Fermentative Production Of Succinic Acid Research Articles

Mitochondrial membrane transporters as attractive targets for the fermentative production of succinic acid from glycerol in Saccharomyces cerevisiae.

Previously, we reported an engineered Saccharomyces cerevisiae CEN.PK113-1A derivative able to produce succinic acid (SA) from glycerol with net CO2 fixation. Apart from an engineered glycerol utilization pathway that generates NADH, the strain was equipped with the NADH-dependent reductive branch of the TCA cycle (rTCA) and a heterologous SA exporter. However, the results indicated that a significant amount of carbon still entered the CO2-releasing oxidative TCA cycle. The current study aimed to tune down the flux through the oxidative TCA cycle by targeting the mitochondrial uptake of pyruvate and cytosolic intermediates of the rTCA pathway, as well as the succinate dehydrogenase complex. Thus, we tested the effects of deletions of MPC1, MPC3, OAC1, DIC1, SFC1, and SDH1 on SA production. The highest improvement was achieved by the combined deletion of MPC3 and SDH1. The respective strain produced up to 45.5g/L of SA, reached a maximum SA yield of 0.66 gSA/gglycerol, and accumulated the lowest amounts of byproducts when cultivated in shake-flasks. Based on the obtained data, we consider a further reduction of mitochondrial import of pyruvate and rTCA intermediates highly attractive. Moreover, the approaches presented in the current study might also be valuable for improving SA production when sugars (instead of glycerol) are the source of carbon.

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.

Application and evaluation of preselection approaches to decide on the use of equipment modules

The idea of module-based plant design is to select equipment modules of-the-peg instead of designing plants completely tailor-made to speed up plant design. Using equipment modules in plant design leads to equipment that does not exactly fit requirements. For some process units of a plant this might result in a moderate increase of investment and/or operating costs; for others using equipment modules of-the-peg might lead to a substantial increase of costs. The key question is: For which process units of a plant can one afford increased costs due to equipment modules not exactly fitting the requirements?In this work the effect of using equipment modules on investment and operating costs is investigated. Three different preselection approaches to decide on the use of equipment modules are applied and evaluated. One is purely based on investment costs, whereas the two other approaches developed consider investment and operating costs, either directly or based on exergoeconomics.Using the fermentative production of succinic acid as case study, it is shown that investment and operating costs are equally important and that the preselection approach based directly on investment and operating costs is found most suitable when deciding on the use of equipment modules.

Life cycle analysis of fermentative production of succinic acid from bread waste

According to the US Department of Energy, succinic acid (SA) is a top platform chemical that can be produced from biomass. Bread waste, which has high starch content, is the second most wasted food in the UK and can serve as a potential low cost feedstock for the production of SA. This work evaluates the environmental performance of a proposed biorefinery concept for SA production by fermentation of waste bread using a cradle-to-factory gate life cycle assessment approach. The performance was assessed in terms of greenhouse gas (GHG) emissions and non-renewable energy use (NREU). Waste bread fermentation demonstrated a better environmental profile compared to the fossil-based system, however, GHG emissions were about 50% higher as compared to processes using other biomass feedstocks such as corn wet mill or sorghum grains. NREU for fermentative SA production using waste bread was significantly lower (~ 46%) than fossil-based system and about the same as that of established biomass-based processes, thus proving the great potential of waste bread as a valuable feedstock for bioproduction of useful chemicals. The results show that steam and heating oil used in the process were the biggest contributors to the NREU and GHG emissions. Sensitivity analyses highlighted the importance of the solid biomass waste generated in the process which can potentially be used as fish feed. The LCA analysis can be used for targeted optimization of SA production from bread waste, thereby enabling the utilization of an otherwise waste stream and leading to the establishment of a circular economy.

