Insights into the degradation capacities of Amycolatopsis tucumanensis DSM 45259 guided by microarray data

The analysis of catabolic capacities of microorganisms is currently often achieved by cultivation approaches and by the analysis of genomic or metagenomic datasets. Recently, a microarray system designed from curated key aromatic catabolic gene families and key alkane degradation genes was designed. The collection of genes in the microarray can be exploited to indicate whether a given microbe or microbial community is likely to be functionally connected with certain degradative phenotypes, without previous knowledge of genome data. Herein, this microarray was applied to capture new insights into the catabolic capacities of copper-resistant actinomycete Amycolatopsis tucumanensis DSM 45259. The array data support the presumptive ability of the DSM 45259 strain to utilize single alkanes (n-decane and n-tetradecane) and aromatics such as benzoate, phthalate and phenol as sole carbon sources, which was experimentally validated by cultivation and mass spectrometry. Interestingly, while in strain DSM 45259 alkB gene encoding an alkane hydroxylase is most likely highly similar to that found in other actinomycetes, the genes encoding benzoate 1,2-dioxygenase, phthalate 4,5-dioxygenase and phenol hydroxylase were homologous to proteobacterial genes. This suggests that strain DSM 45259 contains catabolic genes distantly related to those found in other actinomycetes. Together, this study not only provided new insight into the catabolic abilities of strain DSM 45259, but also suggests that this strain contains genes uncommon within actinomycetes.Fil: Bourguignon, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; ArgentinaFil: Bargiela, Rafael. Consejo Superior de Investigaciones Científicas; EspañaFil: Rojo, David. Centro de Metabolómica y Bioanálisis; EspañaFil: Chernikova, Tatyana N.. Bangor University; Reino UnidoFil: de Rodas, Sara A. López. Universidad Complutense de Madrid; EspañaFil: García-Cantalejo, Jesús. Universidad Complutense de Madrid; EspañaFil: Näther, Daniela J.. Goethe Universitat Frankfurt; AlemaniaFil: Golyshin, Peter N.. Bangor University; Reino UnidoFil: Barbas, Coral. Centro de Metabolómica y Bioanálisis; EspañaFil: Ferrero, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; ArgentinaFil: Ferrer, Manuel. Consejo Superior de Investigaciones Científicas; Españ

Bacterial benz(a)anthracene catabolic networks in contaminated soils and their modulation by other co-occurring HMW-PAHs

Polycyclic aromatic hydrocarbons (PAHs) are major environmental pollutants in a number of point source contaminated sites, where they are found embedded in complex mixtures containing different polyaromatic compounds. The application of bioremediation technologies is often constrained by unpredictable end-point concentrations enriched in recalcitrant high molecular weight (HMW)-PAHs. The aim of this study was to elucidate the microbial populations and potential interactions involved in the biodegradation of benz(a)anthracene (BaA) in PAH-contaminated soils. The combination of DNA stable isotope probing (DNA-SIP) and shotgun metagenomics of 13C-labeled DNA identified a member of the recently described genus Immundisolibacter as the key BaA-degrading population. Analysis of the corresponding metagenome assembled genome (MAG) revealed a highly conserved and unique genetic organization in this genus, including novel aromatic ring-hydroxylating dioxygenases (RHD). The influence of other HMW-PAHs on BaA degradation was ascertained in soil microcosms spiked with BaA and fluoranthene (FT), pyrene (PY) or chrysene (CHY) in binary mixtures. The co-occurrence of PAHs resulted in a significant delay in the removal of PAHs that were more resistant to biodegradation, and this delay was associated with relevant microbial interactions. Members of Immundisolibacter, associated with the biodegradation of BaA and CHY, were outcompeted by Sphingobium and Mycobacterium, triggered by the presence of FT and PY, respectively. Our findings highlight that interacting microbial populations modulate the fate of PAHs during the biodegradation of contaminant mixtures in soils

