from the Birla Institute of Technology and Science (BITS-Pilani, Goa, India), Enrichment cultures under sulphate-reducing and methanogenic conditions were set up using contaminated sediments from Goa, using oil as substrate to avoid the loss of metabolic potential. Some of the microcosms exhibited degradation potential, but the biomass is still low to conduct further experiments.
• An experiment using linear alkylbenzenes (LAB) as carbon sources and groundwater from a LAB-contaminated aquifer was set up in collaboration with people from the Microbiology Department of the University of Barcelona. The method for Compound Specific Isotope Analysis (CSIA) by GC-IRMS of these compounds was wet up. However, after several months of incubation no fractionation (C or H) on the different compounds was observed. Nevertheless, the cultures are being maintained in the lab for future investigations.
• Finally, the fruitful collaboration with Prof Jun Yao, from the University of Science and technology of Beijing, gave me the chance to obtain samples from an oil reservoir. This system can be used as model for hydrocarbon anaerobic (in this case methanogenic) polluted sites, like groundwaters and marine sediments, as it is being constantly supplied water. I studied it in collaboration with the Chinese research group and the group of the Department of Geomicrobiology, Federal Institute for Geosciences and Natural Resources (BGR).
Isotopic measurements of reservoir fluids and GC-analysis of the oil confirmed that the reservoir is highly methanogenic and that PAH degradation is taking place in situ. In addition to the field measurements, microcosms containing 13C-labelled aromatic hydrocarbons (methylnaphthalene, ethylbenzene and m-xylene) were set up and we detected production of labelled methane in some of them, which confirms that methanogenic degradation of aromatic hydrocarbons (including PAH) is a feasible process in oil reservoirs. Microcosms amended with oil exhibited a high rate of methane production and the consortia could extensively degrade oil under these conditions. Moreover, the results of the isotopic measurements of the enrichment cultures at different temperatures and some additional experiments with 13C-labelled acetate suggest that the metabolic pathway for the mineralization of PAH is depending, among other factors, on the temperature. Finally, the microbial communities from the reservoir were characterized. High amounts of methanogenic archaea and putative syntrophic bacteria were detected, indicating syntrophic oxidation of hydrocarbons or fermentation products coupled to hydrogenotrophic methanogenesis is likely occurring. As a result of this work, a manuscript has been published (Jiménez et al., 2012. Org Geochem 2: 44-54), another one is to be submitted and some more are under preparation.
Regarding the use of BACTRAPs®, preliminary exposure experiments have been done in Gölzau using activated charcoal as carrying material. The performance (regarding the sorption and desorption properties) of this material had been previously tested in vitro and it had proven to be suitable to be used in situ. In the first step, naphthalene and phenanthrene have been selected for the experiments in situ, because their metabolism has already been studied.