Economical Succinic Acid Production Research Articles

Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification.

Succinic acid (SA) is one of the fermentative products of anaerobic metabolism, and an important industrial chemical that has been much studied for its bio-based production. The key to the economically viable bio-based SA production is to develop an SA producer capable of producing SA with high yield and productivity without byproducts. Mannheimia succiniciproducens is a capnophilic rumen bacterium capable of efficiently producing SA. In this study, in silico genome-scale metabolic simulations were performed to identify gene targets to be engineered, and the PALK strain (ΔldhA and Δpta-ackA) was constructed. Fed-batch culture of PALK on glucose and glycerol as carbon sources resulted in the production of 66.14 g/L of SA with the yield and overall productivity of 1.34 mol/mol glucose equivalent and 3.39 g/L/h, respectively. SA production could be further increased to 90.68 g/L with the yield and overall productivity of 1.15 mol/mol glucose equivalent and 3.49 g/L/h, respectively, by utilizing a mixture of magnesium hydroxide and ammonia solution as a pH controlling solution. Furthermore, formation of byproducts was drastically reduced, resulting in almost homo-fermentative SA production. This allowed the recovery and purification of SA to a high purity (99.997%) with a high recovery yield (74.65%) through simple downstream processes composed of decolorization, vacuum distillation, and crystallization. The SA producer and processes developed in this study will allow economical production of SA in an industrial-scale. Biotechnol. Bioeng. 2016;113: 2168-2177. © 2016 Wiley Periodicals, Inc.

Economical Succinic Acid Production from Sugarcane Juice by Actinobacillus succinogenes Supplemented with Corn Steep Liquor and Peanut Meal as Nitrogen Sources

Production of succinic acid by Actinobacillus succinogenes GXAS137 using sugarcane juice as a low-cost carbon source was developed. The optimum substrate concentration, expressed in its glucose content, was 70 g/L. As yeast extract (YE) supplementation is not economically attractive, various nitrogen sources were compared with YE in terms of their efficiency for succinic acid production. When the elemental nitrogen ratio of corn steep liquor power (CSLP) to peanut meal (PM) was 3:1, the succinic acid concentration was 56.88 g/L. The yield is almost the same supplemented with YE. When batch fermentation was carried out in a 1.3 L stirred bioreactor, 57.06 g/L of succinic acid was produced. Fed-batch fermentation was performed to minimize the substrate inhibition effect, producing 62.06 g/L of succinic acid. These results suggest that sugarcane juice supplemented with CSLP and PM as nitrogen sources could be utilized for the economical and efficient production of succinic acid.

Economically enhanced succinic acid fermentation from cassava bagasse hydrolysate using Corynebacterium glutamicum immobilized in porous polyurethane filler

An immobilized fermentation system, using cassava bagasse hydrolysate (CBH) and mixed alkalis, was developed to achieve economical succinic acid production by Corynebacterium glutamicum. The C. glutamicum strains were immobilized in porous polyurethane filler (PPF). CBH was used efficiently as a carbon source instead of more expensive glucose. Moreover, as a novel method for regulating pH, the easily decomposing NaHCO3 was replaced by mixed alkalis (NaOH and Mg(OH)2) for succinic acid production by C. glutamicum. Using CBH and mixed alkalis in the immobilized batch fermentation system, succinic acid productivity of 0.42gL−1h−1 was obtained from 35gL−1 glucose of CBH, which is similar to that obtained with conventional free-cell fermentation with glucose and NaHCO3. In repeated batch fermentation, an average of 22.5gL−1 succinic acid could be obtained from each batch, which demonstrated the enhanced stability of the immobilized C. glutamicum cells.

Succinic acid production from hemicellulose hydrolysate by an Escherichia coli mutant obtained by atmospheric and room temperature plasma and adaptive evolution

Atmospheric and room temperature plasma and adaptive evolution were combined to generate Escherichia coli mutants, which can simultaneously and efficiently utilize glucose and xylose to produce succinic acid in chemically defined medium under exclusively anaerobic condition. Compared to the parent strain BA305, a pflB, ldhA, ppc, and ptsG deletion strain overexpressing ATP-forming phosphoenolpyruvate (PEP) carboxykinase (PEPCK), the sugar consumption rate and succinic acid productivity of mutant BA408 were significantly improved with a marked increase in the key enzyme activities. Subsequent anaerobic fermentation of BA408 with corn stalk hydrolysate produced a final succinic acid concentration of 23.1gL−1 with a yield of 0.85gg−1 sugar mixture. The observed synthesis of succinic acid from the corn stalk hydrolysate showed a great potential usage of renewable biomass as a feedstock for an economical succinic acid production using E. coli.

