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

Abstract

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.