Enhanced direct gaseous CO2 fixation into higher bio-succinic acid production and selectivity.

Utilizing CO2 for bio-succinic acid production is an attractive approach to achieve carbon capture and recycling (CCR) with simultaneous production of a useful platform chemical. Actinobacillus succinogenes and Basfia succiniciproducens were selected and investigated as microbial catalysts. Firstly, the type and concentration of inorganic carbon concentration and glucose concentration were evaluated. 6 g C/L MgCO3 and 24 g C/L glucose were found to be the optimal basic operational conditions, with succinic acid production and carbon yield of over 30 g/L and over 40%, respectively. Then, for maximum gaseous CO2 fixation, carbonate was replaced with CO2 at different ratios. The "less carbonate more CO2" condition of the inorganic carbon source was set as carbonate: CO2 = 1:9 (based on the mass of carbon). This condition presented the highest availability of CO2 by well-balanced chemical reaction equilibrium and phase equilibrium, showing the best performance with regarding CO2 fixation (about 15 mg C/(L·hr)), with suppressed lactic acid accumulation. According to key enzymes analysis, the ratio of phosphoenolpyruvate carboxykinase to lactic dehydrogenase was enhanced at high ratios of gaseous CO2, which could promote glucose conversion through the succinic acid path. To further increase gaseous CO2 fixation and succinic acid production and selectivity, stepwise CO2 addition was evaluated. 50%-65% increase in inorganic carbon utilization was obtained coupled with 20%-30% increase in succinic acid selectivity. This was due to the promotion of the succinic acid branch of the glucose metabolism, while suppressing the pyruvate branch, along with the inhibition on the conversion from glucose to lactic acid.

Transcriptional regulation in key metabolic pathways of Actinobacillus succinogenes in the presence of electricity.

The potential of renewable energy application via direct electrode interaction for the production of bio-based chemicals is a promising technology. The utilization of extracellular energy in pure culture fermentations aims in intracellular redox balance regulation in order to improve fermentation efficiency. This work evaluates the impact of a bioelectrochemical system in succinic acid fermentation and the metabolic response of Actinobacillus succinogenes. The metabolic pathway regulation of A. succinogenes was evaluated via RNA expression of the key enzymes that participate in TCA cycle, pyruvate metabolism and oxidative phosphorylation. The genes that were significantly overexpressed in BES compared to non-BES were phosphoenolpyruvate carboxykinase (0.4-fold change), inorganic pyrophosphatase (2.3-fold change) and hydrogenase (2.2-fold change) and the genes that were significantly underexpressed were fumarase (-0.94-fold change), pyruvate kinase (-6.9-fold change), all subunits of fumarate reductase (-2.1 to -1.17-fold change), cytochromes I and II (-1.25 and -1.02-fold change, respectively) and two C4-carboxylic acid transporters.

Microbioreactor (micro-Matrix) potential in aerobic and anaerobic conditions with different industrially relevant microbial strains.

Microscale fermentation systems are important high throughput tools in clone selection, and bioprocess set up and optimization, since they provide several parallel experiments in controlled conditions of pH, temperature, agitation, and gas flow rate. In this work we evaluated the performance of biotechnologically relevant strains with different respiratory requirements in the micro-Matrix microbioreactor. In particular Escherichia coli K4 requires well aerated fermentation conditions to improve its native production of chondroitin-like capsular polysaccharide, a biomedically attractive polymer. Results from batch and fed-batch experiments demonstrated high reproducibility with those obtained on 2 L reactors, although highlighting a pronounced volume loss for longer-term experiments. Basfia succiniciproducens and Actinobacillus succinogenes need CO2 addition for the production of succinic acid, a building block with several industrial applications. Different CO2 supply modes were tested for the two strains in 24 h batch experiments and results well compared with those obtained on lab-scale bioreactors. Overall, it was demonstrated that the micro-Matrix is a useful scale-down tool that is suitable for growing metabolically different strains in simple batch process, however, a series of issues should still be addressed in order to fully exploit its potential.

One step forward, two steps back: Transcriptional advancements and fermentation phenomena in Actinobacillus succinogenes 130Z.

