Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.     

Succinate production from different carbon sources under anaerobic conditions by metabolic engineered Escherichia coli strains

Succinic acid has drawn much interest as a precursor of many industrially important chemicals. Using a variety of feedstocks for the bio-production of succinic acid would be economically beneficial to future industrial processes. Escherichia coli SBS550MG is able to grow on both glucose and fructose, but not on sucrose. Therefore, we derived a SBS550MG strain bearing both the pHL413 plasmid, which contains Lactococcus lactis pycA gene, and the pUR400 plasmid, which contains the scrK, Y, A, B, and R genes for sucrose uptake and catalyzation. Succinic acid production by this modified strain and the SBS550pHL413 strain was tested on fructose, sucrose, a mixture of glucose and fructose, a mixture of glucose, fructose and sucrose, and sucrose hydrolysis solution. The modified strain can produce succinic acid efficiently from all combinations of different carbon sources tested with minimal byproduct formation and with high molar succinate yields close to that of the maximum theoretic values. The molar succinic acid yield from fructose was the highest among the carbon sources tested. Using the mixture of glucose and fructose as the carbon source resulted in slightly lower yields and much higher productivity than using fructose alone. Fermenting sucrose mixed with fructose and glucose gave a 1.76-fold higher productivity than that when sucrose was used as the sole carbon source. Using sucrose pretreated with sulfuric acid as carbon source resulted in a similar succinic acid yield and productivity as that when using the mixture of sucrose, fructose, and glucose. The results of the effect of agitation rate in aerobic phase on succinate production showed that supplying large amount of oxygen in aerobic phase resulted in higher productions of formate and acetate, and therefore lower succinate yield. This study suggests that fructose, sucrose, mixture of glucose and fructose, mixture of glucose, fructose and sucrose, or sucrose hydrolysis solution could be used for the economical and efficient production of succinic acid by our metabolic engineered E. coli strain.     

Enhanced production of succinic acid by Actinobacillus succinogenes with reductive carbon source

Carbon sources with different oxidation states were used to investigate the possibility increasing the availability of NADH and the NADH/NAD⁺ ratio and to determine the effect of this manipulation on the distribution of metabolites in Actinobacillus succinogenes NJ113. The sugars glucose, sorbitol and gluconate were each used at an initial concentration of 40 g/L.The yield of succinic acid (0.75) and the ratio of succinic acid to acetic acid (5.06) were both higher for sorbitol than the values obtained with glucose (0.66 and 2.68, respectively). In contrast, with gluconate as the carbon source the yield of succinic acid was 0.54 and the ratio of succinic acid to acetic acid was only 1.70. This work showed that different levels of NADH availability and the NADH/NAD⁺ ratio can be achieved by using carbon sources that have different oxidation states.Highly reduced sorbitol was examined as a possible carbon substrate for maximizing the redox potential during the production of succinic acid.     

Succinic acid production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source

Succinic acid was produced by fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source. When cells were anaerobically cultured in a medium containing 6.5 g/L glycerol, a high succinic acid yield (133%) was obtained while avoiding the formation of by-product acetic acid. The gram ratio of succinic acid to acetic acid was 25.8:1, which is 6.5 times higher than that obtained using glucose (ca. 4:1) as a carbon source. Therefore, succinic acid can be produced with much less by-product formation by using glycerol as a carbon source, which will facilitate its purification. When glucose and glycerol were cofermented with the increasing ratio of glucose to glycerol, the gram ratio of succinic acid to acetic acid and succinic acid yield decreased, suggesting that glucose enhanced acetic acid formation irrespective of the presence of glycerol. Glycerol consumption by A. succiniciproducens required unidentified nutritional components present in yeast extract. By intermittently feeding yeast extract along with glycerol, a high succinic acid yield (160%) could be obtained while still avoiding acetic acid formation. This resulted in the highest ratio of succinic acid to acetic acid (31.7:1).     

