Diversity of Cold-Adapted Bacteria from Batura Glacier Pakistan and use of Exopolysaccharide from Pseudomonas sp. BGI-2 for Cryopreservation

Abstract

Bacterial diversity of Batura glacier was investigated using culture-dependent and culture- independent methods. 27 cold-adapted bacteria (mostly psychrotrophic) were isolated using culture-dependent method. Majority of the isolates exhibited growth at a wide range of temperature (4-35˚C), pH (5.0–12.0) and salinity (1-6%). Culture-dependent diversity results revealed isolates from the three glacier samples belonged to 3 major phylogenetic groups: Actinobacteria (48%), Bacteroidetes (26%) and Proteobacteria (22%) while Flavobacterium (26%), Arthrobacter (22%) and Pseudomonas (19%) represented the dominant genera. Bacterial 16S rRNA gene sequences in the unculturable community were also dominated by the Proteobacteria, Actinobacteria and Bacteroidetes, while Arthrobacter, Mycoplana, Ochrobactrum, Kaistobacter, Janthinobacterium and Flavobacterium represented the dominant genera. The glacial isolates demonstrated antimicrobial activities against other microorganisms which also included the multidrug resistant strains. Majority of the isolates exhibited activities for more than one extracellular enzymes including lipases, proteases, cellulases and amylases. Almost, all the isolates were sensitive to the broad spectrum antibiotics (ofloxacin and imipenem), while resistance was found against the narrow spectrum antibiotics. The isolate BGI-2 was selected for further studies based on its high abundance, relatively rapid growth at low temperatures and maximum EPS production among the 7 exopolysaccharide (EPS) producing isolates. Molecular identification using 16S rRNA gene sequencing placed BGI-2 in the genus Pseudomonas with the closest species Pseudomonas mandelii (99.59%) and Pseudomonas frederiksbergensis (99.59%). Pseudomonas sp. BGI-2 was able to grow in a wide range of temperature (4-35˚C), pH (5.0-11.0), and salinity (1-5%). The strain was able to use glucose, galactose, mannose, mannitol and glycerol as carbon sources. 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, which is relatively high for a Pseudomonas species compared to previous studies using the same method for quantification. The EPS is a heteropolysaccharide with glucose, galactose and glucosamine as the main sugar monomers. The strain is also able to use molasses as a growth substrate, an alternative for the relatively expensive sugars for large scale EPS production. Freeze-thaw survivability of BGI-2 was significantly higher than the non-EPS producing strains, including a psychrotroph BGI-11 isolated from the same environment and a mesophilic Escherichia coli strain K12. Also, the EPS produced by BGI-2 conferred significant cryoprotection for a mesophilic E. coli K12. Increase in EPS concentration from 1-5% significantly improved survivability of the E. coli K12, which was comparable to 20% glycerol. We used the possibility of employing EPS from BGI-2 as a cryoprotective agent for the cryopreservation of two microalga and three cyanobacterial strains. The EPS extracted from the glacier bacterium Pseudomonas sp., BGI-2 worked well for the cryopreservation of cyanobacterial strains Synechococcus sp. CB0101, Synechococcus sp. CBW1003, and Microcystis aeruginosa PCC 7806 and one microalga strain, Scenedesmus obliquus HTB1. Overall, biomass recovery for the microalgae and cyanobacterial strains in EPS was better than 5% DMSO and 10% glycerol after 9 months of cryopreservation. Moreover, concentration of the EPS used as cryoprotective agent is critical and varies with different strains, depending on their sensitivity. Successful colonization of the harsh cold environments is a result of the molecular evolution and adaptations. Draft genome sequence data revealed BGI-2 genome has 11 EPS-producing genes compared to none in the 6 closely related mesophilic Pseudomonas strains. We also found more stress response genes in the genome of BGI-2 than the closely related mesophilic counterparts. The stress response genes included osmotic stress, oxidative stress, cold shock, detoxification and carbon starvation. The genome also contains genes involved in cold adaptation, including desaturases for maintenance of membrane fluidity, production and uptake of compatible solutes, and production of exopolymers. Finally, another isolate Rhodococcus sp. BGI-11 exhibited ability to utilize long chain hydrocarbon hexadecane as the carbon source. This strain also tolerated the maximum salt concentration (8%) among all the glacial isolates. The strain also demonstrated growth at a wide range of temperature (4-35°C) and pH (4-11). Thus, the cold-adapted halotolerant Rhodococcus sp. BGI-11 can be used as a potential candidate for bioremediation purpose in Deep Ocean, where accidental oil spills are threat to its biodiversity.