Efficient Production Of Succinic Acid Research Articles

Regulation of intracellular level of ATP and NADH in Escherichia coli to promote succinic acid production

Succinic acid is an important C4 platform chemical that is widely used in food, chemical, medicine sectors. The bottleneck of fermentative production of succinic acid by engineered Escherichia coli is the imbalance of intracellular cofactors, which often leads to accumulation of by-products, lower yield and low productivity. Stoichiometric analysis indicated that an efficient production of succinic acid by E. coli FMME-N-26 under micro-aeration conditions might be achieved when the TCA cycle provides enough ATP and NADH for the r-TCA pathway. In order to promote succinic acid production, a serial of metabolic engineering strategies include reducing ATP consumption, strengthening ATP synthesis, blocking NADH competitive pathway and constructing NADH complementary pathway were developed. As result, an engineered E. coli FW-17 capable of producing 139.52 g/L succinic acid and 1.40 g/L acetic acid in 5 L fermenter, which were 17.81% higher and 67.59% lower than that of the control strain, was developed. Further scale-up experiments were carried out in a 1 000 L fermenter, and the titer of succinic acid and acetic acid were 140.2 g/L and 1.38 g/L, respectively.

Engineering Corynebacterium glutamicum for efficient production of succinic acid from corn stover pretreated by concentrated-alkali under steam-assistant conditions

Corynebacterium glutamicum was developed for efficient production of succinic acid from corn stover (CS) pretreated by concentrated-alkali under steam-assistant (CASA) conditions. First, C. glutamicum was engineered by 1) blocking the by-products pathways (deletion of ldh, pta-ackA, and cat), 2) enhancing the carbon flux to succinate (overexpression of pyc and ppc), and 3) releasing the end-product inhibition (overexpression of Ncgl0275). The recombinant strain produced 117.8 g/L succinate in fed-batch fermentation. Second, to fully utilize xylose in lignocellulosic hydrolysate, two xylose utilization pathways—the isomerase pathway and the Weimberg pathway—were introduced into the recombinant strain. Third, CS was pretreated by CASA with a higher sugars yield and a lower black liquid. Finally, 64.16 g/L of succinic acid was obtained from 150 g/L CASA-pretreated CS by engineered C. glutamicum. These results showed a succinate high-producing C. glutamicum strain using glucose and xylose simultaneously as well as an effective and environmentally acceptable pretreatment strategy.

Immobilization of Actinobacillus succinogenes on nano- and micro-fiber membranes for efficient and robust production of succinic acid.

This work aimed to study the efficiency of nano- and micro- fiber membranes in immobilizing Actinobacillus succinogenes CCTCC M2012036 for succinic acid production. Among the four kinds of electrospun nanofiber membranes of cellulose acetate, chitosan, poly(vinyl alcohol) (PVA) and chitosan-PVA, the cellulose acetate nanofiber membrane-immobilized cells performed the best with a succinic acid concentration and yield to be 27.3 ± 3.5g/L and 70.9 ± 5.8%. The cell-immobilized viscose microfiber membrane presented good reuse stability, and 17 batches of fermentation without activity loss were realized with the highest succinic acid yield of 83.20%. A microfiber membrane bioreactor was further constructed with the cell-immobilized viscose microfiber membrane to perform fermentation on a larger scale, and the concentration, yield and productivity of succinic acid were 73.20g/L, 86.50% and 1.49g/(L⋅h) using a fed-batch strategy, which were 124.30%, 127.60% and 124.2% of those obtained in the traditional fermenter. This study provided an approach for improving the practicality of biological succinic acid production.

A novel biocatalyst, Enterobacter aerogenes LU2, for efficient production of succinic acid using whey permeate as a cost-effective carbon source

BackgroundSuccinic acid (SA), a valuable chemical compound with a broad range of industrial uses, has become a subject of global interest in recent years. The bio-based production of SA by highly efficient microbial producers from renewable feedstock is significantly important, regarding the current trend of sustainable development.ResultsIn this study, a novel bacterial strain, LU2, was isolated from cow rumen and recognized as an efficient producer of SA from lactose. Proteomic and genetic identifications as well as phylogenetic analysis were performed, and strain LU2 was classified as an Enterobacter aerogenes species. The optimal conditions for SA production were 100 g/L lactose, 10 g/L yeast extract, and 20% inoculum at pH 7.0 and 34 °C. Under these conditions, approximately 51.35 g/L SA with a yield of 53% was produced when batch fermentation was conducted in a 3-L stirred bioreactor. When lactose was replaced with whey permeate, the highest SA concentration of 57.7 g/L was achieved with a yield and total productivity of 62% and 0.34 g/(L*h), respectively. The highest productivity of 0.67 g/(L*h) was observed from 48 to 72 h of batch fermentation, when E. aerogenes LU2 produced 16.23 g/L SA.ConclusionsThis study shows that the newly isolated strain E. aerogenes LU2 has great potential as a new biocatalyst for producing SA from whey permeate.

