Extracellular Enzymatic Activity of Bacterial Strains Isolated from a Local Hotspring Tarabalo, Nayagarh District, Orissa, India
H Mohanta, C Rath
extracellular enzymes, hotspring, thermophilic bacteria
H Mohanta, C Rath. Extracellular Enzymatic Activity of Bacterial Strains Isolated from a Local Hotspring Tarabalo, Nayagarh District, Orissa, India. The Internet Journal of Microbiology. 2008 Volume 7 Number 2.
Studies on the 49 extracellular enzyme producing thermophilic bacteria isolated from a sulphur hotspring Tarabalo, Orissa, India were carried out. Sediment and water sample of the hotspring were assessed on Nutrient agar and MacConkey agar medium for bacterial strains. Out of 49 isolates, 77.75%, 97.95%, 87.75%, 83.67%, 87.71% of the strains showed amylase, protease, caseinase, lipase, cellulase activities respectively by plate assay method at 54°C.
Thermophilic microorganisms have gained a great deal of attention in the last two decades (Becker
The aim of this study was to asses extracellular enzymatic activities of thermophilic bacteria in Tarabalo a local sulphur Hotspring of Orissa, India.
Materials and Methods
Isolation of bacteria
The samples were collected from three sites of the thermal spring of Tarabalo area of Nayagarh district of Orissa, India. It is the second largest hotspring in India, as per the report of Tourism Development Corporation, India. Tarabalo is still a virgin hotspring in regard to microbial wealth, exploration. Temperature and pH of the sediment and water sample were measured in the field after collection. Soil and water samples were collected from three different sources in Tarabalo Hotspring aseptically and transported in to the laboratory in sterile polythenebags (Sediment samples) and autoclaved glass vials (Water samples) respectively. One gram of the sediment sample was suspended in 5 ml of sterile distilled water that served as stock sample, which was streaked on Nutrient agar (NA) and MacConkey Agar (MA) plates. Where as one loop of water sample was directly streaked on the above said plates. (Media were prepared by addition of NB and MB+1.5%Agar+50% distilled water+50% hotspring water for NA and MA respectively). The plates were packed in polythenebags to avoid drying and were incubated in 540C. After 24-48 hours of incubation, the bacterial colonies were selected based on their morphological characteristics and picked up for pure culture on modified NA, medium. Isolated colonies were preserved on NA slants at room temperature for future use. Further, sediment and water samples were serially diluted (10 fold dilution) and subjected for total plate count through spread plate and pour plate method for enumeration of bacteria on NA plates followings the method of Rath and Subramanyam (1998).
All the 49 isolates were assessed for their extracellular enzymatic activity. The strains were screened on solid agar media at two different temperatures 380C and 540C in triplicates.
Freshly grown cultures of test bacteria were spot inoculated on starch agar plates by the help of a sterile loop. Amylase activity was observed by incubating the plates and exposing to iodine vapours (Rath and Subramanyam 2000). Protease activity of the isolates was studied by growing isolates on skimmed milk agar plates (Rath 1996). Similarly caseinase activity was also studied by observing the zone of clearance around the colonies where the isolates were incubated at 540C, after spot inoculation on casein (1%) nutrient agar plates. Lipase activity of the isolates was reported by inoculating the isolates into tributryn agar plates, following the method of (Rath 1999). Cellulase activity of the isolates was studied on carboxy methyl cellulose (CMC1%) agar plates following the method of (Rath and Subramanyam 1997)
Water and sediments showed a chaterstics hydrogen sulphide odour. During sample collection the temperature and pH of water and sediments were measured, to be 56.60 C, 8.5 and 56.10C, 8.45 respectively. Bacteria were more in sediment in comparison to the hotspring water, (Table-1). Forty-nine bacteria were selected based on morphological features and growth on NA and MA plates named HCTB-1 through HCTB-49. All 49 isolates were screened for different extracellular enzymatic activities. The strains were assessed for enzymatic activity in terms of zone sizes and substrate digestion at the temperature mentioned below Table-2 & Fig.1.
Thirty eight, forty eight, forty three, forty one and forty two isolates out of 49 isolates screened, showed positive activity for amylase, protease, caseinase, lipase and cellulase respectively, though enzymatic activities were observed at 380C, an increased enzymatic activity in terms of zone sizes was reported at higher temperature (540C), indicating the thermophilic nature of the isolates as well as the thermostability of the enzymes. Surprisingly HCTB-13 and HCTB-40 did not show amylase activity at low temperature (380C), but the activity was reported at higher temperature (540C) with a zone diameter of 5mm and 10mm respectively (table-2). Similarly HCTB-24 represented lipase activity at 540C with a zone diameter of 17mm, where as no activity was reported at lower temperature (380C). No such distinctions were reported while studying the protease and cellulase activities of the isolates during the investigation.
But the isolate HCTB-45, which showed lipase activity at 380C, did not show activity at 540C. A maximum of 48 isolates showed protease activity on skimmed milk agar plates. The % enzymatic activities of the isolates are presented in Table-3.
In this investigation we clearly recorded the occurrence of bacterial strains, in the hotspring Tarabalo, in the distinct of Nayagarh in state of Orissa, with industrially important extracellular enzymatic activities. It is worth to mention that most of the amylolytic enzymes produced by various microbial strains are not able to function efficiently under the condition prevailing in industrial process. Hence there is a constant search for better performers (Kelly and Fogarty 1983). In this regarded we recorded amylase activity of 38 bacterial isolates at 540C could be attractive candidates for commercial amylase. The smaller zone sizes on solid agar plates are presumably because of low amounts of lipase molecules released by the colonies (Kouker and Jaeger 1987). It is well established that the logarithim of the lipase activity is linearly related to the zone diameter there by fulfilling a requirement of a valid agar assay (Lawrence
The above investigation clearly revealed thermostable extracellular enzymatic activity of bacterial strains isolated from a local Hotspring. To the best of our knowledge, perhaps this study is first of its kind to explore the bacterial wealth of Tarabalo Hotspring in the state, Orissa, India. Studies such as this is a prerequisite for tapping the biotechnological potential of the microbes from this unique ecosystem.
The authors are highly thankful to Dr. S.K.Das, Principal, Dr. B.C. Gochayat, Controller of examination, Mr. H.H. Das, Head, Department of Microbiology, M.P.C. Autonomous College, Baripada, Orissa for providing necessary laboratory facilities. The author HSM highly acknowledges the help of laboratory staffs Mr. A. Pattnaik and G. Pati of Dept. of Microbiology, during the investigation.