Steady-state analysis of continuous succinic acid fermentations by Actinobacillus succinogenes

Abstract

Succinic acid (SA) is poised to become a significant building-block or platform chemical in the bio-based economy. Of the microbial strains that show promise for biological SA production, the wild-type bacterium Actinobacillus succinogenes is one of the top-contenders. While strides have been made towards understanding the behaviour of the organism and developing the fundamentals for an industrial process based on the organism, there is still a large scope of research required and a multitude of challenges to be addressed. In particular, an improved understanding of the metabolism of the organism under favourable biofilm conditions is required and its potential as a microbial host in a biorefinery setting needs to be established. Therefore, the aim of this thesis is to develop a fundamental understanding of the central metabolism of the organism under biofilm conditions on relevant substrates, and to determine its performance as a SA producer on scalable biorefinery streams. Continuous operation is chosen as the processing mode as it allows for both reactor and metabolic steady-state conditions which facilitates more accurate analysis of fermentation data through metabolic flux balancing, and resembles the foreseeable mode of industrial operation. In addition, fermentations are conducted under biofilm conditions in custom reactors since the organism naturally and unavoidably produces biofilm, and unique and process-favourable metabolic behaviour has been observed in biofilms of A. succinogenes.

The thesis firstly assesses the performance of the organism on xylose in relation to the model substrate glucose, since both these carbohydrates constitute major fractions of renewable feedstocks, especially lignocellulosic biomass. Continuous biofilm fermentations reveal that A. succinogenes is able to effectively ferment xylose to SA. However, although xylose consumption rates are similar to those of glucose at dilution rates of 0.05, 0.10 and 0.30 h-1, lower yields (0.55 – 0.68 g g-1) and SA productivities (1.5 – 3.4 g L-1 h-1) are achieved. SA titres of between 10.9 and 29.4 g L-1 are attained with SA-to-acetic acid ratios between 3.0 and 5.0 g g-1. In addition, pyruvic acid formation is found to be substantially greater in xylose fermentations (1.2 – 1.9 g L-1) as detected by means of a modified HPLC method. In agreement with glucose fermentations, increased SA yields on xylose are observed at increasing SA tires indicating increased carbon flux to SA. Mass balance closures on xylose (80.6 to 85.3%) are shown to be lower than those on glucose, and are incomplete for both substrates. Furthermore, redox balances suggest that the central metabolic network, based on measurements of excreted metabolites, is unable to produce the required reduction power (as NADH) to account for the measured SA concentration. A possible source of the reduction power is the oxidative pentose phosphate pathway (OPPP).

Following the findings of the first portion of the thesis, the second section further investigates the metabolic behaviour of A. succinogenes. To probe the metabolic flux distributions beyond the limitations of metabolite-based flux balancing and to explore the hypothesis of the OPPP serving as the source of additional reduction power, assays of glucose-6-phosphate dehydrogenase (G6PDH) are performed on cell extracts of biofilm removed in situ during fermentations. A kinetic model of the assay data, based on Michaelis-Menten kinetics, is developed in vitro to estimate flux into the OPPP from glycolysis at the glucose-6-phosphate node. From the kinetic model it is found that G6PDH rates, normalised to cell concentration and substrate uptake rate, increase with increasing SA titre, corresponding to increasing deviations in the redox balance. Furthermore, metabolic flux models that include OPPP flux show similar trends to those observed through the kinetic models. Therefore, evidence of the OPPP leading to increased SA yields is provided. The OPPP generates reduction power as NADPH which can be converted to NADH by transhydrogenase, thereby facilitating additional flux through the reductive branch of the TCA cycle, wherein SA is produced, and closing the redox balance.

The final portion of the thesis explores the applied potential of A. succinogenes as a microbial platform for SA production in a biorefinery context. Fermentations of non-detoxified, deacetylated, dilute acid pretreated, corn stover hydrolysate (DDAP-H) are performed in a continuous reactor fitted with a novel agitator fixture capable of supporting biofilm to increase cell density. A. succinogenes is found to achieve competitive yields (0.78 g g-1), titres (39.6 g L-1) and productivities (1.8 g L-1h-1) on DDAP-H at dilution rates of 0.02, 0.03, 0.04 and 0.05 h-1. Furthermore, the organism is able to handle putative microbial inhibitors such as furfural and hydroxymethyfurfural (HMF) through conversion to less inhibitory compounds, likely the alcohol counterparts. However, it is found that continuous operation at reasonable productivities is only possible by starting at low dilution rates (~0.01 h-1) after initial batch operation, then gradually increasing the throughput to allow the culture to adapt – even under complete conversion of furfural and HMF. Various lignin-derived, low molecular weight phenolic compounds are shown to be present in the feed and increase during fermentation suggesting a possible link to the observed inhibition and the need for an acclimatisation phase.

Overall this thesis demonstrates that biofilms of A. succinogenes have the potential to produce SA at enhanced yields and productivities on glucose, xylose and process-relevant biorefinery streams under continuous operation. Furthermore, insight is provided into the unique metabolic behaviour of the organism under non-growth conditions where increased OPPP flux generates reduction power capable of sustaining increased flux to SA. In future work, this advantageous behaviour should be leveraged to develop a process capable of homosuccinate production with A. succinogenes, particularly employing a biorefinery stream as feedstock.