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Universal primers for SSU rRNA genes allow profiling of natural communities by simultaneously amplifying templates from Bacteria, Archaea, and Eukaryota in a single PCR reaction. Despite the potential to show relative abundance for all rRNA genes, universal primers are rarely used, due to various concerns including amplicon length variation and its effect on bioinformatic pipelines. We thus developed 16S and 18S rRNA mock communities and a bioinformatic pipeline to validate this approach. Using these mocks, we show that universal primers (515Y/926R) outperformed eukaryote-specific V4 primers in observed versus expected abundance correlations (slope = 0.88 vs. 0.67–0.79), and mock community members with single mismatches to the primer were strongly underestimated (threefold to eightfold). Using field samples, both primers yielded similar 18S beta-diversity patterns (Mantel test, p < 0.001) but differences in relative proportions of many rarer taxa. To test for length biases, we mixed mock communities (16S + 18S) before PCR and found a twofold underestimation of 18S sequences due to sequencing bias. Correcting for the twofold underestimation, we estimate that, in Southern California field samples (1.2–80 μm), there were averages of 35% 18S, 28% chloroplast 16S, and 37% prokaryote 16S rRNA genes. These data demonstrate the potential for universal primers to generate comprehensive microbiome profiles.

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 (similar to 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. IMPORTANCE Sulfidic environments worldwide are often dominated by sulfur-oxidizing, carbon-fixing Campylobacteria. Environmental factors associated with this group's dominance are now understood, but far less is known about the ecology and physiology of members of subgroups of chemoautotrophic Campylobacteria. In this study, we used a novel method to differentiate the genus-specific chemoautotrophic activity of three subtypes of Campylobacteria. In combination with evidence from microscopic counts, chemical consumption/production during incubations, and DNA-based measurements, our data show that oxygen concentration affects both community composition and chemoautotrophic function in situ. These results help us better understand factors controlling microbial diversity at deep-sea hydrothermal vents, and provide first-order insights into the ecophysiological differences between these distinct microbial taxa.

Marine intertidal sediments fluctuate in redox conditions and nutrient availability, and they are also known as an important sink of nitrogen mainly through denitrification, yet how denitrifying bacteria adapt to this dynamic habitat remains largely untapped. Here, we investigated novel intertidal benthic ecotypes of the model pelagic marine bacterium Ruegeria pomeroyi DSS-3 with a population genomic approach. While differing by only 1.3% at the 16S rRNA gene level, members of the intertidal benthic ecotypes are complete denitrifiers whereas the pelagic ecotype representative (DSS-3) is a partial denitrifier lacking a nitrate reductase. The intertidal benthic ecotypes are further differentiated by using non-homologous nitrate reductases and a different set of genes that allow alleviating oxidative stress and acquiring organic substrates. In the presence of nitrate, the two ecotypes showed contrasting growth patterns under initial oxygen concentrations at 1 vol% versus 7 vol% and supplemented with different carbon sources abundant in intertidal sediments. Collectively, this combination of evidence indicates that there are cryptic niches in coastal intertidal sediments that support divergent evolution of denitrifying bacteria. This knowledge will in turn help understand how these benthic environments operate to effectively remove nitrogen.

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 CO(2) 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. IMPORTANCE Sulfidic environments worldwide are often dominated by sulfur-oxidizing, carbon-fixing Campylobacteria. Environmental factors associated with this group’s dominance are now understood, but far less is known about the ecology and physiology of members of subgroups of chemoautotrophic Campylobacteria. In this study, we used a novel method to differentiate the genus-specific chemoautotrophic activity of three subtypes of Campylobacteria. In combination with evidence from microscopic counts, chemical consumption/production during incubations, and DNA-based measurements, our data show that oxygen concentration affects both community composition and chemoautotrophic function in situ. These results help us better understand factors controlling microbial diversity at deep-sea hydrothermal vents, and provide first-order insights into the ecophysiological differences between these distinct microbial taxa.

