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Rapid detection of microbial contamination in conventional and alternative fuels is hampered by the lack of sensitive and cost-effective assays to detect total and specific microorganisms in fuels. Here, we report a simple and highly sensitive TaqMan quantitative polymerase chain reaction (qPCR) assay for universal detection and quantification of fungi, bacteria, and archaea in fuel in a single multiplexed reaction. Universal primers and probes targeting conserved regions of the 16S and 18S rRNA genes were designed and validated for specific amplification of total fungi, bacteria, and archaea in fuel. The assay is able to detect as low as 10 pg of fungal and bacterial DNA. The combination of a simple liquid–liquid extraction to recover cells from fuel with a freeze-and-heat method to release DNA for direct qPCR amplification eliminates the need for DNA extractions from contaminated samples, thus making the assay much faster, inexpensive, and less laborious. The universal microbial multiplex qPCR assay demonstrated a high capacity to detect and quantify a wide range of microbial contaminants. The assay was validated to accurately detect and quantify the intended microorganisms in the presence of high levels of nontarget DNA and in fuel from field samples. This robust microbial qPCR assay can be applied to microbial detection in environmental and industrial settings to facilitate risk assessment and mitigation of microbial contamination.

To reduce the impact of biodeterioration in fuel systems, effective methods for early detection and monitoring of microbial growth in the fuel are required. This study presents the development of broad-range peptide biorecognition elements (BREs) that target cell surface determinants produced by hydrocarbon-degrading microorganisms during growth in fuel. BREs were used to biofunctionalize fluorescent semiconductor particles (quantum dots, QDs) to produce a sensitive quantitative fluorescence-based assay for detection of microbial growth in fuel. A biopanning method in the presence of fuel was used to select phage-displayed heptameric peptide BREs against epitopes conserved in fuel-degrading Gram-negative bacteria including Pseudomonasspp. and Acinetobacterspp. Fluorescence microscopy analysis and fluorescence signal measurements relative to colony-forming units (CFUs) demonstrated the binding and specificity of the BRE-QDs for fuel-degrading Gram-negative bacteria. Cross-reactivity with Gram-positive bacteria Arthrobacterand Lysinibacilluswas not observed. The assay was shown to be specific for detection of Gram-negative bacteria. Jet fuel samples amended with different concentrations of fuel-degrading bacteria were used to determine the sensitivity and limit of detection (LOD) of the assay; an LOD of 5 × 104colony forming units (CFUs) with detection levels as low as 5 × 103CFUs was established for the best performing BRE–QD conjugate. The peptide BRE–QD chemistry effectively detected biocontaminated fuel samples from fuel tanks. The peptide BREs may serve to biofunctionalize various fluorescent, chemiluminescent, and colorimetric molecules as well as optical and electrical transducers to developed effective biosensors for detection of microbial contamination in fuel.

There have been multiple conflicting reports about the biocompatibility and antimicrobial activity of graphene oxide. To address this, we conducted a study to characterize the antimicrobial properties of graphene oxide (GO) and its biocompatibility with mammalian cells. When GO was added to a bacterial culture at 25 μg/mL, the results showed that bacteria grew faster and to a higher optical density than cultures without GO. Scanning electron microscopy indicated that bacteria formed dense biofilms in the presence of GO. This was shown by a large mass of aggregated cells and extracellular polymeric material. Bacterial growth on filters coated with 25 and 75 μg of GO grew 2 and 3 times better than on filters without GO. Closer analysis showed that bacteria were able to attach and proliferate preferentially in areas containing the highest GO levels. Graphene oxide films failed to produce growth inhibition zones around them, indicating a lack of antibacterial properties. Also, bacteria were able to grow on GO films to 9.5 × 109cells from an initial inoculation of 1.0 × 106, indicating that it also lacks bacteriostatic activity. Thus, silver-coated GO films were able to produce clearing zones and cell death. Also, graphene oxide was shown to greatly enhance the attachment and proliferation of mammalian cells. This study conclusively demonstrates that graphene oxide does not have intrinsic antibacterial, bacteriostatic, and cytotoxic properties in both bacteria and mammalian cells. Furthermore, graphene oxide acts as a general enhancer of cellular growth by increasing cell attachment and proliferation.