GC-MS hydrocarbon degradation profile data of Pseudomonas frederiksbergensis SI8, a bacterium capable of degrading aromatics at low temperatures.

The ability of the psychrotrophic bacterium Pseudomonas frederiksbergensis SI8 to grow and degrade aromatic hydrocarbons efficiently at low temperature is shown in this study. The robust growth of P. frederiksbergensis SI8 was demonstrated in jet fuel and an aromatic blend. The bacterium showed 2.5 to 3-fold faster growth in the aromatic blend than in jet fuel. The hydrocarbons degradation profile of P. frederiksbergensis SI8 at ambient temperature (i.e., 28 °C) and low temperature (i.e., 4 °C) was characterized by Gas Chromatography-Mass Spectrometry (GC-MS) analysis. GC-MS data demonstrated that P. frederiksbergensis SI8 is a novel psychrotrophic bacterium with the ability to degrade aromatic hydrocarbons at temperatures as low as 4 °C. Specifically, P. frederiksbergensis SI8 consumed toluene, ethylbenzene, n-propylbenzene and methyl ethyl benzene efficiently. The data presented here serves to characterize the hydrocarbon degradation profile of P. frederiksbergensis SI8 and corroborates the capacity of this bacterium to degrade aromatic hydrocarbons at low temperatures. The raw GC-MS data for the degradation of hydrocarbons by P. frederiksbergensis SI8 grown at 4 °C and 28 °C for 14 days have been deposited in Mendeley Data and can be retrieved from https://dx.doi.org/10.17632/z9292bvdmh.1 and https://dx.doi.org/10.17632/dp3sgwpj23.1. The datasets and raw data presented here were associated with the main research work "Metagenomic characterization reveals complex association of soil hydrocarbon-degrading bacteria" [1].

Toxicity and human health assessment of an alcohol-to-jet (ATJ) synthetic kerosene.

A toxicological investigation was conducted for alcohol-to-jet (ATJ) fuels intended as a 50:50 blend with petroleum-derived fuel Jet Propulsion (JP)-8. The ATJ synthetic paraffinic kerosene (SPK) fuel was produced by Gevo (Englewood CO) and derived either from biomass (bio) or non-biomass sources. All toxicity tests were performed with one or both ATJ fuels following addition of a standard additive package required for JP-8. The primary fuel, Gevo (bio) ATJ SPK produced from biomass-derived iso-butanol, exhibited the same dermal irritation potential in rabbits as JP-8; the non-biomass-derived fuel was less irritating. The Gevo (bio) fuel was non-clastogenic in micronucleus testing with rats and neither version was mutagenic in the bacterial reverse mutation assay. A 90-day study was performed with Gevo (bio) ATJ SPK by exposing male and female Fischer 344 rats to target concentrations of 0, 200, 700 or 2000 mg/m3 of fuel, 6 hr per day, 5 days a week for 69 exposure days and included neurobehavioral assays and reproductive health evaluations in the study design. Results were negative or limited to irritant effects in the respiratory system due to exposure to a vapor and aerosol mixture in the 2000 mg/m3 exposure group. Occupational exposure limits for JP-8 were proposed for these ATJ fuels since these fuels display similar or somewhat lower toxicity than JP-8. As both versions of the Gevo ATJ jet fuel were similar, handling of either fuel alone or in a blend with petroleum-derived JP-8 appears unlikely to increase human health risks for workers.

Toxicity and occupational exposure assessment for hydroprocessed esters and fatty acids (HEFA) alternative jet fuels.

The U.S. Air Force (USAF) has pursued development of alternative fuels to augment or replace petroleum-based jet fuels. Hydroprocessed esters and fatty acids (HEFA) renewable jet fuel is certified for use in commercial and USAF aircraft. HEFA feedstocks include camelina seed oil (Camelina sativa, HEFA-C); rendered animal fat (tallow, HEFA-T); and mixed fats and oils (HEFA-F). The aim of this study was to examine potential toxic effects associated with HEFA fuels exposures. All 3 HEFA fuels were less dermally irritating to rabbits than petroleum-derived JP-8 currently in use. Inhalation studies using male and female Fischer-344 rats included acute (1 day, with and without an 11-day recovery), 5-, 10- or 90-day durations. Rats were exposed to 0, 200, 700 or 2000 mg/m3 HEFA-F (6 hr/day, 5 days/week). Acute, 5 - and 10-day responses included minor urinalysis effects. Kidney weight increases might be attributed to male rat specific hyaline droplet formation. Nasal cavity changes included olfactory epithelial degeneration at 2000 mg/m3. Alveolar inflammation was observed at ≥700 mg/m3. For the 90-day study using HEFA-C, no significant neurobehavioral effects were detected. Minimal histopathological effects at 2000 mg/m3 included nasal epithelium goblet cell hyperplasia and olfactory epithelium degeneration. A concurrent micronucleus test was negative for evidence of genotoxicity. All HEFA fuels were negative for mutagenicity (Ames test). Sensory irritation (RD50) values were determined to be 9578 mg/m3 for HEFA-C and greater than 10,000 mg/m3 for HEFA-T and HEFA-F in male Swiss-Webster mice. Overall, HEFA jet fuel was less toxic than JP-8. Occupational exposure levels of 200 mg/m3 for vapor and 5 mg/m3 for aerosol are recommended for HEFA-based jet fuels.

