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Algae hold great potential as a source of renewable fuel due to their ability to produce refinery-compatible diesel and jet fuel precursors. Significant effort has been made to maximize productivity under photoautotrophic growth conditions; however, little progress has been made to discover and understand reduced carbon assimilation pathways or enzymatic degradation of complex carbon substrates in algae. We purport that utilization of plant-based carbon substrates in addition to photosynthesis (mixotrophic growth) for biochemical assimilation into biomass, biofuels, and bioproducts, can increase cultivation productivity and improve the economic viability of algal-derived biofuels. Herein we report that a freshwater production strain of microalgae, Auxenochlorella protothecoides UTEX 25, is capable of directly degrading and utilizing non-food plant substrates, such as switchgrass, for cell growth. Glycome profiling of plant substrates before and after addition to A. protothecoides cultures demonstrates the utilization of xyloglucans. Genomic, proteomic and transcriptomic analyses revealed the identity of many enzymes that are hypothesized to be involved in complex carbohydrate degradation, including several family 5 and 9 glycosyl hydrolases. This work paves the way for future designer engineering of plant‑carbon utilization to further improve productivity of algal production strains.

We characterize the distribution of oceanic phosphorus-containing lipids (PL) in the Northeast Atlantic by Iatroscan thin layer chromatography and high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Phospholipids are a small but significant fraction of oceanic particulate organic carbon (POC) (1.5%). We describe the distribution of 1862 PL compounds in total, of which only ~27% have elemental compositions that match those found in the Nature Lipidomics Gateway database (e.g., phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidyl serine (PS), and phosphatidylinositol (PI)). The highest phospholipid concentration is found in the epipelagic, which reflects primary production in that depth horizon. Depth-related PL removal was the strongest for PL signals that match database-reported (known) lipids and was lower for saturated non-database (novel) matched PL. The transformation of known PL is marked by depth-related increase in saturation with PA that is assumed to be generated as the earliest transformation product of PL. Novel unsaturated P-lipids likely originate from both PL transformation processes and in-situ biological production at the surface layer. Novel PL are dominated by unsaturated compounds for which unsaturation increased between the epipelagic (average molecular double bond equivalents, DBE = 5) and the abyssopelagic (average DBE = 6.7) zones. Additionally, those compounds increase in both average molecular weight and contribution to all lipid content with increasing depth, likely from cross-linking of unsaturated compounds. Our data indicate that novel PL are selectively preserved with depth and therefore are P and C carriers to the deep Atlantic. We demonstrate that a full appreciation of phosphorus cycling requires additional data on phospholipid composition and especially the ecological role and depth-related molecular change of these compounds.

We report the co-liquefaction performance of unicellular, red alga Cyanidioschyzon merolae and Galdieria sulphuraria under sub-critical water conditions within a stainless-steel batch reactor under different temperatures (150-300°C), residence time (15-60min), and Cyanidioschyzon merolae to Galdieria sulphuraria mass loading (0-100%). Individual liquefaction of C. merolae and G. sulphuraria at 300°C achieved maximum biocrude oil yield of 18.9 and 14.0%, respectively. The yield of biocrude oil increased to 25.5%, suggesting a positive synergistic effect during the co-liquefaction of 80-20mass loading of C. merolae to G. sulphuraria. The biocrude oils were analyzed by FT-ICR MS which showed that co-liquefaction did not significantly affect the distribution of product compounds compared to individual oils. The co-liquefied biocrude and biochar have a higher-heating-value of 35.28 and 7.96MJ/kg. Ultimate and proximate analysis were performed on algae biomass, biocrude and biochar.

