Increase In Succinic Acid Production Research Articles

Production of Succinic Acid by Metabolically Engineered Actinobacillus succinogenes from Lignocellulosic Hydrolysate Derived from Barley Straw.

Succinic acid is an industrially important component that plays a key role in food additives, dietary supplements, and precursors for biodegradable polymers. Due to environmental and economic issues, succinic acid production has become increasingly attractive. This work aimed to improve succinic acid production from lignocellulosic biomass in Actinobacillus succinogenes through genetic modifications and fermentation strategies. Firstly, the effects on succinic acid production by overexpressing genes encoding phosphoenol carboxylase, malate dehydrogenase, and fumarase were evaluated in batch fermentations of engineered A. succinogenes strains. The engineered A. succinogenes expressing PCK, MDH, and FUM (AS-PMF) showed a 1.3-fold increase in succinic acid production compared to the wild-type strain. Subsequently, the fed-batch fermentation with MgCO3 was carried out using AS-PMF, which led to producing 50 g/l of succinic acid with 0.79 g/g of yield. Finally, 22.2 g/l of succinic acid with 0.64 g/g of yield was achieved in batch fermentation from lignocellulosic hydrolysate of barley straw. These results support that sustainable succinic acid from agricultural wastes might be a promising strategy for industrial applications.

Mutation breeding of high-stress resistant strains for succinic acid production from corn straw

The production of succinic acid from corn stover is a promising and sustainable route; however, during the pretreatment stage, byproducts such as organic acids, furan-based compounds, and phenolic compounds generated from corn stover inhibit the microbial fermentation process. Selecting strains that are resistant to stress and utilizing nondetoxified corn stover hydrolysate as a feedstock for succinic acid production could be effective. In this study, A. succinogenes CICC11014 was selected as the original strain, and the stress-resistant strain A. succinogenes M4 was obtained by atmospheric and room temperature plasma (ARTP) mutagenesis and further screening. Compared to the original strain, A. succinogenes M4 exhibited a twofold increase in stress resistance and a 113% increase in succinic acid production when hydrolysate was used as the substrate. By conducting whole-genome resequencing of A. succinogenes M4 and comparing it with the original strain, four nonsynonymous gene mutations and two upstream regions with base losses were identified.Key points• A high-stress-resistant strain A. succinogenes M4 was obtained by ARTP mutation• The production of succinic acid increased by 113%• The mutated genes of A. succinogenes M4 were detected and analyzedGraphical

Enhanced Succinic Acid Production and Electronic Utilization Efficiency by Actinobacillus succinogenes 130Z in an ORP-Controlled Microbial Electrolysis Cell System

Microbial electrochemical systems have shown great value as a means of enhancing the efficiency of fermentation reactions, but at present, there is no reliable means to balance the extracellular electron supply and corresponding intracellular demands in these systems. The current work describes the unique use of an oxidation–reduction-potential (ORP)-level-controlled microbial electrolysis cell (MEC) system to successfully balance the extracellular electron supply and succinic acid fermentation via A. succinogenes (130Z). The ORP-controlled MEC system with neutral red (NR) yielded a significant increase in succinic acid production (17.21%). The utilization of NR in this MEC system improved the ORP regulatory sensitivity. The optimal approach to the ORP level control was the use of a −400 mV high-voltage electric pulse-based strategy, which increased the yield of succinic acid by 13.08% compared to the control group, and reduced the energy consumption to 52.29% compared to the potentiostatic method. When compared to the −1 V constant potential MEC system, the high-voltage electric pulse-based ORP strategy for the MEC system control provided sufficient electrons to this system while using less electricity (11.96%) and producing 12.48% (74.43 g/L) more succinic acid during fed-batch fermentation. The electronic utilization efficiency of the ORP-controlled MEC system was 192.02%, which was 15.19 times that of the potentiostatic system. The electronic utilization efficiency is significantly increased in the ORP-controlled MEC system. Succinic acid production is ensured by a high-voltage electric pulse-based method, while the influence on cell growth and power consumption are minimized. Fed-batch fermentation with the high-voltage electric pulse-based ORP strategy for MEC system control is noted to be ideal to achieve a further increase in succinic acid concentration and electronic utilization efficiency.