Internal mass transfer considerations in biofilms of succinic acid producing Actinobacillus succinogenes

The rumen bacterium Actinobacillus succinogenes is reputable for its high productivity, -yield and -titre fermentative production of succinic acid under biofilm conditions. The paper presents an analysis of internal mass transfer effects in biofilm fermentations of A. succinogenes. Internal mass transfer effects were investigated by batch fermentations using attached- and resuspended biofilms as biocatalysts. In the latter, the biofilms were resuspended after initial development to simulate mass transfer free (free cell) fermentations. Intrinsic kinetics for succinic production obtained from resuspended free cell fermentations predicted faster production rates than for the attached biofilms runs (biofilm thicknesses in the range of 120–200 µm), indicating internal mass transfer restrictions. A developed biofilm reaction diffusion model gave good predictions of attached biofilm batch results by accounting for internal mass transfer in the biofilm. Biofilm effectiveness factors ranged from 75% to 97% for all batches at the inception of batch conditions but increased with progression of batch operation due to increased succinic acid titres which inhibited production rates. Biofilm thickness and succinic acid concentrations were shown to have a significant effect on internal mass transfer. A simplified algorithm was developed to estimate the pseudo-steady state glucose penetration and biofilm effectiveness of A. succinogenes biofilms without the requirement to solve the overall mass transfer model. The results clearly showed that internal mass transfer need to be considered in biofilm fermentations involving A succinogenes as high biomass concentrations may not always equate to increased productivities if mass transfer effects dominate.

Promising advancement in fermentative succinic acid production by yeast hosts

As a platform chemical with various applications, succinic acid (SA) is currently produced by petrochemical processing from oil-derived substrates such as maleic acid. In order to replace the environmental unsustainable hydrocarbon economy with a renewable environmentally sound carbohydrate economy, bio-based SA production process has been developed during the past two decades. In this review, recent advances in the valorization of solid organic wastes including mixed food waste, agricultural waste and textile waste for efficient, green and sustainable SA production have been reviewed. Firstly, the application, market and key global players of bio-SA are summarized. Then achievements in SA production by several promising yeasts including Saccharomyces cerevisiae and Yarrowia lipolytica are detailed, followed by calculation and comparison of SA production costs between oil-based substrates and raw materials. Lastly, challenges in engineered microorganisms and fermentation processes are presented together with perspectives on the development of robust yeast SA producers via genome-scale metabolic optimization and application of low-cost raw materials as fermentation substrates. This review provides valuable insights for identifying useful directions for future bio-SA production improvement.

Recovery of succinic acid from whey fermentation broth by reactive extraction coupled with multistage processes

Abstract Fermentative production of succinic acid (SA) from renewable resources such as whey is environmentally sustainable compared to petroleum-based synthesis. However, a major drawback of fermentation is the concurrent production of SA with byproducts such as lactic acid (LA), formic acid (FA) and acetic acid (AA). Therefore, appropriate downstream SA recovery and purification steps are significant in ensuring sustainable SA production. In this study, SA was fermented by Actinobacillus succinogenes and recovered in an integrated process consisting of ultrafiltration, vacuum distillation and reactive extraction. The extractant used was tri-n-octylamine (TOA) with 1-octanol as a diluent for both liquid-liquid (LLE) extraction and supported liquid membrane (SLM). The produced SA titer and yield was 11.16 g/L and 0.44 g/g, respectively. The steady state ultrafiltration permeate flux ranged from 31.18 to 33.42 L/m2h, and complete decolorization of the fermentation broth was achieved with 10 % (w/v) of powdered activated carbon. The extraction efficiency for LLE was 51.5 %, whereas SLM achieved 57.3 % recovery. SA exhibited transport and permeability coefficient of 0.00697 h−1 (R2 > 0.92) and 0.08605 cm h−1, respectively. Extraction of SA tremendously decreased as the aqueous pH was increased from 2 to 5. In SLM, initial SA flux was calculated as 9.65 g/m2h and doubled that of lactic acid. Selective extraction of only SA was not achieved; however, residue biological material and macromolecular substances were effectively removed. Herein, we clearly demonstrated that process integration applied in reactive extraction is a promising approach for recovery of SA from fermentation broth.

Engineering Oleaginous Yeast as the Host for Fermentative Succinic Acid Production From Glucose.