Efficient succinic acid production from high-sugar-content beverages by Actinobacillus succinogenes.

This study presents the production of succinic acid (SA) by Actinobacillus succinogenes using high-sugar-content beverages (HSCBs) as feedstock. The aim of this study was the valorization of a by-product stream from the beverage industry for the production of an important building block chemical, such as SA. Three types of commercial beverages were investigated: fruit juices (pineapple and ace), syrups (almond), and soft drinks (cola and lemon). They contained mainly glucose, fructose, and sucrose at high concentration-between 50 and 1,000 g/L. The batch fermentation tests highlighted that A. succinogenes was able to grow on HSCBs supplemented with yeast extract, but also on the unsupplemented fruit juices. Indeed, the bacteria did not grow on the unsupplemented syrup and soft drinks because of the lack of indispensable nutrients. About 30-40 g/L of SA were obtained, depending on the type of HSCB, with yield ranging between 0.75 and 1.00 gSA /gS . The prehydrolysis step improved the fermentation performance: SA production was improved by 6-24%, depending on the HSCB, and sugar conversion was improved of about 30-50%.

Efficient succinic acid production using a biochar-treated textile waste hydrolysate in an in situ fibrous bed bioreactor

Textile waste contains biodegradable fraction which can be used as an alternative feedstock for succinic acid production. The feasibility of bioconversion of biochar-treated textile waste hydrolysate into succinic acid was evaluated. A substrate loading of 9% was applied to hydrolyze mixed textile waste and the resultant glucose-rich hydrolysate with dyestuff was collected after enzymatic hydrolysis. Biochar from different pyrolysis temperatures (400–700 °C) and different dosages of the selected biochar (1–5%) were applied to remove colorants. The results showed that biochar had a good performance at a dosage of 2 w/w % on removal of colorant inhibitors. There was no negative effect observed during the subsequent fermentation. After optimization of fermentation media in shake flasks, the resultant succinic acid titer reached 19.6 g/L with a SA yield of 0.76 g/g, supplemented with 30 g/L tryptone. The in situ fibrous bed bioreactor (isFBB) can further improve SA titer, up to 28.8 g/L corresponding to a yield of 0.61 g/g without pH control. Furthermore, a shorter lag phase during succinic acid fermentation in isFBB was observed. It can be concluded that a novel textile waste-based biorefinery approach for succinic acid production aided by biochar sorption was successfully developed.

Co-fermentation of glucose and xylose from sugarcane bagasse into succinic acid by Yarrowia lipolytica

This study focused on the feasibility of Y. lipolytica PSA02004 co-utilising glucose and xylose from sugarcane bagasse hydrolysate in succinic acid (SA) fermentation. Optimum pH, temperature and cellulase dosage of enzymatic hydrolysis through optimisation were pH 5, 50 °C and 40 FPU/g, respectively. The hydrolysis yields for glucose and xylose were 0.35 ± 0.00 g/g and 0.15 ± 0.00 g/g, respectively. A feasibility study using a 2.5-L bioreactor demonstrated that mixed glucose and xylose could be utilised for SA production, with the resultant SA titre, yield and productivity of 28.2 ± 0.6 g/L, 0.55 ± 0.01 g/g and 0.36 ± 0.01 g/L/h, respectively. Then, the investigation of nutrient-limiting factor showed 40 g/L of initial glucose concentration and 8.4 g/L of initial xylose concentration containing hydrolysate and 2% CSL were the optimum medium composition in SA fermentation. SA production using sugarcane bagasse hydrolysate was conducted, in which SA titre, yield and productivity were 33.2 ± 0.3 g/L, 0.58 ± 0.01 g/g and 0.33 ± 0.01 g/L/h, respectively. The experimental results reported in this study show that glucose and xylose in sugarcane bagasse hydrolysate were successfully co-utilised by Y. lipolytica PSA02004 in SA production. This implies a sugarcane waste biorefinery could lead to a generic feedstock for the efficient production of succinic acid.

