Isolation, characterization and exploring biotechnological potential of halophilic archaea from salterns of western India

Abstract

Thirteen halophilic archaea were isolated from Kandla and Bhayander salt pans. These isolates were grouped into three different genera Halobacterium, Haloferax and Haloarcula based on morphological and biochemical characterization, polar lipid analysis, Amplified 16S rDNA restriction analysis (ARDRA) and 16S rDNA sequence analysis. Biochemical characterization suggested the ability of isolates to produce protease, amylase and poly-hydroxybutyrate (PHB) indicating their biotechnological potential. The isolates were further screened for the amount of extracellular protease produced. Halobacterium sp. SP1(1) showed significant protease production compared to other isolates. Protease producing ability of the isolate was influenced by several factors such as NaCl concentration, type of protein source, metal ions and surfactants, and presence of amino acid supplements in the production medium. Soybean flour, FeCl₃ and dicotylsulfosuccinate were found to increase protease production by 2.36, 1.54 and 1.26 folds, respectively compared to production in basal medium. Effect of organic solvents used in paints (n-decane, n-undecane and n-dodecane) was also investigated on protease production by the isolate. Protease production by Halobacterium sp. SP1(1) was enhanced by 1.2 folds in presence of n-decane compared to control. Furthermore, the ability of isolate to hydrolyse fish protein was investigated using three different edible fishes (Pomfret, Flat fish and Seer fish) as sole protein source. Pomfret was found to be a good protein source for protease production by the isolate. These results revealed that Halobacterium sp. SP1(1) may have potential for paint-based antifouling coating preparations and fish sauce preparation by virtue of its extracellular protease.

Keywords: ARDRA; Fish protein; Halobacterium; Halophilic archaea; Organic solvents; Protease; Salt pans.

Fig. 1

a Map location of sampling area. b Kandla slat pan. c Bhayandar slat pan

Fig. 2

a Thin layer chromatography of polar lipids from haloarchaeal isolates. Lanes 1 and 8 represent reference Halobacterium salinarum and Haloferax mediterranei; lanes 2–7 represent isolates SP1(1), SP1(2b), SP3(2), SP2(1), SP2(2) and SP4, respectively. Blue-purple/pink spots represent glycolipids and yellow–brown spot represent PGS. Spots of PGS are seen in lane 1–7. b Thin layer chromatography of polar lipids from haloarchaeal isolates. Lanes 1 and 9 represent reference Halobacterium salinarum and Haloferax mediterranei; lanes 2–8 represent isolates SP1(2a), SP2(3), SP3(1), SOP, BS1, BS2a and BS2b, respectively. Blue-purple/pink spots represent glycolipids and yellow–brown spot represent PGS. Faint spot of PGS is seen in lane 1

Fig. 3

Dendogram based on ARDRA showing diversity among 13 haloarchaeal isolates and four reference strains

Fig. 4

Protease production (U/ml) by Halobacterium sp. SP1(1), SP1(2b) and SP3(2) as a function of time (h)

Fig. 5

Protease production (U/ml) by Halobacterium sp. SP1(1) at different NaCl concentrations as a function of time (h)

Fig. 6

Effect of protein sources on growth and protease production (U/ml) by Halobacterium sp. SP1(1)

Fig. 7

Effect of metal ions on growth and protease production (U/ml) by Halobacterium sp. SP1(1)

Fig. 8

Effect of surfactants on growth and protease production (U/ml) by Halobacterium sp. SP1(1)

Fig. 9

Effect of amino acid supplementation on growth and protease production (U/ml) by Halobacterium sp. SP1(1)

Fig. 10

Growth and protease (U/ml) production in presence of organic solvents by Halobacterium sp. SP1(1)

Fig. 11

Growth and protease production (U/ml) by Halobacterium sp. SP1(1) using edible fish powder as protein source