Integrating Shotgun Metagenomics, 16s Rrna Gene Metabarcoding and Culture Approaches: A Better Outlook for Functional Profiling of a Pah-Contaminated Soil

Understanding bacterial diversity and function is critical for designing bioremediation strategies. This research aimed to assess chronically hydrocarbon contaminated soil bacterial diversity and their aromatic compound degradation (ACD) potential by integrating shotgun metagenomic, 16S rRNA gene metabarcoding and culture approaches. While soil metabarcoding showed dominance of Proteobacteria, metagenomics indicated that 99,5% of the sequences were taxonomically assigned to Streptomycetales order and that almost all genes related to ACD were assigned to the latter. To inspect other phyla contribution to ACD, a functional prediction was delved, and two culture approaches were used. PICRUSt2 revealed that ACD pathways were mostly found in Alphaproteobacteria, Actinobacteria and Gammaproteobacteria classes. An enrichment culture (r-EFP) was obtained with pyrene as sole carbon and energy source and a bacterial strain (S19P6), identified as a member of Mycolicibacterium genus, was isolated. Both cultures demonstrated the ability to degrade more than 90% of the supplemented pyrene after 21 days of incubation. 16S rRNA gene metabarcoding and shotgun metagenomics approaches in r-EFP indicated predominance of Proteobacteria Phylum and the presence of genes responsible for the degradation of ACD mostly assigned to the predominant phyla. Complementing different methodologies enable the recognition of the metabolic potential of soil Proteobacteria related to ACD.Fil: Festa, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Granada, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Irazoqui, José Matías. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Cuadros Orellana, Sara. Universidad Catolica de Maule; ChileFil: Quevedo, Claudio. Universidad Catolica de Maule; ChileFil: Coppotelli, Bibiana Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Morelli, Irma Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentin

Efecto de la contaminación crónica, factores geoquímicos y bioestimulación en el catabolismo de hidrocarburos en ambientes marinos contaminados

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19 de febrero de 201

Towards biorecycling of plastics: Isolation and characterization of Pseudomonas capeferrum TDA1, a bacterium capable to degrade polyurethane mono- and oligomers

