original scientific paper UDK 579.63(262.3-17) MICROBIA L DIVERSITY IN TH E GUL F O F TRIESTE: CHARACTERIZATIO N O F AEROBI C HETEROTROPHI C BACTERIA L STRAINS Maria SAB EC Dipl. ing., researcher, C.E.T.A., 34 ! 70 Gorizia, Via V. Veneto 19, IT dip!, inl., raziskovalka, C.E.T.A., 34170 Gorica, Via V. Veneto 19, IT Stefano CARDINAL! Ph.D., laboratory director, C.E.T.A., 3417 0 Gorizia, Via V. Veneto 19, IT, dr., direktor laboratorija, C.E.T.A., 34170 Gorica, Via V. Veneto 19, IT, Paulo RAMANI BSc., researcher, Laboratory of Marine Biology, 34100 Trieste, Strada costiera 336, IT dipl. biolog, raziskovalec, Laboratorio di biología marina, 34100 Trst, Strada costiera 336, IT Paoia DEL NEGRO BSc., researcher, Laboratory of Marine Biology, 34100 Trieste, Strada costiera 336, IT dipl. biolog, raziskovalka. Laboratorio di biología marina, 34100 Trst, Strada costiera 336, IT ABSTRACT Generally the microbial ecology studies do not stress the strains taxonomic characterization, and this is the reason why microbial diversity in marine waters is poorly understood. In this paper, the results of a preliminary study on microbial diversity in the Gulf of Trieste is presented. The 61% of the isolated strains are Gram negative and, from a taxonomic point of view, are very heterogeneus. The Gram positive strains belong to Bacillus, Staphilococcus, Micrococcus, Rhodococcus, Streptomyces and Sporasarcina. Furthermore, isolation methods, with particular reference to substrata and incubation periods, are considered. Key words: Gulf of Trieste, microbial diversity, marine microbiology Ključne besede: Tržaški zaliv, mikrobna diverziteta, mikrobiologija morja INTRODUCTION the autochthonous microbial flora, not only in terms of biomass and potential activity, but also of community Bacteria often dominate plankton biomass and are structure and species composition, is needed. The most recognized as important agents of biogeocbemicat frequent types of organisms must be isolated and exam-changes in marine ecosystems, but bacteria! diversity is ined for their metabolic activities and their ecological poorly understood yet because conventional isolation significance. methods neglect about 90% of the micro-organisms Since marine microbiologists are seldom interested (Pickup, 1991; Fuhrman eta!., 1992). in taxonomy and identification of new bacterial isolates, To understand the role that heterotrophic bacteria the compositions of bacterial communities are little play in a certain marine environment the knowledge on known (Austin, 1988). M. SABEC . 5. CARDINAL! , P, RAMAN! , P. DE L NEGRO : MICROBIA L DIVERSIT Y I N TH E GULF... . 27-3 2 Microbiologicaf researches in the Gulf of Trieste studied the influence of river outflows and sewage efflu­ents on bacteria! density and bacteriological contami­nation (Del Negro et ai, 1993). Some researches exam­ined the dynamics in the pelagic microbial food web and the dependence of bacteria! abundance on different ecological factors (temperature, depth, availability of nutrients and oxygen, etc.) (Herndl eta/., 1989). The species composition of the bacterial population in the Adriatic Sea is little known (Turk, 1991): to our knowledge, the only taxonomic research in heterotro­phic bacteria was related to the Centra! Adriatic (Sobot, 1981) and proved the presence of representatives of the genus Pseudomonas, Vibrio, Acinetobacter, Flavobac­terium and Micrococcus. These results partly corre­spond with those from other parts of the world (e.g. Austin, 1979). The concern of the present study was the identifica­tion and characterization of bacteria! cultures isolated from the Gulf of Trieste that could contribute to the elucidation of the diversity of the predominant heterot­rophic bacterial populations in this area. MATERIALS AND METHODS The study area The Gulf of Trieste, the farthest north-eastern part of the Adriatic Sea, is a shallow water area characterized by a limited water exchange (Stravisi, 1983). The hy~ drodynamism of the whole Gulf is primarily linked to the ascending eastern current coming from the Istrian coast and carrying higher salinity waters from the Mid­dle Adriatic into the northern basin. This current leads to anti-clockwise circulatory