Project description:In comparison with provision of either ammonium or nitrate alone, simultaneously supplying both forms of N results in superior growth and yield for the majority of plants including rice. Using a rice 22K oligo-array, we performed transcriptome analysis to identify genes of rice (Oryza sativa L. ssp. japonica) responsive to change of N-supply forms and N-starvation. Using the supply of ammonium nitrate (one to one molar ratio) as control, the total number of root genes that were equal or more than two fold up- or down- regulated was 445, 324, and 781 by upon supply of either ammonium or nitrate or continuous N starvation, respectively for 96 h. In the shoot the equivalent numbers were much smaller only 32, 58, and 165, respectively. Clustering of the rice genes associated with different environmental stresses revealed substantial organ specificity of the root and shoot to N starvation, and also to the N supply form. Genes encoding transporters for ammonium and nitrate, nitrate reductase, glutamate dehydrogenase, and aspartate amino transferase, showed great response to change of the N supply form, especially to N starvation. Some of the genes involved in chlorophyll metabolism, carbon fixation and assimilation, were enhanced by ammonium supply only, but significantly suppressed by N-starvation. In the shoot there was increased expression of more general stress genes under nitrate when compared to ammonium nutrition. In the root the reverse situation was true with more apparent stress under ammonium nutrition. The microarray approach has revealed new levels of complexity in the response of rice to the form of N supply. Experiment Overall Design: Rice seedlings were grown in a hydroponic system in a growth chamber with control of both temperature and light. After normal growth for two weeks and for one week with completely avoiding any N source, the plants at four and a half leaf stage were re-supplied with ammonium nitrate (equal amount of either form of N), or only ammonium (ammonium sulphate and ammonium chloride), or only nitrate (calcium nitrate and magnesium nitrate), and continuing at zero N (N starvation). All the other essential nutrients were the same for all four treatments, except for sulfate and chloride which ranged between 1.1 â 2.6 mM and 0.5 â 2.5 mM, respectively. In Arabidopsis, addition of 3 mM Cl to âcontrolsâ did not produce any changes in the expression of genes in an experiment investigating the re-supply of 3 mM nitrate (Scheible et al., 2004). In other microarray experiments, extra addition of 5 mM Cl for Arabidopsis (Wang et al., 2000; Wang et al., 2004) and 1.4 mM sulphate for tomato (Wang et al., 2001) were used to replace nitrate for identifying the N deprivation responsive genes. Therefore, we assume that the relative smaller difference in sulfate and chloride concentrations among the four treatments in the normal supply range for plants would not significantly affect the expression profile of genes responsible for changes in N supply form and starvation in our experiments. Experiment Overall Design: For identifying the genes responding to the various N conditions by micro-array analysis, we extracted total RNA respectively from the roots and shoots at the 96 h after initiation of the four respective treatments. We used amplified and labeled cRNA from ammonium nitrate treatment as control, we performed array-hybridization for the cRNA from the treatment of single ammonium form, single nitrate form, or continuous N depletion, respectively. Agilent 60-mer oligonucleotide arrays containing 21,938 unique transcription units (Agilent Technologies, Tokyo, Japan) were used. Two biological replicates of each treatment were performed for microarray analyses.

Project description:Background: The global demand for affordable carbon has never been stronger, and there is an imperative in many industrial processes to use waste streams to make products. Gas-fermenting acetogens offer a potential solution and several commercial gas fermentation plants are currently under construction. As energy limits acetogen metabolism, supply of H2 should diminish substrate loss to CO2 and facilitate production of reduced and energy-intensive products. However, the effects of H2 supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions. Results: Here, we quantify the effects of H2 supplementation by comparing growth on CO, syngas, and a high-H2 CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model (GEM). CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H2. We show that H2 supply strongly impacts carbon distribution with a four-fold reduction in substrate loss as CO2 (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H2 supplementation lowers the molar acetate/ethanol ratio by five-fold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H2 availability provided reducing power via H2 oxidation and saved redox as cells reduced all the CO2 to formate directly using H2 in the Wood-Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin-reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity. Conclusions: Our quantitative molecular analysis revealed that H2 drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H2 supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses.

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Nitrogen gas supply to lactate-based chain elongation

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