Bio-refinery of waste streams for green and efficient succinic acid production by engineered Yarrowia lipolytica without pH control

This paper presents an efficient, green and sustainable approach for succinic acid (SA) fermentation, in which glucose-rich hydrolysates from various waste streams were used as the substrate for SA production by Yarrowia lipolytica without pH control for the first time. First, Y. lipolytica PGC202 was found to be able to consume glucose-rich mixed food waste (MFW) hydrolysate for SA production with high efficiency. After medium optimization, a SA titer at 31.7 g/L with a yield of 0.52 g/g and productivity of 0.60 g/L/h was obtained from isFBB fermentation when using 60 g/L glucose-containing MFW hydrolysate as the carbon source supplemented with 3% tryptone. When glucose-rich hydrolysate from other waste streams was used, the highest SA yield of 0.61 g/g via yeast fermentation at low pH was achieved, which demonstrated the extensive substrate adaptability and great potential of Y. lipolytica. By feeding concentrated MFW hydrolysate, SA titer up to 71.6 g/L was achieved from fed-batch fermentation with pH decreased naturally to 2.8. Finally, with the revised recovery method, 68.3% of the total SA with purity between 89.5% and 91.0% was recovered from the acidic fermentation broth with only low input of acid for acidification, which demonstrated the great advantages of SA production at low pH over that under neutral conditions in terms of economic improvement and environmental sustainability. In general, this study overcomes one of the major economic drawbacks for future feasible large-scale fermentative SA production, and it also illustrates a promising prospective for bio-SA production.

Efficient production of succinic acid in engineered Escherichia coli strains controlled by anaerobically-induced nirB promoter using sweet potato waste hydrolysate

Succinic acid has attracted interest worldwide as a precursor of many industrially crucial chemicals. Biosynthesis of succinic acid from biomass is developing as an environmentally friendly strategy now. Conversion of sweet potato waste (SPW) to succinic acid could implement high-value utilization of biomass, cut cost of the fermentation process and reduce the pollution of environment. Engineered Escherichia coli (E. coli) strain HD134 under the control of anaerobically-induced nirB promoter from Salmonella enterica (PSnirB) could produce about 16.30 g/L succinic acid with a yield of 0.83 g/g after 48 h on glucose. With SPW hydrolysate as the substrate, 18.65 g/L succinic acid with a yield of 0.94 g/g after 48 h fermentation achieved. Compared to SD134 under Trc control induced with Isopropyl β-D-Thiogalactoside (IPTG), this concentration and yield represented an 8.56% and 6.82% increase, respectively. The use of anaerobically-induced PSnirB not only could attain higher production of succinic acid than IPTG-induced Trc promoter, but omit cost of expensive exogenous inducers. The efficient production of succinic acid from SPW was firstly studied by anaerobically-induced PSnirB control, which achieved relative lower cost compared to glucose as substrate and IPTG as the inducer. This novel fermentation process conduces to the cosmically industrial succinic acid bioproduction.

Efficient production of succinic acid from herbal extraction residue hydrolysate

In this study, six different herbal-extraction residues were evaluated for succinic acid production in terms of chemical composition before and after dilute acid pretreatment (DAP) and sugar release performance. Chemical composition showed that pretreated residues of Glycyrrhiza uralensis Fisch (GUR) and Morus alba L. (MAR) had the highest cellulose content, 50% and 52%, respectively. Higher concentrations of free sugars (71.6 g/L total sugar) and higher hydrolysis yield (92%) were both obtained under 40 FPU/g DM at 10% solid loading for GUR. Using scanning electron microscopy (SEM), GUR was found to show a less compact structure due to process of extraction. Specifically, the fibers in pretreated GUR were coarse and disordered compared with that of GUR indicated by SEM. Finally, 65 g/L succinic acid was produced with a higher yield of 0.89 g/g total sugar or 0.49 g/g GUR. Our results illustrate the potential of GUR for succinic acid production.