During the last 50 years, plastic industry has grown exponentially with an estimated 8300 million metric tonnes of plastic produced to date. Regardless of the large variety of polymers available, 99% are entirely fossil-fuel based which compromises its degradability after use. Major synthetic polymers in use today are polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyurethane (PU) and polyethylene terephthalate (PET). The current methods for disposing of plastic waste mainly include landfilling, incineration, mechanical and chemical recycling. Despite of the significant improvement of these technologies, it is still necessary to overcome several limitations and deficiencies. Polyurethane (PU) is a synthetic polymer used as raw material in several industries. In 2015, PU global production reached 27 million metric tons, making the sixth most-used plastic worldwide. The main constituents of polyurethane are isocyanates, polyols and chain extenders. Unfortunately, the mismanaged plastic has spread out in different habitats across our planet including cold marine areas and uninhabited places, threatening wildlife and ecosystems. In order to avoid further contamination, it is necessary to transform plastic waste by restoring functional properties, providing added value and exploring new application areas that could provide economic benefits in a long- term perspective. In the last 10 years, a transition from a linear economy to a sustainable, bio-based circular economy has become fundamental to cope the fossil fuel-driven climate change and global plastic pollution. This transformation involves industrial and basic research strongly focused on biotechnology and bioprocesses. Within this transition, microorganisms are key players due to the wide diversity of enzymes and metabolic pathways that could be used for the development of sustainable processes and biomaterials. Recently, microorganisms with plastic-degrading potential have been regularly identified in different environments such as waste disposal, landfills, plastic refineries, open dumps, etc. Selective pressure and evolution of genetically flexible mechanisms have contribute to metabolize anthropogenic compounds, which it has been noted in several enzymatic reactions designed for the efficient degradation of a wide variety of recalcitrant substrates, leading to novel metabolic pathways. Even though several bacterial genera have been reported in the degradation of environmental pollutants, Pseudomonas species are amongst the most cited degraders of aromatic hydrocarbons and plastic polymers. The genus Pseudomonas incorporates one of the most complex, diverse, and ecologically significant group of bacteria on the planet. Members of this genus are found in large numbers in all the major natural environments (terrestrial, freshwater, and marine) and form intimate associations with plants and animals. This universal distribution suggests a remarkable degree of physiological and genetic adaptability. In fact, Pseudomonas have been most frequently linked with PU degradation. Chemically, polyester-based PUs are semi-crystalline structures containing hydrolysable ester and urethane bonds that are fragmented by extracellular enzymes (hydrolases), releasing oligomeric and monomeric building blocks. For instance, amines, alcohols, acids, aromatics, and other residues, such as EG (ethylene glycol), 1,4-butanediol (BDO), adipic acid (AA) ,4′-methylenedianiline (MDA) and 2,4- toluene diamine (2,4-TDA) are constantly present during PU degradation. However, MDA and 2,4-TDA are considered environmental pollutants, which represent a major risk for species in the aquatic and terrestrial areas. This fragmentation of the polymer is known as depolymerization and it is essential for strengthening recycling processes that use plastic waste as feedstock. The broad spectrum of building blocks might be used as carbon and energy source for microorganisms that degrade these compounds and/or use them for the production of higher-value elements. This latter is considered a promising upcycling strategy to reduce fossil-fuel plastic waste and promote new waste management strategies. Previous studies have revealed that extracellular enzymes are essential for biofilm formation on the polymer surface, reducing the resistance and durability of plastic materials. This first step promotes microbial attachment and further degradation. Enzymes with hydrolytic and proteolytic activity have been detected in spherical structures called outer membrane vesicles (OMVs) in several Pseudomonas