Abstract:Succinic acid is an important platform chemical for synthesis of C4 compounds. We applied genome shuffling to improve fermentative production of succinic acid by A. succinogenes. Using a screening strategy composed of selection in fermentation broth, cultured in 96-deep-well plates, and condensed HPLC screening, a starting population of 11 mutants producing a higher succinic acid concentration was selected and subjected to recursive protoplasts fusion. After three rounds of genome shuffling, strain F3-II-3-F w… Show more
“…The FA/AA decrease corresponds to the observations of Bradfield & Nicol [15] where low D in a biofilm reactor resulted in a similar trend. The decrease in FA is most likely attributed to formate dehydrogenase converting FA to CO 2 and NADH since Zheng et al [24], Xi et al [27] and Du et al [29] observed a FA decrease against time after initial formation in a batch fermenter.Figure 3
Chemostat mass-based yield ratios (SA/AA, FA/AA and Y
Glc,SA
) plotted against D. The average Y Glc,SA is relatively stable at 0.48 ± 0.047 g.g −1 . FA/AA is close to the equimolar value (0.77 g.g −1 ) at a D of 0.8 h −1 , decreasing to zero as D is decreased.…”
Section: Resultsmentioning
confidence: 99%
“…This clearly indicates that non-growth or maintenance production of SA plays an important role and that yield differences exist between the growth and maintenance modes. In some studies, the consumption of FA is reported in the non-growth phase of the batch fermentation [24,27,29]. With regard to substrate inhibition, differences between biofilm and suspended cultures are reported [12] where established biofilms appear to be unaffected by the initial Glc concentration while the lag time of the suspended cultures (no lag for biofilm cultures) is clearly influenced by the initial Glc concentrations, with higher lag times reported for higher Glc concentrations [18].…”
BackgroundSuccinic acid is well established as bio-based platform chemical with production quantities expecting to increase exponentially within the next decade. Actinobacillus succinogenes is by far the most studied wild organism for producing succinic acid and is known for high yield and titre during production on various sugars in batch culture. At low shear conditions continuous fermentation with A. succinogenes results in biofilm formation. In this study, a novel shear controlled fermenter was developed that enabled: 1) chemostat operation where self-immobilisation was opposed by high shear rates and, 2) in-situ removal of biofilm by increasing shear rates and subsequent analysis thereof.ResultsThe volumetric productivity of the biofilm fermentations were an order of magnitude more than the chemostat runs. In addition the biofilm runs obtained substantially higher yields. Succinic acid to acetic acid ratios for chemostat runs were 1.28±0.2 g.g-1, while the ratios for biofilm runs started at 2.4 g.g-1 and increased up to 3.3 g.g-1 as glucose consumption increased. This corresponded to an overall yield on glucose of 0.48±0.05 g.g-1 for chemostat runs, while the yields varied between 0.63 g.g-1 and 0.74 g.g-1 for biofilm runs. Specific growth rates (μ) were shown to be severely inhibited by the formation of organic acids, with μ only 12% of μmax at a succinic acid titre of 7 g.L-1. Maintenance production of succinic acid was shown to be dominant for the biofilm runs with cell based production rates (extracellular polymeric substance removed) decreasing as SA titre increases.ConclusionsThe novel fermenter allowed for an in-depth bioreaction analysis of A. succinogenes. Biofilm cells achieve higher SA yields than suspended cells and allow for operation at higher succinic acid titre. Both growth and maintenance rates were shown to drastically decrease with succinic acid titre. The A. succinogenes biofilm process has vast potential, where self-induced high cell densities result in higher succinic acid productivity and yield.
