Suitability of soxhlet extraction to quantify microalgal fatty acids as determined by comparison with in situ transesterification

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Lipids 47 (2012): 195-207, doi:10.1007/s11745-011-3624-3.To assess Soxhlet extraction as a method for quantifying fatty acids (FA) of microalgae, crude lipid, FA content from Soxhlet extracts and FA content from in-situ transesterification (ISTE) were compared. In most cases, gravimetric lipid content was considerably greater (up to 7-fold) than the FA content of the crude lipid extract. FA content from Soxhlet lipid extraction and ISTE were similar in 12/18 samples, whereas in 6/18 samples, total FA content from Soxhlet extraction was less than the ISTE procedure. Re-extraction of residual biomass from Soxhlet extraction with ISTE liberated a quantity of FA equivalent to this discrepancy. Employing acid hydrolysis before Soxhlet extraction yielded FA content roughly equivalent to ISTE, indicating that acidic conditions of ISTE are responsible for this observed greater recovery of FA. While crude lipid derived from Soxhlet extraction was not a useful proxy for FA content for the species tested, it is effective in most strains at extracting total saponifiable lipid. Lipid class analysis showed the source of FA was primarily polar lipids in most samples (12/18 lipid extracts contained 15%). This investigation confirms the usefulness of ISTE, reveals limitations of gravimetric methods for projecting biodiesel potential of microalgae, and reinforces the need for intelligent screening using both FA and lipid class analysis.2012-11-0

Triacylglycerol profiling of microalgae strains for biofuel feedstock by liquid chromatography–high-resolution mass spectrometry

Biofuels from photosynthetic microalgae are quickly gaining interest as a viable carbon-neutral energy source. Typically, characterization of algal feedstock involves breaking down triacylglycerols (TAG) and other intact lipids, followed by derivatization of the fatty acids to fatty acid methyl esters prior to analysis by gas chromatography (GC). However, knowledge of the intact lipid profile could offer significant advantages for discovery stage biofuel research such as the selection of an algal strain or the optimization of growth and extraction conditions. Herein, lipid extracts from microalgae were directly analyzed by ultra-high pressure liquid chromatography–mass spectrometry (UHPLC-MS) using a benchtop Orbitrap mass spectrometer. Phospholipids, glycolipids, and TAGs were analyzed in the same chromatographic run, using a combination of accurate mass and diagnostic fragment ions for identification. Using this approach, greater than 100 unique TAGs were identified over the six algal strains studied and TAG profiles were obtained to assess their potential for biofuel applications. Under the growth conditions employed, Botryococcus braunii and Scenedesmus obliquus yielded the most comprehensive TAG profile with a high abundance of TAGs containing oleic acid

Genus-specific carbon fixation activity measurements reveal distinct responses to oxygen among hydrothermal vent campylobacteria

Author Posting. © American Society for Microbiology, 2022. This article is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 88(2),(2022): e02083-21, https://doi.org/10.1128/AEM.02083-21.Molecular surveys of low temperature deep-sea hydrothermal vent fluids have shown that Campylobacteria (previously Epsilonproteobacteria) often dominate the microbial community and that three genera, Arcobacter, Sulfurimonas, and Sulfurovum, frequently coexist. In this study, we used replicated radiocarbon incubations of deep-sea hydrothermal fluids to investigate activity of each genus under three experimental conditions. To quantify genus-specific radiocarbon incorporation, we used newly designed oligonucleotide probes for Arcobacter, Sulfurimonas, and Sulfurovum to quantify their activity using catalyzed-reporter deposition fluorescence in situ hybridization (CARD-FISH) combined with fluorescence-activated cell sorting. All three genera actively fixed CO2 in short-term (∼ 20 h) incubations, but responded differently to the additions of nitrate and oxygen. Oxygen additions had the largest effect on community composition, and caused a pronounced shift in community composition at the amplicon sequence variant (ASV) level after only 20 h of incubation. The effect of oxygen on carbon fixation rates appeared to depend on the initial starting community. The presented results support the hypothesis that these chemoautotrophic genera possess functionally redundant core metabolic capabilities, but also reveal finer-scale differences in growth likely reflecting adaptation of physiologically-distinct phylotypes to varying oxygen concentrations in situ. Overall, our study provides new insights into how oxygen controls community composition and total chemoautotrophic activity, and underscores how quickly deep-sea vent microbial communities respond to disturbances.This research was funded by the U.S. National Science Foundation grants OCE-1131095 (S.M.S.) and OCE-1136727 (S.M.S., J.S.S.). Further support was provided by the WHOI Investment in Science Fund (S.M.S.). Funding for J.M. was further provided by doctoral fellowships from the Natural Sciences and Engineering Research Council of Canada (PGSD3-430487-2013, PGSM-405117-2011) and the National Aeronautics and Space Administration Earth Systems Science Fellowship (PLANET14F-0075), an award from the Canadian Meteorological and Oceanographic Society, and the WHOI Academic Programs Office

