JOURNAL OF SCIENCE OF HNUE Natural Sci., 2011, Vol. 56, No. 7, pp. 109-116 STUDY ON SOME ACTINOMYCETES ISOLATED FROM MANGROVE SOIL IN GIAO THUY, NAM DINH PROVINCE Duong Minh Lam ( ) and Nguyen Thanh Dat Hanoi National University of Education ( ) Email: duong.minhlam@gmail.com Abstract. In this study we examined diversity and activities of some actinomycetes isolated from mangroves in Giao Thuy, Nam Dinh Province. The distribution of actinomycetes in soil is strongly affected by external factors. Extra-cellular enzyme producing ability of mangrove actinomycetes is very high, particularly the ability of producing cellulase degrading filter paper (122/128 strains), CMC (110/128 strains), protease (83/128 strains) and amylase(78/128 strains). Mangrove actinomycetes often produce antibiotics of narrow activity range, few strains are able to produce broad ranged antibiotics. The ability of producing antibiotics inhibiting test micro-organisms worked best on Fusarium oxysporium (34 strains or 34%), or at worst on Pseudomonas aeruginosa (2 strains or 2%). All the studied strains grew well in NaCl free environment or at low NaCl concentrations, and grew slower at 20%o NaCl, grew poorly or no growth at 50%o NaCl. The studied actinomycetes are halotolerant, not halophilic. The selected actinomycetes were all able to assimilate different N and C sources. They were able to produce quite strong degrading enzymes for filter paper. Keywords: Mangrove, actinomycetes, microbial ecology. 1. Introduction At present, there are about 18.10 6 hectares of mangroves worldwide, Vietnam has mangroves of approximately 200,000 hectares. Besides their main functions such as coast protection, wind and wave shielding and silt trapping, mangroves are able to recreate and supply many resources to coastal people, helping conserve biodiversity and regulate the climate [3]. In the mangroves there is not only flora and fauna but also an abundant and diverse microbiota, among which actinomycetes plays a very significant role. Microorganisms decompose organic and inorganic compounds, closing the material and energy cycles in ecosystems, helping to stabilise the ecosystems [1]. However, there has been limited understanding about mangrove micro-organisms. It is therefore necessary to have a study on the actinomycetes in mangrove ecosystems. This 109
Duong Minh Lam and Nguyen Thanh Dat study sheds light on the isolation and some bio-characteristics of actinomycetes through which their role in the mangrove ecosystem of Giao Thuy, Nam Dinh can be elucidated. 2. Content 2.1. Materials and methods *Materials of study: Soil samples were taken from the mangroves of Giao Thuy, Nam Dinh in the period from April 2001-September 2002. Chemicals: glucose, saccarose, starch, meat extract, peptone, BAB (blood agar base). KH 2 PO 4, FeSO 4.7H 2 O, CaCO 3, KNO 3, NaCl, MgSO 4.2H 2 O, KCl, NaNO 3, H 2 SO 4, BaCl 2.2H 2 O, Glycerine (China). All chemicals are pure at analysis level. The test microbes included: Bacillus subtilis; Escherichia coli; Salmonella typhymurium; Candida albicans bought at Quarantine Institute, Ministry of Health, Hai Ba Trung, Hanoi; Pseudomonas aeruginosa and Staphylococcus aureus received from Bach Mai Hospital, which had been isolated from urine, blood or sputum of patients who had urethra or lung disease. These bacteria have been resistant to a wide range of antibiotics such as cezet (CAZ), gentamycin (GM), tobramycin (Tb), amikacin (AM), ciproffloxacin (Cip), chloramphenicol (C), trimetoprin, sulfamethoxadol, ceptiazol (Cro), biseptol (SXT) (data provided by Microbiology Department, Bach Mai Hospital); Fusarium oxysporum that caused many diseases in plants, received from the National Breed Centre, Hanoi National University; Mucor sp., Aspergillus niger isolated from mangroves by staff of Biotechnology Microbiology Department, Faculty of Biology, Hanoi Naitonal University of Education. *Methods of study: - Isolation method: Actinomycetes strains were isolated using serial dilution methods on both Gause I and Czapek-glucose media, in which de-ionized water was replaced with soil-free sea water to ensure minerals needed for microbes [1]. - Enzyme screening: Screening for enzymatic activity such as cellulase, amylase, protease using diffusion method on medium Gause I, with corresponding materials being paper powder or CMC (0.5%), soluble starch (1%), and Gelatin (1%); exogenic enzymatic activity was also screened for using the hole punching method on relevant materials [5]. - Determination of salt-tolerance: The selected actinomycetes strains were inoculated on medium Gauze I supplemented with different salt concentrations. After 7-10 days, the growth and development of actinomycetes was assessed [2]. - Determination of carbon source assimilation: Actinomycetes strains were inoculated on ISP media using different C sources. The culture broth was kept 110