Diverse Profile of Fermentation Byproducts From Thin Stillage.

The economy of biorefineries is influenced not only by biofuel production from carbohydrates but also by the production of valuable compounds from largely underutilized industrial residues. Currently, the demand for many chemicals that could be made in a biorefinery, such as succinic acid (SA), medium-chain fatty acids (MCFAs), and lactic acid (LA), is fulfilled using petroleum, palm oil, or pure carbohydrates as raw materials, respectively. Thin stillage (TS), the residual liquid material following distillation of ethanol, is an underutilized coproduct from the starch biofuel industry. This carbon-rich material has the potential for chemical upgrading by microorganisms. Here, we explored the formation of different fermentation products by microbial communities grown on TS using different bioreactor conditions. At the baseline operational condition (6-day retention time, pH 5.5, 35°C), we observed a mixture of MCFAs as the principal fermentation products. Operation of a bioreactor with a 1-day retention time induced an increase in SA production, and a temperature increase to 55°C resulted in the accumulation of lactic and propionic acids. In addition, a reactor operated with a 1-day retention time at 55°C conditions resulted in LA accumulation as the main fermentation product. The prominent members of the microbial community in each reactor were assessed by 16S rRNA gene amplicon sequencing and phylogenetic analysis. Under all operating conditions, members of the Lactobacillaceae family within Firmicutes and the Acetobacteraceae family within Proteobacteria were ubiquitous. Members of the Prevotellaceae family within Bacteroidetes and Lachnospiraceae family within the Clostridiales order of Firmicutes were mostly abundant at 35°C and not abundant in the microbial communities of the TS reactors incubated at 55°C. The ability to adjust bioreactor operating conditions to select for microbial communities with different fermentation product profiles offers new strategies to explore and compare potentially valuable fermentation products from TS and allows industries the flexibility to adapt and switch chemical production based on market prices and demands.

Enhanced succinic acid production from corncob hydrolysate by microbial electrolysis cells

In this study, Actinobacillus succinogenes NJ113 microbial electrolysis cells (MECs) were used to enhance the reducing power responsible for succinic acid production from corncob hydrolysate. During corncob hydrolysate fermentation, electric MECs resulted in a 1.31-fold increase in succinic acid production and a 1.33-fold increase in the reducing power compared with those in non-electric MECs. When the hydrolysate was detoxified by combining Ca(OH)2, NaOH, and activated carbon, succinic acid production increased from 3.47 to 6.95g/l. Using a constant potential of −1.8V further increased succinic acid production to 7.18g/l. A total of 18.09g/l of succinic acid and a yield of 0.60g/g total sugar were obtained after a 60-h fermentation when NaOH was used as a pH regulator. The improved succinic acid yield from corncob hydrolysate fermentation using A. succinogenes NJ113 in electric MECs demonstrates the great potential of using biomass as a feedstock to cost-effectively produce succinate.

Regulation of NAD(H) pool and NADH/NAD+ ratio by overexpression of nicotinic acid phosphoribosyltransferase for succinic acid production in Escherichia coli NZN111

Succinic acid is not the dominant fermentation product from glucose in wild-type Escherichia coli W1485. To reduce byproduct formation and increase succinic acid accumulation, pyruvate formate-lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, E. coli NZN111, the ldhA and pflB deletion strain, could not utilize glucose anaerobically due to the block of NAD+ regeneration. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase, a rate limiting enzyme of NAD(H) synthesis encoded by the pncB gene, resulted in a significant increase in cell mass and succinic acid production. Furthermore, the results indicated a significant increase in NAD(H) pool size, and decrease in the NADH/NAD+ ratio from 0.64 to 0.13, in particular, the concentration of NAD+ increased 6.2-fold during anaerobic fermentation. In other words, the supply of enough NAD+ for NADH oxidation by regulation of NAD(H) salvage synthesis mechanism could improve the cell growth and glucose utilization anaerobically. In addition, the low NADH/NAD+ ratio also change the metabolite distribution during the dual-phase fermentation. As a result, there was a significant increase in succinic acid production, and it is provided further evidence that regulation of NAD(H) pool and NADH/NAD+ ratio was very important for succinic acid production.