movements, which are also enhanced by the winds blowing from the NNE and SE (Mosetti, 1968). The seawater temperatures range from 7 to 27°C and the salinity between 12 to 36 psu, subject to the rainfall in the area (Fonda Umani, 1991), Sampling A superficial sea water sample was collected asepti­caily in May 1993 in a station 300 m off shore located near the National Marine Park "Riserva Parco Marino di Miramare" in the Gulf of Trieste (fig. 1), It was stored with ice and processed within few hours. Enumeration of bacteria As an indicator of water quality, coliforms were de­tected with Brilliant green bile (2%) broth (S dilutions in 3 replicates), incubated at 35°C for 48 hours. The enumeration of bacterial population was per­formed by: /- direct counting with epifluorescence microscopy: 5 ml of the collected sampie were filtered on 0,22 pm polycarbonate black membrane (Nucleopore) stained with DAP1, and the bacteria were counted under fluorescent microscope (Porter & Feig, 1980) 2. plate counts of viable heterotrophic bacteria: Serial dilutions with artificial sea water (ASW - g/l: CaC12 2; KBr 0,15; KCI 0,75; MgCI2 5,71; MgS0 4 '7H2 0 5,8; NaCI 23,8; NaHCQ3 0,3; Na 2 S04 3,6; SrCI 2 6H2 0 0,05) from 10"1 to 10"6 were inoculated in 6 replicates by spreading on 90 mm Petri dishes with 10 different media: - MAR: marine agar 2216, Difco - PCA plate count agar, Difco - PCA prepared with sea water - PCA prepared with ASW - PYA: peptone yeast extract agar (Schneider & Rheinheimer, 1988) - PYA prepared with ASW - PYS (g/l: peptone 1; yeast extract 1; KCI 0,75; MgS04-7H20 6,98; NaCI 23,8; agar 20) - CEL: cellulose agar (Schneider & Rheinheimer, 1988) - CH I 1: chitin agar (Schneider & Rheinheimer, 1988) - CH! 2: modified chitin agar - two layer plates with basal medium ((NH4)2S04 2 g; Agar 15 g; ASW 1000 ml) and 3 g/l of crude chitine added to the upper layer. The counts of CFU (colony forming units) were made after incubation at 25°C for 48 hours. Isolation and preservation of bacterial strains From the enumeration plates that were incubated at 25°C for 3-4 further days, 100 colonies were picked on the bases of colony morphology and streaked onto MAR plates, with serial restreaking of each culture being done until the purity of the strains was ensured. Heat-fixed smears of the cultures were Gram stained and the prepa­rations examined under the microscope. After the purifi­cation, the cultures were inoculated onto MAR slants, incubated at 25°C and subcultured every 4-6 weeks. For the isolation of Actinomycetes the membrane method (Hirsch & Christensen, 1983) on 3 special media (Okazaki & Okami, 1976) was used (filtered sample: 500 m!): -MYS (maltose yeast extract agar), -G G (glycerol glycine agar) -SC (soluble starch casein agar). After all the isolation and purification steps had been followed, the isolates were examined for the phenotypic. characters. Those unable to grow on MAR were not considered typical marine bacteria and were eliminated from further study. For the preservation, ali the strains were freeze dried and are now preserved at the National Cultures Bank in Gorizia. M . SABEC , S . CARDINAL! , P. RAMAN! , P. DE L NEGRO : MICROBIA L DIVERSIT Y I N TH E GULF... , 27-3 2 Fig. 1: Study area with the sampling station (near Mira­mare). Slika 1: Obravnavano območje z vzorčevalno postajo (Miramare). Characterization of the bacterial strains Each strain was examined for at least 28 phenotypic characters. All test media were inoculated using cultures that had been grown on MAR for a week at 25°C. Whenever possible, ASW or MAR were used for the preparation of the media, and the incubation tempera­ture was 25°C. The characters observed were: 1. Colonial morphology and staining reactions: - presence of pigment on MAR (after 7 days); - motility and cell shape were determined micro­scopically from wet mounts of 24 hours cultures; - Gram-staining reaction {Difco staining solutions); - the presence of spores was examined on wet preparations under a phase contrast microscope and on Schaeffer-Fulton stained smears (Doetsch, 1981); 2. Biochemical tests: - oxidase reaction was determined with the applica­tion of 3-day cuitures and Oxo'td oxidase sticks; - catalase activity was tested with transferring growth from MAR to a drop of 3% H 2 0 2 (Kaneko et a!., 1979) - fermentative or oxidative metabolism of glucose and 5 other carbohydrates (lactose, maltose, mannitoi, mannose and sucrose) was determined following the modified Leifson's method (Smibert & Krieg, 1981); - indole production was detected with the standard procedure (3 days at 25°C) (Smibert & Krieg, 1981); - nitrate and nitrite reduction was recorded after 7 days (Smibert & Krieg, 1981); - Voges-Proskauer and methyl red tests were carried out in MR-VP Difco broth at 30°C in 48 hours and in 5 days (Smibert & Krieg, 1981). 