Study on some actinomycetes isolated from mangrove soil in Giao Thuy... in an incubator at 28-30 C, after 7-10 days the growth and development of actinomycetes were observed to assess their C-source assimilation [4]. - Determination of nitrogen source assimilation: Actinomycetes strains were inoculated on Czapeck glucose medium replaced with different nitrogen-containing compounds. The culture broth was kept at 28-30 C. The growth and development of actinomycetes were observed after 7-10 days to assess their N-source assimilation [6]. - Determination of filter paper activity: Actinomycetes strains were inoculated in Gauze I medium with filter paper as a single carbon source, after 20-30 days, the filter paper activity of actinomycetes was assessed [2]. 2.2. Results and discussions 2.2.1. Isolation The soil samples collected in Giao Thuy, Nam Dinh were diluted to 10 2 %o concentration in sterile physiological water, after that 0.05 ml samples were platedon to surface of agar medium in Petri dishes. Each experiment was replicated three times. The results of isolating actinomycetes are shown in Table 1. Table 1. Number of actinomycetes in the mangroves of Giao Thuy, Nam Dinh Total Landscape Temperature NaCl Depth number characteristics ( ph CFU/g C) (%o) (cm) of strains Well-grown forest, dominant trees included 25-30 15-20 0 7-8 55 0.622 10 4 Sonneratia, Kandelia candel, etc. 5 6.5-7 73 1.732 10 4 There are some notes during the isolation process as followed: The average number of actinomycetes found in 1 g of surface soils was consistently lower than that in 1 g of 5 cm deep soil. Soil samples mixed with a little sand contained more actinomycetes than those mixed with a lot of sand, samples taken at a position far away from the rivulet contained more actinomycetes than those taken at a position closer to the rivulet. Thus, the distribution of actinomycetes is greatly influenced by external conditions. In particular, there were more actinomycetes in 5 cm deep soil than in surface soil layers. That is because the mangrove forest in Giao Thuy, Nam Dinh is newly planted forest, as a result it is low and scattered. Therefore the surface soil layer is directly exposed to the sunlight, which is not favourable 111
Duong Minh Lam and Nguyen Thanh Dat to the development of actinomycetes. On the other hand, the diurnal tidal regime wipes away part of organic remains on the surface soil, hence reducing its fertility. At 5 cm depth, due to the prolonged organic accumulation of the forest, organic substances are retained and are subjected to many groups of micro-organisms such as fungi and bacteria, which create intermediate substances favouring the growth of actinomycetes. On the Gauze I medium (with the presence of starch) the number of actinomycetes was always higher than that on Czapek-glucose medium, since starch showed better effect on actinomycetes activity than glucose. The actinomycetes that are well adapted to mangrove forest must have developed polysaccharide hydrolases such as amylases and cellulases, which are inhibited in the presence of glucose. Isolation of actinomycetes was also conducted from decayed branch, leaf and trunk samples, however, there were not any actinomycetes found. Thus it is clear that actinomycetes only act as secondary decomposers, after some primary decomposers such as some fungi and bacteria. 