Effect of nutrient limitation in chemostat cultures on amino acid excretion in Clostridium thermocellum

Sammanfattning: Introduction: Clostridium thermocellum is considered a model organism forconsolidated bioprocessing, due to its ability to hydrolyze lignocellulosicbiomass more efficiently than many other organisms and to produce ethanol.In order to meet the industrial requirements of ethanol yield and titer, metabolicengineering efforts have been made resulting in a strain that successfullydisplays increased ethanol yield with reduced amount of some byproducts.However, the ethanol yield in this engineered strain still does not meet theindustrial requirements and significant amounts of amino acids are stillproduced. To attempt to decrease the level of amino acid excretion intended toimprove the ethanol yield in C. thermocellum, it is essential to understand itsmetabolism and how it is affected by different cultivation conditions and mediumcompositions. This study aimed to gain an insight in how carbon- and nitrogenlimitation affect amino acid excretion in C. thermocellum, with the hypothesisthat excess of carbon and nitrogen yields more amino acid excretion. Methods: Mass-balance based calculations of rates and yields were used toanalyze the metabolism of a wild-type of C. thermocellum (DSM 1313) grownanaerobically in carbon- or nitrogen-limiting chemostats. For this, Low-Carbonmedium containing, respectively, cellobiose (5 g/L) and urea (0.15 g/L) as thelimiting nutrient was used. Both cultivations were performed at 55 °C, pH 7.0and 400 RPM shaking at a dilution rate of 0.1 h-1. Conclusion:  Considering yields of total amino acids excreted in bothlimitations, it was hypothesized that C. thermocellum exploited the amino acidexcretion to maintain carbon balance around the pyruvate node caused byexcess of the carbon. Based on yield of valine excreted in particular, it washypothesized that amino acid excretion was used to maintain redox balance inthe metabolism of C. thermocellum, where malate shunt could play a major role.However, results of the Carbon-limitation did not allow any conclusion ofnitrogen excess having an effect on amino acid excretion in C. thermocellum.