Characterization of a Robust Enzymatic Assay for Inhibitors of 2-Oxoglutarate-Dependent Hydroxylases

The prolyl-4-hydroxylase proteins regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylation of proline residues targeting HIF-1alpha for proteasomal degradation. Using the purified catalytic domain of prolyl hydroxylase 2 (PHD2(181-417)), an enzymatic assay has been developed to test inhibitors of the enzyme in vitro. Because PHD2 hydroxylates HIF-1alpha, with succinic acid produced as an end product, radiolabeled [5-(14)C]-2-oxoglutaric acid was used and formation of [14C]-succinic acid was measured to quantify PHD2(181-417) enzymatic activity. Comparison of the separation of 2-oxoglutaric acid and succinic acid by either ion exchange chromatography or precipitation with phenylhydrazine showed similar results, but the quantification and throughput were vastly increased using the latter method. The PHD2 reaction was substrate and concentration dependent. The addition of iron to the enzyme reaction mix resulted in an increase in enzymatic activity. The Km value for 2-oxoglutaric acid was determined to be 0.9 microM, and known PHD2 inhibitors were used to validate the assay. In addition, the authors demonstrate that this assay can be applied to other 2-oxoglutaric acid-dependent enzymes, including the asparaginyl hydroxylase, factor-inhibiting HIF-1alpha (FIH). A concentration-dependent increase in succinic acid production using recombinant FIH enzyme with a synthetic peptide substrate was observed. The authors conclude that a by-product enzyme assay measuring the conversion of 2-oxoglutaric acid to succinic acid using the catalytic domain of the human PHD2 provides a convenient method for the biochemical evaluation of inhibitors of the 2-oxoglutaric acid-dependent hydroxylases.

Succinic acid production from Bacteroides fragilis: Process optimization and scale up in a bioreactor

We report the effect of different physiological and nutritional parameters on succinic acid production from Bacteroides fragilis. This strain initially produced 0.70 g L −1 of succinic acid in 60 h. However, when process optimization was employed, 5.4 g L −1 of succinic acid was produced in medium consisting of glucose (1.5%); tryptone (2.5%); Na 2CO 3 (1.5%), at pH 7.0, when inoculated with 4% inoculum and incubated at 37 °C, 100 rpm for 48 h. A marked enhancement in succinic acid production was observed when the optimized conditions were employed in a 10 L bioreactor. A total of 12.5 g L −1 of succinic acid was produced in 30 h. This is approximately 12-fold increase in succinic acid production when compared to the initial un-optimized medium production. This enhancement in succinic acid production may be due to the control of CO 2 supply and the impeller speed. This is also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.

Effect of process parameters on succinic acid production in Escherichia coli W3110 and enzymes involved in the reductive tricarboxylic acid cycle

The effect of process optimization on succinic acid production by Escherichia coli W3110 and on enzymes involved in the reverse tricarboxylic acid cycle was studied. Approximately, 7.02 g L-1 of succinic acid was produced in 60 h at pH 7.0 in 500 mL anaerobic bottles containing 300 mL of the medium, wherein the sucrose concentration was 2.5%, the ratio of tryptone to ammonium hydrogen phosphate was 1:1, and the concentration of magnesium carbon ate was 1.5%. When these optimized fermentation conditions were employed in a 10 L bioreactor, 11.2 g L-1 of succinic acid was produced in 48 h. This is a 10-fold increase in succinic acid production from the initial titer of 0.94 g L-1. This clearly indicates the importance of process optimization, where by manipulating the media composition and production conditions, a remarkable increase in the production of the desired biomolecule can be obtained. The production of succinic acid is a multi-step reaction through the reverse tricarboxylic acid cycle. A linear relationship was observed between succinic acid production and the enzyme activities. The enzyme activities were found to increase in the order phospho-enol-pyruvate carboxylase<malate dehydrogenase<fumarase<fumarate reductase. The activity of phospho-enol-pyruvate carboxykinase was also estimated. Results indicate that this enzyme was not a very active participant in the production of succinic acid, since it catalyzes the phosphorylation of oxaloacetic acid to yield phospho-enol-pyruvate.