Small subunit rRNA (SSU rRNA) amplicon sequencing can quantitatively and comprehensively profile natural microbiomes, representing a critically important tool for studying diverse global ecosystems. However, results will only be accurate if PCR primers perfectly match the rRNA of all organisms present. To evaluate how well marine microorganisms across all 3 domains are detected by this method, we compared commonly used primers with >300 million rRNA gene sequences retrieved from globally distributed marine metagenomes. The best-performing primers compared to 16S rRNA of bacteria and archaea were 515Y/926R and 515Y/806RB, which perfectly matched over 96% of all sequences. Considering cyanobacterial and chloroplast 16S rRNA, 515Y/926R had the highest coverage (99%), making this set ideal for quantifying marine primary producers. For eukaryotic 18S rRNA sequences, 515Y/926R also performed best (88%), followed by V4R/V4RB (18S rRNA specific; 82%)—demonstrating that the 515Y/926R combination performs best overall for all 3 domains. Using Atlantic and Pacific Ocean samples, we demonstrate high correspondence between 515Y/926R amplicon abundances (generated for this study) and metagenomic 16S rRNA (median R2 = 0.98, n = 272), indicating amplicons can produce equally accurate community composition data compared with shotgun metagenomics. Our analysis also revealed that expected performance of all primer sets could be improved with minor modifications, pointing toward a nearly completely universal primer set that could accurately quantify biogeochemically important taxa in ecosystems ranging from the deep sea to the surface. In addition, our reproducible bioinformatic workflow can guide microbiome researchers studying different ecosystems or human health to similarly improve existing primers and generate more accurate quantitative amplicon data. IMPORTANCE PCR amplification and sequencing of marker genes is a low-cost technique for monitoring prokaryotic and eukaryotic microbial communities across space and time but will work optimally only if environmental organisms match PCR primer sequences exactly. In this study, we evaluated how well primers match globally distributed short-read oceanic metagenomes. Our results demonstrate that primer sets vary widely in performance, and that at least for marine systems, rRNA amplicon data from some primers lack significant biases compared to metagenomes. We also show that it is theoretically possible to create a nearly universal primer set for diverse saline environments by defining a specific mixture of a few dozen oligonucleotides, and present a software pipeline that can guide rational design of primers for any environment with available meta’omic data.

Molecular surveys of low temperature deep-sea hydrothermal vent fluids have shown that Campylobacteria (prev. Epsilonproteobacteria) often dominate the microbial community and that three subgroups - Arcobacter, Sulfurimonas and Sulfurovum - frequently coexist. In this study, we used replicated radiocarbon incubations of deep-sea hydrothermal fluids to investigate the activities of each group under three distinct incubation conditions. In order to quantify group-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 groups actively fixed CO2 in short-term (~ 20 h) incubations with either nitrate, oxygen, or no additions (control) at similar per-cell carbon fixation rates. Oxygen additions had the largest effect on community composition and overall cell numbers, and caused a pronounced shift in community composition at the amplicon sequence variant (ASV) level after only 20 h of incubation for all three groups. Interestingly, the effect of oxygen on carbon fixation rates appeared to depend on the initial starting community. Higher carbon fixation rates in oxygen-amended treatments were noted for all three taxa after an unintended disturbance to the sample site that may have selected for more oxygen-tolerant phylotypes. When viewed from a coarse taxonomic level, our data support assertions that these chemoautotrophic groups are functionally redundant in terms of their core metabolic capabilities since they were simultaneously active under all incubation conditions. In contrast, the higher resolution of amplicon sequencing allowed us to reveal finer-scale differences in growth that likely reflect adaptation of physiologically-distinct subtypes to varying oxygen concentrations in situ. Despite this progress, we still know remarkably little about the factors that maintain genomic diversity and allow for stable co-existence among these three campylobacterial groups. Moving forward, we suggest that more subtle biological factors such as enzyme substrate specificity, motility, cell morphology, and tolerance to environmental stress should be more thoroughly investigated to better understand ecological niche differentiation at deep-sea hydrothermal vents.

