Several recent studies have suggested that control of isoprene emission rate is in part exerted by supply of extrachloroplastic phosphoenolpyruvate to the chloroplast. To test this hypothesis, we altered PEP supply by differential induction of cytosolic nitrate reductase (NR) and PEP carboxylase (PEPC) in plants of Populus deltoides grown with NO3- or NH4+ as the sole nitrogen source. Growth with 8 mM NH4+ produced a high leaf nitrogen concentration, compared with 8 mM NO3-, as well as slightly elevated rates of photosynthesis and significantly enhanced rates of isoprene emission and content of dimethylallyl diphosphate (DMAPP, a precursor to isoprene biosynthesis), chlorophyll (a+b) and carotenoids. Growth with 8 mM NO3- resulted in parallel reductions in both leaf isoprene emission rate and DMAPP. The differential effects of growth with NH4+ or NO3- were not observed when plants were grown with 4 mM nitrogen. The effects of reduced DMAPP availability were specific to isoprene emission and were not propagated to higher isoprenoids, as the correlations between nitrogen content and either leaf chlorophyll (a+b) or total carotenoids were unaffected by nitrogen source. Biochemical analysis revealed significantly higher levels of NR and PEPC activity in leaves of 8 mM NO3- -grown plants, consistent with their fundamental roles in nitrate assimilation. Taken together, these results support the hypothesis that foliar assimilation of NO3- reduces isoprene emission rate by competing for carbon skeletons (mediated by PEPC) within the cytosol and possibly reductant within the chloroplast. Cytosolic competition for PEP is a major regulator of chloroplast DMAPP supply, and we offer a new "safety valve" hypothesis to explain why plants emit isoprene.

Continuous cultures of Botryococcus braunii race B were maintained at photosynthetic cell densities as high as 20 g dry weight per liter for up to 3 months. Growth associated triterpene hydrocarbon accumulation was nearly constant at 22.5% of dry weight for a range of growth rates maintained by daily replacement of 5-15% of the respective cultures. The ability to achieve high cell concentrations and oil levels of roughly 5 g triterpene oil/L resulted from a combination of high light (∼ 1/4 full sun for 15 h/day) and replenishing stoichiometrically balanced growth medium. Due to light-limited growth conditions, cell concentration dropped nearly linearly with increased dilution rate. This reduction in cell number resulted in increased productivity per cell at higher dilution rates and was accompanied by a dramatic increase in algae colony size from 0.09 to 0.343 mm at high dilution rate. This change in colony size resulted in an equally dramatic change in optical density (OD) per gram dry weight, which precluded use of simple correlations of OD and cell concentration. A trickle-film photobioreactor was also demonstrated as a scalable approach to achieving these ultra-high cell concentrations. Additional media analysis revealed a steady increase in photobioreactor conductivity suggesting an accumulation of ions may be the reason for rapid culture crash and washout observed at all dilution rates after several months of continuous operation. The volumetric productivity of 22.5 mg oil/L/photo-h reported here is more than an order of magnitude higher than previous reports for B. braunii race B, reflecting the high cell densities used in this work and substantiating a higher metabolic rate for B. braunii race B than previously surmised from its relatively long doubling times.