Enhancing biodesulfurization by engineering a synthetic dibenzothiophene mineralization pathway.

A synthetic dibenzothiophene (DBT) mineralization pathway has been engineered in recombinant cells of Pseudomonas azelaica Aramco J strain for its use in biodesulfurization of thiophenic compounds and crude oil. This functional pathway consists of a combination of a recombinant 4S pathway responsible for the conversion of DBT into 2-hydroxybiphenyl (2HBP) and a 2HBP mineralization pathway that is naturally present in the parental P. azelaica Aramco J strain. This novel approach allows overcoming one of the major bottlenecks of the biodesulfurization process, i.e., the feedback inhibitory effect of 2HBP on the 4S pathway enzymes. Resting cells-based biodesulfurization assays using DBT as a sulfur source showed that the 2HBP generated from the 4S pathway is subsequently metabolized by the cell, yielding an increase of 100% in DBT removal with respect to previously optimized Pseudomonas putida biodesulfurizing strains. Moreover, the recombinant P. azelaica Aramco J strain was able to use DBT as a carbon source, representing the best characterized biocatalyst harboring a DBT mineralization pathway and constituting a suitable candidate to develop future bioremediation/bioconversion strategies for oil-contaminated sites.

Simultaneous valorization and biocatalytic upgrading of heavy vacuum gas oil by the biosurfactant-producing Pseudomonas aeruginosa AK6U.

Heavy vacuum gas oil (HVGO) is a complex and viscous hydrocarbon stream that is produced as the bottom side product from the vacuum distillation units in petroleum refineries. HVGO is conventionally treated with thermochemical process, which is costly and environmentally polluting. Here, we investigate two petroleum biotechnology applications, namely valorization and bioupgrading, as green approaches for valorization and upgrading of HVGO. The Pseudomonas aeruginosa AK6U strain grew on 20% v/v of HVGO as a sole carbon and sulfur source. It produced rhamnolipid biosurfactants in a growth-associated mode with a maximum crude biosurfactants yield of 10.1 g l-1 , which reduced the surface tension of the cell-free culture supernatant to 30.6 mN m-1 within 1 week of incubation. The rarely occurring dirhamnolipid Rha-Rha-C12 -C12 dominated the congeners' profile of the biosurfactants produced from HVGO. Heavy vacuum gas oil was recovered from the cultures and abiotic controls and the maltene fraction was extracted for further analysis. Fractional distillation (SimDist) of the biotreated maltene fraction showed a relative decrease in the high-boiling heavy fuel fraction (BP 426-565 °C) concomitant with increase in the lighter distillate diesel fraction (BP 315-426 °C). Analysis of the maltene fraction revealed compositional changes. The number-average (Mn) and weight-average (Mw) molecular weights, as well as the absolute number of hydrocarbons and sulfur heterocycles were higher in the biotreated maltene fraction of HVGO. These findings suggest that HVGO can be potentially exploited as a carbon-rich substrate for production of the high-value biosurfactants by P. aeruginosa AK6U and to concomitantly improve/upgrade its chemical composition.

Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds.

The 4S pathway is the most studied bioprocess for the removal of the recalcitrant sulfur of aromatic heterocycles present in fuels. It consists of three sequential functional units, encoded by the dszABCD genes, through which the model compound dibenzothiophene (DBT) is transformed into the sulfur-free 2-hydroxybiphenyl (2HBP) molecule. In this work, a set of synthetic dsz cassettes were implanted in Pseudomonas putida KT2440, a model bacterial "chassis" for metabolic engineering studies. The complete dszB1A1C1-D1 cassette behaved as an attractive alternative - to the previously constructed recombinant dsz cassettes - for the conversion of DBT into 2HBP. Refactoring the 4S pathway by the use of synthetic dsz modules encoding individual 4S pathway reactions revealed unanticipated traits, e.g., the 4S intermediate 2HBP-sulfinate (HBPS) behaves as an inhibitor of the Dsz monooxygenases, and once secreted from the cells it cannot be further taken up. That issue should be addressed for the rational design of more efficient biocatalysts for DBT bioconversions. In this sense, the construction of synthetic bacterial consortia to compartmentalize the 4S pathway into different cell factories for individual optimization was shown to enhance the conversion of DBT into 2HBP, overcome the inhibition of the Dsz enzymes by the 4S intermediates, and enable efficient production of unattainable high added value intermediates, e.g., HBPS, that are difficult to obtain using the current monocultures.

Biocatalytic desulfurization of thiophenic compounds and crude oil by newly isolated bacteria.