Control of acetic acid metabolism of recombinant Yarrowia lipolytica for efficient succinic acid production

Succinic acid is a high value-added organic acid widely used in food, chemical and pesticide industries. As a new robust non-conventional yeast, Yarrowia lipolytica attracts more and more attention due to its potential for industrial applications. Previously, we obtained a succinic acid-producing strain through gene deletion of succinic acid dehydrogenase subunit encoding gene Ylsdh5, resulting in the strain of PGC01003. However, the recombinant strain produced large amount of acetic acid due to imbalance between glycolysis and TCA cycle which hindered the efficient production of succinic acid. PDH bypass was interfered to decrease the overflow of acetic acid and produce succinic acid under natural pH. Acetic acid was reduced to 4.6 g/L through heterologous expression of acetyl coenzyme A synthase from Salmonella enteric, which was 75.4% of the control strain. Deletion of CoA-transferase gene Ylach1 eliminated acetate formation and improved succinic acid production, and the resulting strain produced as high as 7.0 g/L succinic acid. Our study provides foundation for further construction of efficient cell factory of succinic acid production.

Hydrolysis of fruit and vegetable waste for efficient succinic acid production with engineered Yarrowia lipolytica

Abstract Bio-based production of succinic acid (SA) is regarded to supplement the petro-based processing for environmental reasons. However, in order to enhance the industrial competitiveness of bio-based SA, it is necessary to develop a method that is cost-effective, environmental friendly and with high efficiency. In this study, the utilization of fruit and vegetable waste (FVW) hydrolysate for SA production via engineered Yarrowia lipolytica was reported for the first time. In the study, the hydrolysis conditions were firstly optimized and the final glucose concentration of 56.7 g L−1 and a yield of 0.46 g g−1 were obtained with 2% glucoamylase, 1% cellulase, 2% hemicellulase and 0.25% pectinase at pH 5.0 and 55 °C. Then, the feasibility of using the hydrolysate for SA production via Y. lipolytica PSA02004 was demonstrated. SA titer and yield at 43.1 g L−1 and 0.46 g g−1, respectively were obtained using 100 g L−1 glucose-containing FVW hydrolysate and 4% corn steep liquor (CSL) as fermentation medium by free cell batch fermentation. Finally, in situ fibrous bed bioreactor (isFBB) and fed batch fermentation were demonstrated to be effective approach for improved SA production, from which SA productivity of 0.69 g L−1 h−1 and the resultant SA concentration of 140.6 g L−1 were achieved, respectively. In summary, this study achieved the anticipated target of turning waste into value-added products with high efficiency and cost-effectiveness.

Green and sustainable succinic acid production from crude glycerol by engineered Yarrowia lipolytica via agricultural residue based in situ fibrous bed bioreactor

In situ fibrous bed bioreactor (isFBB) for efficient succinic acid (SA) production by Yarrowia lipolytica was firstly developed in our former study. In this study, agricultural residues including wheat straw, corn stalk and sugarcane bagasse were investigated for the improvement of isFBB, and sugarcane bagasse was demonstrated to be the best immobilization material. With crude glycerol as the sole carbon source, optimization for isFBB batch fermentation was carried out. Under the optimal conditions of 20g sugarcane bagasse as immobilization material, 120gL−1 crude glycerol as carbon source and 4Lmin−1 of aeration rate, the resultant SA concentration was 53.6gL−1 with an average productivity of 1.45gL−1h−1 and a SA yield of 0.45gg−1. By feeding crude glycerol, SA titer up to 209.7gL−1 was obtained from fed batch fermentation, which was the highest value that ever reported.

Efficient production of succinic acid from duckweed (Landoltia punctata) hydrolysate by Actinobacillus succinogenes GXAS137

A novel process of enzyme pretreatment and semi-simultaneous saccharification and fermentation (SSSF) was developed in this work to improve succinic acid (SA) productivity from duckweed (Landoltia punctata) and achieve low viscosity. Viscosity (83.86%) was reduced by the pretreatment with combined enzymes at 50 °C for 2 h to a greater extent than that by single enzyme (26.19–71.75%). SSSF was an optimal combination with 65.31 g/L of SA content, which was remarkably higher than those obtained through conventional separate hydrolysis and fermentation (62.12 g/L) and simultaneous saccharification and fermentation (52.41 g/L). The combined approach was effective for SA production. Approximately 75.46 g/L of SA content with a yield of 82.87% and a productivity of 1.35 g/L/h was obtained after 56 h in a 2 L bioreactor. Further studies will focus on increasing the working scale of the proposed method.