Oleaginous yeast Yarrowia lipolytica is a prospective host for production of succinic acid. The interruption of tricarboxylic acid cycle through succinate dehydrogenase gene (SDH) deletion was reported to result in strains incapable of glucose utilization and this ability had to be restored by chemical mutation or long adaptive laboratory evolution. In this study, a succinate producing strain of Y. lipolytica was engineered by truncating the promoter of SDH1 gene, which resulted in 77% reduction in SDH activity but did not impair the ability of the strain to grow on glucose. The flux toward succinic acid was further improved by overexpressing the genes in the glyoxylate pathway and the oxidative TCA branch, and expressing phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes. A short adaptation on glucose reduced the lag phase of the strain and increased its tolerance to high glucose concentrations. The resulting strain produced 7.8 ± 0.0 g/L succinic acid with a yield of 0.105 g/g glucose in shake flasks without pH control, while mannitol (11.8 ± 0.8 g/L) was the main by-product. Further investigations showed that mannitol accumulation was caused by low pH stress and buffering the fermentation medium eliminated mannitol formation. In a fed-batch bioreactor in mineral medium at pH 5, at which point according to Ka values of succinic acid, the major fraction of product was in acidic form rather than dissociated form, the strain produced 35.3 ± 1.5 g/L succinic acid with 0.26 ± 0.00 g/g glucose yield.

From second generation feed-stocks to innovative fermentation and downstream techniques for succinic acid production

Succinic acid (SA) is one of the most important biobuilding blocks in biorefinery. Its production from fermentation of renewable biomass sources is becoming a consolidated alternative that is more sustainable and potentially more economic than the traditional petroleum-based path for SA production. Fermentative production of SA has been successfully commercialized and a large and increasing number of SA-derivatives are promoting the economic stability of this production. However, the companies producing SA from fermentation are targeting specialized markets and the production is far from large-scale bulk chemical synthesis. In order to develop optimized and economic processes, the best candidates in every step of the SA production process must be identified. In this paper, the most promising biomass sources, pretreatment methods, fermentation conditions (i.e. host microorganism, fermenter design and operative mode) and separation techniques for industrial SA production are critically reviewed. Selection of the host microorganism is a key factor for SA production. However, the availability, potential and sustainability of feedstocks, fermentation and separation process must also be carefully evaluated for a cost-effective and environmentally sustainable SA production.

Direct electrochemical extraction increases microbial succinic acid production from spent sulphite liquor

The fermentative production of succinic acid leads to increasing toxicity over time, and requires continuous succinic acid removal from the broth to improve fermentation efficiency.

A novel riboregulator switch system of gene expression for enhanced microbial production of succinic acid.

In this paper, a novel riboregulator Switch System of Gene Expression including an OFF-TO-ON switch and an ON-TO-OFF switch was designed to regulate the expression state of target genes between "ON" and "OFF" by switching the identifiability of ribosome recognition site (RBS) based on the thermodynamic stability of different RNA-RNA hybridizations between RBS and small noncoding RNAs. The proposed riboregulator switch system was employed for the fermentative production of succinic acid using an engineered strain of E. coli JW1021, during which the expression of mgtC gene was controlled at "ON" state and that of pepc and ecaA genes were controlled at the "OFF" state in the lag phase and switched to the "OFF" and "ON" state once the strain enters the logarithmic phase. The results showed that using the strain of JW1021, the yield and productivity of succinic acid can reach 0.91gg-1 and 3.25gL-1h-1, respectively, much higher than those using the strains without harboring the riboregulator switch system.

Performance and mechanism analysis of succinate production under different transporters in Escherichia coli

Succinic acid is a platform chemical with potential for bio-based synthesis. However, the production of bio-based succinate is limited because of insufficient succinate efflux capacity in the late stage of fermentation. In the present study, three different transporters, which have been reported to be responsible for C4-dicarboxylates transport, were employed for investigation of the transport capacity of succinate in Escherichia coli. After engineered strains were constructed, the fermentative production of succinic acid was studied in serum bottles and 3 L of fermentor. The results demonstrated that engineered strain showed better efflux capacity than control strain under high concentration of succinate. The highest production of succinate was 68.66 g/L, while the NCgl2130 transporter may be the best candidate for succinate export in E. coli. Further research showed that the expression levels and relative enzyme activities involved in the metabolic pathway all increased markedly, and the maximum activities of PPC, PCK, PYK, and MDH increased by 1.50, 1.38, 1.28, and 1.27-fold in recombinant E. coli AFP111/pTrc99a-NCgl2130, respectively. Moreover, the maximum level of intracellular ATP increased by 23.79% in E. coli AFP111/pTrc99a-NCgl2130. Taken together, these findings indicated that engineered transporters can improve succinate production by increasing key enzyme activities and intracellular ATP levels. To the best of thew authors’ knowledge, this is the first report on a mechanism to improve succinate production by engineered transporters. This strategy set up a foundation for improving the biosynthesis of other C4-dicarboxylates, such as fumaric acid and malic acid.

