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Application of Metagenomics for Identification of Novel Petroleum Hydrocarbon Degrading Enzymes in Natural Asphalts from the Rancho La Brea Tar Pits

  • Baquiran, Jean-Paul Mendoza
Publication Date
Jan 01, 2010
eScholarship - University of California
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Recent studies on the biodiversity of asphalt deposits at the Rancho La Brea Tar Pits in Los Angeles, California have revealed the existence of several hundred new species of bacteria and gene sequences encoding putative novel degradative enzymes (Kim and Crowley, 2007). The presence of fossilized extinct animal remains in the La Brea Tar Pits has led to estimations that these natural asphalt seeps have existed for at least 40,000 years (Akersten et al., 1983). These deposits consist of petroleum that has been degraded to the extent that the remaining material is comprised mainly of asphalts and heavy oils, which have saturated into the soil matrix. Since petroleum hydrocarbons are both a target and a product of microbial metabolism (Hamme et al., 2003), the role of microorganisms that inhabit this and other similar environments is directly relevant to development of technology for bioremediation, biotransformation of petroleum hydrocarbons, and microbial enhanced oil recovery for extracting and refining heavy oil.In this research, both culture-dependent and culture-independent methods were used to characterize microorganisms and consortia from the La Brea Tar Pits, which are able to degrade a variety of polycyclic aromatic hydrocarbons (PAHs) and BTEX (benzene, toluene, ethyl benzene and xylene). Initial studies applied PCR-DGGE methods to identify the microbial consortia that degrade selected petroleum hydrocarbons during enrichment cultures on pure compounds or mixtures of substances found in petroleum. This study revealed a single Pseudomonas sp. that may be able to degrade multiple PAHs and biphenyl. In addition, PCR based techniques identified a naphthalene dioxygenase from Pseudomonas stutzeri that appears to function as a major degradative enzyme in this system. DNA microarray analysis further revealed the presence of a wide variety of petroleum hydrocarbon degrading genes and has verified that naphthalene dioxygenase is the most commonly found degradative enzyme in this microbial community.To further investigate the true microbial diversity of the La Brea Tar Pits, the microbial community associated with the heavy oil at Rancho La Brea was studied used a metagenomics approach. A fosmid clone library was constructed using 38 Kb fragments of DNA extracted from the asphalt-soil samples of Pit 101. This library of about 3,000 clones was then screened for DNA inserts, which contained specific genes that could be targeted using PCR based methods. One selected clone contained the 16S rRNA gene of an unclassified Rhodospirallacaea and a putative 2-nitropropane dioxygenase, which suggest that this new organism degrades persistent nitroalkanes that are common in asphalt.The current investigation of the La Brea Tar pits concluded using Illumina technologies (Illumina, Inc.) to deep sequence the metagenomic library of over 3,000 clones using a high throughput sequencer. Over 75 MB of DNA has been sequenced, from which over 650 contigs with an average length of 500 bp were assembled. Bioinformatics analysis indicated the presence of genes encoding three types of dioxygenases, one of which encoded a naphthalene dioxygenase from Pseudomonas sp. along with two other genes that are most similar to previously reported genes encoding biphenyl and toluene dioxygenases. However, sequence analysis revealed that these genes were not significantly similar to these known dioxygenases and may thus be novel. The results of this research provide a foundation for further studies on the evolution and assembly of metabolic pathways in bacteria that have undergone long term adaptation to survival in natural asphalts.

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