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) have 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 shape 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. Viability of the HA biofilms were 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.

A. succinogenes is well known for utilising various catabolic pathways. A multitude of batch fermentation studies confirm flux shifts in the catabolism as time proceeds. It has also been shown that continuous cultures exhibit flux variation as a function of dilution rate. This indicates a direct influence of the external environment on the proteome of the organism. In this work, ATP production efficiency was explored to evaluate the extent of bio-available energy on the production behaviour of A. succinogenes. It was found that the microbe successively utilised its most-to-least efficient energy extraction pathways, providing evidence of an energy optimisation survival strategy. Moreover, data from this study suggest a pyruvate overflow mechanism as a means to throttle acetic and formic acid production, indicating a scenario in which the external concentration of these acids play a role in the energy extraction capabilities of the organism. Data also indicates a fleeting regime where A. succinogenes utilises an oxidised environment to its advantage for ATP production. Here it is postulated that the energy gain and excretion cost of catabolites coupled to the changing environment is a likely mechanism responsible for the proteome alteration and its ensuing carbon flux variation. This offers valuable insights into the microbe’s metabolic logic gates, providing a foundation to understand how to exploit the system.

Biofilms of Actinobacillus succinogenes have demonstrated exceptional capabilities as biocatalysts for high productivity, titre, and yield production of succinic acid. This work gauges the effectiveness of these biofilms through analysis of the structure and cell viability of biofilms by using confocal scanning laser microscopy, scanning electron microscopy and image analysis software. The structure of the mature six-day-old biofilms of A. succinogenes was characterized by amorphous cell microcolonies of variable area and shape, with a mean thickness of 92±35 µm and low surface area to volume ratios of 0.25±0.07 µm 2 µm-3. Biofilm surface area coverage was low near the base (33±13%) but increased towards the outer layer. Despite the presence of water channels at the deeper portions of the biofilm, a greater portion of inactive cells was closest to the attachment surface and 65±2% of the biofilm consisted of dead cells. In this way, a cell viability gradient existed where the outer layer exhibits a greater fraction of active cells compared to the base layer. Biofilm cells underwent a phenotypic change, expressing a filamentous cell morphology different from rod-like morphology of suspended cells. The filamentous morphology permits extensive cell entanglements within a microcolony which may add to the intactness of microcolonies.

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 −1 DCW h −1 compared to the 0.8 g g −1 DCW 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.

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