Small subunit ribosomal RNA (SSU rRNA) amplicon sequencing can quantitatively and comprehensively profile natural microbiomes, representing a critically important tool for studying diverse global ecosystems. However, results will only be accurate if PCR primers perfectly match the rRNA of all organisms present. To evaluate how well marine microorganisms across all 3 domains are detected by this method, we compared commonly-used primers with > 300 million rRNA gene sequences retrieved from globally-distributed marine metagenomes. The best-performing primers when comparing to 16S rRNA of Bacteria and Archaea were 515Y/926R and 515Y/806RB, which perfectly matched over 96% of all sequences. Considering Cyanobacteria and Chloroplast 16S rRNA, 515Y/926R had the highest coverage (99%), making this set ideal for quantifying marine primary producers. For eukaryotic 18S rRNA sequences, 515Y/926R also performed best (88%), followed by V4R/V4RB (18S rRNA-specific; 82%) – demonstrating that the 515Y/926R combination performs best overall for all 3 domains. Using Atlantic and Pacific Ocean samples, we demonstrate high correspondence between 515Y/926R amplicon abundances (generated for this study) and metagenomic 16S rRNA (median R2=0.98, n=272), indicating amplicons can produce equally accurate community composition data versus shotgun metagenomics. Our analysis also revealed that expected performance of all primer sets could be improved with minor modifications, pointing toward a nearly-completely universal primer set that could accurately quantify biogeochemically-important taxa in ecosystems ranging from the deep-sea to the surface. In addition, our reproducible bioinformatic workflow can guide microbiome researchers studying different ecosystems or human health to similarly improve existing primers and generate more accurate quantitative amplicon data.Significance StatementPCR amplification and sequencing of marker genes is a low-cost technique for monitoring prokaryotic and eukaryotic microbial communities across space and time, but will only work optimally if environmental organisms match PCR primer sequences exactly. In this study, we evaluated how well primers match globally-distributed short-read oceanic metagenomes. Our results demonstrate primer sets vary widely in performance, and that at least for marine systems, rRNA amplicon data from some primers lack significant biases compared to metagenomes. We also show that it is possible to create a nearly universal primer set for diverse saline environments by defining a specific mixture of a few dozen oligonucleotides and present a software pipeline that can guide rational design of primers for any environment with available meta’omic data.

Universal SSU rRNA primers allow comprehensive quantitative profiling of natural communities by simultaneously amplifying templates from Bacteria, Archaea, and Eukaryota in a single PCR reaction. Despite the potential to show all rRNA gene relative gene abundances, they are rarely used due to concerns about length bias against 18S amplicons and bioinformatic challenges converting mixed 16S/18S sequences into amplicon sequence variants. We thus developed 16S and 18S rRNA mock communities and a bioinformatic pipeline to validate this three-domain approach. To test for length biases, we mixed eukaryotic and prokaryotic mocks before PCR, and found consistent two-fold underestimation of longer 18S sequences due to sequencing but not PCR bias. Using these mocks, we show universal V4-V5 primers (515Y/926R) outperformed eukaryote-specific V4 primers in observed vs. expected abundance correlations and sequences with single mismatches to the primer were strongly underestimated (3-8 fold). A year of monthly time-series data from a protist-enriched 1.2-80 μm size fraction yielded an average of 9% 18S, 17% chloroplast 16S, and 74% prokaryote 16S rRNA gene amplicons. These data demonstrate the potential for universal primers to generate quantitative and comprehensive microbiome profiles, although gene copy and genome size variability should be considered - as for any quantitative genetic analysis.

Obligately chemoautotrophicCampylobacteriadominate productivity in dark, sulfidic, and oxygen-depleted environments. However, biochemical mechanisms underlying their growth remain poorly known, limiting understanding of their physiology, ecology, and biogeochemical impact. In this study, we used comparative genomics, conceptual modeling of core metabolism, and chemostat growth yields to derive a model of energy conservation consistent with experimental data for the versatile chemoautotrophSulfurimonas denitrificans. Our model rests on three core mechanisms: Firstly, to allow electrogenic sulfur-based denitrification, we predict that the campylobacterial-type sulfur oxidation enzyme complex must donate electrons to the membrane quinone pool, possibly via a sulfide:quinone oxidoreductase. Secondly, to account for the unexpectedly low growth efficiency of aerobic sulfur oxidation compared to denitrification, we posit the high-affinity campylobacterial-type cbb3cytochrome c oxidase has a relatively low H+/e− of 1, likely due to a lack of proton pumping under physiological conditions. Thirdly, we hypothesize that reductant for carbon fixation by the reverse tricarboxylic acid cycle is produced by a non-canonical complex I that reduces both ferredoxin and NAD(P)H. This complex is conserved among relatedCampylobacteriaand may have allowed for the radiation of organisms likeS. denitrificansinto sulfur-rich environments that became available after the great oxidation event. Our theoretical model has two major implications. Firstly, it sets the stage for future experimental work by providing testable hypotheses about the physiology, biochemistry, and evolution of chemoautotrophicCampylobacteria. Secondly, it provides constraints on the carbon fixation potential of chemoautotrophicCampylobacteriain sulfidic environments worldwide by predicting theoretical ranges of chemosynthetic growth efficiency.SignificanceChemoautotrophicCampylobacteriaare abundant in many low-oxygen, high-sulfide environments where they contribute significantly to dark carbon fixation. Although the overall redox reactions they catalyze are known, the specific biochemical mechanisms that support their growth are mostly unknown. Our study combines conceptual modeling of core metabolic pathways, comparative genomics, and measurements of physiological growth yield in a chemostat to infer the most likely mechanisms of chemoautotrophic energy conservation in the model organismSulfurimonas denitrificans. The hypotheses proposed herein are novel, experimentally falsifiable, and will guide future biochemical, physiological, and environmental modelling studies. Ultimately, investigating the core mechanisms of energy conservation will help us better understand the evolution and physiological diversification of chemoautotrophicCampylobacteriaand their role in modern ecosystems.

