[Microbiology Research 2011; 2:e10] [page 37] An agar degrading diazotrophic actinobacteria from hyperalka- line meteoric lonar crater lake - a primary study Avinash A. Raut, Shyam S. Bajekal Department of Microbiology, Yashwantrao Chavan College of Science, Karad, Vidyanagar, Maharashtra, India Abstract There are very few reports on agarases being produced by actinobacteria, Streptomyces coelicolor being the only one known since decades for its agar degrading property. Here we report an agar degrading diazotrophic actinobacterium other than Streptomyces coelicolor, isolated from the lit- toral soil of Lonar Lake situated in Buldhana district of Maharashtra, India, a lake charac- terised by high alkalinity, carbonates, bicar- bonates, and algal blooms. The lake has a mean diameter of 1800 meters. The Gram-pos- itive filamentous rod grew in a simple medium of pH 10.5 containing agar as a sole source of carbon. The agar degrading property was detected by the appearance of depressions around each colony after 48 h of growth. The enzyme responsible for this degradation, agarase was also detected and estimated. The isolate also grew on Ashby’s Nitrogen free Mannitol Medium aerobically and fixed nitro- gen. Morphological, physiological and bio- chemical characteristics of the isolate are pre- sented in this paper. Introduction Agar, a complex polysaccharide is extracted from marine red algae belonging to the family Rhodophyceae, mainly from Gelidium and Gracilaria species. It is composed of two frac- tions agarose and agaropectin. Agarose, the main constituent, is a neutral polysaccharide that forms a linear chain structure consisting of repeating units of agarobiose, which is an alternating polymer of D-galactose and 3,6- anhydro-L-galactose linked by alternating β- (1,4) and α-(1,3) bonds. 1 It is widely employed as a gelling agent for microbiological culture media. Agarases are enzymes that hydrolyse the polymer agarose and have potential appli- cations in the food, cosmetic and medical industries for breaking down agar compo- nents. 2 Agar is attacked by relatively few species of microorganisms, including bacteria and actin- omycetes. Most of the previously reported agar degrading bacteria such as Alteromonas species, Bacillus cereus, Cytophaga, Pseudoalteromonas, Pseudomonas, Vibrio sp. and Zobella sp. have quite been isolated from marine environment. 2-14 Since agar is a poly- saccharide produced by marine red algae, it is natural that most agar degrading bacteria are inhabitants of marine habitats. However, there are very few reports of nitrogen fixing bacteria degrading agar except for an agar degrading marine nitrogen fixing bacterium placed in the family Vibrionaceae 15 There are also no reports of any actinobacteria other than Streptomyces coelicolor reported by Stanier. 16 Lonar Lake situated in Buldhana district of Maharashtra, India is known to be formed due to a meteorite impact some 52,000 years ago and has a mean diameter of 1800 meters Figure 1. It is an alkaline closed basin lake with large masses of algal blooms floating on the water and on the banks. Hence, the occurrence of agar degrading bacteria was a distinct possibil- ity in this environment. During the course of a study on nitrogen fixing bacteria from this environment we came across an agar degrad- ing organism. We report the morphological, physiological and biochemical characteristics of this organism in this paper. Materials and Methods Enrichment and isolation Soil samples collected from the littoral zone of Lonar Lake were subjected to enrichment of nitrogen fixing bacteria on Ashby’s Nitrogen free Mannitol broth with pH-10.5 and NaCl-2% amended as per the chemical characteristics of Lonar Lake soil. Tubes were incubated at 40°C for 48 h in a shaking incubator. After incuba- tion, loopfuls of enriched medium from each tube were streak inoculated on Ashby’s Nitrogen free Mannitol solid medium contain- ing 2.0% agar, pH-10.5 and incubated at 40°C for 48 h. Colony characteristics and morpho- logical characters of isolates Isolate was studied for its colony characters, Gram nature by Hucker and Conn modified Gram’s staining method. Mycelium arrange- ment was observed under microscope at by coverslip culture technique. 17 Agar degrading ability The organism was initially isolated as a nitrogen fixing actinobacterium from Lonar Lake. It was detected to be agar degrading by the appearance of depressions in agar around the colonies after 48 hours incubation. Confirmation was done by growing the isolate on a simple medium of composition Agar-20.0 g/L, NaCl-20.0g/L and incubated at 40°C for 48 h. Agarase activity of the isolate was also determined by the method of Shieh et al. 18 on semi solid PY medium of composition 2.0 g Peptone, 0.5 g yeast extract and agar 15.0g in 1 litre Lonar Lake water (instead of 90% sea water), with pH 10.5. Phylogenetic analysis of the 16s rRNA gene The partial 16s rRNA sequence was deter- mined in the Molecular Biology Unit of National Centre for Cell Sciences (NCCS), Pune using Universal Eubacteria-specific primers 16F27 (5’-CCA GAG TTT GAT CMT GGC TCA G-3’) and 16R1525XP (5’-TTC TGC AGT CTA GAA GGA GGT GWT CCA GCC-3’). 19 Its analysis was done using the web based SeqMatch of RDP-II (http.//www.rdp.cme. msu.edu). The derived sequence was submit- ted to DDBJ database and an accession num- ber was obtained for the same sequence. Agarase assay This assay was performed in triplicates according to the procedure of Miller et al. 20 Fifty (50) μL of the enzyme was added to 0.8% agarose solution (in carbonate buffer, pH 10.5) and allowed to react at 40°C for 30 minutes. The reaction was then stopped by addition of 3.0 mL DNS (3, 5-dinitrosalicylic acid) reagent. Microbiology Research 2011; volume 2:e10 Correspondence: Avinash A. Raut, Department of Microbiology, Yashwantrao Chavan College of Science, Karad,Vidyanagar, - 415 124, Maharashtra, India. E-mail: avinashraut@aol.in Key words: actinobacterium, agarase, Lonar Lake, algal blooms, depressions. Acknowledgements: the authors would like to thank the Management and Principal of Yashwantrao Chavan College of Science, Karad for the facilities provided in the laboratory. They also would like to thank Dr. Yogesh S. Shouche, Scientist, and Mr. Girish Kulkarni, Senior Research Fellow of Molecular Biology Unit, National Centre for Cell Sciences, Pune for the phylogenetic analysis of the isolate. Received for publication: 11 June 2011. Revision received: 29 July 2011. Accepted for publication: 29 July 2011. This work is licensed under a Creative Commons Attribution NonCommercial 3.0 License (CC BY- NC 3.0). ©Copyright A.A. Raut and S.S. Bajekal, 2011 Licensee PAGEPress, Italy Microbiology Research 2011; 2:e10 doi:10.4081/mr.2011.e10 Non-commercial use only