Efficient production of succinic acid in immobilized fermentation with crude glycerol from Escherichia coli

Efficient production of succinic acid in immobilized fermentation with crude glycerol from Escherichia coli

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7g/L succinic acid with a yield of 0.53g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.

Influence of pH and neutralizing agent on anaerobic succinic acid production by a Corynebacterium crenatum strain

Environmental conditions, particularly pH, have significant effects on the efficiency and final titers of bio-based products. Therefore, these factors need to be identified to ensure the fermentation process is economically attractive. In this study, strategies for controlling pH were optimized to enhance succinic acid production by Corynebacterium crenatum J-2. The results indicate that pH 6.8 is the optimal value for anaerobic succinic acid production by C.crenatum J-2 in terms of productivity and titer. The use of Mg(OH)2 as the neutralizing agent for pH control resulted in the highest levels of succinic acid concentration, yield, and productivity; superior to the levels obtained with Ca(OH)2, KOH, and NaOH. Under conditions of pH 6.8 and Mg(OH)2 as the neutralizing agent, 45.7g/L succinic acid was produced within 12h during the prophase of anaerobic fermentation, resulting in a succinic acid productivity of 3.8g/(L·h). Succinic acid concentration reached 53.8g/L at 22h, with a productivity of 2.45g/(L·h). The results of this study will be useful for the development of highly efficient succinic acid production processes utilizing industrial Corynebacterium spp. strains.

Metabolic engineering of Mannheimia succiniciproducens for succinic acid production based on elementary mode analysis with clustering.

Mannheimia succiniciproducens, a capnophilic gram-negative rumen bacterium, has been employed for the efficient production of succinic acid. Although M. succiniciproducens metabolism was previously studied using a genome-scale metabolic model, more metabolic characteristics are to be understood. To this end, elementary mode analysis accompanied with clustering ('EMC' analysis) is used to gain further insights on metabolic characteristics of M. succiniciproducens allowing efficient succinic acid production. Elementary modes (EMs) generated from the central carbon metabolic network of M. succiniciproducens are clustered to systematically analyze succinic acid production routes. Based on the results of EMC analysis, zwf gene is identified as a novel overexpression target for the improved succinic acid production. This gene is overexpressed in a previously constructed succinic acid-overproducing M. succiniciproducens LPK7 strain. Heterologous NADPH-dependent mdh is later intuitively selected for overexpression to synergistically improve succinic acid production by utilizing abundant NADPH pool mediated by the overexpressed zwf. The LPK7 strains co-expressing mdh alone and both zwf and mdh genes are subjected to fed-batch fermentation to better examine their succinic acid production performances. Strategies of EMC analysis will be useful for further metabolic engineering of M. succiniciproducens and other microorganisms to improve production of succinic acid and other chemicals of interest.

Enterobacter sp. LU1 as a novel succinic acid producer - co-utilization of glycerol and lactose.

SummarySuccinic acid is an important C4‐building chemical platform for many applications. A novel succinic acid‐producing bacterial strain was isolated from goat rumen. Phylogenetic analysis based on the 16S rRNA sequence and physiological analysis indicated that the strain belongs to the genus Enterobacter. This is the first report of a wild bacterial strain from the genus Enterobacter that is capable of efficient succinic acid production. Co‐fermentation of glycerol and lactose significantly improved glycerol utilization under anaerobic conditions, debottlenecking the utilization pathway of this valuable biodiesel waste product. Succinic acid production reached 35 g l−1 when Enterobacter sp. LU1 was cultured in medium containing 50 g l−1 of glycerol and 25 g l−1 of lactose as carbon sources.

High efficiency succinic acid production from glycerol via in situ fibrous bed bioreactor with an engineered Yarrowia lipolytica

In this study, in situ fibrous bed bioreactor (isFBB) was developed at the first time for efficient succinic acid (SA) production by Yarrowia lipolytica. After optimization, SA titer, productivity and yield of 51.9g/L, 1.46g/L/h and 0.42g/g were obtained respectively via isFBB fermentation under conditions of 750cm2 cotton towel, 120g/L initial glycerol and 3L/min aeration rate. By fed batch strategy, SA titer raised up to 198.2g/L was achieved, which was the highest value ever reported. In operation stability study, SA productivity showed no obvious decrease after 12 repeated batches of 460h fermentation, and cell viability even recovered within two repeated batches after intentional interruption. This study successfully attained a highly efficient and stable isFBB for enhanced SA production by Y. lipolytica.