John J. Kilbane

Sulfur speciation of desulfurized coals by XANES spectroscopy

Abstract Least square analysis of the X-ray absorption near edge structure (XANES) region of sulfur K edge X-ray absorption fine structure (XAFS) spectra can provide a quantitative analysis of both the organic and inorganic functional forms of sulfur in coal. In the current article, this method is applied to speciation of the sulfur forms in a number of desulfurized coals. The samples investigated include biologically desulfurized coals, samples subjected to selective chemical reactions, samples subjected to molten caustic leaching and specimens treated with boiling perchloroethylene. In all cases, analysis of XANES provides a reasonably quantitative speciation of the changes in the sulfur forms resulting from the various treatments.

Characterization of the dszABC genes of Gordonia amicalis F.5.25.8 and identification of conserved protein and DNA sequences

Gordonia amicalis F.5.25.8 has the unique ability to desulfurize dibenzothiophene and to metabolize carbazole [Santos et al., Appl Microbiol Biotechnol 71:355–362, 2006]. Efforts to amplify the dsz genes from G. amicalis F.5.25.8 based on polymerase chain reaction (PCR) primers designed using the dsz gene sequences of Rhodococcus erythropolis IGTS8 were mostly unsuccessful. A comparison of the protein sequences of dissimilar desulfurization enzymes (DszABC, BdsABC, and TdsABC) revealed multiple conserved regions. PCR primers targeting some of the most highly conserved regions of the desulfurization genes allowed us to amplify dsz genes from G. amicalis F.5.25.8. DNA sequence data that include nearly the entirety of the desulfurization operon as well as the promoter region were obtained. The most closely related dsz genes are those of G. alkinovorans strain 1B at 85% identity. The PCR primers reported here should be useful in microbial ecology studies and the amplification of desulfurization genes from previously uncharacterized microbial cultures.

Biodesulfurization: a model system for microbial physiology research

Biological desulfurization (biodesulfurization) of dibenzothiophene (DBT) by the 4S pathway is a model system for an enviromentally benign way to lower the sulfur content of petroleum. Despite a large amount of effort the efficiency of the 4S pathway is still too low for a commercial oil biodesulfurization process, but the 4S pathway could potentially be used now for commercial processes to produce surfactants, antibiotics, polythioesters and other chemicals and for the detoxification of some chemical warfare agents. Proteins containing disulfide bonds are resistant to temperature, pH, and solvents, but the production of disulfide-rich proteins in microbial hosts is challenging. The study of the 4S pathway can provide insights as to how to maximize the production of disulfide-rich proteins. Engineering of the operon encoding the 4S pathway to contain a greater content of methionine and cysteine may be able to link use of DBT as a sole sulfur source to increasing 4S pathway activity by increasing the nutritional demand for sulfur. This strategy could result in the development of biocatalysts suitable for use in an oil biodesulfurization process, but the study of the 4S pathway can also lead to a better understanding of microbial physiology to optimize activity of a mult-step co-factor-requiring pathway, as well as the production of highly stable industrially relevant enzymes for numerous applications.

Future Applications of Biotechnology to the Energy Industry

There has been a dramatic growth in the production of biofuels in recent times. Global biofuel production tripled between 2000 and 2007, and biofuels accounted for about 1.6% of global transportation fuel in 2012 (International Energy Agency). At the time of this writing, 2015, ethanol production is by far the greatest contribution by biotechnology to energy production, with revenue accounting for

Enhancement of Microbial Biodesulfurization via Genetic Engineering and Adaptive Evolution

40.9 billion worldwide in 2014 vs.

Roles of sulfite oxidoreductase and sulfite reductase in improving desulfurization by Rhodococcus erythropolis

3.8 billion for biodiesel and

Enhancement of desulfurization activity by enzymes of the Rhodococcus dsz operon through coexpression of a high sulfur peptide and directed evolution

0.019 billion for bio-methane. It is logical to imagine the future contribution of biotechnology to world energy production may increase not only in the area of biofuel production, but also in petroleum production, petroleum upgrading, biogas production, chemical production, crop improvement, bioremediation, microbiologically influenced corrosion, space travel, and other topics. However, the future contributions of biotechnology to the energy industry are not only influenced by technical advances in biotechnology, but also by the price of fossil fuels, the development of renewable energy generally, politics, global population growth, and other factors. Concerns about the use of crops for food versus fuel production, environmental effects of land use related to biofuel production, decreased oil prices, ever-increasing advances in the generation and use of wind and solar energy, and political will to promote/subsidize the development of alternative energy are also influencing factors.

Biodesulfurization: How to Make it Work?

In previous work from our laboratories a synthetic gene encoding a peptide (“Sulpeptide 1” or “S1”) with a high proportion of methionine and cysteine residues had been designed to act as a sulfur sink and was inserted into the dsz (desulfurization) operon of Rhodococcus erythropolis IGTS8. In the work described here this construct (dszAS1BC) and the intact dsz operon (dszABC) cloned into vector pRESX under control of the (Rhodococcus) kstD promoter were transformed into the desulfurization-negative strain CW25 of Rhodococcus qingshengii. The resulting strains (CW25[pRESX-dszABC] and CW25[pRESX-dszAS1BC]) were subjected to adaptive selection by repeated passages at log phase (up to 100 times) in minimal medium with dibenzothiophene (DBT) as sole sulfur source. For both strains DBT metabolism peaked early in the selection process and then decreased, eventually averaging four times that of the initial transformed cells; the maximum specific activity achieved by CW25[pRESX-dszAS1BC] exceeded that of CW25[pRESX-dszABC]. Growth rates increased by 7-fold (CW25[pRESX-dszABC]) and 13-fold (CW25[pRESX-dszAS1BC]) and these increases were stable. The adaptations of CW25[pRESX-dszAS1BC] were correlated with a 3-5X increase in plasmid copy numbers from those of the initial transformed cells; whole genome sequencing indicated that during its selection processes no mutations occurred to any of the dsz, S1, or other genes and promoters involved in sulfur metabolism, stress response, or DNA methylation, and that the effect of the sulfur sink produced by S1 is likely very small compared to the cells’ overall cysteine and methionine requirements. Nevertheless, a combination of genetic engineering using sulfur sinks and increasing Dsz capability with adaptive selection may be a viable strategy to increase biodesulfurization ability.