Biorefinery approach towards greener succinic acid production from oil palm frond bagasse

Oil palm frond (OPF) bagasse, being one of the cheapest biomass ever reported ($19.4/t), was hydrolyzed and used as a raw material for fermentative production of succinic acid for the first time. Results indicated that glucose and xylose were released as the main carbon source after alkaline pretreatment followed by enzymatic hydrolysis. The resultant hydrolysate from the pretreated OPF bagasse was used directly as the carbon source in the fermentation without any purification. The pretreatment using alkaline followed by enzymatic hydrolysis of the OPF bagasse had revealed a total sugar yield of 50.3g out of 100g raw biomass. After 60h of anaerobic batch fermentation using OPF bagasse hydrolysate, 86% of total sugar was consumed by Actinobacillus succinogenes 130Z which led to a succinic acid yield of 0.71g/g of sugar. This indicates that the largest residue from palm oil industry, OPF bagasse, can be used effectively as a carbon source for succinic acid production. A total of 36.6g/L of succinic acid was produced with an exogenous supply of formate at 25mM, indicating that the re-utilization of formate in the waste stream could enhance succinic acid production.

Enhanced succinic acid productivity by expression of mgtCB gene in Escherichia coli mutant.

In this study, a novel engineering Escherichia coli strain (CBMG111) with the expression of mgtCB gene was constructed for the enhanced fermentative production of succinic acid by utilizing the synergetic effect of mgtC gene to improve the growth of strains at the environment of low Mg(2+) concentration and mgtB to enhance the transport of Mg(2+) into cells. After the effect of the expression of the individual genes (mgtA, mgtB, mgtC) on the growth of E. coli was clarified, the fermentative production of succinic acid by CBMG111 was studied with the low-price mixture of Mg(OH)2 and NH3·H2O as the alkaline neutralizer and the biomass hydrolysates as the carbon sources, which demonstrated that the expression of mgtCB gene can significantly increase the productivity of succinic acid (2.97gL(-1)h(-1)) compared with that by using the engineering strain with the overexpression of mgtA gene.

Bio-generation of succinic acid by fermentation of Physaria fendleri seed polysaccharides

The bio-oils research unit at the national center for agricultural utilization research in Peoria, Illinois produces over 70% of crushed oilseed as press cake annually as tons of seed are crushed for oil. A large amount of this press cake cannot be used as animal feed because of anti-nutritional properties and generally winds up landfilled. Lesquerella fendleri now reclassified as Physaria fendleri and its press cake belongs in this category of oilseed processed here and its meal is unsuitable as animal feed. Hence the purpose of this study was to explore use of its press cake especially its polysaccharide content as renewable substrate for fermentative production of succinic acid when the crop is commercialized. Success with P. fendleri would lead to utilization of similar oilseed press cakes. Succinic acid, an important platform chemical is mainly produced industrially from maleic anhydride, a petrochemical intermediate as substrate. But because most research in succinic acid fermentative production is based on glucose, our goal here was to explore expansion of sugar substrate use beyond glucose so the polysaccharides from oilseed crushes could become renewable feedstuff for succinic acid production. Our initial trials found that the ruminal microorganism strain Actinobacillus succinogenes DSM 22,257 was capable of producing succinate using standard sugars known to be components of P. fendleri polysaccharides, i.e., glucose, arabinose, galactose, galacturonic acid and xylose. Subsequent trials with hydrolyzates of P. fendleri press cake using this strain successfully produced succinate at 93 to >98% conversion. The succinic acid produced was readily quantitatively recovered by solvent extraction of the heterogeneous fermentation broth. The crude extract was purified by recrystallization to >99% purity.