Within the field of bioproduction, non-model organisms offer promise as bio-platform candidates. Non-model organisms can possess natural abilities to consume complex feedstocks, produce industrially useful chemicals, and withstand extreme environments that can be ideal for product extraction. However, non-model organisms also come with unique challenges due to lack of characterization. As a consequence, developing synthetic biology tools, predicting growth behavior, and building computational models can be difficult. There have been many advancements that have improved work with non-model organisms to address broad limitations, however each organism can come with unique surprises. Here we share our work in the non-model bacterium Actinobacillus succinognes 130Z, which includes both advancements in synthetic biology toolkit development and pitfalls in unpredictable fermentation behaviors. To develop a synthetic biology "tool kit" for A. succinogenes, information gleaned from a growth study and antibiotic screening was used to characterize 22 promoters which demonstrated a 260-fold range of fluorescence protein expression. The strongest of the promoters was incorporated into an inducible system for tunable gene control in A. succinogenes using the promoter for the lac operon as a template. This system flaunted a 481-fold range of expression and no significant basal expression. These findings were accompanied by unexpected changes in fermentation products characterized by a loss of succinic acid and increase in lactic acid after approximately 10 months in the lab. During evaluation of the fermentation shifts, new tests of the synthetic biology tools in a succinic acid producing strain revealed a significant loss in their functionality. Contamination and mutation were ruled out as causes and further testing is needed to elucidate the driving factors. The significance of this work is to share a successful tool development strategy that could be employed in other non-model species, report on an unfortunate phenomenon that needs addressed for further development of A. succinogenes, and provide a cautionary tale for those undertaking non-model research. In sharing our findings, we seek to provide tools and necessary information for further development of A. succinogenes as a platform for bioproduction of succinic acid and to illustrate the importance of diligent and long-term observation when working with non-model bacteria.

Adaptation of Methanosarcina barkeri 227 as acetate scavenger for succinate fermentation by Actinobacillus succinogenes.

Acetate is the main by-product from microbial succinate production. In this study, we performed acetate removal by Methanosarcina barkeri 227 for succinate fermentation by Actinobacillus succinogenes 130Z. The acetoclastic methanogen M. barkeri requires similar environmental factors to A. succinogenes, and the conditions required for co-cultivation were optimized in this study: gas used for anaerobicization, strain adaptation, medium composition, pH adjustment, and inoculation time points. M. barkeri 227 was adapted to acetate for 150 days, which accelerated the acetate consumption to 9-fold (from 190 to 1726 mmol gDW-1 day-1). In the acetate-adapted strain, there was a noticeable increase in transcription of genes required for acetoclastic pathway-satP (acetate transporter), ackA (acetate kinase), cdhA (carbon monoxide dehydrogenase/acetyl-CoA synthase complex), and mtrH (methyl-H4STP:CoM methyltransferase), which was not induced before the adaptation process. The activities of two energy-consuming steps in the pathway-acetate uptake and acetate kinase-increased about 3-fold. This acetate-adapted M. barkeri could be successfully applied to succinate fermentation culture of A. succinogenes, but only after pH adjustment following completion of fermentation. This study suggests the utility of M. barkeri as an acetate scavenger during fermentation for further steps towards genetic and process engineering.

Effect of shear on morphology, viability and metabolic activity of succinic acid-producing Actinobacillus succinogenes biofilms.

Two custom-designed bioreactors were used to evaluate the effect of shear on biofilms of a succinic acid producer, Actinobacillus succinogenes. The first bioreactor allowed for in situ removal of small biofilm samples used for microscopic imaging. The second bioreactor allowed for complete removal of all biofilm and was used to analyse biofilm composition and productivity. The smooth, low porosity biofilms obtained under high shear conditions had an average cell viability of 79% compared to 57% at the lowest shear used. The maximum cell-based succinic acid productivity for high shear biofilm was 2.4 g g-1DCW h-1 compared to the 0.8 g g-1DCW h-1 of the low shear biofilm. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays confirmed higher cell metabolic activities for high shear developed biofilm compared to biofilm developed at low shear conditions. Results clearly indicated that high shear biofilm cultivation has beneficial morphological, viability, and cell-based productivity characteristics.

Biochemical conversion of sweet sorghum bagasse to succinic acid.

Succinic acid, an important intermediate in the manufacture of plastics and other commodity and specialty chemicals, is currently made primarily from petroleum. We attempted to biosynthesize succinic acid through microbial fermentation of cellulosic sugars derived from the bagasse of sweet sorghum, a renewable feedstock that can grow in a wide range of climates around the world. We investigated pretreating sweet sorghum bagasse (SSB) with concentrated phosphoric acid at mild conditions (40-85°C) at various residence times and biomass concentrations. We then subjected the pretreated SSB to enzymatic hydrolysis with a commercial cellulase to release glucose. The highest glucose yield was obtained when SSB was pretreated at 50°C for 43 min at 130 g/L biomass concentration on dry basis. Fermentation was carried out with Actinobacillus succinogenes 130Z, which readily converted 29.2 g/L of cellulosic glucose to 17.8 g/L of succinic acid in a 3.5-L bioreactor sparged with CO2 at a rate of 0.5 vvm, thus reducing the carbon footprint of the process. Overall, we demonstrated, for the first time, the use of SSB for production of succinic acid using practices that lower energy use, future equipment cost, waste generation, and carbon footprint.