“…The FA/AA decrease corresponds to the observations of Bradfield & Nicol [15] where low D in a biofilm reactor resulted in a similar trend. The decrease in FA is most likely attributed to formate dehydrogenase converting FA to CO 2 and NADH since Zheng et al [24], Xi et al [27] and Du et al [29] observed a FA decrease against time after initial formation in a batch fermenter.Figure 3
Chemostat mass-based yield ratios (SA/AA, FA/AA and Y
Glc,SA
) plotted against D. The average Y Glc,SA is relatively stable at 0.48 ± 0.047 g.g −1 . FA/AA is close to the equimolar value (0.77 g.g −1 ) at a D of 0.8 h −1 , decreasing to zero as D is decreased.…”
Section: Resultsmentioning
confidence: 99%
“…This clearly indicates that non-growth or maintenance production of SA plays an important role and that yield differences exist between the growth and maintenance modes. In some studies, the consumption of FA is reported in the non-growth phase of the batch fermentation [24,27,29]. With regard to substrate inhibition, differences between biofilm and suspended cultures are reported [12] where established biofilms appear to be unaffected by the initial Glc concentration while the lag time of the suspended cultures (no lag for biofilm cultures) is clearly influenced by the initial Glc concentrations, with higher lag times reported for higher Glc concentrations [18].…”
BackgroundSuccinic acid is well established as bio-based platform chemical with production quantities expecting to increase exponentially within the next decade. Actinobacillus succinogenes is by far the most studied wild organism for producing succinic acid and is known for high yield and titre during production on various sugars in batch culture. At low shear conditions continuous fermentation with A. succinogenes results in biofilm formation. In this study, a novel shear controlled fermenter was developed that enabled: 1) chemostat operation where self-immobilisation was opposed by high shear rates and, 2) in-situ removal of biofilm by increasing shear rates and subsequent analysis thereof.ResultsThe volumetric productivity of the biofilm fermentations were an order of magnitude more than the chemostat runs. In addition the biofilm runs obtained substantially higher yields. Succinic acid to acetic acid ratios for chemostat runs were 1.28±0.2 g.g-1, while the ratios for biofilm runs started at 2.4 g.g-1 and increased up to 3.3 g.g-1 as glucose consumption increased. This corresponded to an overall yield on glucose of 0.48±0.05 g.g-1 for chemostat runs, while the yields varied between 0.63 g.g-1 and 0.74 g.g-1 for biofilm runs. Specific growth rates (μ) were shown to be severely inhibited by the formation of organic acids, with μ only 12% of μmax at a succinic acid titre of 7 g.L-1. Maintenance production of succinic acid was shown to be dominant for the biofilm runs with cell based production rates (extracellular polymeric substance removed) decreasing as SA titre increases.ConclusionsThe novel fermenter allowed for an in-depth bioreaction analysis of A. succinogenes. Biofilm cells achieve higher SA yields than suspended cells and allow for operation at higher succinic acid titre. Both growth and maintenance rates were shown to drastically decrease with succinic acid titre. The A. succinogenes biofilm process has vast potential, where self-induced high cell densities result in higher succinic acid productivity and yield.
“…A 96-well plate assay was used to screen for mutant strains of high succinate production18. Mutants were spread on LB plates that contained 100 μg/mL ampicillin, 50 μg/mL kanamycin, and 10 μg/mL chloramphenicol, and then incubated at 37 °C for 12 h, after which the colonies were transferred to 96-well plates containing 1 mL of fermentation medium.…”
This study was initiated to improve E. coli succinate production by engineering the E. coli global transcription factor, Cra (catabolite repressor/activator). Random mutagenesis libraries were generated through error-prone PCR of cra. After re-screening and mutation site integration, the best mutant strain was Tang1541, which provided a final succinate concentration of 79.8 ± 3.1 g/L: i.e., 22.8% greater than that obtained using an empty vector control. The genes and enzymes involved in phosphoenolpyruvate (PEP) carboxylation and the glyoxylate pathway were activated, either directly or indirectly, through the mutation of Cra. The parameters for interaction of Cra and DNA indicated that the Cra mutant was bound to aceBAK, thereby activating the genes involved in glyoxylate pathway and further improving succinate production even in the presence of its effector fructose-1,6-bisphosphate (FBP). It suggested that some of the negative effect of FBP on Cra might have been counteracted through the enhanced binding affinity of the Cra mutant for FBP or the change of Cra structure. This work provides useful information about understanding the transcriptional regulation of succinate biosynthesis.
“…The yield of succinate is strongly related to NADH availability. Therefore, a flux distribution which supports higher NADH production is desirable for succinate formation (Singh et al, 2011;Zheng et al, 2013;Liu et al, 2010;Zheng et al, 2013;Li et al, 2010).…”
Section: Effect Of Deletions On Growth Of a Succinogenes And Succinamentioning
-Attention has been focused on microbial succinic acid production as an alternative for conventional chemical synthesis that is associated with environmental pollution. A metabolic model for Actinobacillus succinogenes 130Z was developed with a mixture of glucose and xylose as substrate. The metabolic fluxes during succinicate production were determined using flux balance analysis by linear programming optimization in the MATLAB environment. Different glucose ratios (0.3, 0.4 and 0.7 mol.mol -1 substrate) were used as model assumptions to calculate optimal fluxes, maximum growth and succinate production. The model revealed that higher growth rates and product yields were correlated with higher glucose content in the substrate mixture. When glucose constituted 0.5 mol.mol -1 substrate, a lower succinate yield (0.64 mol.mol -1 substrate) was obtained, compared to 0.73 mol.mol -1 substrate when glucose was used individually. Deletion of different unessential reactions in the model showed that a knockout of the acetate formation pathway would increase the succinate yield by 21% when glucose and xylose were used in equal molar ratios.
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