At deep-sea hydrothermal vents, a large source of potential chemical energy is created when reducing vent fluid and oxidizing seawater mix. In this environment, chemolithoautotrophic microbes catalyze exergonic redox reactions which in turn provide the energy needed to fuel their growth and the fixation of CO 2 into biomass. In addition to producing new organic matter, this process also consumes compounds contained both in vent fluid and entrained seawater ( e.g. H 2 , NO 3 − ). Despite their biogeochemical importance, such reactions have remained difficult to quantify due to methodological limitations. To address this knowledge gap, this study reports a novel application of isobaric gas-tight fluid samplers for conducting incubations of hydrothermal vent fluids at in situ temperature and pressure. Eighteen ~24 h incubations were carried out, representing seven distinct conditions that examine amendments consisting of different electron donors and acceptors. Microbial activity was observed in all treatments, and time series chemical measurements showed that activity was limited by electron acceptor supply, confirming predictions based on geochemical data. Also consistent with these predictions, the presence of nitrate increased rates of hydrogen consumption and yielded ammonium as a product of nitrate respiration. The stoichiometry of predicted redox reactions was also determined, revealing that the sulfur and nitrogen cycles are incompletely understood at deep-sea vents, and likely involve unknown intermediate redox species. Finally, the measured rates of redox processes were either equal to or far greater than what has been reported in previous studies where in situ conditions were not maintained. In addition to providing insights into deep-sea hydrothermal vent biogeochemistry, the methods described herein also offer a practical approach for the incubation of any deep-sea pelagic sample under in situ conditions.

Chemoautotrophic bacteria belonging to the genus Sulfurimonas (class Campylobacteria) were previously identified as key players in the turnover of zero-valence sulfur, a central intermediate in the marine sulfur cycle. S. denitrificans was further shown to be able to oxidize cyclooctasulfur (S8 ). However, at present the mechanism of activation and metabolism of cyclooctasulfur is not known. Here, we assessed the transcriptome and proteome of S. denitrificans grown with either thiosulfate or S8 as the electron donor. While the overall expression profiles under the two growth conditions were rather similar, distinct differences were observed that could be attributed to the utilization of S8 . This included a higher abundance of expressed genes related to surface attachment in the presence of S8 , and the differential regulation of the sulfur-oxidation multienzyme complex (SOX), which in S. denitrificans is encoded in two gene clusters: soxABXY 1 Z 1 and soxCDY 2 Z 2 . While the proteins of both clusters were present with thiosulfate, only proteins of the soxCDY 2 Z 2 were detected at significant levels with S8 . Based on these findings a model for the oxidation of S8 is proposed. Our results have implications for interpreting metatranscriptomic and -proteomic data and for the observed high level of diversification of soxY 2 Z 2 among sulfur-oxidizing Campylobacteria.

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.