2.2.2. Extra-cellular hydrolase activity of the isolated actinomycetes strains The isolated actinomycetes were screened for their capacity of producing extracellular enzymes, the results are shown in Table 2. Table 2. Exogenous enzymatic capacity of the isolated actinomycetes Protease Amylase Cellulase CMC Filter paper Number of tested strains 128 128 128 128 Number of strains having the capacity 83 78 110 122 Percentage (%) 64.85 60.93 85.9 95.31 Actinomycetes from mangroves are good extra-cellular hydrolase producers. Most of studied strains produced cellulase with 122 strains which decayed filter paper (95.31 percent of the total studied strains); 110 strains were able to decompose CMC (85.9%). There were 83 strains (64.85%) excreted proteases into the surrounding environment. The number of strains that could produce amylase was the 78 (60.93%). Among 128 studied actinomycetes strains, there were 32 strains that were able to produce all the three enzymes mentioned above (accounted for 25%), none of the isolates were not able to produce any enzyme. Enzyme producing capacity helps actinomycetes adapted to their living conditions in the mangroves, where life is very hard under strong influences of salt concentration, temperature and tidal regimes. The great enzyme producing capacity of mangrove actinomycetes 112
Study on some actinomycetes isolated from mangrove soil in Giao Thuy... is significant not only to the growth and development of actinomycetes, but also in ecological terms, helping strengthen the material and energy processes in mangrove ecosystem. 2.2.3. Antibiotics production of mangrove actinomycetes The isolated actinomycetes were screened for their antagonisms against diseasecausing microbes in human beings and in animals. For the sources of test microbes, see materials and methods section. The results are presented in Table 3. Table 3. Antibiotic activity of mangrove actinomycetes No. Test microbes Number of strains having the activity Degree of the activity Weak Moderate Strong 1 P. aeruginosa 2/100 1 0 1 2 S. typhimurium 14/100 7 5 2 3 E. coli 16/100 7 9 0 4 C. albicans 6/100 5 1 0 5 F. oxysporium 34/100 15 10 9 6 A. niger 9/100 9 0 0 7 Penicillium sp. 10/100 7 1 2 8 Mucor sp. 29/100 6 16 7 9 S. aureus 10/100 3 5 2 Note. Weak: D - d 10 mm; Moderate: 10 mm < D - d 20 mm; Strong: 20 mm < D - d, where D - d is diameter of inhibition zone. It can be seen that most of the mangrove actinomycetes in the study were able to produce antibiotics in a narrow range - it means that they could only inhibit one test micro-organism. Very few strains were able to produce multiple target antibiotics. The mangrove actinomycetes were more likely to produce antibiotics against mold than against bacteria. This was particularly true in the case of F. oxysporium (34%) the number of actinomycetes could produce antibiotics against this test microbe. There were very few strains that produced antibiotics against P. aeruginosa (2%). This is probably because in mangrove ecological conditions, actinomycetes have been encountering and competing with moulds more than they have with bacteria. 2.2.4. Influence of salt concentration to the growth and development of some actinomycetes strains Strains that strongly produced extracellular hydrolases were selected for the affection of salt on the growth. The research results are shown in Table 4. Nine strains had different tolerance for salt. Strain D2 and TD32 were the most tolerant, and at the same time had strong exogenous enzymatic activity. Strain TD29 was 113
Duong Minh Lam and Nguyen Thanh Dat the least salt tolerant and had the weakest exogenous enzymatic activity. Table 4. Influence of salt concentration to the growth and development of some actinomycetes strains Salt concentration (%o) Strain 0 10 20 30 40 50 D2 ++++ ++++ +++ +++ ++ + D4 ++++ +++ +++ ++ + +/- D4d ++++ +++ +++ ++ + +/- TD1 ++++ +++ ++ ++ + +/- TD5 ++++ +++ ++ + + +/- TD16 ++++ +++ ++ + + +/- TD26 +++ ++++ ++ + + +/- TD29 ++++ +++ + + +/- - TD32 +++ +++ ++++ ++ ++ + Note. ++++: Very strong growth; +++ : strong growth; ++: moderate growth; +: poor growth; +/-: very poor growth. The chosen actinomycetes strains grew well at 0-10%o salt concentrations; at higher salt concentration from 20-40%o the development of actinomycetes gradually decreased and no growth was observed at 50%o salt concentration. As low as 0-10%o salt concentrations and so there had been few strain variations. Thus high salt concentration had negative effects on the growth of actinomycetes. Effect of salt concentration on the growth of actinomycetes can be explained by the following mechanism: When salt concentration of the environment is high, Na + ions are transported into the cell through the cell membrane, at this stage there would be H + ions flow moving outwards in the opposite direction (antiport). This leads to change in the intracellular ph from being neutral to being more basic. The intracellular ph change would in turn