Hyaluronic acid (HA) is a high value, naturally occurring polysaccharide that has widespread biomedical application. Although most HA is commercially extracted from animal tissues, there has been an increasing trend towards microbial production, since the capsules of certain streptococci are composed of human-identical HA. The main objective of this thesis was to investigate the effect of selected culture conditions on the partitioning of HA between capsular and soluble forms and the growth and production of HA in cultures of Streptococcus zooepidemicus.
To determine accurately the effect of a culture variable on HA synthesis, it was essential to develop a method of measuring total HA (tHA) concentration, comprising both solubilised HA (sHA) and capsular HA. From a study of capsule removal treatments, incubation of samples in 0.05% (w/v) sodium dodecyl sulfate achieved complete and non-destructive capsule removal in 5 minutes. Using this technique, a quantitative measure of the specific capsule content (SCC) was obtained. Batch fermentation studies in which tHA concentration was measured showed that two variables, culture pH and aeration, had a profound effect on HA biosynthesis. In contrast, agitation (between 300 and 600 rpm) had no effect. The optimum pH for HA production from glucose-yeast extract medium by S. zooepidemicus was 6.7 (± 0.2) under anaerobic conditions, with specific glucose consumption and tHA synthesis rates of 4.0 and 0.26 g g-1 h-1, respectively. Outside this range, tHA production was poor. Aeration improved the tHA yield from glucose by as much as 20%, compared to anaerobic cultures. However, tHA production was insensitive to aeration rates between 0.1 to 1.0 L min-1.
Using transmission electron microscopy and high performance liquid chromatography-size exclusion column techniques, capsule size of S. zooepidemicus cells from stirred batch cultures was found to vary significantly depending on culture conditions. Whereas cells from anaerobic cultures had only small capsules (SCC = 0.1 gHA gDW HA-free-1), those from cultures with aeration or low agitation (300 rpm) possessed very large capsules, with the SCC as high as 0.3 g g-1. However, the presence of large capsules presented little barrier to the transport of substrates and products, into and out of the cells since the specific rates of glucose consumption were similar. Solubilisation of capsules into the broth was found to be mostly shear dependent as the rate of sHA production at 300 rpm was 80% that at 600 rpm (0.35 g L-1 h-1), however the tHA production rates were identical. This is in contradiction to previous reports by others who concluded that lower agitation resulted in lower rates of HA synthesis, but who only mentioned sHA concentrations. It is clear that analysis based on sHA measurement alone is misleading.
Growth and product formation, including HA biosynthesis, were inhibited by lactate, the main catabolic product of S. zooepidemicus, in a concentration-dependent manner. The inhibition was observed under both anaerobic and aerated conditions. At broth lactate concentrations above 39 g L-I, HA biosynthesis was reduced significantly. Inhibition was also characterised by a reduction in glucose uptake rate and metabolic energy production (based on the concomitant fall of lactate production rate). Although lactate production was significantly suppressed under glucose limited conditions, the productivity of the culture, including HA synthesis, was markedly reduced.
HA production was higher under aerated conditions, however biomass yield and specific growth rate of S. zooepidemicus was lower compared to anaerobic cultures. The reduced biomass growth under aerated conditions was hypothesised to be due to H202 inhibition, since H202 was qualitatively detected in S. zooepidemicus aerated cultures and its growth in anaerobic culture with added H202 was inhibited. A structured model, which successfully described anaerobic HA fermentation by S. zooepidemicus, was extended to describe HA fermentation under aerated conditions. The model incorporated oxygen metabolism and growth inhibition by H202. The model was shown to fit experimental data better when H202 inhibition kinetics were included than without. Model-based studies suggests that the toxic effect of H202 on S. zooepidemicus was severe and the removal rate of this metabolite would be high, therefore the level of hydrogen peroxide in the medium was expected to be small. Since growth and HA production compete for energy as well as glucose-derived precursors, the improved HA production observed under aerated conditions was attributed to the partial inhibition of growth, most probably by H202.