Continuous anaerobic fermentations were performed in a novel external-recycle, biofilm reactor using D-glucose and CO 2 as carbon substrates. Succinic acid (SA) yields were found to be an increasing function of glucose consumption with the succinic acid to acetic acid ratio increasing from 2.4 g g -1 at a glucose consumption of 10 g L -1 , to 5.7 g g -1 at a glucose consumption of 50 g L -1 . The formic acid to acetic acid ratio decreased from an equimolar value (0.77 g g -1 ) at a glucose consumption of 10 g L -1 to a value close to zero at 50 g L -1 .The highest SA yield on glucose and highest SA titre obtained were 0.91 g g -1 and 48.5 g L -1respectively. Metabolic flux analysis based on the established C 3 and C 4 metabolic pathways of Actinobacillus succinogenes revealed that the increase in the succinate to acetate ratio could not be attributed to the decrease in formic acid and that an additional source of NADH was present. The fraction of unaccounted NADH increased with glucose consumption, suggesting that additional reducing power is present in the medium or is provided by the activation of an alternative metabolic pathway. KeywordsFermentation, biofilms, bioreactors, succinic acid, metabolic flux analysis.Continuous succinic acid production by Actinobacillus succinogenes in a biofilm reactor: Steady-state metabolic flux variation 2

BackgroundCo-production of chemicals from lignocellulosic biomass alongside fuels holds promise for improving the economic outlook of integrated biorefineries. In current biochemical conversion processes that use thermochemical pretreatment and enzymatic hydrolysis, fractionation of hemicellulose-derived and cellulose-derived sugar streams is possible using hydrothermal or dilute acid pretreatment (DAP), which then offers a route to parallel trains for fuel and chemical production from xylose- and glucose-enriched streams. Succinic acid (SA) is a co-product of particular interest in biorefineries because it could potentially displace petroleum-derived chemicals and polymer precursors for myriad applications. However, SA production from biomass-derived hydrolysates has not yet been fully explored or developed.ResultsHere, we employ Actinobacillus succinogenes 130Z to produce succinate in batch fermentations from various substrates including (1) pure sugars to quantify substrate inhibition, (2) from mock hydrolysates similar to those from DAP containing single putative inhibitors, and (3) using the hydrolysate derived from two pilot-scale pretreatments: first, a mild alkaline wash (deacetylation) followed by DAP, and secondly a single DAP step, both with corn stover. These latter streams are both rich in xylose and contain different levels of inhibitors such as acetate, sugar dehydration products (furfural, 5-hydroxymethylfurfural), and lignin-derived products (ferulate, p-coumarate). In batch fermentations, we quantify succinate and co-product (acetate and formate) titers as well as succinate yields and productivities. We demonstrate yields of 0.74 g succinate/g sugars and 42.8 g/L succinate from deacetylated DAP hydrolysate, achieving maximum productivities of up to 1.27 g/L-h. Moreover, A. succinogenes is shown to detoxify furfural via reduction to furfuryl alcohol, although an initial lag in succinate production is observed when furans are present. Acetate seems to be the main inhibitor for this bacterium present in biomass hydrolysates.ConclusionOverall, these results demonstrate that biomass-derived, xylose-enriched hydrolysates result in similar yields and titers but lower productivities compared to clean sugar streams, which can likely be improved via fermentation process developments and metabolic engineering. Overall, this study comprehensively examines the behavior of A. succinogenes on xylose-enriched hydrolysates on an industrially relevant, lignocellulosic feedstock, which will pave the way for future work toward eventual SA production in an integrated biorefinery.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0425-1) contains supplementary material, which is available to authorized users.

BackgroundBio-manufacturing of high-value chemicals in parallel to renewable biofuels has the potential to dramatically improve the overall economic landscape of integrated lignocellulosic biorefineries. However, this will require the generation of carbohydrate streams from lignocellulose in a form suitable for efficient microbial conversion and downstream processing appropriate to the desired end use, making overall process development, along with selection of appropriate target molecules, crucial to the integrated biorefinery. Succinic acid (SA), a high-value target molecule, can be biologically produced from sugars and has the potential to serve as a platform chemical for various chemical and polymer applications. However, the feasibility of microbial SA production at industrially relevant productivities and yields from lignocellulosic biorefinery streams has not yet been reported.ResultsActinobacillus succinogenes 130Z was immobilised in a custom continuous fermentation setup to produce SA on the xylose-enriched fraction of a non-detoxified, xylose-rich corn stover hydrolysate stream produced from deacetylation and dilute acid pretreatment. Effective biofilm attachment, which serves as a natural cell retention strategy to increase cell densities, productivities and resistance to toxicity, was accomplished by means of a novel agitator fitting. A maximum SA titre, yield and productivity of 39.6 g L−1, 0.78 g g−1 and 1.77 g L−1 h−1 were achieved, respectively. Steady states were obtained at dilution rates of 0.02, 0.03, 0.04, and 0.05 h−1 and the stirred biofilm reactor was stable over prolonged periods of operation with a combined fermentation time of 1550 h. Furthermore, it was found that a gradual increase in the dilution rate was required to facilitate adaptation of the culture to the hydrolysate, suggesting a strong evolutionary response to the toxic compounds in the hydrolysate. Moreover, the two primary suspected fermentation inhibitors, furfural and HMF, were metabolised during fermentation with the concentration of each remaining at zero across all steady states.ConclusionsThe results demonstrate that immobilised A. succinogenes has the potential for effective conversion of an industrially relevant, biomass-derived feed stream to succinic acid. Furthermore, due to the attractive yields, productivities and titres achieved in this study, the process has the potential to serve as a means for value-added chemical manufacturing in the integrated biorefinery.