affect the physiological, biochemical reactions in the cell hence the effect on the growth and development of actinomycetes. In addition, at high NaCl concentration there would be an unbalanced concentration gradient between the intracellular environment and the extra-cellular environment, as a result, water from cytoplasm would go outwards leading to the plasmolysis that makes the cell stop or be slow to divide. Actinomycetes grew poorly at salt concentrations of over 20%o, this shows that actinomycetes are halotolerant strains, not halophilic strains. Thus it can be said that actinomycetes have a freshwater origin and have migrated to mangroves in the not too distant past. 114
Study on some actinomycetes isolated from mangrove soil in Giao Thuy... 2.2.5. Assimilation of N and C of the selected Actinomycetes It is very important to know nitrogen and carbon assimilation capacity of the actinomycetes as it shows their importance in mangrove ecosystems and helps to control fermentation processes. The results of the screening of actinomycetes for their ability to assimilate different nitrogen and carbon sources are shown in Table 5 and Table 6. Table 5. Assimilation of N sources of some selected Actinomycetes strains Strain KNO 3 Yeast extract Pepton NaNO 3 NH 4 Cl Urine NH 4 NO 3 (NH 4 ) 2 SO 4 D2 +++ +++ +++ +++ ++ ++ ++ ++ D4 +++ +++ +++ +++ + + + + D4d +++ +++ +++ +++ + + + ++ TD1 +++ +++ +++ +++ + + + + TD5 +++ +++ +++ +++ + + ++ ++ TD16 +++ +++ ++ ++ + + + + TD26 +++ +++ +++ +++ ++ ++ ++ ++ TD29 +++ +++ +++ +++ + ++ + + TD32 +++ +++ +++ +++ ++ +++ ++ ++ Note. +++: good growth; ++: moderate growth; +: poor growth. All the selected actinomycetes strains were able to assimilate different N sources including organic and inorganic compounds in vitro to different extents. They all assimilated N organic sources and NO 3 well, but had poor assimilation for NH + 4. Thus NH + 4 ions have unfavourable effect on the development of actinomycetes since the reduction of NH + 4 ions causes a change in the intracellular ph. The ability of assimilating different carbon sources of nine selected actinomycetes strains was all very good and in which starch was the best. This shows that mangrove actinomycetes have very effective polysaccharide digestible enzymes. This finding also came up with isolating actinomycetes from Touchien River in Taiwan [7]. Table 6. Assimilation of C source of some selected Actinomycetes strains Strain Glucose Saccharose Lactose Manitol Galactose Arabinose Starch D2 +++ ++ +++ +++ ++ +++ ++++ D4 +++ ++ +++ ++ ++ ++ ++++ D4d ++ + ++ + +++ +++ ++++ TD1 +++ ++ +++ +++ ++ ++ ++++ TD5 +++ +++ +++ +++ ++ ++ ++++ TD16 +++ ++ ++ ++ + + ++++ TD26 +++ +++ +++ +++ +++ +++ ++++ 115
Duong Minh Lam and Nguyen Thanh Dat TD29 +++ +++ +++ +++ ++ ++ ++++ TD32 +++ +++ +++ +++ +++ +++ ++++ Note. ++++: very good growth; +++: good growth; ++: moderate growth; + poor growth. 3. Conclusion Actinomycetes distribution in soils is strongly affected by external factors. They possess high extra-cellular enzyme producing capacity and antibiotics. All the studied strains grew well in NaCl free environment or at low NaCl concentrations. That proved they are terrestrial origin. Diverse nitrogen and carbon source assimilation of the studied mangrove actinomycetes showed their great importance in the ecosystem. REFERENCES [1] H. Bredholt, E. Fjrvik, G. Johnsen, and S. B. Zotchev, 2008. Actinomycetes from sediments in the Trondheim Fjord, Norway: diversity and biological activity. Marine Drugs 6, pp. 12-24. [2] N.T. Dat, M.T. Hang, 2000. Biology of microorganisms. Education Publishing House. pp. 213-273. [3] FAO, 2001. Global Forest Resources Assessment 2000: Main report. FAO Forestry Paper 140. Rome. www.fao.org/forestry/fo/fra/main/index.jsp. [4] E.B. Shirling, D. Gottlieb, 1968. Cooperative description of type cultures of Actinomycetes II. Species descriptions from first study. International Journal of Systematic Bacteriology. 18(2): pp. 69-189. [5] A. G. Williams, 1983. Staining reactors for detection of hemicellulose degrading bacteria. Microbiology Letters 20: pp. 253-258. [6] A. G. Williams and L. Winkins, 1989. Bergey s manual of systematic Bacteriology. 4: pp. 2451-2492. [7] K. C. Hoang, C. Y. Lee, M. Tseng, W. F. Chu, 2007. Polyester-degrading actinomycetes isolated from Touchien river of Taiwan. World Journal of Microbiology and Biotechnology 23: pp. 201-205. 116