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Thermotoga neapolitana is a marine hyperthermophilic bacterium that produces hydrogen as a growth-associated product. The objectives of this research were to quantify hydrogen production by T. neapolitana using alternative organic carbon and nitrogen sources derived from agricultural feedstocks in the fermentation medium to replace glucose and yeast extract/Trypticase peptone, and to investigate growth and product formation in glucose medium. Organic carbon sources tested were beet pulp pellet, cellobiose, cellulose, corn starch, rice flour, starch, sucrose, xylan, and xylose, and organic nitrogen sources tested were canola meal, cottonseed meal, fish meal, linseed meal, and soybean meal. Hydrogen concentrations measured after batch incubations of T. neapolitana at 77C were not significantly different for media containing glucose, sucrose, or xylan, suggesting that feedstocks or hydrolysates with these sugars have potential for biohydrogen production. Soybean meal and canola meal were found to be promising organic nitrogen alternatives to yeast extract with or without Trypticase peptone for hydrogen production by T. neapolitana, an important result since prior investigators reported no growth in medium without yeast extract. The cell and hydrogen product yields for T. neapolitana cultured in glucose medium at incubation temperature of 77C were 0.233 g cell mass g-1 glucose and 0.0326 g H2 g-1 glucose (2.91 mol H2 mol-1 glucose), respectively, and the growth-associated product constant was 0.140 g H2 g-1 cell mass.