Culturable Diversity of Psychrophilic Bacteria from Different Glaciers of Karakoram Mountain Range and Role of Cell Membrane Fatty Acids in Cold Adaptation

Abstract

Temperature of approximately 85% of Earth is under 5°C. These low temperature territories are distributed throughout the world ranging from Artic to Antarctica, European Alps to Deep Oceans and Hindu Kush-Karakoram-Himalaya Mountains. Cold environments are accompanied by a wide range of organisms covering all three domains of life including bacteria as well. Bacteria inhabiting such low temperature environments, are exposed to variety of extreme challenges. To thrive under such extreme conditions, bacteria adapt various strategies, most importantly pigment production and alterations of fatty acids exist in cell membranes to maintain normal membrane fluidity. The current study was aimed to study glaciochemistry of Ghulmet, Hopar (Bualtar) and Ghulkin glaciers, located in Karakorum Mountain Range, Pakistan, diversity of pigment producing pyschrotolerant bacteria in these glaciers and to determine the alteration in cell membrane fatty acids of bacterial species in response to different temperature. Glacial samples (ice, sediment and water) were collected from Ghulmet, Hopar and Ghulkin glaciers. Glaciochemistry of these samples was carried out to determine total organic carbon (TOC), total phosphorous (TP) and total nitrogen (TN) as well as major anions and cations. Bacteria were isolated from samples by all glaciers on basis of distinct colony morphology. Bacterial isolates were identified using two biomarkers 16S rRNA and rpoB genes. Pigments were extracted from pigment producing bacteria and identified through High-performance liquid chromatography (HPLC). Effect of temperature (5, 15, 25 and 35°C) on distribution of fatty acids existed in cell membranes of studied bacterial species was assessed. Methanolic HCl was used to extract fatty acid methyl esters (FAMEs) from all bacterial cells which were subsequently identified and quantified via Gas chromatography/mass spectroscopy (GC/MS). Cyanobacterium species CHS1 was recovered from Hopar glacier, Pakistan, identified by 16S-23S sequencing and studied for production of pigment and cell membrane fatty acids. Sediments of all glaciers were found the most enriched form of sample with all major ions, TOC, TN and TP followed by meltwater and ice. Hopar glacier sediments found to have highest concentrations of Ca2+ (10016 μg kg-1), K+ (363 μg kg-1), Mg2+ (1081 μg kg-1) and PO42– (0.7 μg kg-1), Ghulkin glacier sediments with NO – (248 μg kg-1), while sediment of Ghulmet glacier were enriched with Na+ (1595 μg kg-1). The highest TOC, TN and TP observed in sediments of all glaciers followed by meltwater and ice. Based on 16S rRNA and rpoB gene sequencing, all bacterial isolates were found to belong to 5 different bacterial phyla. Proteobacteria was predominantly found bacterial phylum covering 66% of total bacterial isolates followed by Actinobacteria (14% bacterial isolates), Firmicutes (10% isolates), Deinococcus-Thermus (6% bacterial isolates) and Bacteroidetes (4% isolates). In addition, out of 50 bacterial isolates, 39 were confirmed after HPLC to produce 11 different types of pigments including 2 unknown pigments as well. A total of 8 pigments were identified using analytical standards as alloxanthin, astaxanthin, bacterioruberin, β,β-carotene, β,ε-Carotene, 19'-hexanoyloxyfucoxanthin, peridinin, violacein and zeaxanthin. The fatty acids analysis in cell membranes of bacterial species revealed monounsaturated fatty acids (n-MUFAs) and fatty acids with branched chains as priniciple groups of fatty acids of bacterial cell membranes. The n-C15:1(cis-10), n-C16:1(cis-9) and n-C18:1(tr-9) were the main types of individual n-MUFAs witnessed in cell membranes of bacterial species, whereas ai-C15:0 and ai-C17:0 were the chief types of distinct branched fatty acids (br-FAs). Experiments related to observe effect of different temperature on cell membrane fatty acids of 10 bacterial strains belonged to Gram negative and Gram positive groups, were also carried out in this research work. Results revealed that Gram negative bacterial species have accumulated n-MUFAs and Gram positive bacteria produced br-FAs as major groups of cell membrane fatty acids. The quantity of n-MUFAs in Gram negative bacteria increased with lowering temperature (5 and 15°C) but decreased with raising temperature (25 and 35°C). Nevertheless, bacterial strains were found constant in production of high quantity of saturated branched chain fatty acids (br-SFAs) in their cell membranes at all temperature. In addition, Gram positive species were observed to accumulate saturated fatty acids in very low quantities in cell membranes at all temperatures. However, br-SFAs in Gram positive group of bacteria were detected in highest quantity in their cell membranes at high temperature (25 and 35°C) as compare to lower temperature (5 and 15°C). Likewise, branched chain monounsaturated fatty acid were observed in lowest concentration at high temperature (25 and 35°C) but in highest concentration at low temperature (5 and 15°C). Similarily, polyunsaturated fatty acids (PUFAs) in very low quantity were found to produce by few bacterial species. The bacterium species HI6 was recovered from Hopar glacier using Lauria Bertoni (LB) as culture medium and identified as Serratia marcescens after 16S rRNA sequencing. Experiments of physiological characterization revealed the ability of isolate HI6 to show optimum growth at neutral pH and 25°C in LB broth. In addition, GC/MS analysis of cell membrane fatty acids of isolate HI6 revealed branched chain fatty acids as most abundant type of fatty acids. Similarly, n-C18:2(cis-9), a polyunsaturated fatty acid, was also detected in cell membrane of isolate HI6. A cyanobacterium species CHS1 was isolated from the sediment of Hopar glacier using BG11 medium and recognized as Nodularia spumigena after subjecting to sequencing of 16S-23S intergenetic segment. Physiological characterization of isolate CHS1 showed its ability to grow optimally at pH 7, 2-4/L NaCl, and 15°C. Likewise, chlorohypll-a was major pigment produced by isolate CHS1, identified and quantified by High-performance liquid chromatography. In addition, all major types of cell membrane fatty acids including saturated fatty acids (SFAs), br-FAs, MUFAs and PUFAs, were detected by Gas chromatography/mass spectroscopy in isolate CHS1. This study provides a detailed view about the shift response of cell membrane fatty acids to various temperature in wide range cold-adapted bacterial species for the first time. This would provide basis in future to recognize the utmost potent role of fatty acids to maintain normal fluidity of cell membranes in bacterial species residing outside polar glacial habitats. Likewise, comprehensive outline of fatty acids present in bacterial cell membrane achieved via this study would also be used in upcoming time as biomarker for identification of cold-tolerant bacteria inhabiting cold environments. Furthermore, glaciochemistry would help to monitor any detrimental outcome of global warming on the health of glaciers located in Karakorum Mountain Range, (Pakistan) as they are at high risk to global warming. Similarly, this research work gives an overview of bacterial diversity in the glaciers under study with unique abilities to produce variety of carotenoid pigment that would be helpful in future as an alternative to synthetic and other (e.g. plants) pigment producing sources.