A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae.

A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors.

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2-2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD(D485G) variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad(D485G)) ABE products resulted in a blend with nearly 50wt%≤C9 products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9gL(-1) while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80wt% of catalysis products having a carbon chain length≥C11 which amounts to 9.8gL(-1) of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production.

Candida keroseneae sp. nov., a novel contaminant of aviation kerosene.

AIMS: To characterize and identify a novel contaminant of aviation fuel. METHODS AND RESULTS: Micro-organisms (yeasts and bacteria) were isolated from samples of aviation fuel. A yeast that proved to have been unrecorded previously was isolated from more than one fuel sample. This novel yeast proved to be a new species of Candida and is described here. Ribosomal RNA gene sequence analyses of internal transcribed spacer (ITS) regions (including 5·8S subunit) plus the 26S D1/D2 domains showed the strains to cluster within the Candida membranifaciens clade nearest to, but distinct from, Candida tumulicola. Phenotypic tests were identical for both isolates. Physiological and biochemical tests supported their position as a separate taxon. The yeast was assessed for its effect on the main constituent hydrocarbons of aviation fuel. CONCLUSIONS: Two strains (IMI 395605(T) and IMI 395606) belonging to the novel yeast species, Candida keroseneae, were isolated from samples of aircraft fuel (kerosene), characterized and described herein with reference to their potential as contaminants of aviation fuel. SIGNIFICANCE AND IMPACT OF THE STUDY: As a result of isolating a novel yeast from aviation fuel, the implications for microbial contamination of such fuel should be considered more widely than previously thought.

Evaluación de la actividad desulfurizadora de aislados nativos de Pseudomonas spp. en presencia de hidrocarburo / Desulfurization activity evaluation of native strains of Pseudomonas spp. in the presence of hydrocarbon

El principal inconveniente en la combustión de los hidrocarburos es la conversión del azufre y el nitrógeno a sus respectivos óxidos, los cuales participan en la formación de lluvia acida y deterioran el medio ambiente e infraestructuras. La remoción de azufre a partir de compuestos órgano-azufrados mediante el uso de microorganismos ha surgido como una alternativa frente al proceso catalítico de hidrodesulfurización (HDS). En el presente trabajo se evaluó la actividad desulfurizadora de veintitrés aislados nativos de Pseudomonas spp. sobre dibenzotiofeno (DBT), usando un sistema de fermentación con igual proporción de fase acuosa y orgánica (n-hexano) en presencia de oleato de etanolamina. Los aislados 02,05 y 06 conservaron su viabilidad en este medio y presentaron una remoción de azufre entre 6,0 y 9,4%, generando los metabolitos DBT-sulfona, DBT-sulfóxido, 2-hidroxibifenilo (2-HBP) y sulfato presentes en la ruta metabólica 4S. Con estos aislados se evaluó la actividad desulfurizadora sobre keroseno y se observó una remoción de azufre entre 19,9 y 62,6% y una disminución del poder calorífico entre 0,45 y 5,55%.

The main difficulty with fossil fuel combustión lies in sulphur and nitrogen becoming converted to their respective oxides, forming part of the acid rain which deteriorates the environment and infrastructure. Removing sulphur from organo-sulfur compounds by using micro-organisms has become an alternative to hydrodesulphurisation (HDS). Twenty-three Pseudomonas spp. native strains' desulphurisation activity on dibenzothiophene (DBT) was evaluated by using a fermentation system having equal proportions of aqueous and organic (n-hexane) phases in the presence of ethanolamine oléate. The 02, 05 and 06 strains maintained their viability in this médium, presenting 6,0% to 9,4% sulphur removal, producing DBT-sulphone, DBT-sulphoxide, 2-hydroxybiphenyl (2-HBP) metabolites and sulphate belonging to the 4S pathway. These native strains' desulphurisation activity was evaluated on kerosene, presenting 19,9% to 62,2% sulphur removal having 0,45% to 5,55% calorific power loss.

Characterization of JP-7 jet fuel degradation by the bacterium Nocardioides luteus strain BAFB.

In the fall of 1996, numerous bacteria capable of degrading JP-7 jet fuel were isolated from soil collected at Beale Air Force Base in northern California. The most prevalent organism, identified as Nocardioides luteus by16s rRNA sequencing (MIDI Labs, Inc.), was selected for further analysis. Analysis of JP-7 following inoculation with N. luteus demonstrated degradation of the C(11) alkane component of the fuel. Growth rates of N. luteus were determined with alkanes of various lengths as the sole carbon and energy source. The organism grew best on shorter length alkanes (C(8) and C(10)). Growth was measurably slower on C(11), and minimal on C(12), C(13), and C(14).