琥珀酸放线杆菌发酵培养基的优化

对琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC1593发酵产生琥珀酸培养基的主要成分,及其含量进行优化。通过单因素试验,得出发酵培养基中葡萄糖、酵母膏和玉米浆的含量对产生琥珀酸有显著影响;采用响应面法(RSM),得出多元二次回归方程拟合的三种因素与琥珀酸含量间的函数关系,并根据优化结果与实验,CGMCC1593产琥珀酸达到41.69g/L。     

Process development of succinic acid production by Escherichia coli NZN111 using acetate as an aerobic carbon source

Escherichia coli strain NZN111 could convert glucose to succinic acid efficiently in anaerobic conditions after the induction of gluconeogenic carbon sources in aerobic conditions. Acetate shows a strong effect on both yield and productivity of succinic acid. In this study, the fed-batch process of succinic acid production by NZN111 using acetate in a chemically defined medium in the aerobic stage was investigated and developed. Increasing cell density could increase succinic acid with a productivity of 3.97 g/(L h) in the first 8 h of the anaerobic phase with an overall yield of 1.42 mol/mol glucose in a 5 L fermentor. However, there was strong repression from succinic acid in the later anaerobic stage. When succinic acid exceeded 30 g/L, the glucose consumption rate began to drop sharply along with the succinic acid production rate. Supplementation with glucose from 30 to 70 g/L in the anaerobic stage showed little effect on succinic acid production. Acetic acid and pyruvic acid accumulated had no effect on succinic acid formation because of their low concentration. With acetate as the sole carbon source for aerobic cultivation in the following scale-up, 60.09 g/L of succinic acid was produced with a yield of 1.37 mol/mol in a 50 L bioreactor.     

Uptake and efflux of succinic acid by uninduced mycelium of Claviceps purpurea.

Claviceps purpurea PRL 1980 grew on partially dissociated succinic acid (pH 4) but not on fully dissociated succinic acid (pH 7.2). Myeclium suspended in 42 mM solution of partially ionized succinic acid (pH 4; 60.1% nonionized, 39% monoanion, and 0.9% dianion, K+ salt) over a period of 25 min accumulated more succinic acid carbon than mycelium suspended in highly ionized solution (pH 6.8; 0.01% nonionized, 4.8% monoanion, and 95% dianion). The greater accumulation from partially ionized solution was not attributable solely to metabolism of succinic acid nor to the lower external concentration of potassium ion. Rate of uptake by sodium azide and iodoacetate-treated mycelium was proportional to external concentration at least up to 200 mumol/ml. External potassium or sodium ion was not required for uptake by inhibited or uninhibited mycelium and external sodium ion and glucose did not allow concentration of succinic acid. The internal concentrations of succinic acid carbon expressed as succinic acid in cell water were about the same as the external concentrations. Uptake was not appreciably affected by extent of ionization of external succinic acid but accumulation was markedly affected. A plot of accumulated succinic acid carbon against external pH produced a bimodal curve with the two maxima corresponding to the maximal concentrations of nonionized and monoanion succinic acid. The bimodal curve probably results from overlapping of two separate curves; the nonionized form accumulating efficiently because of one interaction with the cell and the monoanion form accumulating efficiently because of another interaction. Uptake from concentrated solution is by diffusion and efflux is rapid but not complete. Efflux is not retarded by presence of phosphate in the external solution.     

Effects of dissolved CO2 levels on the growth of Mannheimia succiniciproducens and succinic acid production

A capnophilic rumen bacterium Mannheimia succiniciproducens produces succinic acid as a major fermentation end product under CO(2)-rich anaerobic condition. Since succinic acid is produced by carboxylation of C3 compounds during the fermentation, intracellular CO(2) availability is important for efficient succinic acid formation. Here, we investigated the metabolic responses of M. succiniciproducens to the different dissolved CO(2) concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO(2) concentration was below 8.74 mM. On the other hand, cell growth and succinic acid production increased proportionally as the dissolved CO(2) concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO(2) concentration of 141 mM were 1.49 and 1.52 times higher, respectively, than those obtained at the dissolved CO(2) concentration of 8.74 mM. It was also found that the additional CO(2) source provided in the form of NaHCO(3), MgCO(3), or CaCO(3) had positive effects on cell growth and succinic acid production. However, growth inhibition was observed when excessive bicarbonate salts were added. By the comparison of the activities of key enzymes, it was found that PEP carboxylation by PEP carboxykinase (PckA) is the most important for succinic acid production as well as the growth of M. succiniciproducens by providing additional ATP.     