species. Generally, OMVs play a key role in establishing inter- and intra-species communication, acquisition of nutrients, stress response, delivery of toxins, adhesion and virulence factors, biofilm formation, etc. Even though numerous bacterial strains and enzymes are involved in degradation processes, the complete catabolic mechanism is not totally understood yet. This thesis also centers on the characterization of outer membrane vesicles for extracellular degradation of a polyurethane oligomer and elucidation of the degradation pathway for the polyurethane monomer 2,4-diaminotoluene (2,4-TDA) by Pseudomonas capeferrum TDA1. In the first chapter, bacterial isolation from soil samples and the subsequent protocols to quantify biodegradation of polyurethane building blocks were fully described. The isolated strain was able to use a PU oligomer and 2,4-TDA as sole source of carbon. The latter compound also served as nitrogen source. These results provided a key insight into the catabolic mechanism of the soil bacterium as a potential PU monomer and oligomer-degrader. The second chapter described the identification of the isolated strain as Pseudomonas sp. by partial 16S rRNA gene sequencing, membrane fatty acid profile and structural gene for the cis/trans isomerase (cti). In addition, genomic DNA was isolated from bacterial cells grown on succinate and utilized for whole genome sequencing in order to detect catabolic genes related to aromatic compounds degradation. Preliminary, enzymes involved in the metabolic pathway were identified, which eventually led to a suggested degradation pathway for Pseudomonas sp. grown on 2,4-TDA. The strain was identified as Pseudomonas capeferrum (type strain WCS358) using the full 16S rRNA gene sequence. The third chapter reported a new method of RNA extraction from Pseudomonas capeferrum TDA1 growing on 2,4-TDA. Phenols and catechols are central intermediates of the aromatics biodegradation that can be easily oxidized to yield the corresponding quinones, which interfere with nucleic acids and tend to co- precipitate or degrade RNA. The chemical process is regulated by the activity of polyphenol oxidases enzymes, which have been identified in several Pseudomonas species previously. This optimized protocol incorporated several modifications including the use of a carrier, pooled samples and a final cleaning up step that could improve it significantly, yielded a high-quality RNA measured by A260/A280, A260/230 ratios (2.02 ± 0.16, 1.95 ± 0.01, respectively) from cells grown on 2,4-TDA compared to standard assays. Moreover, RIN (RNA integrity number) values were analyzed and samples with a RIN higher than 7.0 were selected for downstream applications, confirming the RNA quality. Finally, the fourth chapter evaluated the transcriptional changes in Pseudomonas capeferrum TDA1 grown on 2,4-TDA using RNA-seq. From all the expressed genes, one third were overexpressed in comparison to the control (succinate). These alterations in the gene expression demonstrates that aromatic compounds trigger adaptive responses that modify the transcriptional regulation mechanism including important changes not only in the catabolic system, but also in other patterns related to bacterial cell physiology and biofilm formation. In order to evaluate extracellular degradation, OMVs isolated from P. capeferrum TDA1 grown on a PU oligomer were tested for hydrolytic activity. Purified OMVs showed higher esterase activity compared to cell pellets. Relative OMV yields in TDA1 raised significantly in PU oligomer (0.28 ± 0.05%) compared to succinate (0.09 ± 0.01%). This three-fold increased activity could demonstrate that the release of OMV is part of the adaptive mechanisms of bacteria to stressful environmental conditions. The macromolecular degradation may occur through the action of both periplasmic and membrane-bound hydrolases harbored inside of OMVs and can be considered as a supporting mechanism for biodegradation. The results of this thesis present a further understanding of the transcriptome response in P. capeferrum TDA1 exposed to a PU monomer, suggest a model for extracellular degradation involving OMVs and propose a complete catabolic mechanism for the biodegradation of polyester-based PU containing intra and extracellular enzymes. Moreover, further studies on biological degradation of PU will contribute to redesign plastic polymers considering biodegradable building blocks and improving biocatalytic degradation, which could provide a sustainable use of PU plastic waste in the future