We apply Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) for direct characterization of iron-porphyrins in hydrothermal liquefaction (HTL) biocrude oils derived from two algae: Tetraselmis sp. and cyanobacteria. The iron porphyrin compounds are shown to cause catalyst bed plugging during hydroprocessing due to iron deposition. Inductively-coupled plasma optical emission spectrometry (ICP-OES) was utilized for iron quantitation in the plugged catalyst beds formed through hydroprocessing of the two HTL biocrudes and identifies an enrichment of iron in the upper five centimeters of the catalyst bed for Tetraselmis sp. (Fe = 100,728 ppm) and cyanobacteria (Fe = 115,450 ppm). Direct infusion FT-ICR MS analysis of the two HTL biocrudes with optimized instrument conditions facilitates rapid screening and identification of iron porphyrins without prior chromatographic separation. With FT-ICR MS we identify 138 unique iron porphyrin compounds in the two HTL biocrudes that have similar carbon number and double bond equivalent distributions to the metal porphyrins (e.g. Ni and V) reported for petroleum. No iron porphyrins are observed in the cyanobacteria HTL biocrude after hydroprocessing, which indicates that iron porphyrin structures in the HTL biocrude are degraded during hydrotreatment. Hydrodemetallization reactions that occur through hydroprocessing of HTL biocrudes could be responsible for the decomposition of iron porphyrin structures leading to metal deposition in the catalyst bed that result in catalyst deactivation and bed plugging, and must be addressed for effective upgrading of algal HTL biocrudes.

Temperature effect on hydrothermal liquefaction (HTL) of Nannochloropsis gaditana and Chlorella sp. was investigated with 10% biomass loading at HTL temperatures of 180–330 °C, and reaction time of 30 min. Maximum yields of 47.5% for Nannochloropsis sp. and 32.5% biocrude oil yields for Chlorella sp. were obtained at 300 °C. The higher heating values of biocrude oils produced in this work ranged between 34 and 39 MJ/kg. 79% of energy in the Nannochloropsis sp. was recovered at 300 °C and 62% of energy recovery from Chlorella sp. was achieved at 200 °C. Valuable nutrients (NH3–N and PO43−) produced during the HTL process were quantified from the aqueous phase for both strains of biomass. The aqueous phase samples obtained at all temperatures were also analyzed for amino acids and carbohydrates. The suitable temperatures for extraction of lipids, amino acids and carbohydrates have been identified. Sequential HTL experiments conducted have shown the prospect of recovering nutrients and other valuable byproducts along with biocrude oil. The experimental results and analysis indicate that sustainable biofuel production requires the development of strain based strategies for the hydrothermal liquefaction process.

There are major gaps in our understanding of the distribution and role of lipids in the open ocean especially with regard to sulfur-containing lipids (S-lipids). Here, we employ a powerful analytical approach based on high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to elucidate depth-related S-lipid production and molecular transformations in suspended particulate matter from the Northeast Atlantic Ocean in this depth range. We show that within the open-ocean environment S-lipids contribute up to 4.2% of the particulate organic carbon, and that up to 95% of these compounds have elemental compositions that do not match those found in the Nature Lipidomics Gateway database (termed “novel”). Among the remaining 5% of lipids that match the database, we find that sulphoquinovosyldiacylglycerol (SQDG) are efficiently removed while sinking through the mesopelagic zone. The relative abundance of other assigned lipids (sulphoquinovosylmonoacylglycerol (SQMG), sulfite and sulfate lipids, Vitamin D2 and D3 derivatives, and sphingolipids) did not change substantially with depth. The novel S-lipids, represented by hundreds of distinct elemental compositions (160–300 molecules at any one depth), contribute increasingly to the lipid and particulate organic matter pools with increased depth. Depth-related transformations cause (i) incomplete degradation/transformation of unsaturated S-lipids which leads to the depth-related accumulation of the refractory saturated compounds with reduced molecular weight (average 455 Da) and (ii) formation of highly unsaturated S-lipids (average abyssopelagic molecular double bond equivalents, DBE=7.8) with lower molecular weight (average 567 Da) than surface S-lipids (average 592 Da). A depth-related increase in molecular oxygen content is observed for all novel S-lipids and indicates that oxidation has a significant role in their transformation while (bio)hydrogenation possibly impacts the formation of saturated compounds. The instrumentation approach applied here represents a step change in our comprehension of marine S-lipid diversity and the potential role of these compounds in the oceanic carbon cycle. We describe a very much higher number of compounds than previously reported, albeit at the level of elemental composition and fold-change quantitation with depth, rather than isomeric confirmation and absolute quantitation of individual lipids. We emphasize that saturated S-lipids have the potential to transfer carbon from the upper ocean to depth and hence are significant vectors for carbon sequestration.