3. Degradative capabilities: - Casein and chitin degradations were tested with a modified Smibert and Krieg (1981) method; the tests were carried out with two layer plates (the bottom one of MAR and the upper one of casein or chitine agar) and the results were recorded after 2 weeks of incubation; - The degradation of agar, Tween 20 and DNA were tested with MAR, MAR supplemented with 0,1 g CaCl2 and 10 m! Tween 20 (autoclaved separately) and DNAse test agar DIFCO prepared with ASW; the results were registered after 1 week of incubation (Kaneko ef al, 1979); - after 4 days the degradation of urea (Oxoid broth base, prepared with half strength ASW) was detected. 4. Physiology tests: - growth at 37°C (MAR, 5 days); - growth on 0% and 4% of NaCI (7 days) (Kaneko et ai, 1979). 5. Resistance to: Penicillin (10 U.I.) and to 0-129 was determined on MAR plates after 24 hours by the agar diffusion tech­nique using commercial discs The isolated Streptomycetes were identified with numerical techniques (Langham eta!., 1989) comprising characterization by 50 different tests. RESULTS Enumeration The Brilliant green bile (2%) broth test for the detec­tion of the coli-aerogenes group showed no presence of this kind of bacteria. Using epifluorescence technique 5,5 x 106 cells/ml were detected. The average counts obtained on the 10 media em­ployed are presented in Table 1: medi a MA R PC A PC A PC A PY A PY A PY S O L CH I CH I (0% NaCI) (sea (ASW) (sea (ASW) 1 2 water) water) CPU/ml 200 300 23 50 10 20 20 10 1000 100 Tab. T; Bacteria enumeration on 10 different media. Tabela 1: Število bakterij, zraslih na 10 različnih me­dijih. isolation Among the cultures isolated many were not able to M. SABEC , S . CARDINAL! , P. RAMANI , P, DE L NEGRO : MICROBIA L DIVERSIT Y I N TH E GULF... , 27-3 2 grow on MAR and some lost viability, so a total of 84 pure cultures were obtained. The quantity of strains isolated from every media is presented in the table 2: Isolation media No. of strains MAR 24 PCA (0% NaCI) 13 PCA (sea water) 13 PCA (ASW) 4 PYA (sea water) 12 A (ASW) 3 PYS 6 CEL 5 CHI 1 2 CH I 2 2 Tab, 2: Number of strains isolated from each medium. Tabefa 2: Število sojev bakterijskih vrst, izoliranih iz vsakega medija. The special media and the technique used for the isolation of Actinomycetes proved to be inefficient, since no bacterial colony, with a myceliar type of growth could be found. But 4 Streptornyces and 2 Rho­dococcus strains were isolated by the less selective method on 4 media: MAR, CHI 1, CHi 2 and PCA. Description of the isolates The isolates showed to be very diverse, belonging to many different bacterial groups. Some of them were identified to the genus level. 51 isolates (61%) were Gram negative and 33 (39%) Gram positive. More than half of the cultures were col­ourless (46) and motility was observed only by 27. Most of the isolates are able to grow also without salt (76%) or with 4% of NaCI (85%). The majority was also able to grow at 37°C (85%) and was sensitive to penicillin (82%) and 0-12 9 (70%). Among Cram positive strains, on the basis of typical colonies forming substrate and aerial mycelium, 4 were recognized as Streptornyces and 2 as nocardiforms, probably Rbodococcus, showing elementary branching and a marked rod-coccus growth cycle. The Streptomy­cetes were identified as Streptornyces anulatus (2 strains) and 5. exfoliatus (1 strain); due to the high value of the standard error of taxonomic distance, the fourth Streptornyces strain is tentatively assigned to S. anulatus. Among 22 Gram positive rods, 12 were spore form­ing, catalase positive with fermentative metabolism of glucose and were identified as Bacillus. Only 5 Gram positive cocci were isolated and only 1 was oxidative, probably a Micrococcus. 