Influence of nutritional factors on the nature, yield, and composition of exopolysaccharides produced by Gluconacetobacter xylinus I-2281

Abstract

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.

FIG. 1.

Proposed pathway for the biosynthesis of sugar nucleotides and the production of gluconacetan in G. xylinus I 2281 with glucose and fructose used as the substrate. Activated sugar nucleotides are sequentially added to a lipid carrier to form repeating units of the polysaccharide. The last step involves transport of the repeating units across the cell membrane to the outer layer and polymerization to form the EPS. 1, glucose permease; 2, glucose dehydrogenase; 3, glucose hexokinase; 4, phosphoglucomutase; 5, UDP-glucose pyrophosphorylase; 6, UDP-glucose dehydrogenase; 7, d-gluconate dehydrogenase (flavin adenine dinucleotide dependent); 8, d-gluconate dehydrogenase (pyrroloquinoline quinone dependent); 9, phosphoglucoisomerase; 10, glucose-1-phosphate thymidylyl transferase; 11, glucose dehydratase; 12, rhamnose synthetase; 13, fructose permease; 14, fructose hexokinase; 15, mannose isomerase; 16, mannose mutase; 17, mannose-1-phosphate guanylyl transferase; 18, levansucrase; 19, polymerase. PPP, penthose phosphate pathway.

FIG. 2.

Batch fermentation of G. xylinus on 6 g of acetate per liter and 15 g of sucrose per liter. Concentration profiles can be divided into two main phases with respect to substrate consumption. Phase 1 corresponds to the uptake of acetate; phase 2 corresponds to consumption of sucrose. (A) Δ, acetate; −, carbon dioxide evolution rate; X, biomass. (B) ▴, (keto)-gluconate, which represents the sum of 5-ketogluconate, 2-ketogluconate, and gluconate; ♦, 5-ketogluconic acid; ○, gluconic acid; ⋄, 2-ketogluconic acid. (C) ▪, sucrose; •, gluconacetan.

FIG. 3.

Composition of EPS resulting from harsh (panels a, b, and c) and mild (panel d) hydrolysis of samples collected at the beginning (white columns), middle (grey columns), and end (black columns) of the second phase (Fig. 2) during growth on glucose (a), fructose (b), and sucrose (c and d). Monosaccharide concentrations were normalized with respect to rhamnose concentration (panels a, b, and c).