An engineering Escherichia coli mutant with high succinic acid production in the defined medium obtained by the atmospheric and room temperature plasma

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose in nutrient-rich or minimal media anaerobically. Co-expression of heterologous pyruvate carboxylase and nicotinic acid phosphoribosyltransferase in BA002 resulted in a significant increase in cell mass and succinic acid production. Nevertheless, the resultant strain, BA016, still could not grow in a defined medium without tryptone and yeast extract. To solve the problem, a novel atmospheric and room temperature plasma mutation method was employed to generate mutants which can grow in the defined medium. A mutant designated as LL016 was observed to be the best strain that regained the capacity of cell growth and glucose utilization in a defined medium anaerobically. After 120h of fermentation in the defined medium, 35.0g/L of glucose was consumed to generate 25.2g/L of succinic acid. Furthermore, with the highest glucose consumption rate in the dual-phase fermentation, the yield of succinic acid in LL016 reached 0.87g/g, which was higher than that observed in other strains. From an industrial standpoint, the defined medium is much cheaper than LB medium, which shows a great potential usage for the economical production of succinic acid by LL016.

Ultrasonic pretreatment and acid hydrolysis of sugarcane bagasse for succinic acid production using Actinobacillus succinogenes

Immense interest has been devoted to the production of bulk chemicals from lignocellulose biomass. Diluted sulfuric acid treatment is currently one of the main pretreatment methods. However, the low total sugar concentration obtained via such pretreatment limits industrial fermentation systems that use lignocellulosic hydrolysate. Sugarcane bagasse hemicellulose hydrolysate is used as the carbon and nitrogen sources to achieve a green and economical production of succinic acid in this study. Sugarcane bagasse was ultrasonically pretreated for 40 min, with 43.9 g/L total sugar obtained after dilute acid hydrolysis. The total sugar concentration increased by 29.5 %. In a 3-L fermentor, using 30 g/L non-detoxified total sugar as the carbon source, succinic acid production increased to 23.7 g/L with a succinic acid yield of 79.0 % and a productivity of 0.99 g/L/h, and 60 % yeast extract in the medium could be reduced. Compared with the detoxified sugar preparation method, succinic acid production and yield were improved by 20.9 and 20.2 %, respectively.

Spirit-based distillers’ grain as a promising raw material for succinic acid production

Spirit-based distillers' grain (SDG) is the main by-product of the Chinese liquor industry, with an annual output of approx. 100 million tons. The economical potential of fermentative production of succinic acid from SDG was investigated using Actinobacillus succinogens. Use of pretreated SDG (PSDG) as the sole source of C and N yielded succinic acid at 35.5 g l(-1) with a yield of 19.7 % (g per 100 g PSDG) after 48 h in a 3 l stirred bioreactor. SDG is thus a promising feedstock for the economical production of succinic acid.

A complete industrial system for economical succinic acid production by Actinobacillus succinogenes

An industrial fermentation system using lignocellulosic hydrolysate, waste yeast hydrolysate, and mixed alkali to achieve high-yield, economical succinic acid production by Actinobacillus succinogenes was developed. Lignocellulosic hydrolysate and waste yeast hydrolysate were used efficiently as carbon sources and nitrogen source instead of the expensive glucose and yeast extract. Moreover, as a novel method for regulating pH mixed alkalis (Mg(OH) 2 and NaOH) were first used to replace the expensive MgCO 3 for succinic acid production. Using the three aforementioned substitutions, the total fermentation cost decreased by 55.9%, and 56.4 g/L succinic acid with yield of 0.73 g/g was obtained, which are almost the same production level as fermentation with glucose, yeast extract and MgCO 3. Therefore, the cheap carbon and nitrogen sources, as well as the mixed alkaline neutralize could be efficiently used instead of expensive composition for industrial succinic acid production.

Efficient production of succinic acid from corn stalk hydrolysates by a recombinant Escherichia coli with ptsG mutation

Succinic acid is considered to be one of the key platform chemicals used in a variety of industrial applications. The exploitation of biomass to produce succinic acid requires a microbial type that can ferment the mixture of reducing sugars derived from lignocellulose. The recombinant Escherichia coli strains with homologous or cyanobacterial ppc overexpression and ldhA, pflB, ptsG mutations were constructed, and the mixed sugar fermentations were carried out with the prominent strain SD121. Then, a modeled corn stalk hydrolysates containing 30 g l −1 glucose, 10 g l −1 xylose and 2.5 g l −1 arabinose was applied for succinic acid fermentation with SD121. A yield of 0.77 g succinic acid g −1 total sugar was achieved. Fermentation of corn stalk hydrolysates with SD121 produced a final succinic acid concentration of 36.55 g l −1 with a higher yield of 0.83 g g −1 total sugar in anaerobic bottles. In two-stage fermentation process in bioreactor, initial aerobic growth facilitated the subsequent anaerobic succinic acid production with a final concentration of 57.81 g l −1, and a yield of 0.87 g g −1 total sugar. This was the first report of succinic acid production from corn stalk hydrolysates by metabolically engineered Escherichia coli. The higher succinic acid yield from corn stalk hydrolysates compared to modeled sugar mixtures, showed a great potential usage of renewable biomass as a feedstock for an economical succinic acid production using E. coli.

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l −1. In batch fermentation, 45.5 g l −1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l −1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l −1 at a rate of 1.21 g l −1 h −1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.

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.