Cultivation of microalgae has the potential to provide lipid-derived feedstocks for conversion to liquid transportation fuels. Lipid extracts from microalgae are significantly more complex than those of traditional seed oils, and their composition changes significantly throughout the microalgal growth period. With three acyl side chains per molecule, triglycerides (TAGs) are an important fuel precursor, and the distribution of acyl chain composition for TAGs has a significant impact on fuel properties and processing. Therefore, determination of the distribution of microalgal TAG production is needed to assess the value of algal extracts designed for fuel production and to optimize strain, cultivation, and harvesting practices. Methods utilized for TAG speciation commonly involve complicated and time-consuming chromatographic techniques. We present a method for TAG speciation and quantification based on direct-infusion mass spectrometry, which provides rapid characterization of TAG profiles without chromat...

Induction of oil accumulation in algae for biofuel production is often achieved by withholding nitrogen. How- ever, withholding nitrogen often reduces total biomass yield. In this report, it is demonstrated that Chlorella sorokiniana will not only accumulate substantial quantities of neutral lipids when grown in the absence of nitrogen but will also exhibit unimpeded growth rates for up to 2 weeks. To deter- mine the physiological basis for the observed increase in oil and biomass accumulation, we compared photosynthetic and respiration rates and chlorophyll, lipid, and total energy con- tent under ammonia replete and deplete conditions. Under N- depleted growth conditions, there was a 64 % increase in total energy density and a ∼20-fold increase in oil accumulation relative to N-replete growth leading to a 1.6-fold greater total energy yield in N-depleted than in N-replete cultures. We pro- pose that the higher energy accumulation in N-depleted cul- tures is due to enhanced photosynthetic energy capture and conversion associated with reduced chlorophyll levels and reduced self-shading as well as a shift in metabolism leading to the accumulation of oils.

We illustrate a detailed compositional characterization of hydrothermal liquefaction (HTL) oils derived from two biochemically distinct microalgae, Nannochloropsis gaditana and Chlorella sp. (DOE 1412), for a range of reaction temperature as observed by high-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). The unique capability to unequivocally derive molecular formulae directly from FT-ICR MS-measured mass-to-charge ratio (for several thousand compounds in each oil) shows that lipids are completely reacted/converted for any reaction temperature above 200 °C and reveals the formation of nonlipid reaction products with increasing temperature. Specifically, lipid-rich oil is obtained at low reaction temperature (

Hydrothermal liquefaction (HTL) crude oil and hydrotreated product from pine tree farm waste (forest product residual, FPR) have been analyzed by direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) in both positive- and negative-ionization modes and high-resolution twodimensional heteronuclear 1H-13C NMR spectroscopy. FT-ICR MS resolves thousands of compounds in complex oils and provides unparalleled compositional details for individual molecules for identification of compound class (heteroatom content), type (number of rings plus double bonds to carbon or double bond equivalents (DBE) and carbon number (degree of alkylation). Heteronuclear 1H-13C NMR spectroscopy provides one-bond and multiple-bond correlations between pairs of 1H and 13C chemical shifts that are characteristic of different organic functional groups. Taken together this information provides a picture of the chemical composition of these oils. Pyrolysis crude oil product from pine wood was characterized for comparison. Generally, pyrolysis oil is comprised of a more diverse distribution of heteroatom classes with higher oxygen number relative to HTL oil as shown by both positive- and negative-ion ESI FT-ICR MS. A total of 300 N1, 594 O1 and 267 O2 compounds were observed as products of hydrotreatment. The relative abundance of N1O1, N1O2, N1O3, N2, N2O1, N2O2more » and O3 compounds are reduced to different degrees after hydrotreatment and other higher heteroatom containing species (O4-O10, N1O4, N1O5 and N2O3) are completely removed by hydrotreatment.« less