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%.

Impact of metabolite accumulation on the structure, viability and development of succinic acid-producing biofilms of Actinobacillus succinogenes.

Biofilms of Actinobacillus succinogenes have demonstrated exceptional capabilities as biocatalysts for high productivity, titre and yield production of succinic acid (SA). The paper presents a microscopic analysis of A. succinogenes biofilms developed under varied fermenter conditions. The concentration of excretion metabolites is controlled by operating the fermenter in a continuous mode where the liquid throughput is adjusted. It is clearly illustrated how the accumulation of excreted metabolites (concomitant with the sodium build-up due to base dosing) has a severe effect on the biofilm structure and physiology. Under high accumulation (HA) conditions, some cells exhibit severe elongation while maintaining a cross-sectional diameter like the rod/cocci-shaped cells predominantly found in low accumulation (LA) conditions. The elongated cells formed at high accumulation conditions were found to be more viable than the clusters of rod/cocci-shaped cells and appear to form connections between the clusters. The global microscopic structure of the HA biofilms also differed significantly from the LA biofilms. Although both exhibited shedding after 4 days of growth, the LA biofilms were more homogenous (less patchy), thicker and with high viability throughout the biofilm depth. The viability of the HA biofilms was threefold lower than the corresponding LA biofilms towards the end of the fermentation. Visual observations were supported by quantitative analysis of multiple biofilm samples and strengthened the main observations. The work presents valuable insights on the effect of metabolite accumulation on biofilm structure and growth.

Miscanthus straw as substrate for biosuccinic acid production: Focusing on pretreatment and downstream processing.

The main aim of this study was to optimize pretreatment strategies of Miscanthus × giganteus for biosuccinic acid production. A successful pretreatment with organosolv method (80% w/w of glycerol, 1.25% of H2SO4), prevented sugars conversion to furfurals and organic acids, and thereby resulted in high sugar recovery (glucan > 98%, xylan > 91%) and biomass delignification (60%). Pretreated biomass was subjected to hydrolysis with various cellulolytic enzyme cocktails (Viscozyme® L, Carezyme 1000L®, ß-Glucanase, Cellic® CTec2, Cellic® HTec2). The most effective enzymes mixture composed of Cellic® CTec2 (10% w/w), ß-Glucanase (5% w/w) and Cellic® HTec2 (1% w/w) resulted in high glucose (93.1%) and xylose (69.2%) yields after glycerol-based pretreatment. Succinic acid yield of 75-82% was obtained after hydrolysates fermentation, using Actinobacillus succinogenes 130Z. Finally a successful downstream concept for succinic acid purification was proposed. The succinic acid recovery with high purity (>98%) was developed.

Investigation of putative invasion determinants of Actinobacillus species using comparative genomics.

Actinobacillus spp. are Gram-negative bacteria associated with mucosal membranes. While some are commensals, others can cause important human and animal diseases. A. pleuropneumoniae causes severe fibrinous hemorrhagic pneumonia in swine but not systemic disease whereas other species invade resulting in septicemia and death. To understand the invasive phenotype of Actinobacillus spp., complete genomes of eight isolates were obtained and pseudogenomes of five isolates were assembled and annotated. Phylogenetically, A. suis isolates clustered by surface antigen type and were more closely related to the invasive A. ureae, A. equuli equuli, and A. capsulatus than to the other swine pathogen, A. pleuropneumoniae. Using the LS-BSR pipeline, 251 putative virulence genes associated with serum resistance and invasion were detected. To our knowledge, this is the first genome-wide study of the genus Actinobacillus and should contribute to a better understanding of host tropism and mechanisms of invasion of pathogenic Actinobacillus and related genera.

High-affinity l-malate transporter DcuE of Actinobacillus succinogenes catalyses reversible exchange of C4-dicarboxylates.