Microorganisms possess enormous highly specific metabolic activities, which enable them to utilize and transform nearly every known chemical class present in crude oil. In this context, one of the most studied biocatalytic processes is the biodesulfurization (BDS) of thiophenic sulfur-containing compounds such as benzothiophene (BT) and dibenzothiophene (DBT) in crude oils and refinery streams. Three newly isolated bacterial strains, which were affiliated as Rhodococcus sp. strain SA11, Stenotrophomonas sp. strain SA21, and Rhodococcus sp. strain SA31, were enriched from oil contaminated soil in the presence of DBT as the sole S source. GC-FID analysis of DBT-grown cultures showed consumption of DBT, transient formation of DBT sulfone (DBTO2) and accumulation of 2-hydroxybiphenyl (2-HBP). Molecular detection of the plasmid-borne dsz operon, which codes for the DBT desulfurization activity, revealed the presence of dszA, dszB, and dszC genes. These results point to the operation of the known 4S pathway in the BDS of DBT. The maximum consumption rate of DBT was 11 µmol/g dry cell weight (DCW)/h and the maximum formation rate of 2-HBP formation was 4 µmol/g DCW/h. Inhibition of both cell growth and DBT consumption by 2-HBP was observed for all isolates but SA11 isolate was the least affected. The isolated biocatalysts desulfurized other model DBT alkylated homologs. SA11 isolate was capable of desulfurizing BT as well. Resting cells of SA11 exhibited 10% reduction in total sulfur present in heavy crude oil and 18% reduction in total sulfur present in the hexane-soluble fraction of the heavy crude oil. The capabilities of the isolated bacteria to survive and desulfurize a wide range of S compounds present in crude oil are desirable traits for the development of a robust BDS biocatalyst to upgrade crude oils and refinery streams.

Rate-limiting step analysis of the microbial desulfurization of dibenzothiophene in a model oil system.

A mechanistic analysis of the various mass transport and kinetic steps in the microbial desulfurization of dibenzothiophene (DBT) by Rhodococcus erythropolis IGTS8 in a model biphasic (oil-water), small-scale system was performed. The biocatalyst was distributed into three populations, free cells in the aqueous phase, cell aggregates and oil-adhered cells, and the fraction of cells in each population was measured. The power input per volume (P/V) and the impeller tip speed (vtip ) were identified as key operating parameters in determining whether the system is mass transport controlled or kinetically controlled. Oil-water DBT mass transport was found to not be limiting under the conditions tested. Experimental results at both the 100 mL and 4 L (bioreactor) scales suggest that agitation leading to P/V greater than 10,000 W/ m(3) and/or vtip greater than 0.67 m/s is sufficient to overcome the major mass transport limitation in the system, which was the diffusion of DBT within the biocatalyst aggregates.

Exploring the mechanism of biocatalyst inhibition in microbial desulfurization.

Microbial desulfurization, or biodesulfurization (BDS), of fuels is a promising technology because it can desulfurize compounds that are recalcitrant to the current standard technology in the oil industry. One of the obstacles to the commercialization of BDS is the reduction in biocatalyst activity concomitant with the accumulation of the end product, 2-hydroxybiphenyl (HBP), during the process. BDS experiments were performed by incubating Rhodococcus erythropolis IGTS8 resting-cell suspensions with hexadecane at 0.50 (vol/vol) containing 10 mM dibenzothiophene. The resin Dowex Optipore SD-2 was added to the BDS experiments at resin concentrations of 0, 10, or 50 g resin/liter total volume. The HBP concentration within the cytoplasm was estimated to decrease from 1,100 to 260 µM with increasing resin concentration. Despite this finding, productivity did not increase with the resin concentration. This led us to focus on the susceptibility of the desulfurization enzymes toward HBP. Dose-response experiments were performed to identify major inhibitory interactions in the most common BDS pathway, the 4S pathway. HBP was responsible for three of the four major inhibitory interactions identified. The concentrations of HBP that led to a 50% reduction in the enzymes' activities (IC50s) for DszA, DszB, and DszC were measured to be 60 ± 5 µM, 110 ± 10 µM, and 50 ± 5 µM, respectively. The fact that the IC50s for HBP are all significantly lower than the cytoplasmic HBP concentration suggests that the inhibition of the desulfurization enzymes by HBP is responsible for the observed reduction in biocatalyst activity concomitant with HBP generation.

Aerobic metabolism of phenylacetic acids in Azoarcus evansii.

The aerobic metabolism of phenylacetic acid (PA) and 4-hydroxyphenylacetic acid (4-OHPA) was investigated in the beta-proteobacterium Azoarcus evansii. Evidence for the existence of two independent catabolic pathways for PA and 4-OHPA is presented. 4-OHPA metabolism involves the formation of 2,5-dihydroxyphenylacetate (homogentisate) and maleylacetoacetate catalyzed by specifically induced 4-OHPA 1-monooxygenase and homogentisate 1,2-dioxygenase. The metabolism of PA starts by its activation to phenylacetyl-CoA (PA-CoA) via an aerobically induced phenylacetate-coenzyme A ligase. Phenylalanine (Phe) aerobic metabolism in this bacterium proceeds also via PA and PA-CoA. Whole cells of A. evansii transformed [1-(14)C]PA to (14)C-phenylacetyl-CoA and subsequently to a number of unknown labeled products, which were also observed in PA-degrading bacteria from different phylogenetic groups, i.e. Escherichia coli, Rhodopseudomonas palustrisand Bacillus stearothermophilus. A chromosomal region from A. evansiiof 11.5 kb containing a cluster of 11 phenylacetic acid catabolic ( paa) genes ( paaYZGHIKABCDE) was sequenced and characterized. The derived gene products were similar to the characterized putative gene products involved in PA catabolism in E. coli and Pseudomonas putida and to other putative PA catabolic gene products of diverse bacteria. RT-PCR analysis of the paa genes of A. evansiigrowing aerobically with PA showed a probable organization of the paa genes in three operons. The similarity of the PA metabolic products pattern and of gene sequences suggests a common aerobic bacterial PA pathway.