We utilized high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT- ICR MS) to describe the depth-dependent distribution and molecular nature of nitrogen containing lipids (N-lipids) from suspended particles in an oceanic environment. Samples were collected at the Porcupine Abyssal Plain (PAP) sustained observatory in the northeast Atlantic (49.0°N, 16.5°W). Approximately 12.5% of FT-ICR MS observed lipids contain N. Only 19% of the lipids we recorded have elemental compositions that match those in the Nature Lipidomics Gateway database. Our results illustrate: (i) the proportional and selective accumulation of N-lipids with increased depth ; (ii) that N-lipids which contain phosphorus are more stable than those without P ; (iii) the majority of the deep Atlantic unsaturated N- lipids are highly unsaturated and (iv) there is depth-related increase in the saturated N- lipids which indicates that saturation is an important process for the export of lipid N and C to the deep ocean. These observations provide a description of N-lipid characteristics, transformation and preservation potential through the water column in the mesotrophic area of the North Atlantic Ocean.

We report a detailed compositional characterization of a bio-crude oil and aqueous by-product from hydrothermal liquefaction of Nannochloropsis salina by direct infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) in both positive- and negative-ionization modes. The FT-ICR MS instrumentation approach facilitates direct assignment of elemental composition to >7000 resolved mass spectral peaks and three-dimensional mass spectral images for individual heteroatom classes highlight compositional diversity of the two samples and provide a baseline description of these materials. Aromatic nitrogen compounds and free fatty acids are predominant species observed in both the bio-oil and aqueous fraction. Residual organic compounds present in the aqueous fraction show distributions that are slightly lower in both molecular ring and/or double bond value and carbon number relative to those found in the bio-oil, albeit with a high degree of commonality between the two compositions.

Photosynthetic microbes are considered promising biofactories for transforming inorganic carbon from the atmosphere into a renewable source of chemicals and precursors of industrial interest; however, there continues to be a need for strains that demonstrate high productivity, environmental robustness, and the potential to be genetically manipulated. Genome sequencing and biochemical characterization of promising culture collection microalgae strains, as well as the isolation of previously unidentified strains from the environment or mixed cultures, bring us closer to the goal of decreasing the cost-per-gallon of algal biofuels by identifying new and promising potential production strains. The halotolerant alga Picochlorum soloecismus was isolated from the culture collection strain, Nannochloropsis salina CCMP 1776. Here, we show that P. soloecismus accumulates moderate levels of fatty acids and high levels of total carbohydrates and that it can effectively grow in a range of salinities. In addition, we make use of its sequenced genome to compare it to other biofuel production platforms and to validate the capacity for engineering this strain's genome. Our work shows that Picochlorum soloecismus is a candidate production strain for the generation of renewable bioproducts.

Recalcitrant organic groundwater contaminants, such as 1,4-dioxane, may require strong oxidants for complete mineralization. However, their efficacy for in-situ chemical oxidation (ISCO) is limited by oxidant decay and reactivity. Hydroxypropyl-β-cyclodextrin (HPβCD) was examined for its ability to stabilize aqueous-phase ozone (O3) and prolong oxidation potential through inclusion complex formation. Partial transformation of HPβCD by O3 was observed. However, HPβCD proved to be sufficiently recalcitrant, because it was only partially degraded in the presence of O3. The formation of a HPβCD:O3 clathrate complex was observed, which stabilized decay of O3. The presence of HPβCD increased the O3 half-life linearly with increasing HPβCD:O3 molar ratio. The O3 half-life in solutions increased by as much as 40-fold relative to HPβCD-free O3 solutions. Observed O3 release from HPβCD and indigo oxidation confirmed that the formation of the inclusion complex is reversible. This proof-of-concept study demonstrates that HPβCD can complex O3 while preserving its reactivity. These results suggest that the use of clathrate stabilizers, such as HPβCD, can support the development of a facilitated-transport enabled ISCO for the O3 treatment of groundwater contaminated with recalcitrant compounds.

