It is widely established that nutritional nitrogen deprivation increases lipid accumulation but severely decreases growth rate in microalgae. To understand the regulatory branch points that determine the partitioning of carbon among its potential sinks, we analyzed metabolite and transcript levels of central carbon metabolic pathways and determined the average fluxes and quantum requirements for the synthesis of protein, carbohydrates and fatty acid in the diatom Phaeodactylum tricornutum. Under nitrate-starved conditions, the carbon fluxes into all major sinks decrease sharply; the largest decrease was into proteins and smallest was into lipids. This reduction of carbon flux into lipids together with a significantly lower growth rate is responsible for lower overall FA productivities implying that nitrogen starvation is not a bioenergetically feasible strategy for increasing biodiesel production. The reduction in these fluxes was accompanied by an 18-fold increase in a-ketoglutarate (AKG), 3-fold increase in NADPH/NADP(+), and sharp decreases in glutamate (GLU) and glutamine (GLN) levels. Additionally, the mRNA level of acetyl-CoA carboxylase and two type II diacylglycerol-acyltransferases were increased. Partial suppression of nitrate reductase by tungstate resulted in similar trends at lower levels as for nitrate starvation. These results reveal that the GS/GOGAT pathway is the main regulation site for nitrate dependent control of carbon partitioning between protein and lipid biosynthesis, while the AKG/GL(N/U) metabolite ratio is a transcriptional signal, possibly related to redox poise of intermediates in the photosynthetic electron transport system. (C) 2013 Elsevier Ltd. All rights reserved.