Actinobacillus succinogenes is a natural succinate producer, which is the result of fumarate respiration. Succinate production from anaerobic growth with C4 -dicarboxylates requires transporters catalysing uptake and efflux of C4 -dicarboxylates. Transporter Asuc_1999 (DcuE) found in A. succinogenes belongs to the Dcu family and was considered the main transporter for fumarate respiration. However, deletion of dcuE affected l-malate uptake of A. succinogenes rather than fumarate uptake. DcuE complemented anaerobic growth of Escherichia coli on l-malate or fumarate; thus, the transporter was characterized in E. coli heterologously. Time-dependent uptake and competitive inhibition assays demonstrated that l-malate is the most preferred substrate for uptake by DcuE. The Vmax of DcuE for l-malate was 20.04 µmol/gDW·min with Km of 57 µM. The Vmax for l-malate was comparable to that for fumarate, whereas the Km for l-malate was 8 times lower than that for fumarate. The catalytic efficiency of DcuE for l-malate was 7.3-fold higher than that for fumarate, showing high efficiency and high affinity for l-malate. Furthermore, DcuE catalysed the reversible exchange of three C4 -dicarboxylates - l-malate, fumarate and succinate - but the preferred substrate for uptake was l-malate. Under physiological conditions, the C4 -dicarboxylates were reduced to succinate. Therefore, DcuE is proposed as the l-malate/succinate antiporter in A. succinogenes.

Production of various organic acids from different renewable sources by immobilized cells in the regimes of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SFF).

The study was aimed at production of different organic acids (OA) (lactic, fumaric, or succinic) by various microbial cells (filamentous fungi Rhizopus oryzae (F-814, F-1127) and bacteria Actinobacillus succinogenes B-10111) immobilized into poly(vinyl alcohol) (PVA) cryogel from diverse renewable raw materials (wheat and rice straw, aspen and pine sawdust, Jerusalem artichoke stems and tubers, biomass of macro- and microalgae) under batch conditions. The process productivity, bulk output and OA concentrations were higher in case of using immobilized cells than in case of free cells under identical conditions. A higher OA productivity was reached via simultaneous enzymatic saccharification and microbial fermentation (SSF) of same raw materials as compared to their separate enzymatic hydrolysis and fermentation of accumulated reducing sugars (SHF). Maximal concentrations of all OAs studied were obtained for bioconversion of Jerusalem artichoke tubers. The immobilized cells were used in long-term conversion of various renewable materials to OAs in SSF.

Homogeneous solid dispersion (HSD) system for rapid and stable production of succinic acid from lignocellulosic hydrolysate.

Continuous bio-production of succinic acid was reported in homogeneous solid dispersion (HSD) system utilizing porous coconut shell activated carbon (CSAC) as immobilization carrier. The aim of the present work was to implement the HSD system to increase the area of cell immobilization and the rate of succinic-acid production from the lignocellulosic medium. The ratio of the two enzymes (cellulase-to-hemicellulase) was initially optimized to break down the lignocellulose into its free monomers, wherein the best ratio was determined as 4:1. Succinic-acid production was evaluated in the HSD system by varying the substrate loading and dilution rate. The results showed that high productivities of succinic acid were obtained when 60 g/L glucose was fed over a dilution rates ranging from 0.03 to 0.4/h. The titer of succinic acid decreased gradually with higher dilution rate, whereas the residual substrate concentration increased with it. Critical dilution rate was determined to be 0.4/h at which the best productivity of succinic acid of 6.58 g/L h and its yield of 0.66 g/g were achieved using oil palm fronds (OPF) hydrolysate. This work lends evidence to the use of CSAC and lignocellulosic hydrolysate to further exploit the potential economies of scale.

Continuous Succinic Acid Fermentation by Actinobacillus Succinogenes: Assessment of Growth and Succinic Acid Production Kinetics.

Succinic acid is one of the most interesting platform chemicals that can be produced in a biorefinery approach. The paper reports the characterization of the growth kinetics of Actinobacillus succinogenes DSM 22257 using glucose as carbon source. Tests were carried out in a continuous bioreactor operated under controlled pH. Under steady-state conditions, the conversion process was characterized in terms of concentration of glucose, cells, acids, and pH. The effects of acid-succinic, acetic, and formic-concentration in the medium on fermentation performance were investigated. The fermentation was interpreted according to several models characterized by substrate and product inhibition. The selected kinetic model of biomass growth and of metabolite production described the microorganism growth rate under a broad interval of operating conditions. Under the investigated operating conditions, results pointed out that: no substrate inhibition was observed; acetic acid did not inhibit the cell growth and succinic acid production.

Opportunities, challenges, and future perspectives of succinic acid production by Actinobacillus succinogenes.

