Project Overview

Summary:

Dr. Melanie Beazley at the University of Central Florida was awarded an RFP-VI grant at $425,012 to conduct the RFP-VI project titled, “Biodegradation of ‘Hidden’ High Molecular Weight Polycyclic Aromatic Hydrocarbons: Closing Critical Research Gaps”. The project consisted of 1 principal investigator (Beazley), 1 co-PI (Dr. Campiglia), 5 PhD students (Lewis, Chehelamirani, Santana, Chandana, Comas), and 5 undergraduate students (Benke, da Silva, Fotiadis, Kadriu, Stillword).

Polycyclic aromatic hydrocarbons (PAHs) are a main component of petroleum that includes some of the most toxic, carcinogenic, genotoxic, and mutagenic hydrocarbons. Their molecular structures vary as combinations of multiple fused benzene rings that become more complex with size and aromaticity. As the molecular weight (MW) of PAHs increases, so does their potency with dibenzo[a,l]pyrene, a six-ring PAH with MW of 302 g mol^-1, considered to be the most potent carcinogen of all PAHs. To date there is a critical gap in the research regarding the environmental occurrence and distribution of these high molecular weights (HMW) PAHs due to difficulties in analytical separation and detection. As a result, the behavior of these "hidden" organic compounds in a natural ecosystem and the microbial metabolic activities that may degrade them are as yet unknown. Recent advances, however, in state-of-the-art detection and quantification of HMW-PAHs using high-resolution vibrational spectroscopy by Co-PI Campiglia open new and exciting avenues of study for these "hidden" PAHs. As microorganisms are the primary drivers of petroleum degradation in the environment, microbial biodegradation studies of HMW-PAHs MW ≥ 302 will be conducted in conjunction with analytical identification and quantification of these PAHs and their metabolites. This project will address research Theme Two with the goal to examine the biodegradation of "hidden" HMW-PAHs MW ≥ 302 by (1) individual microbial isolates and (2) sediment microbial communities to determine biological degradation pathways and identify metabolic intermediates using both culture-dependent and culture-independent (i.e., metagenomics) methods.

Research Objectives and Goals: The objectives of the proposed study are to (1) determine the ability of known PAH-degrading bacterial species to degrade HMW-PAH MW ≥ 302 compounds in pure cultures and identify the metabolic degradation products using fluorescence spectroscopy with emphasis on PAH-metabolites that are known to be carcinogenic; and (2) identify new, environmentally relevant bacteria capable of degrading HMW-PAH MW ≥ 302 compounds through sediment enrichment incubations using both culture-dependent isolation techniques and culture-independent techniques (i.e., metagenomics). These objectives will be achieved through an integrated research program coupling microbiological and state-of-the-art analytical techniques in order to investigate the microbial degradation of recalcitrant HMW-PAH organic compounds with a goal to better understand the microbial response to oil spills.

Potential Scientific and Societal Impact: The extent to which human activities are influencing coastal ecosystems is an issue of regional and national importance. Oil spills cause considerable alteration to the functioning of these systems and improving our understanding of how recalcitrant, highly toxic hydrocarbons, which may build up in sediments over long periods of time, are naturally degraded will add to our overall knowledge of their fate in natural systems. This project will build on and leverage contributions of our prior research activities directed at understanding microbial responses to oil in the Gulf of Mexico as well as developing state-of-the-art analytical techniques to measure these "hidden" PAHs and their metabolites. The project will provide training opportunities for two Ph.D. graduate students and 4-6 undergraduate students. This proposed research study will contribute directly to the ultimate goal of the Gulf of Mexico Research Initiative, which is "to improve society's ability to understand and respond to the impacts of petroleum pollution and related stressors of the marine and coastal ecosystems, with an emphasis on conditions in the Gulf of Mexico."

Research Highlights

     Dr. Beazley’s research to date resulted in 1 peer-reviewed publication to, 4 scientific conference presentations and 9 datasets submitted to the GoMRI Information and Data Cooperative (GRIIDC), which are available to the public.  Significant outcomes of their research (all related to GoMRI Research Theme 2) are highlighted below.

