[Show abstract][Hide abstract] ABSTRACT: We determined the quantum requirements for growth (1/phi) and fatty acid (FA) biosynthesis (1/phi FA) in the marine diatom, Phaeodactylum tricornutum, grown in nutrient replete conditions with nitrate or ammonium as nitrogen sources, and under nitrogen limitation, achieved by transferring cells into nitrogen free medium or by inhibiting nitrate assimilation with tungstate. A treatment in which tungstate was supplemented to cells grown with ammonium was also included. In nutrient replete conditions, cells grew exponentially and possessed virtually identical 1/phi of 4044mol photonsmol C1. In parallel, 1/phi FA varied between 380 and 409mol photonsmol C1 in the presence of nitrate, but declined to 348mol photonsmol C1 with ammonium and to 250mol photonsmol C1 with ammonium plus tungstate, indicating an increase in the efficiency of FA biosynthesis relative to cells grown on nitrate of 8% and 35%, respectively. While the molecular mechanism for this effect remains poorly understood, the results unambiguously reveal that cells grown on ammonium are able to direct more reductant to lipids. This analysis suggests that when cells are grown with a reduced nitrogen source, fatty acid biosynthesis can effectively become a sink for excess absorbed light, compensating for the absence of energetically demanding nitrate assimilation reactions. Our data further suggest that optimal lipid production efficiency is achieved when cells are in exponential growth, when nitrate assimilation is inhibited, and ammonium is the sole nitrogen source.
Journal of Phycology 04/2013; 49(2):381-388. DOI:10.1111/jpy.12046 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One approach to achieve continuous overproduction of lipids in microalgal “cell factories” relies upon depletion or removal of nutrients that act as competing electron sinks (e.g., nitrate and sulfate). However, this strategy can only be effective for bioenergy applications if lipid is synthesized primarily de novo (from CO2 fixation) rather than from the breakdown and interconversion of essential cellular components. In the marine diatom, Phaeodactylum tricornutum, it was determined, using 13C-bicarbonate, that cell growth in nitrate (NO
3−)-deprived cultures resulted predominantly in de novo lipid synthesis (60 % over 3 days), and this new lipid consisted primarily of triacylglycerides (TAGs). Nearly complete preservation of 12C occurred in all previously existing TAGs in NO
3−-deprived cultures and thus, further TAG accumulation would not be expected from inhibition of TAG lipolysis. In contrast, both high turnover and depletion of membrane lipids, phosphatidylcholines (PCs), were observed in NO
3−-deprived cultures (both the headgroups and fatty acid chains), while less turnover was observed in NO
3− replete cultures. Liquid chromatography-tandem mass spectrometry mass spectra and 13C labeling patterns of PC headgroups provided insight into lipid synthesis in marine diatoms, including suggestion of an internal pool of glycine betaine that feeds choline synthesis. It was also observed that 16C fatty acid chains incorporated into TAGs and PCs contained an average of 14 13C carbons, indicating substantial incorporation of 13C-bicarbonate into fatty acid chains under both nutrient states.
BioEnergy Research 12/2012; 5(4):876-885. DOI:10.1007/s12155-012-9201-7 · 3.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One factor limiting biosolar hydrogen (H(2)) production from cyanobacteria is electron availability to the hydrogenase enzyme. In order to optimize 24-h H(2) production this study used Response Surface Methodology and Q2, an optimization algorithm, to investigate the effects of five inhibitors of the photosynthetic and respiratory electron transport chains of Synechocystis sp. PCC 6803. Over 3 days of diurnal light/dark cycling, with the optimized combination of 9.4 mM KCN (3.1 μmol 10(10) cells(-1)) and 1.5 mM malonate (0.5 μmol 10(10) cells(-1)) the H(2) production was 30-fold higher, in EHB-1 media previously optimized for nitrogen (N), sulfur (S), and carbon (C) concentrations (Burrows et al., 2008). In addition, glycogen concentration was measured over 24 h with two light/dark cycling regimes in both standard BG-11 and EHB-1 media. The results suggest that electron flow as well as glycogen accumulation should be optimized in systems engineered for maximal H(2) output.
[Show abstract][Hide abstract] ABSTRACT: The nitrogen (N) concentration and pH of culture media were optimized for increased fermentative hydrogen (H(2)) production from the cyanobacterium, Synechocystis sp. PCC 6803. The optimization was conducted using two procedures, response surface methodology (RSM), which is commonly used, and a memory-based machine learning algorithm, Q2, which has not been used previously in biotechnology applications. Both RSM and Q2 were successful in predicting optimum conditions that yielded higher H(2) than the media reported by Burrows et al., Int J Hydrogen Energy. 2008;33:6092-6099 optimized for N, S, and C (called EHB-1 media hereafter), which itself yielded almost 150 times more H(2) than Synechocystis sp. PCC 6803 grown on sulfur-free BG-11 media. RSM predicted an optimum N concentration of 0.63 mM and pH of 7.77, which yielded 1.70 times more H(2) than EHB-1 media when normalized to chlorophyll concentration (0.68 +/- 0.43 micromol H(2) mg Chl(-1) h(-1)) and 1.35 times more when normalized to optical density (1.62 +/- 0.09 nmol H(2) OD(730) (-1) h(-1)). Q2 predicted an optimum of 0.36 mM N and pH of 7.88, which yielded 1.94 and 1.27 times more H(2) than EHB-1 media when normalized to chlorophyll concentration (0.77 +/- 0.44 micromol H(2) mg Chl(-1) h(-1)) and optical density (1.53 +/- 0.07 nmol H(2) OD(730) (-1) h(-1)), respectively. Both optimization methods have unique benefits and drawbacks that are identified and discussed in this study.
