A Glacier Bacterium Produces High Yield of Cryoprotective Exopolysaccharide

Abstract

Pseudomonas sp. BGI-2 is a psychrotrophic bacterium isolated from the ice sample collected from Batura glacier, Pakistan. This strain produces highly viscous colonies on agar media supplemented with glucose. In this study, we have optimized growth and production of exopolysaccharide (EPS) by the cold-adapted Pseudomonas sp. BGI-2 using different nutritional and environmental conditions. Pseudomonas sp. BGI-2 is able to grow in a wide range of temperatures (4-35°C), pH (5-11), and salt concentrations (1-5%). Carbon utilization for growth and EPS production was extensively studied and we found that glucose, galactose, mannose, mannitol, and glycerol are the preferable carbon sources. The strain is also able to use sugar waste molasses as a growth substrate, an alternative for the relatively expensive sugars for large scale EPS production. Maximum EPS production was observed at 15°C, pH 6, NaCl (10 g L^-1), glucose as carbon source (100 g L^-1), yeast extract as nitrogen source (10 g L^-1), and glucose/yeast extract ratio (10/1). Under optimized conditions, EPS production was 2.01 g L^-1, which is relatively high for a Pseudomonas species compared to previous studies using the same method for quantification. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis of EPS revealed glucose, galactose, and glucosamine as the main sugar monomers. Membrane protection assay using human RBCs revealed significant reduction in cell lysis (∼50%) in the presence of EPS, suggesting its role in membrane protection. The EPS (5%) also conferred significant cryoprotection for a mesophilic Escherichia coli k12 which was comparable to glycerol (20%). Also, improvement in lipid peroxidation inhibition (in vitro) resulted when lipids from the E. coli was pretreated with EPS. Increased EPS production at low temperatures, freeze thaw tolerance of the EPS producing strain, and increased survivability of E. coli in the presence of EPS as cryoprotective agent supports the hypothesis that EPS production is a strategy for survival in extremely cold environments such as the glacier ice.

Keywords: Karakoram; cryopreservation; exopolysaccharide; glacier bacteria; psychrotrophs.

FIGURE 1

Culture and phylogeny (a) Mucoid phenotype of BGI-2 on tryptic soya agar supplemented with 2% glucose, (b) Ethanol precipitation of exopolysaccharide, and (c) phylogenetic tree of Pseudomonas sp. BGI-2 using neighbor joining method with bootstrap value (%) greater than 50 from 1000 replicates. Numbers in the brackets are GenBank accession numbers for the 16S rRNA gene sequences and Acinetobacter baumannii DSM 30007 is used as an outgroup to root the tree.

FIGURE 2

Monosaccharide analysis of the exopolysaccharide: (A) High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) chromatogram of nine sugars used as standard. (B) HPAEC-PAD chromatogram of monosaccharides present in EPS.

FIGURE 3

NMR analysis of the exopolysaccharide. (A) ¹H NMR spectra of EPS preparation BGI-2 (800 MHz, D₂O), with expansion of carbohydrate-derived NMR resonances shaded in orange (anomeric O₂CH-units) and green (OCH and OCH₂ units); major anomeric ¹H NMR resonances a–g are annotated (cf. text); numbers denote relative ¹H NMR section integrals (total: 100%), and (B) ¹³C DEPT NMR spectra of EPS preparation BGI-2 (125 MHz, D₂O), with expansion of carbohydrate-derived ¹³C NMR resonances shaded in orange (anomeric O₂CH-units) and green (OCH-units) and blue (OCH₂-units) units; red numbers indicate half-width (Hz) of anomeric and OCH₂ ¹³C NMR resonances, most probably resulting from overlap of related but different chemical environments; blue numbers denote relative ¹³C NMR section integrals (total: 100%, with respect to section shown).

FIGURE 4

Role of EPS in cryoprotection: (A) Freeze thaw survivability of Pseudomonas sp. BGI-2 compared to Rhodococcus sp. BGI-11 and E. coli k12 using plate count method. (B) Cryoprotective effect of different concentrations of EPS (1–5%) on E. coli k12 subjected to seven freeze thaw cycles using plate count method.

FIGURE 5

Membrane protection assay (RBCs lysis) using human RBCs shows significant reduction in% hemolysis when RBCs were pretreated with EPS compared to the controls with no EPS but only the surfactants (sodium dodecyl sulfate and titron x).