• High molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) specifically benzo[a]pyrene and the family of dibenzopyrene isomers were studied in pure culture and within a consortium of microorganisms to determine the mechanism of degradation of these 5- and 6-ring PAHs. Our results suggest that these HMW-PAHs are highly refractory and resistant to degradation by not only a single microorganism but also a consortium of microorganisms, specifically those isolated from oil-contaminated sediment. Pure culture assays with the PAH-degrading bacterial strain Mycobacterium vanbaalenii PYR-1 and consortium assays of bacteria isolated from Deepwater Horizon oil-impacted sediment indicated that benzo[a]pyrene only partially degraded after three months of incubation, and that all five isomers of the dibenzopyrenes including dibenzo[a,l]pyrene, dibenzo[a,h]pryene, dibenzo[e,l]pyrene, dibenzo[a,e]pyrene, and dibenzo[a,i]pyrene, did not degrade after incubation supporting the hypothesis that these toxic HMW-PAHs are very recalcitrant and possibly build up in sediment over time. The extremely low solubility of these HMW-PAHs in seawater most likely played a role in their resistance to biodegradation along with their large 5- and 6-ring PAH structures.
  
  
• High throughput DNA sequencing of microbial consortium assays indicated that the abundance of known oil-degrading bacteria from orders Oceanospirillales, Caulobacterales, Sphingomonadales, and Nitrosococcales were enhanced in incubations with HMW-PAHs even while no measurable degradation of the dibenzopyrenes was observed. Of the five isomers studied, dibenzo[a,h]pyrene supported the fewest number of microbial species suggesting the isomer was more toxic compared to the other dibenzopyrene isomers.
  
  
• To identify new and environmentally relevant bacteria capable of degrading HMW-PAH compounds, diffusion chambers amended with benzo[a]pyrene were placed in situ in sediment and water collected from a coastal marsh impacted by the Deepwater Horizon oil spill. Seven unique bacterial isolates most closely related phylogenetically at the genus level to Algoriphagus, Paracoccus, Joostella, Thalassospira, and Muricauda were cultured from the diffusion chambers after four weeks of placement in Gulf of Mexico sediment and water. These results suggest that oil-contaminated environments may harbor and select for novel microorganisms capable of degrading and/or tolerating highly contaminated environments. Further sequencing and culture studies are needed to identify these organisms and their ability to degrade HMW-PAHs.
  
  
• There has been a critical gap in the research regarding the environmental occurrence and distribution of these HMW-PAHs due to difficulties in analytical separation and detection. As a result, the behavior of these "hidden" organic compounds in natural ecosystems has been unknown. Our state-of-the-art methodology using high-resolution vibrational spectroscopy was successful in analytically separating and identifying metabolites of these HMW-PAHs. Metabolites of benzo[a]pyrene, including dihydrodiols, tetrahydrotriols and tetrahydrotetrols were detected at trace concentration levels using photoluminescence spectroscopy.
  
  
• Benzo[a]pyrene metabolites were successfully extracted from water samples using solid-phase extraction and room temperature fluorescence spectroscopy (SPE-RTF). Metabolites were extracted with an octadecyl (C-18) silica membrane and determined directly on the surface of the solid membrane via RTF spectroscopy. Since metabolite quantification does not require elution steps, excellent recoveries (~ 100%) were often obtained with the straightforward procedure. The strong fluorescence of benzo[a]pyrene metabolites on C-18 membranes provided SPE-RTF with competitive limits of detection (LODs) at the pg mL^-1 concentration levels. This methodology is applicable to fluorescence metabolites of other PAHs including dibenzopyrenes with molecular weight 302Da.
  
  
• The main limitation of the SPE-RTF results was the spectral overlapping of co-extracted fluorophores that might also exist in samples of unknown composition. The selectivity of this technique was considerably improved by processing multidimensional data formats with multiway deconvolution methods. The combination of either RTF excitation-emission matrices (EEMs) or total synchronous fluorescence spectra (SFS) with unfolded-partial least squares/residual bi-linearization (U-PLS/RBL) allows for the determination of targeted metabolites in the presence of unknown interference. Both data formats – EEMs or SFS – were generated with the aid of a commercial spectrofluorimeter equipped with a continuous wave (CW) excitation source for steady state spectroscopy.  
  
  
• A selectivity enhancement was obtained by adding the temporal dimension to excitation and fluorescence spectra. The complete data sets were known as wavelength-time matrices (WTMs) and time-resolved excitation emission matrices (TREEMs). In addition to spectral information, WTMs and TREEMs provided fluorescence lifetime information for the identification of spectrally overlapped metabolites. This methodology has proven useful for the determination of co-eluted benzo[a]pyrene metabolites in HPLC fractions. The same is true for fluorescence metabolites of other PAHs.
  
  
• The ultimate selectivity was obtained by recording WTMs and TREEMs in 1-octanol at 77K or 4.2K. This approach provided narrow excitation and fluorescence spectra with vibrational information for the identification of closely related metabolites – such as tetrahydrotetrols - in HPLC fractions.

 Proposal Abstract - RFP-VI PI Melanie Beazley