[Show abstract][Hide abstract] ABSTRACT: By optimizing concentrations of key nutrients in the media of Synechocystis sp. PCC 6803, we achieved nearly 150-fold greater photofermentative hydrogen (H2) production than was achieved by analogous, sulfur-deprived cultures, which are well known to produce much more H2 than cultures grown on complete media. This was associated with a 44-fold increase in glycogen concentration. Using response surface methodology to determine optimum conditions, we found that, instead of completely starving cells of sulfur or another essential nutrient, the highest H2 production (0.81 ± 0.36 μmol H2 mg Chl−1 h−1) occurred with 0.52 mM NH4+, 20.1 μM SO42−, and 46 mM HCO3−. H2 profiling experiments provided initial screening of NH4+, HCO3−, SO42−, and PO43− concentrations and identified the significant variables in H2 production to be NH4+, SO42−, and the interactions of both NH4+ and SO42− with HCO3−. Our results indicate that optimized amounts of nitrogen and sulfur in the nutrient media are superior to total deprivation of these nutrients for H2 production.
International Journal of Hydrogen Energy 11/2008; 33(21-33):6092-6099. DOI:10.1016/j.ijhydene.2008.07.102 · 3.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper describes a screening assay, compatible with high-throughput bioprospecting or molecular biology methods, for assessing biological hydrogen (H2) production. While the assay is adaptable to various physical configurations, we describe its use in a 96-well, microtiter plate format with a lower plate containing H2-producing cyanobacteria strains and controls and an upper, membrane-bottom plate containing a color indicator and a catalyst. H2 produced by cells in the lower plate diffuses through the membrane into the upper plate, causing a color change that can be quantified with a microplate reader. The assay is reproducible, semiquantitative, sensitive down to at least 20 nmol of H2, and largely unaffected by oxygen, carbon dioxide, or volatile fatty acids at levels appropriate to biological systems.
[Show abstract][Hide abstract] ABSTRACT: Graduation date: 2009 Many conditions affecting hydrogen (H₂) production by the cyanobacterium, Synechocystis sp. PCC 6803, were optimized to yield maximum H₂ accumulation. Biological H₂ production from photosynthetic species is a promising form of renewable energy since an abundant supply of sunlight hits the Earth every day, and photosynthetic bacteria can harness this solar energy and efficiently split water to produce H₂ in a safe, clean manner. The H₂ could then be used in fuel cells in a closed cycle, with water and heat as the only byproducts. There are many techniques currently in development to maximize H₂ production. We chose to use statistical optimization procedures to identify the factors which have the greatest impact on H₂ production, and simultaneously optimize them. Initially we optimized concentrations of NH₄⁺, HCO₃⁻, and SO₄²⁻, and achieved a 148-fold increase in H₂ production over sulfur deprived cultures, which have been shown to produce more H₂ than cultures grown on complete BG-11 media. With 0.52 mM NH₄⁺, 20.1 μM SO₄²⁻, and 46 mM HCO₃⁻, 0.81±0.36 μmol H₂ mg Chl⁻¹ h⁻¹ was obtained. This increase was associated with a 44-fold increase in glycogen concentration over cultures grown on BG-11. Glycogen breakdown provides substrate to the hydrogenase enzyme under dark, anaerobic conditions. Since interaction effects are strong, we then optimized pH and NH₄⁺ simultaneously, and achieved another 1.94-fold increase over the previously optimized media. This was achieved with an advanced optimization algorithm, which had never been applied to biotechnological applications. Both of these increases in H₂ production were accomplished under optimal glycogen accumulation conditions, which include acclimation to the media formulation over an extended light period, followed by immediate anaerobic, dark fermentative conditions. In an additional study we explored 24-hour H₂ production under natural, diurnal light/dark cycling, and examined glycogen accumulation dynamics as well as electron availability to the hydrogenase. Electron availability was manipulated by exposing the cultures to various inhibitors of enzymes in the photosynthetic and respiratory electron transport chains. Over 3 days, with 9.4 mM KCN and 1.5 mM malonate in the previously optimized media we were able to increase H2 production 30-fold over standard BG-11 without inhibitors.