Characteristics of Bacteria Isolated from Mangrove Estuary of Nakama River, Iriomote Island, Okinawa

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1 Nihon Biseibutsu Seitai Gakkaiho (Bulletin of Japanese Society of Microbial Ecology) Vol. 3, No 2, 59-66, 1989 Characteristics of Bacteria Isolated from Mangrove Estuary of Nakama River, Iriomote Island, Okinawa SOUWAPA ANGSUPANICH1, HIDEO MIYOSHI2 and YOSHIHIKO HATA1 1. Faculty of Agriculture, Kochi University, Nankoku city, Kochi 783, Japan. 2. Usa Marine Biological Institute, Kochi University, Usa-cho, Tosa city, Kochi , Japan. Abstract The mangrove estuary of Nakama River, Okinawa is almost free from the substances produced by human activities and the area is characterized as a novel, oligotrophic estuary in Japan A total of 333 aerobic heterotophic bacteria were isolated from water, leaf litter and mud of the estuary They were examined for morphological, biochemical and cultural characters. The results on 57 unit characters were analyzed by numerical taxonomic method, and 28 phenons were defined at similarity levels of 62-82% The number of phenons increased in the order of water, leaf litter and mud The spectra of bacterial potentials involved in organic metabolism became wider in the following order the isolates from water, those from mud, those from old leaf litter, and those from new leaf litter Key words mangrove estuary, heterotrophic bacteria, numerical taxonomic analysis Introduction The mangrove forest of Nakama River in Iriomote Island, Okinawa has been proclaimed as a conserved natural area by the Japanese government, and the area has not been affected by the pollutants resulting from human activities. Therefore, bacterial community characteristic of only this type of environment is expected. In addition, most microbial examinations for mangrove environment were focused only upon fungal community (Kohlmeyer and Kohlmeyer, 1979), and studies on bacterial community in mangrove environment have been grossly neglected in the past. The present work described here was undertaken to characterize bacteria and to confirm their function with special reference to decomposition in the estuary. To our knowledge, this work is the first, extensive study on the taxonomic structure of bacteria and their function in mangrove environment. Samples Materials and Methods Water, mangrove leaf litter and mud samples were collected in August, 1986 and January, 1987 from the mangrove estuary of Nakama River, Iriomote Island, Okinawa. Using sterilized bottles, surface water was collected at Stations 7 and 11 (Fig. 1) during high tide. By using a corer or a sterilized pincette, samplings of mud, new leaf litter (yellowish brown colour) and old one (black colour) were made at Stn. A. Media EA medium (Weiner et al., 1980) was used for counting, isolation and maintenance of bacteria. The media used for the taxonomic tests were enriched with one third strength of seawater or equivalent strength of a four salt solution (Colwell and

2 Morita, 1964), if necessary. Counting and isolation of bacteria To eliminate sampling error, a reratively large amount of water sample (50ml) or mud sample (20 g) was thoroughly homogenized prior to dilution. For leaf litter samples, three pieces of 1x1cm sample were ground aceptically in a mortar with 1/3 strength sterilized seawater. After appropriate dilution with 1/3 strength sterilized seawater, viable bacteria were counted using the conventional spread-plate method (Gerhardt et al., 1981). Characterization of bacterial isolates A total of 333 bacterial strains were randomly Fig. 1. Map showing the sampling stations in mangrove swamps of Nakama River. Table 1. Origin of bacterial strains employed for the study

3 isolated from the counting plates. After checking the purity of the isolates, each bacterial strain was examined for the following characters. 1. Morphology of isolates: Colony characteristics, micromorphology and Gram stain were examined using the conventional methods (Gerhardt et al., 1981). 2. Biochemical reactions: The following tests were carried out: catalase test, O-F test, nitrate reduction, denitrification, hydrogen sulfide production from cysteine, methyl red test, indole production, V.P. test, and phosphatase production (Gerhardt et al., 1981); ammonia production from peptone (Lewin and Lounsbery, 1969); and urease test (Collies and Patricia, 1984). 3. Degradation tests: The following tests were conducted: hydrolyses of esculin, casein, gelatin and starch (Gerhardt et al., 1981); hydrolysis of tributyrin (Collins and Patricia, 1984); and digestions of tyrosine and crude chitin (Lewin and Lounsbery, 1969). 4. Growth tests: Carbon sources, nitrogen sources, survival at 3C, and temperature limits were tested according to the procedures of Lewin and Lounsbery (1969), and anaerobic growth was examined using the Gas-Pak (Gerhardt et al., 1981). 5. Tolerance tests: Sensitivity to antibiotic agents (penicillin, chloramphenicol, oxytetracycline, kanamycin, polymyxin B, tetracycline, streptomycin and 0/129) was examined according to the methods described by Colwell and Morita (1964). Lauryl sulfate and salinity tolerances were examined by the procedures proposed by Lewin and Lounsbery (1969). Numerical taxonomic analysis The data on 57 unit characters were analyzed using the simple matching types of similarity coefficient. Clustering was done by weighted average linkage method, and a simplified dendrogram was prepared. Results Counting and isolation of bacteria Viable counts of aerobic heterotrophic bacteria for each sample are shown in Table 1. Bacterial densities ranged cfu/ml in water, cfu/g in leaf litter and 106 cfu/g in mud samples. Apparently, bacterial densities were greater in