1 formed spe­cific. cubical packets of 8 cells, producing endospores, and was by some specific additional tests (Holt et at., 1994) confirmed as Sporosarcina ureae. 3 of Gram positive, catalase positive, oxidase negative strains, with fermentative metabolism of glucose, were able to grow on 10% NaCI agar and were identified as Staphylococ­cus { Baird-Parker, 1963). Among Gram negative isolates, 3 had a helical form and were fermentative, MR-VP, indole and urease nega­tive. The 5 Gram negative vibrios, 15 cocci and 28 rods were very heterogeneous and a lot of additional work should be done in order to identify them. DISCUSSION The results of many numerical taxonomy studies show the predominant presence of Gram negative rods along the water column with higher proportion of Gram positive organisms occurring in the sediments (Kaneko ef a/., 1979; Austin, 1982). In our study more than half of the isolates (61%) were Gram negative, although not predominant, probably due to the limited depth of the monitored station. The highest counts were obtained using the standard media for the enumeration of heterotrophic aerobic bacteria (PCA and MAR) and, exceptionally, with two chitin agar media, which gave rise to thousands of Act­inomycetes colonies belonging to the genus Streptorny­ces. Anyway, the high number of streptomycetes colo­nies developed is not indicative of the effective domi­nance of this kind of bacteria (as demonstrated by the lacking of results in the specific tests); the fortuitous in­clusion of few spore chains in the inoculated sample could explain the observed situation: the substantial taxonomic homogeneity of the strains seems to confirm this hypothesis. Although the discrepancy between viable and direct counts may reflect the known presence of unculturabie micro-organisms, the difference observed in this study is greater than previously reported for the area considered (Doizani et a/., 1989). However, a longer incubation period (5-6 days) allowed the development of several colonies on the higher dilution plates, belonging mainly to the genus Rhodococcus and to an unidentified spiril­lum. This indicates the importance to prolong the incu­bation time for determining the total viable counts. As general observation, the isolation methods allowing long incubation periods (high dilution and/or poor and/or difficult media) brought a higher bacteria recov­ery. As many authors report, the majority of the bacteria in marine environment are free living or loosely at­tached to particles, that is why mostly motile bacteria often dominate the isolated strains. In our study motility was observed oniy by 34% of the isolates. This may be due to the method used (wet mounts from solid media grown cultures), or of the shallow Gulf of Trieste typical bacterial population, where sediment bacteria are M. 5ABEC , S, CARDINAL! , P, RAMANI , P, DE L NEGRO : MICROBIA L DIVERSIT Y i N TH E GULF... . 27-3 2 mixed with the free living ones. Not ali of the bacteria characterized in this study re­quired NaCi for their growth. A higher number of colo­nies developed in standard PCA without NaCI and more than half of them were unable to grow on MAR. MacLeod (1986) suggested that the Na-dependency of the transport process permits faster growth and gives or­ganisms possessing it a competitive advantage in a sa­line environment, it is possible that the Na-indipendent bacteria in the marine environment grow best when nu­trients are not limiting, as in the considered period. The presence of bacteria not affected by NaCI might also be due to a terrigen input but the absence of coliforms does not support this hypothesis. This study has shown that the aerobic bacteria with and without dependency are part of the plankton com­munity of the Gulf of Trieste, characterized by low di­versity and high btomass (Fonda Umani etai, 1990). POVZETEK Mikrobna diverziteta morskih voda je slabo raziskana, saj se mikrobne ekološke raziskave ne ozirajo na takso­nomsko pripadnost bakterijskih so je v. 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