RESUMEN
In this study, corn steep liquor powder (CSL) was used as nitrogen source to replace the relatively costly yeast extract typically used for the production of succinic acid with Actinobacillus succinogenes NJ113. Moreover, when heme was added to the fermentation medium and the culture was agitated at a low speed, a maximum succinic acid concentration of 37.9 g/l was obtained from a glucose concentration of 50 g/l, and a productivity of 0.75 g/l/h was achieved. These yields are almost as high as for fermentation with glucose and yeast extract. These results suggest that heme-supplemented CSL may be a suitable alternative nitrogen source for a cost-effective method of producing succinic acid with A. succinogenes NJ113 while consuming less energy than previous methods.
Asunto(s)
Actinobacillus/metabolismo , Biotecnología/métodos , Nitrógeno/metabolismo , Ácido Succínico/metabolismo , Residuos , Zea mays/química , Actinobacillus/efectos de los fármacos , Actinobacillus/crecimiento & desarrollo , Técnicas de Cultivo Celular por Lotes , Medios de Cultivo/farmacología , Fermentación/efectos de los fármacos , Hemo/farmacología , Nitrógeno/farmacología , Polvos , Factores de TiempoRESUMEN
Escherichia coli NZN111 is a double mutant with inactivated lactate dehydrogenase and pyruvate formate-lyase. It cannot utilize glucose anaerobically because of its unusually high intracellular NADH/NAD(+) ratio. We have now constructed a recombinant strain, E. coli NZN111/pTrc99a-mdh, which, during anaerobic fermentation, produced 4.3 g succinic acid l(-1) from 13.5 g glucose l(-1). The NADH/NAD(+) ratio decreased from 0.64 to 0.26. Furthermore, dual-phase fermentation (aerobic growth followed by anaerobic phase) resulted in enhanced succinic acid production and reduced byproduct formation. The yield of succinic acid from glucose during the anaerobic phase was 0.72 g g(-1), and the productivity was 1.01 g l(-1) h(-1).
Asunto(s)
Escherichia coli/fisiología , Mejoramiento Genético/métodos , Glucosa/metabolismo , Malato Deshidrogenasa/metabolismo , Ácido Succínico/metabolismo , Malato Deshidrogenasa/genética , Regulación hacia ArribaRESUMEN
Escherichia coli AFP111, a pflB, ldhA, ptsG triple mutant of E. coli W1485, can be recovered for additional succinate production in fresh medium after two-stage fermentation (an aerobic growth stage followed by an anaerobic production stage). However, the specific productivity is lower than that of two-stage fermentation. In this study, three strategies were compared for reusing the cells. It was found when cells were aerobically cultivated at the end of two-stage fermentation without supplementing any carbon source, metabolites (mainly succinate and acetate) could be consumed. As a result, enzyme activities involved in the reductive arm of tricarboxylic acid cycle and the glyoxylate shunt were enhanced, yielding a succinate specific productivity above g⻹(DCW)h⻹ and a mass yield above 0.90 g g⻹ in the subsequent anaerobic fermentation. In addition, the intracellular NADH of cells subjected to aerobic cultivation with metabolites increased by more than 3.6 times and the ratio of NADH to NAD+ increased from 0.4 to 1.3, which were both favorable for driving the TCA branch to succinate.
Asunto(s)
Biotecnología/métodos , Escherichia coli/metabolismo , Ácido Succínico/química , Aerobiosis , Anaerobiosis , Técnicas de Química Analítica/métodos , Ciclo del Ácido Cítrico , Medios de Cultivo , Fermentación , Modelos Químicos , NAD/química , Factores de TiempoRESUMEN
The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 µg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources.
Asunto(s)
Actinobacillus/metabolismo , Fibras de la Dieta/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/síntesis química , Residuos/análisis , Zea mays/metabolismo , Actinobacillus/efectos de los fármacos , Biotina/farmacología , Carbono/farmacología , Fermentación/efectos de los fármacos , Glucosa/farmacología , Hidrólisis/efectos de los fármacos , Nitrógeno/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Vitaminas/farmacología , Zea mays/efectos de los fármacosRESUMEN
Escherichia coli AFP111 cells recovered from spent two-stage fermentation broth were investigated for additional production of succinic acid under anaerobic conditions. Recovered cells produced succinic acid in an aqueous environment with no nutrient supplementation except for glucose and MgCO(3). In addition, initial glucose concentration and cell density had a significant influence on succinic acid mass yield and productivity. Although the final concentration of succinic acid from recovered cells was lower than from two-stage fermentation, an average succinic acid mass yield of 0.85 g/g was achieved with an average productivity of 1.81 g/l h after three rounds of recycling, which was comparable to two-stage fermentation. These results suggested that recovered cells might be reused for the efficient production of succinic acid.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Redes y Vías Metabólicas/genética , Ácido Succínico/metabolismo , Anaerobiosis , Fermentación , Ingeniería Genética , Glucosa/metabolismo , Magnesio/metabolismoRESUMEN
Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by Escherichia coli AFP111 with knockouts in rpoS, pflAB, ldhA, and ptsG genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.