Iron-binding characterization and polysaccharide production by Klebsiella oxytoca strain isolated from mine acid drainage

Abstract

Aims: To investigate Klebsiella oxytoca strain BAS-10 growth on ferric citrate under anaerobic conditions for exopolysaccharide (EPS) production and localization on cell followed by the purification and the EPS determination of the iron-binding stability constant to EPS or biotechnological applications.

Methods and results: Klebsiella oxytoca ferments ferric citrate under anaerobic conditions and produces a ferric hydrogel, whereas ferrous ions were formed in solution. During growth, cells precipitate and a hydrogel formation was observed: the organic material was constituted of an EPS bound to Fe(III) ions, this was found by chemical analyses of the iron species and transmission electron microscopy of the cell cultures. Iron binding to EPS was studied by cyclic voltammetric measurements, either directly on the hydrogel or in an aqueous solutions containing Fe(III)-citrate and purified Fe(III)-EPS. From the voltammetric data, the stability constant for the Fe(III)-EPS complex can be assumed to have values of approx. 10(12)-10(13). It was estimated that this is higher than for the Fe(III)-citrate complex.

Conclusions: The production of Fe(III)-EPS under anaerobic conditions is a strategy for the strain to survive in mine drainages and other acidic conditions. This physiological feature can be used to produce large amounts of valuable Fe(III)-EPS, starting from a low cost substrate such as Fe(III)-citrate.

Significant and impact of the study: The data herein demonstrates that an interesting metal-binding molecule can be produced as a novel catalyst for a variety of potential applications and the EPS itself is a valuable source for rhamnose purification.

Figure 1

Fe(II) production by Klebsiella oxytoca BAS-10 from Fe(III)-citrate fermentation in FeC ( • ) and in FeKC ( ○ ) media with and without sodium additions, respectively. Bar errors are from double replicates Fe(II) determinations.

Figure 2

Correlation found between proteins (μg ml⁻¹) ( • ) and glucuronic equivalents (μg ml⁻¹) ( ○ ) as an indicator of polysaccharide production, determined in cell pellets during 10 days of incubation in FeC medium under anaerobic conditions. Bar errors are from double replicates analyses.

Figure 3

Determination of CO₂ pressure (hPa) in a sealed 1-l Oxytop bioreactor (plot 1) during citrate fermentation by 100-ml culture of Klebsiella oxytoca BAS-10 and its uninoculated control (plot 2). After ferric hydrogel was precipitated, the culture was shaken (arrow) by magnetic stirring to remove gas entrapped by ferric hydrogel.

Figure 4

Transmission electron microscopy observations of Klebsiella oxytoca BAS-10 (a) growing anaerobically in NaC medium; bar = 1 μm. (b) High electronic dense iron-binding capsular EPS of a cell growing anaerobically in FeC medium. Bar 1 μm was for (a) and (b) photos. (c) A short rod of Kl. oxytoca BAS-10 with detached inner membrane from wall and with very electron dense extrusion such as a bud of Fe(III)-EPS; bar = 0·5 μm. (d) A similar cell but with an overwhelmed production of Fe(III)-EPS like plumes; bar = 1 μm; (e) a detail of EPS emission in some spotted zones of envelope and formation of nano-vesicles out of outer membrane; bar = 0·5 μm.

Figure 5

Cyclic voltammograms performed with a Hg-coated Pt microdisk 12·5 μm radius in the iron hydrogel; (broken line) end potential −0·8 and −1·3 V (solid lane). Scan rate 10 mV s⁻¹.

Figure 6

Cyclic voltammograms of 1 mmol l⁻¹ Fe(III)-citrate in aqueous solutions containing 0·1 mol l⁻¹ NaNO₃ (curves a and b) and 5 mg per 20 ml polysaccharide (curves c and d), 2 mmol l⁻¹ EDTA (curve e). Working electrode: Hg-coated Pt microdisk 12·5 μm radius; reference electrode as indicated in the figure. Scan rate 10 mV s⁻¹.