Primary productivity below the seafloor at deep-sea hot springs

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Proceedings of the National Academy of Sciences.of the United States of America 115 (2018): 6756–6761, doi:10.1073/pnas.1804351115.The existence of a chemosynthetic subseafloor biosphere was immediately recognized when deep-sea hot springs were discovered in 1977. However, quantifying how much new carbon is fixed in this environment has remained elusive. In this study, we incubated natural subseafloor communities under in situ pressure/temperature and measured their chemosynthetic growth efficiency and metabolic rates. Combining these data with fluid flux and in situ chemical measurements, we derived empirical constraints on chemosynthetic activity in the natural environment. Our study shows subseafloor microorganisms are highly productive (up to 1.4 Tg C produced yearly), fast-growing (turning over every 17–41 hours), and physiologically diverse. These estimates place deep-sea hot springs in a quantitative framework and allow us to assess their importance for global biogeochemical cycles.This research was funded by a grant of the Dimensions of Biodiversity program of the US National Science Foundation (NSF-OCE-1136727 to S.M.S. and J.S.S.). Funding for J.M. was further provided by doctoral fellowships from the Natural Sciences and Engineering Research Council of Canada (PGSD3-430487-2013, PGSM-405117-2011) and the National Aeronautics and Space Administration Earth Systems Science Fellowship (PLANET14F-0075), an award from the Canadian Meteorological and Oceanographic Society, and the WHOI Academic Programs Office

Targeted metabolomics reveals proline as a major osmolyte in the chemolithoautotroph Sulfurimonas denitrificans

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in MicrobiologyOpen 7 (2018): e00586, doi:10.1002/mbo3.586.Chemoautotrophic bacteria belonging to the genus Sulfurimonas in the class Campylobacteria are widespread in many marine environments characterized by redox interfaces, yet little is known about their physiological adaptations to different environmental conditions. Here, we used liquid chromatography coupled with tandem mass spectrometry (LC-MS/ MS) in a targeted metabolomics approach to study the adaptations of Sulfurimonas denitrificans to varying salt concentrations that are found in its natural habitat of tidal mudflats. Proline was identified as one of the most abundant internal metabolites and its concentration showed a strong positive correlation with ionic strength, suggesting that it acts as an important osmolyte in S. denitrificans. 2,3-dihydroxypropane- 1- sulfonate was also positively correlated with ionic strength, indicating it might play a previously unrecognized role in osmoregulation. Furthermore, the detection of metabolites from the reductive tricarboxylic acid cycle at high internal concentrations reinforces the importance of this pathway for carbon fixation in Campylobacteria and as a hub for biosynthesis. As the first report of metabolomic data for an campylobacterial chemolithoautotroph, this study provides data that will be useful to understand the adaptations of Campylobacteria to their natural habitat at redox interfaces.NSF, Grant/Award Number: OCE- 1136727 and OCE-1154320; Woods Hole Oceanographic Institution; U.S. Geological Surve

Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Labonte, J. M., Pachiadaki, M., Fergusson, E., McNichol, J., Grosche, A., Gulmann, L. K., Vetriani, C., Sievert, S. M., & Stepanauskas, R. Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system. Frontiers in Microbiology, 10, (2019): 1262, doi:10.3389/fmicb.2019.01262.Phage–host interactions likely play a major role in the composition and functioning of many microbiomes, yet remain poorly understood. Here, we employed single cell genomics to investigate phage–host interactions in a diffuse-flow, low-temperature hydrothermal vent that may be reflective of a broadly distributed biosphere in the subseafloor. We identified putative prophages in 13 of 126 sequenced single amplified genomes (SAGs), with no evidence for lytic infections, which is in stark contrast to findings in the surface ocean. Most were distantly related to known prophages, while their hosts included bacterial phyla Campylobacterota, Bacteroidetes, Chlorobi, Proteobacteria, Lentisphaerae, Spirochaetes, and Thermotogae. Our results suggest the predominance of lysogeny over lytic interaction in diffuse-flow, deep-sea hydrothermal vents, despite the high activity of the dominant Campylobacteria that would favor lytic infections. We show that some of the identified lysogens have co-evolved with their host over geological time scales and that their genes are transcribed in the environment. Functional annotations of lysogeny-related genes suggest involvement in horizontal gene transfer enabling host’s protection against toxic metals and antibacterial compounds.This work was supported by the U.S. National Science Foundation’s Dimensions of Biodiversity Program [OCE-1136488 (to RS), OCE-1136727 (to SMS) and OCE-1136451 (to CV)], as well as DEB-1441717 and OCE-1335810 (to RS), and the DOE JGI CSP project 1477