A comprehensive multi-omics approach uncovers adaptations for growth and survival of Pseudomonas aeruginosa on n-alkanes.

BACKGROUND: Examination of complex biological systems has long been achieved through methodical investigation of the system's individual components. While informative, this strategy often leads to inappropriate conclusions about the system as a whole. With the advent of high-throughput "omic" technologies, however, researchers can now simultaneously analyze an entire system at the level of molecule (DNA, RNA, protein, metabolite) and process (transcription, translation, enzyme catalysis). This strategy reduces the likelihood of improper conclusions, provides a framework for elucidation of genotype-phenotype relationships, and brings finer resolution to comparative genomic experiments. Here, we apply a multi-omic approach to analyze the gene expression profiles of two closely related Pseudomonas aeruginosa strains grown in n-alkanes or glycerol. RESULTS: The environmental P. aeruginosa isolate ATCC 33988 consumed medium-length (C10-C16) n-alkanes more rapidly than the laboratory strain PAO1, despite high genome sequence identity (average nucleotide identity >99%). Our data shows that ATCC 33988 induces a characteristic set of genes at the transcriptional, translational and post-translational levels during growth on alkanes, many of which differ from those expressed by PAO1. Of particular interest was the lack of expression from the rhl operon of the quorum sensing (QS) system, resulting in no measurable rhamnolipid production by ATCC 33988. Further examination showed that ATCC 33988 lacked the entire lasI/lasR arm of the QS response. Instead of promoting expression of QS genes, ATCC 33988 up-regulates a small subset of its genome, including operons responsible for specific alkaline proteases and sphingosine metabolism. CONCLUSION: This work represents the first time results from RNA-seq, microarray, ribosome footprinting, proteomics, and small molecule LC-MS experiments have been integrated to compare gene expression in bacteria. Together, these data provide insights as to why strain ATCC 33988 is better adapted for growth and survival on n-alkanes.

Transcriptomic Analyses Elucidate Adaptive Differences of Closely Related Strains of Pseudomonas aeruginosa in Fuel.

Pseudomonas aeruginosa can utilize hydrocarbons, but different strains have various degrees of adaptation despite their highly conserved genome. P. aeruginosa ATCC 33988 is highly adapted to hydrocarbons, while P. aeruginosa strain PAO1, a human pathogen, is less adapted and degrades jet fuel at a lower rate than does ATCC 33988. We investigated fuel-specific transcriptomic differences between these strains in order to ascertain the underlying mechanisms utilized by the adapted strain to proliferate in fuel. During growth in fuel, the genes related to alkane degradation, heat shock response, membrane proteins, efflux pumps, and several novel genes were upregulated in ATCC 33988. Overexpression of alk genes in PAO1 provided some improvement in growth, but it was not as robust as that of ATCC 33988, suggesting the role of other genes in adaptation. Expression of the function unknown gene PA5359 from ATCC 33988 in PAO1 increased the growth in fuel. Bioinformatic analysis revealed that PA5359 is a predicted lipoprotein with a conserved Yx(FWY)xxD motif, which is shared among bacterial adhesins. Overexpression of the putative resistance-nodulation-division (RND) efflux pump PA3521 to PA3523 increased the growth of the ATCC 33988 strain, suggesting a possible role in fuel tolerance. Interestingly, the PAO1 strain cannot utilize n-C8 and n-C10 The expression of green fluorescent protein (GFP) under the control of alkB promoters confirmed that alk gene promoter polymorphism affects the expression of alk genes. Promoter fusion assays further confirmed that the regulation of alk genes was different in the two strains. Protein sequence analysis showed low amino acid differences for many of the upregulated genes, further supporting transcriptional control as the main mechanism for enhanced adaptation.IMPORTANCE These results support that specific signal transduction, gene regulation, and coordination of multiple biological responses are required to improve the survival, growth, and metabolism of fuel in adapted strains. This study provides new insight into the mechanistic differences between strains and helpful information that may be applied in the improvement of bacterial strains for resistance to biotic and abiotic factors encountered during bioremediation and industrial biotechnological processes.

Draft Genome Sequence of Gordonia sihwensis Strain 9, a Branched Alkane-Degrading Bacterium.

Gordonia sihwensis strain 9 is a Gram-positive bacterium capable of efficient aerobic degradation of branched and normal alkanes. The draft genome of G. sihwensis S9 is 4.16 Mb in size, with 3,686 coding sequences and 68.1% G+C content. Alkane monooxygenase and P-450 cytochrome genes required for alkane degradation are predicted in G. sihwensis S9.