4 Table 2. Summary of the Note: +, 90% of more positive responses; -, 90% or more negative responses; d, 11 to 89%

5 characteristics of phenons in this study positive responses; w, all strains weak positive responses; o, orange; p, pink; y, yellow

6 Table 3. Percent generic compositions of bacterial isolates among the 28 phenons Table 4. Partial list of bacterial potentials to utilize organic substrates summer than in winter. Clustering the isolated bacteria All the isolates were examined for 65 characters, but eight characters, such as the production of ammonia from peptone, utilization of peptone or yeast extract as a sole source of nitrogen, crude chitin digestion, growth at 35C, survival at 3C, and sensitivity to chloramphenicol or oxytetracycline, showed no difference. Thus, these characters were eliminated from the data matrix. The resulting taxonomic grouping of the isolates is illustrated in Fig. 2. Twenty eight phenons, which had 62-82% level of similarity, were recognized. Representative characteristics of each phenon are shown in Table 2. As a whole, each phenon appeared to have relatively high homogeneity. Percent distribution of bacterial isolates among the phenons is presented in Table 3. Seventeen major phenons, which had more than 5% of isolates for each source, contained about 88% of isolates examined, and eleven minor phenons having less than 5% of isolates for each source, contained the rest.

7 In the bacterial isolates from water, the number of phenons recognized was low, but a few of these phenons were large. On the other hand, in the bacterial isolates from mud, there were relatively small phenons as compared with water isolates, and the number of phenons was relatively high. The bacteria from leaf litter were just intermediate between the water and mud isolates. Table 4 shows a partial list of bacterial potentials to utilize organic substrates. The spectra of potentials, as a whole, became narrower in the order of isolates from new leaf litter, old leaf litter, mud and water. Generic description of major phenons Generic grouping of the 17 major phenons described above was carried out using the diagnostic keys proposed by Buchanan and Gibbons (1974), Gibson et al. (1977), Oliver (1982), and Krieg and Holt (1984). Phenons 1, 10, 18, 23 and 25 were identified with Pseudomonas. Phenons 4 and 15 resembled Staphylococcus. Phenons 5 and 8 appeared to be Flexibacter. Phenons 9 and 20 were classified as Alteromonas. Phenons 27 and 28 were identified as Vibrio. Compact phenons 7, 12, 14, and 24 were similar to Flavobacterium, Micrococcus, Bacillus and Moraxella, respectively. The organisms belonging to phenons 19 and 21 were not related to any other genera described in the literatures employed in this study. Discussion The density of aerobic heterotrophic bacteria decreased in the order of old leaf litter, new leaf litter, mud and water (Table 1). The result is probably due to the differences in quantity and quality of nutrients in these microhabitats. The majority of isolates from the estuary were Gram negative rods, and about two thirds of them were motile (Table 2). As shown in Table 3, the major phenons for each source were as follows: Flavobacterium, Pseudomonas, and Moraxella for water; Pseudomonas, Micrococcus, Staphylococcus, Alteromonas and Vibrio for new leaf litter; Pseudomonas, Alteromonas, Flavobacterium, and one unidentified group for old leaf litter; and Pseudomon as, Alteromonas, Flavobacterium, Flexibacter, Staphylococcus, Vibrio, Bacillus and the other unidentified group for mud. Miyoshi (1984) isolated 223 strains from water and mud collected in the mangrove estuary of Shiira River, Iriomote Island, Okinawa, and presumptive identification of genera was carried out using the dichotonous keys proposed by Simidu (1974). The results showed that in the estuary the genera Flavobacterium and Micrococcus were abundant in summer and Pseudomonas, Moraxella and Micrococcus in winter. Except for this study, comparable information is not available for the bacterial flora of mangrove estuary, but the same predominance of pseudomonads was widely confirmed in estuarine and marine environment (Sieburth, 1964). For example, although there is a large number of bacteriological studies of terrestrial forest floors which receive abundant fallen leaves, consistent results have not been obtained due to the differences in mineral soil matrix, vegetation and climate. However, some forest floors are dominated by non-spore-forming, Gram negative rods such as pseudomonads (Research Association of Soil Microorganisms, 1981). Furthermore, Stevenson et al. (1974) isolated 110 strains from water and 165 strains from sediment in semitropical North Inlet Estuary, South Carolina. They detected that the clusters resembling Vibrio, Pseudomonas, Flavobacterium, Achromobacter and cocci in water, and clusters similar to Vibrio, Achromobacter, Alkaligenes, Cytophaga, Pseudomonas and Actinomyces in sediment. Based on our data and the data cited here, some differences in generic composition were noted among water, leaf litter and mud. Particularly, the number of genera gradually increased from water to leaf litter to mud. This tendency may