Comparison of Growth and Primary Shunt Product Formation by Claviceps purpurea Cultured on Succinic Acid and Glucose as Carbon Sources

Growth on a medium containing succinic acid as the sole carbon source produced 1 g (dry weight) of mycelium per liter of medium by 50 days of incubation, whereas 25 g of mycelium was produced in 10 days when glucose was also present in the medium. Primary shunt metabolism took place during growth on succinic acid in spite of the extremely slow growth. Mycelia grown on succinic acid contained a higher percentage of residual mycelium and phosphate, but a lower percentage of mannitol, carbohydrate, lipid, and water-soluble nitrogen, than mycelia grown on a mixture of glucose and succinic acid. Thus, although primary shunt metabolism is favored by rapid growth on a rich, balanced sugar medium, it can also take place during extremely restricted growth in a medium containing succinic acid as the sole carbon source.     

Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO2-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.     

Phosphine Formation from Sewage Sludge Cultures

Gas-chromatographic analysis of the volatile compounds in the headspace of cultures inoculated with sewage sludge indicated phosphine production over a period of several days, a reaction which is clearly microbiologically mediated. The amounts of phosphine recovered from 40-mL specimens sewage sludge enrichment were in a pg-range of 5 to 100 in the presence of glucose as carbon source and phosphate as phosphorus source. Phosphine accumulation was found to be stimulated by increasing the phosphate concentration of the medium. Hypophosphite and lecithin as phosphorus sources instead of phosphate as well as mannitol and succinic acid instead of glucose as carbon sources affected the phosphine formation.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

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.     

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.     

一株琥珀酸产生菌的筛选及鉴定

从牛的瘤胃中筛选获得一株能发酵生产琥珀酸的兼性厌氧菌。对其进行生理生化特性鉴定及16S rRNA基因分析。该菌株短杆状,无鞭毛,革兰氏染色阴性,V-P反应阴性,能发酵多种糖类产酸;其16S rRNA基因与琥珀酸放线杆菌的同源性高达99．8%,认为属于琥珀酸放线杆菌(Actinobacillus succinogenes),并将其命名为琥珀酸放线杆菌(Actinobacillus succinogenes)SW0580,保藏号CGMCC 1593。初步发酵试验表明该菌能发酵60g/L葡萄糖产生25．8g/L的丁二酸。     

Batch and continuous fermentation of succinic acid from wood hydrolysate by Mannheimia succiniciproducens MBEL55E

Batch and continuous cultures of Mannheimia succiniciproducens MBEL55E were carried out in a complex medium containing a NaOH-treated wood hydrolysate for the production of succinic acid. The wood hydrolysate based medium was treated with NaOH before sterilization to reduce the formation of inhibitory compounds. M. succiniciproducens MBEL55E utilized xylose as well as glucose in the wood hydrolysate based medium as a carbon source for the succinic acid production. In batch cultures, the final succinic acid concentration of 11.73 g l⁻¹ was obtained from the pre-treated wood hydrolysate based medium, resulting in a succinic acid yield of 56% and a succinic acid productivity of 1.17 g l⁻¹ h⁻¹, while the corresponding continuous cultures gave the succinic acid yield and productivity of 55% and 3.19 g l⁻¹ h⁻¹, respectively. These results suggest that succinic acid can be produced economically and efficiently by the fermentation of M. succiniciproducens MBEL55E from an inexpensive biomass-based wood hydrolysate.     

