Shiv Mohan Singh

Bacterial diversity and bioprospecting for cold-active enzymes from culturable bacteria associated with sediment from a melt water stream of Midtre Lov´enbreen glacier, an Arctic glacier

Culturable bacterial diversity of Midtre Lovenbreen glacier, an Arctic glacier, was studied using 12 sediment samples collected from different points, along a transect, from the snout of Midtre Lovenbreen glacier up to the convergence point of the melt water stream with the sea. Bacterial abundance appeared to be closer to the convergence point of the glacial melt water stream with the sea than at the snout of the glacier. A total of 117 bacterial strains were isolated from the sediment samples. Based on 16S rRNA gene sequence analyses, the isolates (n=117) could be categorised in to 32 groups, with each group representing a different taxa belonging to 4 phyla (Actinobacteria, Bacilli, Flavobacteria and Proteobacteria). Representatives of the 32 groups varied in their growth temperature range (4-37 degrees C), in their tolerance to NaCl (0.1-1M NaCl) and in the growth pH range (2-13). Only 14 of 32 representative strains exhibited amylase, lipase and (or) protease activity and only one isolate (AsdM4-6) showed all three enzyme activities at 5 and 20 degrees C respectively. More than half of the isolates were pigmented. Fatty acid profile studies indicated that short-chain fatty acids, unsaturated fatty acids, branched fatty acids, cyclic and cis fatty acids are predominant in the psychrophilic bacteria.

Taxonomic characterization, adaptation strategies and biotechnological potential of cryophilic yeasts from ice cores of Midre Lovénbreen glacier, Svalbard, Arctic

Ten strains of cryophilic yeast were studied from glacier ice cores of Svalbard, Arctic. The ice melt samples contained about 3×10(3) - 1×10(4) colony forming unit (CFUs) per ml. Sequence analysis of the isolates, using D1/D2 domain identified five species of yeasts: Cryptococcus adeliensis (MLB-18 JX192655), Cryptococcus albidosimilis (MLB-19 JX192656), Cryptococcus saitoi (MLB-22 JX192659), Rhodosporidium lusitaniae (MLB-20 JX192657), and Rhodotorula mucilaginosa (MLB-27 JX192664). Effect of temperature on growth of these isolates was studied. The strains are able to grow at temperatures ranging between 1 and 20°C. Screening of the cultures for amylase, cellulase, protease, lipase, urease and catalase activity were carried out indicating varying amounts of enzyme production at different temperatures. Characterization of lipase in strain Cryptococcus sp. MLB-24 was performed. Fatty acid methyl ester (FAME) analysis of the cultures grown at four different temperatures (1, 4, 15, and 20°C) was also done. Decrease in temperature was reported to cause increase in concentration of unsaturated fatty acids. High amount of oleic acid accumulated with increase in temperature. These fatty acids possibly help the strains to survive in glacial ice core cold environment. The extracellular and intracellular filtrate of the cultures showed negative antifreeze protein (AFP) activity. The observations indicate that probably the isolates in the present undertaking adapt to low temperatures, by enzyme and PUFA secretion rather than by antifreeze protein secretion.

Atmospheric deposition studies of heavy metals in Arctic by comparative analysis of lichens and cryoconite

Lichens and cryoconite (rounded or granular, brownish-black debris occurring in holes on the glacier surface) from Ny-Ålesund were used for understanding the elemental deposition pattern in the area. Lichen samples collected from low-lying coastal region and cryoconite samples from high altitudinal glacier area were processed and analysed for elements such as aluminium (Al), arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), cesium (Cs), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), vanadium (V) and zinc (Zn) through inductively coupled plasma mass spectrometry. Results showed that heavy metals, Al and Fe, are present in high concentration in the cryoconite samples. Al was also present in high amounts in seven of the eight lichen samples studied. The general scheme of elements in the decreasing order of their concentrations for most of the cryoconite samples was Al > Fe > Mn > Zn > V > Pb > Cr > Ni > Cu > Co > As > Cs > Cd while that for the lichen samples was Al > Fe > Zn > Mn > Pb > Cu > Cs > Cr > Ni > V > Co > As > Cd. Similarity in trends in the two sample types confirms that the environment indeed contains these elements in that order of concentration which overtime got accumulated in the samples. Overall comparison showed most elements to be present in high concentrations in the cryoconite samples as compared to the lichen samples. Within the lichens, elemental accumulation data suggests that the low-lying site (L-2) from where Cladonia mediterranea sample was collected was the most polluted accumulating a number of elements at high concentrations. The probable reasons for such deposition patterns in the region could be natural (crustal contribution and sea salt spray) and anthropogenic (local and long-distance transmission of dust particles). In the future, this data can form a baseline for monitoring quantum of atmospheric heavy metal deposition in lichens and cryoconite of Svalbard, Arctic.

Antarctic ice core samples: culturable bacterial diversity.

Culturable bacterial abundance at 11 different depths of a 50.26 m ice core from the Tallaksenvarden Nunatak, Antarctica, varied from 0.02 to 5.8 × 10(3) CFU ml(-1) of the melt water. A total of 138 bacterial strains were recovered from the 11 different depths of the ice core. Based on 16S rRNA gene sequence analyses, the 138 isolates could be categorized into 25 phylotypes belonging to phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. All isolates had 16S rRNA sequences similar to previously determined sequences (97.2-100%). No correlation was observed in the distribution of the isolates at the various depths either at the phylum, genus or species level. The 25 phylotypes varied in growth temperature range, tolerance to NaCl, growth pH range and ability to produce eight different extracellular enzymes at either 4 or 18 °C. Iso-, anteiso-, unsaturated and saturated fatty acids together constituted a significant proportion of the total fatty acid composition.