Heme nitric oxide/oxygen (H-NOX)-binding proteins act as nitric oxide (NO) sensors among various bacterial species. In several cases, they act to mediate communal behavior such as biofilm formation, quorum sensing, and motility by influencing the activity of downstream signaling proteins such as histidine kinases (HisKa) in a NO-dependent manner. An H-NOX/HisKa regulatory circuit was recently identified in Vibrio cholerae, and the H-NOX protein has been spectroscopically characterized. However, the influence of the H-NOX protein on HisKa autophosphorylation has not been evaluated. This process may be important for persistence and pathogenicity in this organism. Here, we have expressed and purified the V. cholerae HisKa (HnoK) and H-NOX in its heme-bound (holo) and heme-free (apo) forms. Autophosphorylation assays of HnoK in the presence of H-NOX show that the holoprotein in the Fe(II)-NO and Fe(III) forms is a potent inhibitor of HnoK. Activity of the Fe(III) form and aerobic instability of the Fe(II) form suggested that Vibrio cholerae H-NOX may act as a sensor of the redox state as well as NO. Remarkably, the apoprotein also showed robust HnoK inhibition that was dependent on the oxidation of cysteine residues to form disulfide bonds at a highly conserved zinc site. The importance of cysteine in this process was confirmed by mutagenesis, which also showed that holo Fe(III), but not Fe(II)-NO, H-NOX relied heavily upon cysteine for activation. These results highlight a heme-independent mechanism for activation of V. cholerae H-NOX that implicates this protein as a dual redox/NO sensor.

We present the characterisation and vertical distribution of suspended particulate lipids containing C, H and O which have the potential to sequester carbon from the upper ocean when associated with sinking particles. Lipids have been shown to be valuable in a host of environments to provide insights into the sources and processing of organic materials in the oceans. Here we present, direct-infusion, high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with bulk lipid measures for marine lipid characteriazation. We present the water column distribution of free fatty acids, tri-, di- and monoacylglycerols from the surface layer to abyssopelagic depths (4800 m) for samples collected in the Northeast Atlantic at the Porcupine Abyssal Plain sustained observatory (PAP-SO) (49.0 °N, 16.5 °W). Triacylglycerols (TG) with even carbon number (TG) and odd carbon number (oddTG, reflecting bacterial origin), were analysed, while free fatty acids were analysed as unsaturated (UFA), branched (BrFA) and saturated (SAFA) fatty acids. The surface productive layer (euphotic zone) was characterized with the highest incidence of lipids that are not reported in the Nature Lipidomics Gateway database, especially lipids that are highly unsaturated (acyl chain unsaturation was on average 3.8 for TG, oddTG, UFA and diacylglycerols (DG)). Additionally, we observed high lipid degradation at epipelagic depths. Fatty acid markers indicate that diatoms and dinoflagellates were important contributors to the lipid pool. Depth-resolved lipid change includes decreased lipid abundance and molecular diversity together with substantial loss of unsaturation with increasing depth. The major lipid change occurs at upper mesopelagic depths. Unlike other observed lipids, the abundance of SAFA remained essentially constant down the water column whereas the number of SAFAs and their contribution to total lipids increased with depth. Thus, we demonstrate that lipid saturation affects the export of carbon from the atmosphere to the deep ocean.

BACKGROUND Anthocyanin-rich blue corn is an emerging specialty crop in the USA. The antioxidant properties of blue corn offer health benefits in the human diet. The objectives of this study were to identify, characterize and quantify the anthocyanins from blue corn. Hypotheses tested were that total anthocyanin content was similar among southwestern US accessions and that it would vary across locations. It was also examined whether different anthocyanin components were unique to certain genotypes. RESULTS Across all locations and accessions, an average of 0.43 g kg−1 total anthocyanin content (TAC) was observed. Accessions Santa Clara Blue and Ohio Blue displayed the highest TAC. The TAC of accession Flor del Rio was lower by nearly a factor of six. A total of five anthocyanin components were identified. Cyanidin 3-glucoside was the most abundant, followed by pelargonidin and peonidin 3-glucoside. Succinyl and disuccinyl glycosidic forms of cyanidin were also identified. Cyanidin 3-disuccinylglucoside was newly identified as a novel form of anthocyanin. CONCLUSION Quantitative and qualitative anthocyanin expression was determined to be relatively stable across multiple southwestern environments. Increased expression of red and purple pigmentation in accession Flor del Rio appeared to be associated more with reduced TAC and cyanidin 3-glucoside than with elevated pelargonidin per se. A previously unreported anthocyanin component in blue corn, cyanidin 3-disuccinylglucoside, is present in southwestern landraces. © 2016 Society of Chemical Industry