Due to environmental issues and the depletion of fossil-based resources, ecofriendly sustainable biomass-based chemical production has been given more attention recently. Succinic acid (SA) is one of the top value added bio-based chemicals. It can be synthesized through microbial fermentation using various waste steam bioresources. Production of chemicals from waste streams has dual function as it alleviates environmental concerns; they could have caused because of their improper disposal and transform them into valuable products. To date, Actinobacillus succinogenes is termed as the best natural SA producer. However, few reviews regarding SA production by A. succinogenes were reported. Herewith, pathways and metabolic engineering strategies, biomass pretreatment and utilization, and process optimization related with SA fermentation by A. succinogenes were discussed in detail. In general, this review covered vital information including merits, achievements, progresses, challenges, and future perspectives in SA production using A. succinogenes. Therefore, it is believed that this review will provide platform to understand the potential of the strain and tackle existing hurdles so as to develop superior strain for industrial applications. It will also be used as a baseline for identification, isolation, and improvement of other SA-producing microbes.

Effectively converting carbon dioxide into succinic acid under mild pressure with Actinobacillus succinogenes by an integrated fermentation and membrane separation process.

The aim of the present study is to develop an effective bioprocess for converting CO2 into succinic acid (SA) with Actinobacillus succinogenes by an integrated fermentation and membrane separation process. CO2 could be effectively converted into SA using NaOH as the neutralizer under the completely closed exhaust pipe case with self-circulation of CO2 in the bioreactor. Meanwhile, the optimal CO2 partial pressure was 0.4 bar. In addition, a 300 kDa ultrafiltration (UF) membrane was preferred for constructing the membrane bioreactor. Moreover, a high conductivity was toxic to the cells during SA biosynthesis. After removing the high concentration salts by in-situ membrane filtration, the SA productivity and CO2 fixation rate increased by 39.2% compared with the batch culture, reaching 1.39 g·L-1·h-1 and 0.52 g·L-1·h-1 respectively. Furthermore, nanofiltration (NF) was suitable for purifying the SA and recovering the residual substrates in the UF permeate for the next fermentation.

Reconstruction of a genome-scale metabolic model for Actinobacillus succinogenes 130Z.

BACKGROUND: Actinobacillus succinogenes is a promising bacterial catalyst for the bioproduction of succinic acid from low-cost raw materials. In this work, a genome-scale metabolic model was reconstructed and used to assess the metabolic capabilities of this microorganism under producing conditions. RESULTS: The model, iBP722, was reconstructed based on the functional reannotation of the complete genome sequence of A. succinogenes 130Z and manual inspection of metabolic pathways, covering 1072 enzymatic reactions associated with 722 metabolic genes that involve 713 metabolites. The highly curated model was effective in capturing the growth of A. succinogenes on various carbon sources, as well as the SA production under various growth conditions with fair agreement between experimental and predicted data. Calculated flux distributions under different conditions show that a number of metabolic pathways are affected by the activity of some metabolic enzymes at key nodes in metabolism, including the transport mechanism of carbon sources and the ability to fix carbon dioxide. CONCLUSIONS: The established genome-scale metabolic model can be used for model-driven strain design and medium alteration to improve succinic acid yields.

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes.

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass-namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.

Transcriptome analysis and anaerobic C4 -dicarboxylate transport in Actinobacillus succinogenes.

A global transcriptome analysis of the natural succinate producer Actinobacillus succinogenes revealed that 353 genes were differentially expressed when grown on various carbon and energy sources, which were categorized into six functional groups. We then analyzed the expression pattern of 37 potential C4 -dicarboxylate transporters in detail. A total of six transporters were considered potential fumarate transporters: three transporters, Asuc_1999 (Dcu), Asuc_0304 (DASS), and Asuc_0270-0273 (TRAP), were constitutively expressed, whereas three others, Asuc_1568 (DASS), Asuc_1482 (DASS), and Asuc_0142 (Dcu), were differentially expressed during growth on fumarate. Transport assays under anaerobic conditions with [14 C]fumarate and [14 C]succinate were performed to experimentally verify that A. succinogenes possesses multiple C4 -dicarboxlayte transport systems with different substrate affinities. Upon uptake of 5 mmol/L fumarate, the systems had substrate specificity for fumarate, oxaloacetate, and malate, but not for succinate. Uptake was optimal at pH 7, and was dependent on both proton and sodium gradients. Asuc_1999 was suspected to be a major C4 -dicarboxylate transporter because of its noticeably high and constitutive expression. An Asuc_1999 deletion (∆1999) decreased fumarate uptake significantly at approximately 5 mmol/L fumarate, which was complemented by the introduction of Asuc_1999. Asuc_1999 expressed in Escherichia coli catalyzed fumarate uptake at a level of 21.6 µmol·gDW-1 ·min-1 . These results suggest that C4 -dicarboxylate transport in A. succinogenes is mediated by multiple transporters, which transport various types and concentrations of C4 -dicarboxylates.