Screening of Marine Sediments from Bay of Bengal near Pudimadaka Coast of Andhra Pradesh for Isolation of Lipolytic Actinobacteria and Characterization of the Most Potent Isolates

Ten sediment samples collected at various depths of Bay of Bengal near Pudimadaka Coast of Andhra Pradesh were screened for the isolation of lipolytic Actinobacteria by tributyrin agar clearing method. The selected isolates were cultured under submerged fermentation conditions and assayed for their extra cellular lipase producing capabilities using natural triglyceride (triolein) as substrate. Results indicated that 38 of the 88 isolates from 10 sediment samples showed lipolytic activity after primary screening. These isolates were subjected to secondary screening and lipase activity is estimated quantitatively. After preliminary characterization studies the promising isolate BTS-713 was found to belong to genus Streptomycetes and was designated as Streptomyces sp.BTS-713. In conclusion, these results increased the scope of finding industrially important marine actinomycetes from Pudimadaka Coast of Andhra Pradesh.


Introduction
Lipases have widespread occurrence through out the earth's flora and fauna, they are found in many species of animals, plants and microorganisms [Antonian, E., 1988].Commercial lipases are obtained from microorganisms as they are more stable compared to plant or animal lipases and they can be obtained cheaply.In the field of biotechnology, much attention has been paid to the use of lipases of microbial origin.They constitute the most important group of biocatalysts having tremendous biotechnological potential because, firstly, they can be produced in large quantities from microbial sources, secondly, they display exquisite chemo selectivity, regioselectivity and stereo selectivity and thirdly, the crystal structures of many lipases have been solved, facilitating considerably the design of rational engineering strategies.These properties make lipases the most widely used group of biocatalysts in organic chemistry [Jaeger, K.E., 2002].Important applications of lipases in biotechnology include their supplementation to detergents, in the manufacture of food ingredients, pitch control in the pulp and paper industry, and biocatalysis of stereo selective transformations.
Actinomycetes are the most economically and biotechnologically valuable prokaryotes.They are responsible for the production of half of the discovered bioactive secondary metabolites [Berdy, J., 1989].Few members of the actinomycetes have been studied with reference to purification and characterization of lipase enzyme [Sharma, R., 2001].The marine sediments as a source of bioactive actinomycetes was less exploited.In the present study, one natural saline habitat along the Pudimadaka Coast in the East coast of Southern India was selected for the isolation followed by assessment of lipolytic potential of marine actinomycetes.

Sampling Procedure
Ten marine sediment samples were collected from Bay of Bengal near Pudimadaka coast using a core sampler.The maximum depth of collection was 40m and all samples were transferred into sterile zipped polythene bags and transported to the laboratory for the isolation of actinomycetes.The locations and depths of these sampling stations are summarized in Table 1.

Isolation of Actinomycetes colonies from Marine sediments
Isolation and enumeration of actinomycetes were performed by the serial dilution plate technique [Ellaiah, 1996] using starch casein agar medium [Kuster, 1964], malt extract-yeast extract agar medium [Pridham, T.G., 1956-57] and chitin agar medium [Hsu, S.C., 1975].These media containing 50% sea water were supplemented with rifampicin 5μg/ml and nystatin 25μg/ml (Himedia, Mumbai) to inhibit bacterial and fungal contamination, respectively.All plates were incubated at 28±2ºC for 2-3 weeks.After incubation, actinomycetes isolates were distinguished from other microbial colonies which are partially submerged into the agar [Jensen, 1991] and colors of pigmentation including diffusible pigments.Single separated actinomycetes colonies were selected and the sub cultures were maintained on starch casein slants at 4 ºC until further use.

Secondary screening for lipase production
For the production of lipase, the selected isolates were cultivated in a synthetic medium containing olive oil (source of natural triglyceride, triolein) as the sole carbon source under submerged fermentation conditions and assayed for the lipolytic activity of the culture filtrates.45ml of production medium is taken in 250ml Erlenmeyer flask and inoculated with a loopful culture of each isolate.The flasks were incubated at 28ºC for 4 days on a rotary shaker (150 rpm).The culture broth was filtered and the clear filtrate was used as the source of crude enzyme.The composition of the production medium is (g/l): Olive oil, 10;(NH 4 ) 2 SO 4, 5 ; Na 2 HPO 4, 6 ; KH 2 PO 4, 2 ; MgSO 4, 3 ; CaCl 2, 3 with pH 6.0.

Lipase assay
The culture broth was filtered and the lipase activity in the culture filtrate was determined by titrimetry (olive oil substrate emulsion method) as described by Musantra [Musantra, 1992] One unit of enzyme activity is defined as the amount of enzyme required to liberate 1μmole equivalent fatty acid / ml/ min at 40 ºC under the standard assay conditions.All the experiments were carried out in triplicate and the mean of the three values was presented.

