Quantitative Bioreactor Monitoring of Intracellular Bacterial Metabolites in Clostridium autoethanogenum Using Liquid Chromatography–Isotope Dilution Mass Spectrometry

We report a liquid chromatography–isotope dilution mass spectrometry method for the simultaneous quantification of 131 intracellular bacterial metabolites of Clostridium autoethanogenum. A comprehensive mixture of uniformly 13C-labeled internal standards (U-13C IS) was biosynthesized from the closely related bacterium Clostridium pasteurianum using 4% 13C–glucose as a carbon source. The U-13C IS mixture combined with 12C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite. A robust-fitting algorithm was employed to reduce the weight of the outliers on the quantification data. The metabolite calibration curves were linear with R2 ≥ 0.99, limits of detection were ≤1.0 μM, limits of quantification were ≤10 μM, and precision/accuracy was within RSDs of 15% for all metabolites. The method was subsequently applied for the daily monitoring of the intracellular metabolites of C. autoethanogenum during a CO gas fermentation over 40 days as part of a study to optimize biofuel production. The concentrations of the metabolites were estimated at steady states of different pH levels using the robust-fitting mathematical approach, and we demonstrate improved accuracy of results compared to conventional regression. Metabolic pathway analysis showed that reactions of the incomplete (branched) tricarboxylic acid “cycle” were the most affected pathways associated with the pH shift in the bioreactor fermentation of C. autoethanogenum and the concomitant changes in ethanol production

First Documented Attack on a Live Human by a Cookiecutter Shark (Squaliformes, Dalatiidae: Isistius sp.)

v. ill. 23 cm.Also available through BioOne: http://www.bioone.org/doi/abs/10.2984/65.3.365QuarterlyAn adult long-distance swimmer attempting to cross the ‘Alenuihähä Channel between the Hawaiian islands of Hawai‘i and Maui was twice bitten by a cookiecutter shark (Squaliformes, Dalatiidae, Isistius sp.). One of these bites presented as an open, round, concave wound typically observed in cookiecutter shark bites inflicted by members of this genus on a broad spectrum of large biota such as marine mammals, elasmobranchs, and bony fishes. The open wound was debrided, subjected to negative pressure wound therapy, and a split thickness skin graft harvested from the left thigh. Postoperative recovery was complicated by delayed healing of the inferior portion of the graft, and cultures and biopsy were normal skin flora and normal tissue, respectively. At 6 months after the incident, the area appeared to be healing with a stable eschar, and by 9 months the wound was healed. Humans entering pelagic waters at twilight and nighttime hours in areas of Isistius sp. occurrence should do so knowing that cookiecutter sharks are a potential danger, particularly during periods of strong moonlight, in areas of man-made illumination, or in the presence of bioluminescent organisms