Efficient Production Of Succinic Acid Research Articles

Modeling of batch fermentation kinetics for succinic acid production by Mannheimia succiniciproducens

Kinetic models are proposed for the batch production of succinic acid from glucose by Mannheimia succiniciproducens MBEL55E. The models include terms accounting for both substrate and product inhibitions. Experimental data collected from a series of batch fermentations with different initial glucose concentrations were used to estimate parameters and also to validate the models proposed. The optimal values of the parameters were approximated by minimizing the discrepancy between the model predictions and corresponding experimental data. The growth of M. succiniciproducens could be expressed by a modified Monod model incorporating inhibitions of glucose and organic acids accumulated in the culture broth. The Luedeking–Piret model was able to describe the formation of organic acids as the fermentation proceeded, in which succinic, acetic, and formic acids followed a mixed-growth-associated pattern. However, unexpectedly, lactic acid fermentation by M. succiniciproducens was nearly nongrowth-associated. In all cases, the model simulation matched well with the experimental observations, which made it possible to elucidate the fermentation characteristics of M. succiniciproducens during efficient succinic acid production from glucose. These models thus can be employed for the development and optimization of biobased succinic acid production processes.

Platform chemical production from wheat-based biorefining strategy

Development of biorefineries has attracted increasing attention around the world in the past decade. It focuses on alleviating pressure on fossil resources and the environmental burden of current fossil based processes, as well as facilitating sustainable development of the chemical industry. Many chemicals that used to be produced via chemical processes can now potentially be generated biologically from renewable raw materials. At the Satake Centre for Grain Process Engineering, a novel cost-competitive wheat-based biorefining strategy leading to the production of generic microbial feedstocks has been successfully developed (Webb and Wang, 1997 and Webb et al., 2004). These feedstocks are now being evaluated for the production of platform chemicals, biodegradable polymers and biofuels by bacterial fermentation. In the study reported here, succinic acid has been used as a model product for this biorefining strategy to convert wheat flour into fine chemicals. Another advantage of bio-synthesis of succinic acid is the fixation of CO2, which can help towards tackling the global warming challenge. Wheat is one of the predominant renewable resources in Europe. We aim to exploit all components of the wheat grain, producing both value-added end-products and precursors for chemical synthesis. In our process, whole wheat flour firstly undergoes fungal fermentation by Aspergillus awamori. The broth filtrate that is rich in glucoamylase is then used to hydrolyse flour components to generate a glucose-rich stream (flour hydrolysate) while the solid residue is used for fungal autolysis to produce a nutrient-rich stream (fungal autolysate). The possibility of replacing a commercial semi-defined medium by a combination of flour hydrolysate and fungal autolysate was investigated sequentially. Batch fermentations were performed using a complex medium containing wheat flour hydrolysate instead of commercial glucose. This resulted in a similar yield (0.64 g succinic acid per g glucose) and final succinic acid concentration (27 g/L) compared to fermentations with a semi-defined medium. The use of fungal autolysate instead of yeast extract as nitrogen source led to a succinic acid production of 23.2 g/L with a yield of 0.54 g/g. Even fermentations using the wheat-derived flour hydrolysate and fungal autolysate alone resulted in a succinic acid concentration of almost 16 g/L and an overall yield of 0.19 g succinic acid per g wheat flour. When fermentations were carried out with wheat-derived feedstock and an addition of 50 g/L MgCO3, 36.9 g/L succinic acid was produced from 50 g/L initial glucose with a productivity of 1.54 g/L h. The results of these studies demonstrate that a wheat-based biorefinery employing coupled fungal fermentation with subsequent flour hydrolysis and fungal autolysis can produce a suitable feedstock for efficient production of succinic acid.

Succinic acid production from wheat using a biorefining strategy

The biosynthesis of succinic acid from wheat flour was investigated in a two-stage bio-process. In the first stage, wheat flour was converted into a generic microbial feedstock either by fungal fermentation alone or by combining fungal fermentation for enzyme and fungal bio-mass production with subsequent flour hydrolysis and fungal autolysis. In the second stage, the generic feedstock was converted into succinic acid by bacterial fermentation by Actinobacillus succinogenes. Direct fermentation of the generic feedstock produced by fungal fermentation alone resulted in a lower succinic acid production, probably due to the low glucose and nitrogen concentrations in the fungal broth filtrate. In the second feedstock production strategy, flour hydrolysis conducted by mixing fungal broth filtrate with wheat flour generated a glucose-rich stream, while the fungal bio-mass was subjected to autolysis for the production of a nutrient-rich stream. The possibility of replacing a commercial semi-defined medium by these two streams was investigated sequentially. A. succinogenes fermentation using only the wheat-derived feedstock resulted in a succinic acid concentration of almost 16 g l(-1) with an overall yield of 0.19 g succinic acid per g wheat flour. These results show that a wheat-based bio-refinery employing coupled fungal fermentation and subsequent flour hydrolysis and fungal autolysis can lead to a bacterial feedstock for the efficient production of succinic acid.

Economical succinic acid production from cane molasses by Actinobacillus succinogenes

In this work, production of succinic acid by Actinobacillus succinogenes CGMCC1593 using cane molasses as a low cost carbon source was developed. In anaerobic bottles fermentation, succinic acid concentration of 50.6 ± 0.9 g l −1 was attained at 60 h using an optimum medium containing molasses pretreated with sulfuric acid, resulting in a succinic acid yield of 79.5 ± 1.1% and sugar utilization of 97.1 ± 0.6%. When batch fermentation was carried out in a 5-l stirred bioreactor with pretreated molasses, 46.4 g l −1 of succinic acid was attained at 48 h and faster cells growth was also observed. Fed batch fermentation was performed to minimize the substrate (sugar) inhibition effect, giving 55.2 g l −1 of succinic acid and 1.15 g l −1 h −1 of productivity at 48 h. The present study suggests that the inexpensive cane molasses could be utilized for the economical and efficient production of succinic acid by A. succinogenes.

Genome‐scale analysis of Mannheimia succiniciproducens metabolism

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is a capnophilic gram-negative bacterium that efficiently produces succinic acid, an industrially important four carbon dicarboxylic acid. In order to design a metabolically engineered strain which is capable of producing succinic acid with high yield and productivity, it is essential to optimize the whole metabolism at the systems level. Consequently, in silico modeling and simulation of the genome-scale metabolic network was employed for genome-scale analysis and efficient design of metabolic engineering experiments. The genome-scale metabolic network of M. succiniciproducens consisting of 686 reactions and 519 metabolites was constructed based on reannotation and validation experiments. With the reconstructed model, the network structure and key metabolic characteristics allowing highly efficient production of succinic acid were deciphered; these include strong PEP carboxylation, branched TCA cycle, relative weak pyruvate formation, the lack of glyoxylate shunt, and non-PTS for glucose uptake. Constraints-based flux analyses were then carried out under various environmental and genetic conditions to validate the genome-scale metabolic model and to decipher the altered metabolic characteristics. Predictions based on constraints-based flux analysis were mostly in excellent agreement with the experimental data. In silico knockout studies allowed prediction of new metabolic engineering strategies for the enhanced production of succinic acid. This genome-scale in silico model can serve as a platform for the systematic prediction of physiological responses of M. succiniciproducens to various environmental and genetic perturbations and consequently for designing rational strategies for strain improvement.

Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation

Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.