Analysis of defence systems and a conjugative IncP-1 plasmid in the marine polyaromatic hydrocarbons-degrading bacterium Cycloclasticus sp. 78-ME

Marine prokaryotes have evolved a broad repertoire of defence systems to protect their genomes from lateral gene transfer including innate or acquired immune systems and infection-induced programmed cell suicide and dormancy. Here we report on the analysis of multiple defence systems present in the genome of the strain Cycloclasticus sp. 78-ME isolated from petroleum deposits of the tanker 'Amoco Milford Haven'. Cycloclasticus are ubiquitous bacteria globally important in polyaromatic hydrocarbons degradation in marine environments. Two 'defence islands' were identified in 78-ME genome: the first harbouring CRISPR-Cas with toxin-antitoxin system, while the second was composed by an array of genes for toxin-antitoxin and restriction-modification proteins. Among all identified spacers of CRISPR-Cas system only seven spacers match sequences of phages and plasmids. Furthermore, a conjugative plasmid p7ME01, which belongs to a new IncP-1θ ancestral archetype without any accessory mobile elements was found in 78-ME. Our results provide the context to the co-occurrence of diverse defence mechanisms in the genome of Cycloclasticus sp. 78-ME, which protect the genome of this highly specialized PAH-degrader. This study contributes to the further understanding of complex networks established in petroleum-based microbial communities

An Assessment of Microbial Communities and Their Potential Activities Associated with Oil Producing Environments