Preliminary identification of the five most potent actinomycetes isolates
The five most promising actinomycetes isolates were identified up to the genus level by macroscopic and microscopic morphological methods as per Bergey's 2000 manual [Bergey, 2000].The macroscopic method was done by colony characterization on yeast malt extract agar (YMA) medium.Colour of colony and presence of pigmentation were noted.The microscopic characterization was done by cover slip culture method observed after three days.The presence or absence of aerial and substrate mycelium, spore formation, and pigmentation of the vegetative or substrate mycelium were observed.Further characterization of selected isolates was done as per the ISP protocol [Shirling, 1966].The utilization of carbon sources was examined on Pridham and Gottleib's medium [Pridham, 1948] containing miscellaneous carbohydrates to a final concentration of 1%.Cell wall of the selected isolates was purified and analyzed by the methods of Lechevalier and Lechevalier [Lechevalier, 1980].Whole cell sugars were analyzed according to the method of Becker et al. [Becker, B., 1964].The selected isolates were screened for sodium chloride tolerance (1, 4,7,10 and 13% w/v) on Yeast-Malt agar slants according to Trenser et al [Trenser, 1968].Maximum sodium chloride concentration in the medium allowing any growth was recorded.

Isolation of actinomycetes colonies from marine sediments
Using the selective media, 88 actinomycetes strains were isolated (Table 2), of which maximum number of actinomycetes isolated was in Malt extract -Yeast extract agar medium (44) followed by starch casein agar medium (25) and chitin agar medium (19).The entire 10 samples of Pudimadaka are found suitable for the isolation of actinomycetes.High or low number of active strains found depends on many factors like the medium and methods of screening.Moreover, there are so many factors which affect actinomycetes growth and enzyme production, including the chemical and biological environment.

Primary screening for lipolytic actinobacteria
38 of the 88 isolates showed lipolytic activity (Table 3).This suggests that sediments of Pudimadaka are good sources for isolating lipolytic actinomycetes.Only few reports of lipases from marine actinomycetes were made by previous researchers although marine actinomycetes have excellent capacity to elaborate a wide diversity of enzymes [Sharma, 2001].The lipolytic activities of all the stains are shown in Table 3.As indicated in the table, isolate BTS-713 showed maximum lipolytic activity after primary screening (Table 4a and 4b).Screening with the help of tributyrin is a convenient and presumptive test for the detection of lipolytic organisms; hence all the isolates were screened using tributyrin agar clearing method.

Secondary screening for lipase production
All the positive isolates were subjected to submerged fermentation conditions and assayed for lipolytic activity quantitatively.Since tributyrin is not a substrate for lipases alone, all the selected positive isolates are confirmed for their lipolytic activity by the hydrolysis of natural triglyceride (triolein) under submerged fermentation conditions and the results are presented in Table 5a and 5b.As shown in the Table 6, the isolate BTS-713 showed maximum lipolytic activity of 0.0514U among all the positive isolates.Optimization of nutritional and physical parameters for reduction of cost in industrial scale production and presence of other bioactive compounds influencing lipolytic activity of the isolate had to be determined to determine the potency of BTS-713(Fig.1).But our study focuses on screening of marine sediments for lipolytic actinobacteria which has been accomplished.