Economic feasibility of a pilot-scale fermentative succinic acid production from bakery wastes

In this paper, the techno-economic study for a pilot-scale production of succinic acid from food waste via fermentation was evaluated. The pilot plant was based in Hong Kong and designed for converting 1tonne/day of bakery waste into succinic acid. The mass and energy balance of the process was simulated by computer package SuperPro Designer®. The total capital investment for the plant and the total production cost were US$ 1,118,243 and US$ 230,750/year respectively. Overall revenue generated from the process was US$ 374,041/year. The return on investment, payback period and internal rate of return of the project were 12.8%, 7.2 years and 15.3% respectively. The findings indicated that the fermentative succinic acid production from bakery waste was feasible. This is important for attracting investment and industrialization interest on the biorefinery process using domestic wastes as raw materials.

Enhanced succinic acid production by Actinobacillus succinogenes after genome shuffling

Succinic acid is an important platform chemical for synthesis of C4 compounds. We applied genome shuffling to improve fermentative production of succinic acid by A. succinogenes. Using a screening strategy composed of selection in fermentation broth, cultured in 96-deep-well plates, and condensed HPLC screening, a starting population of 11 mutants producing a higher succinic acid concentration was selected and subjected to recursive protoplasts fusion. After three rounds of genome shuffling, strain F3-II-3-F was obtained, producing succinic acid at 1.99 g/l/h with a yield of 95.6 g/l. The genome shuffled strain had about a 73 % improvement in succinic acid production compared to the parent strain after 48 h in fed-batch fermentation. The genomic variability of F3-II-3-F was confirmed by amplified fragment-length polymorphism. The activity levels of key enzymes involved in end-product formation from glucose and metabolic flux distribution during succinic acid production were compared between A. succinogenes CGMCC 1593 and F3-II-3-F. Increased activity of glucokinase, fructose-1,6-bisphosphate aldolase, PEP carboxykinase and fumarase, as well as decreased activity of pyruvate kinase, pyruvate formate-lyase, and acetate kinase explained the enhanced succinic acid production and decreased acetic acid formation. Metabolic flux analysis suggested that increased flux to NADH was the main reason for increased activity of the C4 pathway resulting in increased yields of succinic acid. The present work will be propitious to the development of a bio-succinic acid fermentation industry.

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.

Utilisation of waste bread for fermentative succinic acid production

A novel biorefinery concept of utilising waste bread as a sole nutrient source for the production of a nutrient rich feedstock for the fermentative succinic acid production by Actinobacillus succinogenes has been developed. Waste bread was used in the solid-state fermentations of Aspergillus awamori and Aspergillus oryzae that produce enzyme complexes rich in amylolytic and proteolytic enzymes, respectively. The resulting fermentation solids were added directly to a bread suspension to generate a hydrolysate containing over 100g/L glucose and 490mg/L free amino nitrogen (FAN). A first-order kinetic model was used to describe the effect of initial bread mass ratio on glucose and FAN profiles. The bread hydrolysate was used as the sole feedstock for A. succinogenes fermentations, which led to the production of 47.3g/L succinic acid with a yield and productivity of 1.16g SA/g glucose and 1.12g/Lh. This corresponds to an overall yield of 0.55g succinic acid per g bread. This is the highest succinic acid yield compared from other food waste-derived media reported to date. The proposed process could be potentially utilised to transform no-value food waste into succinic acid, one of the future platform chemicals of a sustainable chemical industry.

Biotechnological production of succinic acid: current state and perspectives

AbstractSuccinic acid has multiple practical applications (e.g. synthesis of 1,4‐butanediol, tetrahydrofuran, gamma‐butyrolactone, and as a monomer of some biodegradable polymers). Bio‐based succinic acid is a potential substitute for current petrochemical production. Facing a shortage of crude oil supply and sharply rising oil prices, biological production of succinic acid from abundant and available biomass has become a topic of worldwide interest. Although great progress has been made in recent decades, much needs to be developed further in order to achieve economic viability. This paper reviews developments in technology and updates research progress of biosuccinate production, including pathways, micro‐organisms, culture conditions, as well as integrated production with other high‐value‐added products. Finally, strategies are proposed for successful commercialization of fermentative succinic acid production. © 2012 Society of Chemical Industry and John Wiley & Sons, Ltd