Microbial populations have been found in oil-associated environments as early as the 1920s. The proliferation and metabolic activities of these microorganisms can have profound deleterious effects on the infrastructure associated with oil reservoirs, production, transport and storage. Biodegradation of hydrocarbons by reservoir microorganisms can lead to the formation of ‘heavy oil’ that is of lower economic value and is more difficult to recover. Some members of reservoir microbial communities also participate in microbial influenced corrosion. By applying modern sequencing technologies, much can be learned about the microorganisms present and their metabolic capabilities. The focus of this dissertation was to provide a comprehensive characterization of microbial communities in two oil production facilities and define their metabolic activity by profiling metabolites of hydrocarbons and sequencing their metagenomes. The most common samples available from oil production facilities are fluids collected at valve openings. These samples are chemically and biologically representative of the bulk fluids at any given location within an oil facility (e.g. pipelines). Microorganisms commonly attach to surfaces and form biofilms that can provide the microbial inhabitants protection from the external environment, allow for localized changes in chemistry, and represent sites of corrosion. Common maintenance of pipelines includes the use of “pigs” which physically disrupt and remove biofilms, corrosion products, and other solids associated with the inner surfaces of a pipeline. Libraries of partial 16S rRNA gene sequences were used to compare the microbial communities in bulk fluids from several locations throughout an oil production facility with the community associated with a “pig envelope”, the fluids enriched with solids removed by a pig. The microbial communities in bulk fluids and biofilms of the oil production facility contained only a few taxa. All samples had similar compositions, but different structure (relative abundances of taxa). An estimation of population density based on qPCR of 16S rRNA gene copy number showed that there was a five-fold increase in the number of bacteria in the pig envelope. The numerically abundant taxa were members of the genera Thermoanaerobacter, Thermacetogenium and Thermovirga, which should be studied further to determine their ability to degrade hydrocarbons and influence corrosion. The community structure, genomic potential, and function of microbial assemblages from two oilfields under different management practices were characterized to measure their potential for hydrocarbon biodegradation. High throughput sequencing of 16S rRNA genes was combined with shotgun metagenomic sequencing and a targeted environmental metabolomics survey to interrogate two oil production facilities. The genomic potential for the abundant taxa was thoroughly interrogated for currently known pathways for hydrocarbon metabolism. Several sequences were identified that are closely related to known hydrocarbon degradation genes; however, there is no conclusive evidence that directly links these taxa and the hydrocarbon metabolites that were identified. The presence of microorganisms and putative signature metabolites in oil-associated environments suggests hydrocarbon degradation is occurring. Hydrocarbon degradation causes souring and ‘heavy oil’ which is harder to extract and of less value. Additionally, when microorganisms are identified in close association with corroded surfaces, they are potentially implicated as participating in surface corrosion. In order to directly associate a particular microorganism with a specific activity, there is still a need for controlled experiments. A better understanding of the microorganisms and their activities in oil production facilities will lead to improved monitoring and mitigation for the future

Towards more complete metagenomic analyses through circularized genomes and conjugative elements

Advancements in sequencing technologies have revolutionized biological sciences and led to the emergence of a number of fields of research. One such field of research is metagenomics, which is the study of the genomic content of complex communities of bacteria. The goal of this thesis was to contribute computational methodology that can maximize the data generated in these studies and to apply these protocols human and environmental metagenomic samples. Standard metagenomic analyses include a step for binning of assembled contigs, which has previously been shown to exclude mobile genetic elements, and I demonstrated that this phenomenon extends to all conjugative elements, which are a subset of mobile genetic elements. I proposed two separate methodologies that could detect contigs that are potential conjugative elements: a curated set of profile hidden Markov models that are very efficient to run, or annotation using the full UniRef90 database, a slower but more sensitive method. I then applied this framework to a large population-based cohort and to a study examining the association of the maternal human gut microbiota and the development of spina bifida. Broadly, the composition and abundances of conjugative elements were discriminatory between the age and geographic cohorts. In the spina bifida cohort, there was an enrichment of Campylobacter hominis and a conjugative element belonging to Campylobacter hominis, which was excluded from the metagenomic bins. Next, I characterized a novel species belonging to the recently discovered manganese-oxidizing genus Manganitrophus growing on oil refinery carbon filters. I successfully circularized the genomes of three strains and got quality assemblies for the remaining two samples. Furthermore, I identified a previously uncharacterized conjugative plasmid belonging to the species using my framework developed in chapter 2. Finally, I developed an assembly pipeline to perform a secondary assembly on binned assemblies using long reads. The secondary assemblies yielded a number of additional circularized sequences that would be useful as scaffolds in future metatranscriptomic, variation analysis, and community dynamic studies. The methodologies and applications in this thesis provide a framework for more complete metagenomic analyses going forward that will aid in our understanding of microbial ecology