Preliminary identification of the five most potent actinomycetes isolates
The five most potent lipolytic actinomycetes isolates were subjected to characterization up to genus level.The culture and morphological properties of the five isolates are shown in Table 7.The results indicate that among the five isolates, three isolates BTS-716, BTS-701, and BTS-711 were characterized by single spores at the tips of sporophores and two isolates BTS-713 and BTS-709 by spiral polyspores.Out of five isolates BTS-716, BTS-701, and BTS-711 showed black spore mass, while BTS-713 isolate showed green-grey spore mass.The form of the colony of isolates BTS-716, BTS-701 and BTS-711 was described to be compact, folded, raised and lichenoid to leathery and that of the isolates BTS-713 and BTS-709 was discrete, floccose and powdery.According to Waksman [1961] such colour and form is exhibited by colonies of both Streptomyces and Micromonospora.However, the colour of the growth and the form of the colony could not serve as basis of pointing out the genus to which actinomycetes isolates belongs to.Hence, its morphological properties must serve as the primary basis of characterization.One distinguish morphological property possessed by the isolates BTS-716, BTS-701, and BTS-711 is absence of an aerial mycelium, a characteristic possessed by all members of Micromonospora [Bergey, 2000].A well developed substrate mycelium that partly penetrates the medium and the formation of single spores at the sporophore tips are two characteristics that may well qualify them as a species of Micromonospora.The formation of a dark-brown pigment by isolates BTS-716 and BTS-701 that dissolves into the medium had also been mentioned by Waksman [1961] to be a possible characteristics of the genus, although not exclusively.Isolates BTS-713 and BTS-709 formed substrate mycelium and abundant aerial mycelium with powdery spore mass.Polysporic actinomycetes, forming characteristic aerial and substrate hyphae represent an important microscopic criterion to identify the genus Streptomyces [Bergey, 2000].The physiological and biochemical characteristics of the isolates are shown in Table 8a and 8b Isolates BTS-716,BTS-709, and BTS-701 exhibited positive reaction to melanin pigmentation and tyrosine reaction while these properties were found to be negative for isolates BTS-711and BTS-713.Isolates BTS-711,BTS-716 and BTS-709 had the ability for H 2 S production and nitrate reductase was secreted by isolate BTS-711,BTS-713, and BTS-709.All the isolates were positive for starch hydrolysis, casein hydrolysis and gelatin hydrolysis.Carbohydrate utilization test plays a prominent role in the taxonomic characterization of actinomycetes strains [Pridham, T.G., 1948].Abundant growth was attained with glucose, lactose, fructose and xylose by all the isolates used.However, little or no growth was attained when isolates BTS-711, BTS-716 and BTS-701 were grown on mannitol, rhamnose, raffinose and glycerol.Isolate BTS-713 efficiently utilized the carbon sources such as, mannitol, rhamnose, raffinose, glycerol and cellobiose.Rhamnose, raffinose, galactose were assimilated by the isolate BTS-709 as the good source of carbon and energy while ribose, galactose, arabinose and cellobiose were well utilized by the isolate BTS-711.All the isolates could coagulate and peptonize milk.Tolerance of the strain to NaCl concentration also serves as an important character for species identification.All the isolates exhibited salt tolerance up to 7%, indicating that the isolates are indigenous to marine environment.All the isolates were resistant to antibiotics used for identification studies and also grew well at 25-35ºC.Isolates BTS-713 and BTS-709 showed growth at pH range of 6-8 and BTS-716, BTS-701 and BTS-711 at pH range 5-9.Cummins and Harris [Cummins, C.S., 1956] stated that actinomycetes have cell wall composition akin to that of Gram-positive bacteria and indicated that the chemical composition of the cell wall might furnish practical methods of differentiating various types of actinomycetes.According to Lechevalier and Lechevalier [1980] the sugar composition often provides valuable information on the classification and identification of actinomycetes.Considering the above, an attempt was made to identify the actinomycetes, by analyzing their cell components.The cell wall peptidoglycan of three isolates BTS-716, BTS-701 and BTS-711 contained Mesodiaminopimelic acid and glycine and the whole cell hydrolysates contained xylose and arabinose.This indicated that they belonged to cell wall type-II, which is a characteristic of the genus Micromonospora [Lechevalier, 1970].However the cell wall peptidoglycan of BTS-713 and BTS-709 contained L L-diaminopimelic acid and glycine and the whole cell hydrolysates contained xylose.This indicated that they belonged to cell wall type-I, which is a characteristic of the genus Streptomyces [Lechevalier, 1970].Sequence based identification and further characterization studies were not performed due to lack of infrastructural facilities.
On the basis of morphological and chemo taxonomical characteristics, the isolates BTS-716, BTS-701and BTS-711 were identified as belonging to the family Steptomycetaceae and the genus Micromonospora while isolates BTS-713 and BTS-709 were assigned to the family of Steptomycetaceae and the genus Streptomyces [Pridham, T.G., 1958;Williams, S.T., 1989].This suggests that the natural substrates such as marine sediments are also good sources for isolation of lipolytic actinobacteria.

Conclusion
Based on the screening results it had been shown that sediments of Pudimadaka, coast of Bay of Bengal possess lipolytic actinomycetes and maybe tapped as one of the potential source for lipase production.The results in general reflect on the lipase production potentiality among the relatively less explored group of marine actinomycetes.It is suggested that frequent and systematic screening for actinomycetes in the Bay of Bengal could provide novel species as well as promising lipolytic Actinobacteria.
.Kamfer et al. suggested the physiological tests as indispensable tools for classification and identification of actinomycetes.

Table 1 .
Details of sampling stations

Table 2 .
Number of Isolates from Sediment Samples

Table 5 .
(a) Lipolytic activity of the isolates under submerged fermentation conditions Station No.

Table 6 .
Ten Most Promising Isolates having Lipase activity

Table 8 .
(a) Physiological and biochemical characteristics of the selected Isolates

Table 8 .
(b) Physiological and biochemical characteristics of the selected Isolates