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Fermentations were performed in an external recycle bioreactor using CO2 and D-glucose at feed concentrations of 20 and 40 g L . Severe biofilm formation prevented kinetic analysis of suspended cell ('chemostat') fermentation, while perlite packing enhanced the volumetric productivity by increasing the amount of immobilised cells.The highest productivity of 6.35 g L h was achieved at a dilution rate of 0.56 h . A constant succinic acid yield of 0.69±0.02 g/(g of glucose consumed) was obtained and found to be independent of the dilution rate, transient state and extent of biofilm buildup -approximately 53% of the carbon that formed phosphoenolpyruvate ended up as succinate. Byproduct analysis indicated that pyruvate oxidation proceeded solely via the formate-lyase pathway. Cell growth and corresponding biofilm formation were rapid at dilution rates higher than 0.35 h when the product concentrations were low (succinic acid<10 g L ), while minimal growth was observed at succinic acid concentrations above this threshold.

The rate inhibiting effect of water as a product on reactions catalysed by cation exchange resins: Formation of mesityl oxide from acetone as a case studyElizabeth Louisa du Toit U Un ni iv ve er rs si it ty y o of f P Pr re et to or ri ia a e et td d - It is known that when water is a product in reactions catalysed by cation exchange resins, it inhibits the reaction rate much more than predicted by the reverse reaction or dilution effects. In this work the inhibiting effect is ascribed to the preferential association of the catalytically active sites with water. In the derivation of the kinetic model, a Freundlich type adsorption isotherm was used to quantify the number of sites occupied by water. This is combined with a power law expression for the reaction rate. The resultant expression can accurately predict the reaction rate for various initial concentrations of water and mesityl oxide. Even when water was initially added to the reaction mixture, this model still gave an absolute average error of 6.5% compared to a 54.6% error when the same approach was followed but with the more popular Langmuir isotherm to describe site deactivation. The kinetic expression previously proposed for this reaction system by Klein and Banchero also failed when water was added to the reaction mixture and gave i U Un ni iv ve er rs si it ty y o of f P Pr re et to or ri ia a e et td d --D Du u T To oi it t, , E E. .L L. . ( (2 20 00 03 3) ) an average error of 71.1%. The procedure used to derive the model is therefore suggested for all cation exchange catalysed reactions where water is one of the products.

BackgroundSuccinic acid is well established as bio-based platform chemical with production quantities expecting to increase exponentially within the next decade. Actinobacillus succinogenes is by far the most studied wild organism for producing succinic acid and is known for high yield and titre during production on various sugars in batch culture. At low shear conditions continuous fermentation with A. succinogenes results in biofilm formation. In this study, a novel shear controlled fermenter was developed that enabled: 1) chemostat operation where self-immobilisation was opposed by high shear rates and, 2) in-situ removal of biofilm by increasing shear rates and subsequent analysis thereof.ResultsThe volumetric productivity of the biofilm fermentations were an order of magnitude more than the chemostat runs. In addition the biofilm runs obtained substantially higher yields. Succinic acid to acetic acid ratios for chemostat runs were 1.28±0.2 g.g-1, while the ratios for biofilm runs started at 2.4 g.g-1 and increased up to 3.3 g.g-1 as glucose consumption increased. This corresponded to an overall yield on glucose of 0.48±0.05 g.g-1 for chemostat runs, while the yields varied between 0.63 g.g-1 and 0.74 g.g-1 for biofilm runs. Specific growth rates (μ) were shown to be severely inhibited by the formation of organic acids, with μ only 12% of μmax at a succinic acid titre of 7 g.L-1. Maintenance production of succinic acid was shown to be dominant for the biofilm runs with cell based production rates (extracellular polymeric substance removed) decreasing as SA titre increases.ConclusionsThe novel fermenter allowed for an in-depth bioreaction analysis of A. succinogenes. Biofilm cells achieve higher SA yields than suspended cells and allow for operation at higher succinic acid titre. Both growth and maintenance rates were shown to drastically decrease with succinic acid titre. The A. succinogenes biofilm process has vast potential, where self-induced high cell densities result in higher succinic acid productivity and yield.

in Wiley InterScience (www.interscience.wiley.com).A novel technique is used to obtain the distribution of particle wetting in trickle-bed reactors for different flow and prewetting conditions. Two prewetting methods were investigated: (1) Levec prewetting, in which the packed catalyst bed is completely flooded and then left to drain before steady state trickle-flow is commenced; (2) Kan prewetting, in which the bed is prewetted by pulsing with the liquid before flow, after which the liquid flow is gradually set back to the required rate. It is shown that the method of prewetting has a major influence on average wetting efficiency and wetting distributions: Average wetting efficiencies differ with as much as 20% between the prewetting modes, and Levec prewetting leads to a considerable fraction of the bed that is not used (up to 7% at the investigated flow conditions), whereas the entire catalyst is used in Kan prewetted beds.It is shown that the particle wetting distributions can influence the modeling of Levec prewetted beds for liquid-and gas-limited conditions.