Microbial Ecology of Coastal Ecosystems: Investigations of the Genetic Potential for Anaerobic Hydrocarbon Transformation and the Response to Hydrocarbon Exposure

Microbial-mediated hydrocarbon transformation plays a vital role in the attenuation of natural and anthropogenic-sourced petroleum contamination in the environment, particularly in marine systems. Indigenous microbial communities in marine habitats are resilient to influxes of petroleum, and it is well documented that many taxa are capable of responding and utilizing these compounds. Coastal ecosystems are often either subjected to or at risk for oil contamination and are of particular concern due to their significant environmental and economic value. The research projects presented here focused on coastal ecosystems and investigated microbial community compositions via next-generation sequencing of 16S rRNA genes, the genetic potential for anaerobic hydrocarbon biodegradation within these communities via molecular surveys of marker genes, and the response of anaerobic populations to exposure of a hydrocarbon via microcosm studies or to products of hydrocarbon transformation processes (i.e. photolysis) via sulfate reduction assays (SRAs). Chesapeake Bay is the largest estuary in the United States, and experiences high nutrient loading and water column hypoxia due to watershed runoff, as well as petroleum contamination from urban runoff, atmospheric deposition, and spills directly into the water column. Past studies have demonstrated that aerobic hydrocarbon-degrading bacteria can be enriched from the water column and from the sediment. However, evidence for anaerobic biodegradation of hydrocarbons had not been demonstrated at the time of our study. Given the recurring seasonal water column hypoxia and the transient exposure to hydrocarbons, we hypothesized that the potential for degradation under anaerobic conditions may exist in Chesapeake Bay sediments. Here, molecular surveys and microcosms were utilized to investigate microbial community composition and the potential for anaerobic hydrocarbon degradation among sites along a transect of the Bay. Sampling locations were chosen both within and outside areas of recurring hypoxia. Distinct geochemical gradients along the transect were revealed. Low oxygen, low sulfate, and high methane concentrations were observed in the upper Bay, as were significantly higher levels of taxa associated with anaerobic processes (e.g., sulfate reducers and methanogens). In contrast, higher oxygen, higher sulfate, and very low methane were measured in the lower Bay. Sulfate-reducers and methanogens decreased in abundance in lower Bay sediments as well. Similarly, molecular surveys showed more frequent detection of marker genes associated with the anaerobic activation of hydrocarbons via the ‘fumarate addition’ pathway (e.g., assA, bssA) in the upper Bay, and microcosms established under sulfate-reducing and/or methanogenic conditions suggested that the model hydrocarbon, hexadecane, was being converted to methane by indigenous sediment communities obtained from the upper Bay sites. These findings illustrate the variability of microbial communities between different locations in Chesapeake Bay as well as differences in their response to a hydrocarbon. Together, the data highlighted the significance that anaerobic processes could potentially play in the event of an oil spill in Chesapeake Bay. The Gulf of Mexico (GoM) is one of the most environmentally and economically important coastal regions in the United States. The Deepwater Horizon (DWH) spill in the GoM was the largest accidental release of crude oil into U.S. waters. Extensive research was carried out on the response of microbial communities to the discharged oil and gas. Collectively, studies emphasized the importance of both aerobic and anaerobic hydrocarbon transformation processes and concluded that native microbial populations responded quickly to the petroleum, promoting contaminant removal from the environment. Two of the research projects presented herein aimed to (1) further study the impact that released oil, once weathered, can have on indigenous anaerobic microbial communities, and to (2) characterize microbial populations associated with weathered oil residues (i.e., sand patties) that have remained in the environment years after the spill and to determine the role these populations have in the attenuation of residual contamination. Once introduced into the environment, oil is subjected to a number of weathering processes, including evaporation, emulsification, and photooxidation. Photooxidation of oil can lead to the incorporation of oxygen molecules into hydrocarbon constituents, which can subsequently result in enhanced bioavailability and/or increased toxicity to certain organisms. Microbial toxicity studies are typically conducted using individual aerobic taxa, as opposed to indigenous communities or anaerobic microorganisms, and little is known with regard to how photolyzed oil affects anaerobes. Experiments presented here assessed the impact that photooxidized hydrocarbons can have on sulfate-reducing communities in coastal sediments. We hypothesized that photolyzed oil or photolyzed oil components would inhibit the sulfate-reducing communities. Three distinct GoM coastal locations were chosen for study. Sediment microbial communities were characterized via 16S rRNA gene sequencing, and the impact of irradiated crude oil or irradiated PAHs (i.e., pyrene, phenanthrene, and a phenanthrene/anthracene mixture) was tested via sulfate reduction assays (SRAs). Sulfate-reducing taxa varied in both abundance and composition across sampling sites. Overall, no impact on sulfate reduction rates was observed for any of the photolyzed compounds at any of the coastal locations investigated. Data suggested that water-soluble photogenerated products did not negatively impact sulfate-reducing communities and that these compounds could potentially be utilized by sulfate-reducing microorganisms. These findings highlight the resilience of native microbial communities in response to an influx of weathered hydrocarbons, as well as the potential of these populations to further mediate hydrocarbon transformation processes. Weathering of oil released during the DWH spill also led to the formation of water-in-oil emulsions. Many of these emulsions washed ashore early after the onset of the spill, whereas an unknown quantity sank in nearshore environments, resulting in the formation of submerged oil mats (SOMs). Fragments of these buried mats continued to wash ashore coastal beaches and marshes years after the spill in the form of oil:sand aggregates (e.g., tar balls, sand patties, etc.). The third research project presented here aimed to use next-generation sequencing of 16S rRNA genes to characterize microbial communities associated with individual oil:sand aggregates collected from different GoM beaches, to use metagenomic sequencing to survey for marker genes associated with hydrocarbon transformation pathways to determine the genetic capacity for biodegradation within the microbial populations, and to conduct targeted metabolomics via mass spectrometry to assess whether these communities mediate transformation of hydrocarbons in situ (i.e., once aggregates are deposited on the beach). Given the presumed differences in residence times and exposure to different environmental conditions, we hypothesized that sand patty microbial communities would be different between sites. Together, molecular surveys demonstrated that individual aggregates had either an anaerobic, facultative anaerobic, or aerobic signature with regard to both the taxonomic composition of communities and the metabolic potential associated with hydrocarbon degradation pathways. Several taxa with known or suspected hydrocarbon-degrading ability were detected (e.g., Marinobacter, Alcanivorax, Mycobacterium), and specific taxa varied among samples. Additionally, profiles of functional genes involved in aerobic and anaerobic hydrocarbon transformation pathways (e.g., assA, alkB) also varied among samples and corresponded with 16S rRNA gene profiles. Results from beach sand and seawater samples confirmed that microbial populations were distinct from those obtained from sand patties. Taxonomic profiles of core communities (i.e., taxa comprising ≥1% of libraries) identified ten shared operational taxonomic units (OTUs) between aggregates and beach sand and seven shared OTUs between aggregates and seawater. Targeted mass spectrometry putatively identified metabolites indicative of aerobic and/or anaerobic hydrocarbon transformation processes (e.g., toluic acid, hydroxybenzoic acid, phenylpropionic acid), and showed that these compounds were not detected in beach sand. These findings provide evidence that aggregate-associated microbes are capable of hydrocarbon degradation and also highlight the potential role that microorganisms likely play in the long-term attenuation of remnant oil present in the environment years after the DWH spill