8 reflect the diversity of environmental conditions. Percentages of strains having the potentials involved in organic metabolism increased in the following order: the isolates from water, those from mud, those from old leaf litter and those from new leaf litter (Table 4). With few exceptional cases, the percentages obtained here are low as compared with those obtained for temperate, eutrophic bay (Miyoshi et al., 1981) or tropical seawaters in the vicinity of Puerto Rico (Pfister and Burkholder, 1965). Therefore, bacterial potentials involved in organic metabolism might be reflected in the differences of available substrates. Acknowledgments We wish to thank Dr. T. Nishijima, Kochi University, for his valuable advice throughout the study. We are also grateful to the Data processing center of Kochi University for help in computer analysis. This study was partly supported by a grant-in-aid from the Ministry of Education, Science and Culture, Japan. References Buchanan, R.E. and N.E. Gibbons, Bergey's manual of determinative bacteriology, 8th ed. Williams and Winkins Co., Baltimore. Collins, C.H. and M.L. Patricia, Microbiological methods, 5th ed. Butterworths, Boston. Colwell, R.R. and R.Y. Morita, Reisolation and emendation of description of Vibrio marina (Russell) Ford. J. Bacteriol., 88, Gerhardt, P., R.G.E. Murray, R.N. Costilow, E.W. Nester, W.A. Wood, N.R. Krieg and G.B. Phillips, Manual of methods for general bacteriology. American Society for Microbiology, Washington D.C. Gibson, D.M., M.S. Hendrie, N.C. Houston and G. Hobbs, The identification some Gram negative heterotrophic aquatic bacteria. In: Aquatic microbiology, (edited by F.A. Skinner and J.M. Shewan), pp Academic press, New York. Kohlmeyer, J. and E. Kohlmeyer, Marine mycology, The higher fungi. Academic Press, New York. Krieg, N.R. and J.G. Holt, Bergey's manual of systematic bacteriology, vol. 1. Williams and Wilkins Co., Baltimore. Lewin, R.A. and D.M. Lounsbery, Isolation, cultivation and characterization of Flexibacteria. J. Gen. Microbiol., 58, Miyoshi, H., T. Handa and Y. Hata, Aerobic heterotrophic bacteria and the decomposition of dead organisms in Uranouchi Bay. Bull. Jpn. Soc. Sci. Fish., 47, Miyoshi, H., Biological processes of mangrove estuary. In: Biological processes in the ocean, (edited by R. Marumo), pp Koseisha- Koseikaku, Tokyo. (in Japanese). Oliver, J.D., Taxonomic scheme for the identification of marine bacteria. Deep-sea Res., 29, Pfister, R.M. and P.R. Burkholder, Numerical taxonomy of some bacteria isolated from Antarctic and tropical seawaters. J. Bacteriol., 90, Research Association of Soil Microorganisms, Microorganisms in soil. Hakuyusha, Tokyo. (in Japanese). Sieburth, J.M., Role of algae in controlling bacterial population in estuarine waters. In: Pollutions Marines par les Microorganismes et les Produits Peteroliers, (edited by Secretarial General de la Commission), pp Commission International Pour L'Exploration Scientifique de la mer Mediteranee Monaco, Paris. Simidu, U., Taxonomy of marine bacteria. In: Marine microbes, (edited by N. Taga), pp Tokyo Univ. Press, Tokyo. (in Japanese). Stevenson, L.H., C.E. Millwood and B.H. Hebeler, Aerobic, heterotrophic bacterial population in estuarine water and sediments. In: Effect of the ocean environment on microbial activities, (edited by R.R. Colwell and R.Y. Morita), pp University Park Press, Baltimore. Weiner, R.M., D. Hussong and R.R. Colwell, An estuarine agar medium for enumeration of aerobic heterotrophic bacteria associated with water, sediment and shellfish. Can. J. Microbiol., 26, (Received July 9, 1988-Accepted October 4, 1988)