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Systems Biology Of Thermotoga Neapolitana For Biological Hydrogen Production

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The hyperthermophile Thermotoga neapolitana was evaluated for hydrogen production in three phases of iterative experimental and computational modeling work. First, the effect of environmental perturbations and fermentation stoichiometry were experimentally determined. Variation of growth temperature within the permissible range did not affect product yields. Maximum H2 production rates were achieved in both 77 and 85 [DEGREE]C experiments. Low pH inhibited glucose consumption; when pH was raised, glucose consumption was complete. Oxygen exposure tests confirmed that O2 addition did not increase H2 production. The fermentation balance at 85 [DEGREE]C was 2.8 mol H2, 2 mol CO2, 1.8 mol acetate, and 0.1 mol lactate per mol of glucose consumed. Constraint-based analysis can increase understanding of metabolic network interactions and can predict effects of metabolic engineering thereby decreasing the need for costly experiments. A comparative reconstruction method was created to convert a constraint -based model for Thermotoga maritima into a model for T. neapolitana based on synteny between the two annotated genome sequences. Flux balance analysis simulations of T. neapolitana batch growth were validated with previously obtained experimental results. The T. neapolitana model was examined to identify mechanisms to allow growth with cysteine as a sole sulfur source, because unlike T. maritima which can only use elemental sulfur in a defined medium, T. neapolitana can also use cysteine. The results were inconclusive ; all genes with known functions related to sulfur metabolism were found in both species. Recent evidence in the literature that both species have a bifurcating hydrogenase that simultaneously utilizes ferredoxin and NADH was also incorporated into the T. neapolitana model. The modified model required the inclu sion of a membrane bound NADH:Ferredoxin oxidoreductase to maintain the correct NAD+/NADH ratio to support growth. Experimental validation of T. neapolitana model-derived hypotheses was conducted. Initial carbon substrate utilization predictions indicated that glycerol, Lrhamnose, and cellotetraose could not support growth. Experimental results showed that both glycerol and L-rhamnose did not support growth, however cellotetraose did support growth. Further analysis comparing protein expression data from cellotetraose and glucose grown cells suggests that the model can be updated to include a more complete cellotetraose pathway.

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2010-10-20

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dissertation or thesis

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