Effect of cell cycle arrest on intermediate metabolism in the marine diatom Phaeodactylum tricornutum.

The inhibitor NU 2058 [6-(cyclohexylmethoxy)-9H-purin-2-amine] leads to G1-phase cell cycle arrest in the marine diatom, Phaeodactylum tricornutum, by binding to two cyclin-dependent kinases, CDKA1 and CDKA2. NU 2058 has no effect on photosynthetic attributes, such as Fv/Fm, chlorophyll a/cell, levels of D2 PSII subunits, or RbcL; however, cell cycle arrest leads to unbalanced growth whereby photosynthetic products that can no longer be used for cell division are redirected toward carbohydrates and triacylglycerols (TAGs). Arrested cells up-regulate most genes involved in fatty acid synthesis, including acetyl-CoA carboxylase, and three out of five putative type II diglyceride acyltransferases (DGATs), the enzymes that catalyze TAG production. Correlation of transcriptomes in arrested cells with a flux balance model for P. tricornutum predicts that reactions in the mitochondrion that supply glycerate may support TAG synthesis. Our results reveal that sources of intermediate metabolites and macromolecular sinks are tightly coupled to the cell cycle in a marine diatom, and that arresting cells in the G1 phase leads to remodeling of intermediate metabolism and unbalanced growth.

High-throughput screening assay for biological hydrogen production.

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.

Quantum requirements for growth and fatty acid biosynthesis in the marine diatom Phaeodactylum tricornutum (Bacillariophyceae) in nitrogen replete and limited conditions.

We determined the quantum requirements for growth (1/ϕµ ) and fatty acid (FA) biosynthesis (1/ϕFA ) 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/ϕµ of 40-44 mol photons · mol C(-1) . In parallel, 1/ϕFA varied between 380 and 409 mol photons · mol C(-1) in the presence of nitrate, but declined to 348 mol photons · mol C(-1) with ammonium and to 250 mol photons · mol C(-1) 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.

Effects of selected electron transport chain inhibitors on 24-h hydrogen production by Synechocystis sp. PCC 6803.

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.

Optimization of pH and nitrogen for enhanced hydrogen production by Synechocystis sp. PCC 6803 via statistical and machine learning methods.

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.