Draft Genome Sequence of Pseudomonas frederiksbergensis SI8, a Psychrotrophic Aromatic-Degrading Bacterium.

Pseudomonas frederiksbergensis strain SI8 is a psychrotrophic bacterium capable of efficient aerobic degradation of aromatic hydrocarbons. The draft genome of P. frederiksbergensis SI8 is 6.57 Mb in size, with 5,904 coding sequences and 60.5% G+C content. The isopropylbenzene (cumene) degradation pathway is predicted to be present in P. frederiksbergensis SI8.

Transcriptional profiling suggests that multiple metabolic adaptations are required for effective proliferation of Pseudomonas aeruginosa in jet fuel.

Fuel is a harsh environment for microbial growth. However, some bacteria can grow well due to their adaptive mechanisms. Our goal was to characterize the adaptations required for Pseudomonas aeruginosa proliferation in fuel. We have used DNA-microarrays and RT-PCR to characterize the transcriptional response of P. aeruginosa to fuel. Transcriptomics revealed that genes essential for medium- and long-chain n-alkane degradation including alkB1 and alkB2 were transcriptionally induced. Gas chromatography confirmed that P. aeruginosa possesses pathways to degrade different length n-alkanes, favoring the use of n-C11-18. Furthermore, a gamut of synergistic metabolic pathways, including porins, efflux pumps, biofilm formation, and iron transport, were transcriptionally regulated. Bioassays confirmed that efflux pumps and biofilm formation were required for growth in jet fuel. Furthermore, cell homeostasis appeared to be carefully maintained by the regulation of porins and efflux pumps. The Mex RND efflux pumps were required for fuel tolerance; blockage of these pumps precluded growth in fuel. This study provides a global understanding of the multiple metabolic adaptations required by bacteria for survival and proliferation in fuel-containing environments. This information can be applied to improve the fuel bioremediation properties of bacteria.

Rapid determination of total sulfur in fuels using gas chromatography with atomic emission detection.

The purpose of this study is to determine whether gas chromatography (GC)-atomic emission detection (AED) can be used in a low-resolution mode for rapid, accurate determinations of total sulfur in fuels at trace levels to complement other popular methods of total sulfur analysis. A method for the rapid determination of total sulfur in fuels (called "fast GC-AED") is developed. The method is tested on gasoline, jet fuel, kerosene, and diesel fuel with sulfur concentrations ranging from 125 mg/L down to 2.5 mg/L. Fast GC-AED shows better performance than traditional GC-AED for total sulfur determinations, especially for complex mixtures containing many different sulfur-containing compounds at trace levels. This method also shows that GC-AED can be used for both rapid determinations of total sulfur and traditional determinations of speciated sulfur without requiring equipment changes. Fast GC-AED is competitive with other popular methods for sulfur analysis. The 5-min program that is developed for fast GC-AED is comparable with the time scale of other methods, such as wavelength dispersive X-ray fluorescence and UV-fluorescence (2 to 5 min). Fast GC-AED also compares favorably with UV-fluorescence for trace sulfur determinations, demonstrating accuracy down to 2.5-mg/L sulfur.

Trace-level measurement of complex combustion effluents and residues using multidimensional gas chromatography-mass spectrometry (MDGC-MS).

The identification and quantitation of non-method-specific target analytes have greater importance with respect to EPA's current combustion strategy. The risk associated with combustion process emissions must now be characterized. EPA has recently released draft guidance on procedures for the collection of emissions data to support and augment site-specific risk assessments (SSRAs) as part of the hazardous waste incineration permitting process. This guidance includes methodology for quantifying total organic (TO) emissions as a function of compound volatility. The ultimate intent is to compare the amount of organic material identified and quantified by target analyte-specific methodologies to organic emissions quantified by the TO methodology. The greater the amount accounted for by the target analyte-specific methodologies, the less uncertainty may be associated with the SSRAs. A limitation of this approach is that the target analyte-specific methodologies do not routinely quantify compounds of low toxicological interest; nor do they target products of incomplete combustion (PICs). Thus, the analysis can miss both toxic and non-toxic compounds. As a result, it is unknown whether the uncharacterized fraction of the TO emission possesses toxic properties. The hypothesis that we propose to test is that organic emissions and organics extracted from particulate matter (PM) are more complex than standard GC-MS-based instrumentation can currently measure. This complexity can affect quantitation for toxic compounds, thereby potentially affecting risk assessments. There is a pressing need to better characterize these organic emissions from hazardous waste incinerators and PM extracts from various other combustion sources. We will demonstrate that multidimensional gas chromatography-mass spectrometry (MDGC-MS) procedures significantly improve chromatographic separation for complex environmental samples. Sequential repetitive heart-cutting MDGC, with coupled mass spectrometry will be shown to be a complete analysis technique. The ability of this technique to disengage components from complex mixtures taken from hazardous and municipal waste incinerators will be shown.