Antifungal activity of Arctic and Antarctic bacteria isolates

Various psychrotolerant bacterial strains with broad-spectrum antimicrobial potential were isolated from a number of Antarctic and Arctic samples (lake sediments, water, as well as faeces, feathers and soils collected in penguin rookeries). Seven isolates from all types of samples exhibited clear antifungal activities against the multidrug-resistant pathogenic yeast strain Candida albicans NCIM 3471. One isolate from the penguin rookery, identified by means of 16S rDNA sequencing as a strain of Enterococcus faecium, showed very strong antimycotic activity against a total of six C. albicans strains. The antibiotic activity was found after ammonium sulphate precipitation and dialysis but was sensitive to proteolytic enzymes.

Isolation and characterization of extracellular polysaccharide Thelebolan produced by a newly isolated psychrophilic Antarctic fungus Thelebolus

The present investigation is on a newly isolated psychrophilic Antarctic filamentous Ascomycetous fungus that has been identified as Thelebolus sp. and given the designation of Thelebolus sp. IITKGP-BT12. The culture was primarily identified through morphological studies, and was further confirmed by 18S rRNA sequencing (GenBank Accession No. KC191572), which revealed its close relatedness with Thelebolus microsporus. The exopolysaccharide (EPS) produced (1.94 g L(-1)) by the fungus was isolated, purified and characterized as glucan having an average molecular mass of 5×10(5)Da. The structure of EPS was determined by gas chromatography with tandem mass spectrometry (GC-MS/MS), Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) studies ((1)H, (13)C and HSQC). NMR analysis indicated the presence of (1→3)-linked β-d-glucan backbone with (1→6)-linked branches of β-d-glucopyranosyl units. Antiproliferative activity of EPS was demonstrated in B16-F0 cells, with IC50 of 275.42 μg m L(-1). Flow cytometry analysis and DNA fragmentation studies revealed that the cytotoxic action of the EPS mediated apoptosis in cancer cells. This is the first ever report on bioactive EPS thelebolan from Thelebolus sp.

Antagonistic interaction networks among bacteria from a cold soil environment

Microbial antagonism in an Arctic soil habitat was demonstrated by assessing the inhibitory interactions between bacterial isolates from the same location. Of 139 isolates obtained from five soil samples, 20 antagonists belonging to the genera, Arthrobacter, Pseudomonas and Flavobacterium were identified. Inter-genus, inter-species and inter-strain antagonism was observed between the interacting members. The extent of antagonism was temperature dependent. In some cases, antagonism was enhanced at 4 °C but suppressed at 18 °C while in some the reverse phenomenon was observed. To interpret antagonism from an ecological perspective, the interacting members were delineated according to their positional roles in a theoretical antagonistic network. When only one antimicrobial producer (P) was present, all the other members permitted grouping into either sensitive (S) or resistant (R). Composite interactive types such as PSR, PS, PR or SR could be designated only when at least two producers were present. Mapping of all possible antagonistic interaction networks based on the individual positional roles of the interactive types illustrates the existence of complex and interconnected networks among microbial communities.

Phosphate solubilizing ability of two Arctic Aspergillus niger strains

Many filamentous fungi were isolated from the soils of Ny-Ålesund, Spitsbergen, Svalbard, and were screened in vitro for their phosphate solubilizing ability. Two strains of Aspergillus niger showed good tricalcium phosphate (TCP) solubilizing ability in Pikovskayas medium. The TCP solubilization index was calculated at varying levels of pH and temperatures. The ability of Aspergillus niger strain-1 to solubilize and release inorganic-P was 285 µg ml–1, while Aspergillus niger strain-2 solubilized 262 µg ml–1 from 0.5% TCP after seven days. This is the first report of TCP solubilization by Arctic strains that may serve as very good phosphate solubilizers in the form of biofertilizer.

Influence of initial pH on ethanol production by the Antarctic basidiomycetous yeast Mrakia blollopis.

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 fermented ethanol between pH 5.0 and pH 10.0 with optimum pH at 8.0–10.0. Knowledge of ethanol fermentability as to the genus Mrakia remains incomplete. Further experiments are required to elucidate the ethanol fermentability of genus e.g., as to optimum fermentation pH, optimum fermentation temperature, and cell viability during fermentation.

Pigment, fatty acid and extracellular enzyme analysis of a fungal strain Thelebolus microsporus from Larsemann Hills, Antarctica

A cold-tolerant fungal strain Thelebolus microsporus was investigated for the first time for its pigment and fatty acid production. High-performance liquid chromatography analysis confirmed the presence of carotenoid pigment. Gas chromatography observations showed the presence of major fatty acids: myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), heptadecanoic acid (17:0), linolenic acid (18:3) and linoleic acid (18:2). Of these, linolenic acid, a polyunsaturated fatty acid, was present in substantial quantity, suggesting that it may have a role in adapting the fungus to low Antarctic temperatures by modulating membrane fluidity. The commercial application of linolenic acid is as a food supplement for humans suffering from eczema, cardiovascular disease and diabetic neuropathy. Another fatty acid, linoleic acid, a precursor of 1-octen-3-ol, is the principal aromatic compound in most fungi and has also been documented in this strain. Screening of the fungal culture for extracellular enzyme activity for amylase, protease, lipase, chitinase and cellulase was carried out. The isolate showed maximum α-amylase activity at 20°C, suggesting effective applications as a detergent additive, in textile processing and in the food industry.