Inability to Find Consistent Bacterial Biocontrol Agents of Pythium aphanidermatum in Cucumber Using Screens Based on Ecophysiological Traits

A collection of 821 rhizobacteria from cucumber, originating from different root locations and stages of plant development, was screened for potential biocontrol agents of Pythium aphanidermatum (Edson) Fitzp. The screening procedure exploited carbon source utilization profiles and growth rates of bacteria as indicators of a partial niche overlap with the pathogen. The bacteria were tested for growth on nine carbon sources (glucose, fucose, sucrose, maltose, asparagine, alanine, galacturonic acid, succinic acid, and linoleic acid), most of which are reported to be used by the zoospores of P. aphanidermatum in the infection process. The isolates were classified as fast- or slow-growing, depending on their growth rate in 1/10 strength TSB. By nonhierarchical cluster analysis, 20 clusters were generated of bacteria with similar profiles of carbon source utilization. Redundancy analysis showed that the type of root sample explained 47% of the variance found in the relative abundance of bacteria from the clusters. Bacteria from clusters using none or few of the carbon sources, e.g., maltose and linoleic acid, with many slow-growing isolates, showed a preference for plants in the vegetative or generative stage, or for old root regions (root base). Bacteria from clusters with fast-growing isolates, using many carbon sources, were relatively abundant in the seedling stage. A selection of 127 bacteria from the different clusters was tested for disease suppressive capabilities in bioassays on young cucumber plants in nutrient solution, inoculated with zoospores of P. aphanidermatum. Nine of these bacteria produced biosurfactants, and 27 showed antibiosis against mycelial growth in plate assays. For 31 isolates, significant positive effects on plant biomass were shown, as analyzed with a general linear regression model. For most isolates, these effects occurred only in one of two replicate assays and no reductions in the degree of root and crown rot were found. Of the isolates that used many of the tested carbon sources, only four had positive effects on plant biomass. The majority of the isolates that positively affected plant biomass used few to moderate numbers of carbon sources and did not produce antibiotics or biosurfactants. In conclusion, competition for the tested carbon sources with the zoospores did not play a decisive role in disease suppression, and no clear relation was found between ecophysiological traits and disease suppression. Only isolate 3.1T8, isolated from root tips in the generative stage of plant growth, significantly increased plant biomass and suppressed root and crown rot symptoms in five out of six bioassays. The isolate produced an antifungal substance in plate assays and showed biosurfactant production in several (cucumber-derived) media.     

Improved Succinic Acid Production in the Anaerobic Culture of an Escherichia coli pflB ldhA Double Mutant as a Result of Enhanced Anaplerotic Activities in the Preceding Aerobic Culture

Escherichia coli NZN111 is a pflB ldhA double mutant which loses its ability to ferment glucose anaerobically due to redox imbalance. In this study, two-stage culture of NZN111 was carried out for succinic acid production. It was found that when NZN111 was aerobically cultured on acetate, it regained the ability to ferment glucose with succinic acid as the major product in subsequent anaerobic culture. In two-stage culture carried out in flasks, succinic acid was produced at a level of 11.26 g/liter from 13.4 g/liter of glucose with a succinic acid yield of 1.28 mol/mol glucose and a productivity of 1.13 g/liter·h in the anaerobic stage. Analyses of key enzyme activities revealed that the activities of isocitrate lyase, malate dehydrogenase, malic enzyme, and phosphoenolpyruvate (PEP) carboxykinase were greatly enhanced while those of pyruvate kinase and PEP carboxylase were reduced in the acetate-grown cells. The two-stage culture was also performed in a 5-liter fermentor without separating the acetate-grown NZN111 cells from spent medium. The overall yield and concentration of succinic acid reached 1.13 mol/mol glucose and 28.2 g/liter, respectively, but the productivity of succinic acid in the anaerobic stage dropped to 0.7 g/liter·h due to cell autolysis and reduced anaplerotic activities. The results indicate the great potential to take advantage of cellular regulation mechanisms for improvement of succinic acid production by a metabolically engineered E. coli strain.     

Metabolism of the dimethyl ester of [2,3-13C]succinic acid in rat hepatocytes

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-13C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-13C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.     

Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO₂-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.