Influence of environmental and nutritional factors on succinic acid production and enzymes of reverse tricarboxylic acid cycle from Enterococcus flavescens

Effect of different environmental and nutritional factors on succinic acid production by batch fermentation of Enterococcus flavescens and enzymes involved in the acid production through reverse tricarboxylic acid cycle (TCA) cycle was investigated. An overall seven-fold increase in succinic acid production (from 0.92 g l −1 in 72 h initially to 6.7 g l −1 in 48 h) was achieved in 300-ml of optimized medium having 3% sucrose, 1:0.5 ratio of tryptone and ammonium hydrogen phosphate, 15 mM MgCO 3 at pH 6.5 when inoculated with 4% (v/v) of seed inoculum and incubated at 39 °C for 48 h, as against initial un-optimized medium. Subsequent scale-up in a 10-l bioreactor using these optimized fermentation conditions under controlled pH and continuous CO 2 supply resulted in 14.25 g l −1 of succinic acid in 30 h. Optimization of the environmental and nutritional parameters resulted in a maximum production of succinic acid by affecting the levels of the enzymes involved in its production via the reverse TCA cycle. A linear relationship was observed between succinic acid production and in the enzyme activities. The enzyme activity was found to be increase in order phosphoenol pyruvate carboxykinase < malate dehydrogenase < fumarase < fumarate reductase < phosphoenol pyruvate carboxylase.

A cost effective fermentative production of succinic acid from cane molasses and corn steep liquor by Escherichia coli

Development and optimization of an efficient and inexpensive medium for succinic acid production by Escherichia coli under anaerobic conditions. Initially, 0.8 gl(-1) of succinic acid was produced in 60 h in 300-ml medium. On optimization, glucose and peptone were replaced by cane molasses and corn steep liquor. Three hundred ml of this medium was inoculated with 4% (v/v) of seed inoculum, incubated at 39 degrees C for 72 h, resulted in 7.1 gl(-1) of succinic acid in 36 h. Scale up in a 10-l fermentor under conditions of controlled pH and continuous CO2 supply in this medium resulted in 17 gl(-1) of succinic acid in 30 h. A ninefold increase in succinic acid production was obtained in 500-ml anaerobic bottles with optimized medium having cane molasses and corn steep liquor as against initial medium containing glucose and peptone. However, a subsequent scale up in a 10-l fermentor resulted in a 2.5-fold increase in succinic acid production as against optimized medium used in 500-ml anaerobic bottles. Succinic acid production was enhanced in medium consisting of inexpensive carbon and nitrogen sources in a shorter span of time.

Succinic Acid Production by Bacteroides fragilis

We previously demonstrated that Bacteroides species elaborate a factor that impairs polymorphonuclear leukocyte (PMN) migration. This factor, active at a pH of 5.5 but not at 7.4, had similar inhibitory characteristics as the Bacteroides metabolite succinic acid. We examined the kinetics of production of this inhibitory factor and correlated its presence with succinic acid concentrations in the culture filtrate. In the four to six hours after Bacteroides fragilis 9032 reached the stationary phase of growth, there was a large increase in succinic acid production with an accompanying reduction in culture pH. This correlated well with the generation of a leukocyte-inhibitory factor. Pure succinic acid in sterile culture medium at concentrations similar to those detected in the 18-hour culture filtrate (20 mmol/L) produced comparable reduction in PMN migration. Following gel filtration chromatography of B fragilis culture filtrate, the fractions containing succinic acid labeled with carbon 14 inhibited PMN migration.