Conventional upgrading of petroleum distillates requires catalysts, high temperatures and high pressures to hydrogenate and cleave fused-ring aromatic hydrocarbons to give alkyl benzenes. A potential alternative is to use bacteria in a two-stage upgrading process: first, bacteria would enzymatically open one ring of di- and tricyclic aromatics to yield polar products under ambient conditions; then mild chemical hydrogenation would reduce the bio-catalytic products to alkyl aromatics. Preliminary trials dealing with the first stage of the potential process have been carried out using the bacterium Pseudomonas fluorescents LP6a. This organism oxidizes a wide range of aromatic hydrocarbons and heterocycles commonly present in petroleum middle distillate fractions, using broad-specificity enzymes. Mutants were generated and shown by mass spectrometry to produce the predicted aromatic ring fission products without carbon loss from pure aromatic substrates. The mutants were prepared as pre-grown, induced, whole cell biocatalysts in aqueous suspension. Aromatic hydrocarbons and heterocycles dissolved in heptamethylnonane (as a synthetic petroleum distillate) were transformed without altering the aliphatic carrier.


Thermo chemical conversion of heavy oils and bitumen's produces middle distillate fractions containing di- and tricyclic aromatics With low fuel value. These aromatics are currently upgraded by expensive high pressure-high temperature chemical hydrogenation involving catalysts which are inactivated by sulfur and nitrogen compounds in the feedstock's. Additionally, consumption of hydrogen is high, contributing to costs. The potential exists for microbes to selectively cleave the aromatic rings enzymatically under near-ambient conditions, yielding products that are more susceptible to chemical hydrogenation than their parent compounds, thereby realizing savings in hydrogenation costs.

A research project was designed to test the feasibility of biological upgrading of certain middle distillates using whole bacterial cells. Theoretically, an enzymatic process could selectively cleave di- and tri-aromatic ring structures, producing phenolics with alkyl side-chains having carboxylic groups. Although these oxygenated products are themselves undesirable in fuel, mild chemical hydrogenation of the products to remove the oxygen (rather than for cracking purposes) could yield alkyl benzenes with better fuel value than the parent fused-ring aromatics. Upgrading would then involve a two-step process: specific enzymatic ring cleavage followed by low temperature, low pressure hydrogenation.

In addition to yielding the desired ring cleavage products, two primary requirements of the biological process were recognized; first, that the enzymatic activity be restricted to aromatic compounds (i.e., not affect aliphatic compounds present in the distillates), yet be effective over a broad range of di- and tricyclic aromatic hydrocarbons, heterocycles, and alkyl-substituted homologues; and second that there be no carbon loss from the aromatic substrates as a consequence of microbial oxidation. Additional requirements included the ability of whole cells to catalyze ring cleavage in a resting state, and to be pre-grown quickly to high density in an active state; that is, to be used as a biocatalyst.

The work described here resulted from preliminary, bench-scale investigations of biocatalytic ring opening of model aromatic compounds both in pure crystalline form and incorporated into "synthetic distillates". The chosen organism, Pseudomonas fluorescents LP6a, had been previously observed to possess several of the properties listed ab

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