Northern regions are generally viewed as unsuitable for microalgal biofuel production due to unfavorable climate and solar insolation levels. However, these conditions can potentially be mitigated by coupling microalgal cultivation to industrial processes such as wastewater treatment. In this study, we have examined the biomass and lipid productivity characteristics of 14 microalgae isolates (Chlorophyta) from the Canadian province of Saskatchewan. Under both photoautotrophic and mixotrophic cultivation, a distinct linear trend was observed between biomass and lipid productivities in the 14 SK isolates. The most productive strain under cultivation in TAP media was Scenedesmus sp.-AMDD which displayed rates of biomass and fatty acid productivities of 80 and 30.7 mg L-1 day-1, respectively. The most productive strain in B3NV media was Chlamydomonas debaryana-AMLs1b which displayed rates of biomass and fatty acid productivities of 51.7 and 5.9 mg L-1 day-1, respectively. In 11 of the isolates tested, secondary municipal wastewater (MCWW) supported rates of biomass productivity between 21 and 33 mg L-1 day-1 with Scenedesmus sp.-AMDD being the most productive. Three strains, Chlamydomonas debaryana-AMB1, Chlorella sorokiniana-RBD8 and Micractinium sp.-RB1b, showed large increases in biomass productivity when cultivated mixotrophically in MCWW supplemented with glycerol. High relative oleic acid content was detected in 10 of the 14 isolates when grown mixotrophically in media supplemented with acetate. There was no detectable effect on the fatty acid profiles in cells cultivated mixotrophically in glycerol-supplemented MCWW. These data indicate that biomass and lipid productivities are boosted by mixotrophic cultivation. Exploiting this response in municipal wastewater is a promising strategy for the production of environmentally sustainable biofuels.

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. FigureOptical microscope image of Botryococcus braunii and high resolution mass spectrum of triacylglycerol 28:2/18:1/18:1 (inset) Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s00216-011-5376-6 and is accessible for authorized users.

This study provides a concise background to the biochemical search for the origin of life, as grounded in the field of prebiotic chemistry. It is intended to provide a good summary of competing theories and place them in a broader context, raising questions about weaknesses in any particular theory. This material is relevant for science educators at all levels, and will stimulate interest in a wide variety of students.

The debate on life in meteorites has entered its eighth episode, with recurrent themes and arguments. We suggest: (1) that deliberate contamination of meteorite specimens be used as a control; (2) that exploration of the solar system proceed apace to obtain pristine samples and check them for life, under stringent isolating conditions that provably avoid contamination of control specimens; and (3) that the combination of macromolecular with inorganic material in carbonaceous chondrites be explored as a possible cause for some of the organized elements and as a possible early step towards protocells in the origin of life.

Most attention for algal biofuel-related schemes has been focused on ‘optimal’ locations, such as the southwestern USA. While these locations have clear advantages such as high yearly insolation and availability of unused land, we believe a case can also be made for adapting algal biofuels for a diversity of ‘suboptimal’ climates. The key, as in other regions, will be to link algaculture with industrial and municipal waste resources, including nutrients from wastewater and CO2 from point source industrial emissions. Productivities comparable to warmer climates may be obtained throughout the year by a combination of factors, including appropriate strain selection for low temperature and waste heat utilization, or by switching to a heterotrophic growth mode when light is insufficient for productive photosynthesis. In this manner, mass algaculture and associated R&D can be justified by offering valuable remediatory functions (i.e. tertiary wastewater treatment and CO2 abatement), rather than relying on optimistic estimations of oil and biomass productivity to spur development in this field.

The biological record suggests that life on Earth arose as soon as conditions were favorable, which indicates that life either originated quickly, or arrived from elsewhere to seed Earth. Experimental research under the theme of “astrobiology” has produced data that some view as strong evidence for the second possibility, known as the panspermia hypothesis. While it is not unreasonable to consider the possibility that Earth’s life originated elsewhere and potentially much earlier, we conclude that the current literature offers no definitive evidence to support this hypothesis. Chladni’s view, that they fall from the skies, pronounced in 1795, was ridiculed by the learned men of the times. (Rachel, 1881) Evidence of life on Mars, even if only in the distant past, would finally answer the age-old question of whether living beings on Earth are alone in the universe. The magnitude of such a discovery is illustrated by President Bill Clinton’s appearance at a 1996 press conference to announce that proof had been found at last. A meteorite chipped from the surface of the Red Planet some 15 million years ago appeared to contain the fossil remains of tiny life-forms that indicated life had once existed on Mars. (Young and Martel, 2010)

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 sevenfold) 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.