Cultivable Ephiphytic Bacteria Obtained from the Unialgal Culture Strain of the Red Alga Gracilariopsis Chorda
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Algal Resources (2014) 7：95-105 Cultivable ephiphytic bacteria obtained from the unialgal culture strain of the red alga Gracilariopsis chorda (Holmes) Ohmi collected from the estuary of Katsuura River in Tokushima Prefecture in southwest Japan Hirotaka KAKITA1 *, Hideki OBIKA1 and Hiroshi KAMISHIMA1 Abstract : To identify the cultivable epiphytic bacteria obtained from the unialgal culture strain of the red alga Gracilariopsis chorda (Holmes) Ohmi collected from the estuary of Katsuura River in Tokushima Prefecture in southwest Japan, morphological and phenotypic classification and genetic analysis were carried out. Eight kinds of cultivable bacteria were isolated. Isolate UGC1-1 (bacterial population: 7.5×107 cfu g-1), UGC1-2 (4.0× 107 cfu g-1)andUGC1-3(5.0× 106 cfu g-1) were isolated from Marine Agar plates. Isolate UGC1-4 (5.0×106 cfu g-1)andUGC1-5(1.0×106 cfu g-1) were isolated from 1/10 ORI and CAM plates, respectively. Isolate UGC1-6 (1.0×105 cfu g-1), UGC1-7 (6.0×104 cfu g-1)andUGC1-8 (2.0×104 cfu g-1) were isolated from CVT plates. The genetic analysis based on partial 16 S rRNA gene sequences (ca. 450 bp) revealed that the Isolate UGC1-1, UGC1-2, UGC1-3, UGC1-4, UGC1-5, UGC1-6, UGC1-7 and UGC1-8 related closely to Ascidianbacter aurantia- cus (Similarity 93.2 %), Ruegeria halocynthiae (99.5 %), Roseovarius sediminilitoris (97.2 %), Litoreibacter janthinus (99.3 %), Zobellia russellii (99.8 %), Marinobacter salarius (99.1 %), Granulosicoccus antarcticvs or G. coccoides (97.9 %) and Labrenzia aggregata (99.8 %), re- spectively. The Isolate UGC1-1 was suggested to be an undescribed taxon assigned to the family Flavobacteriaceae based on genetic analysis. Keywords : Gracilariaceae; cultivable epiphytic bacteria; morphological classification; ge- netic analysis; Flavobacteriaceae Introduction bacteria have already been identified in previ- ous studies (Shiba and Taga 1981; Cole 1982; Some bacterial species are known to associ- Brock and Clyne 1984; Coveney and Wetzel 1989; ate with particular macroalgal species. Positive Weinbweger et al. 1997; Dobretsov and Qian interactions between macroalgae and associ- 2002; Croft et al. 2005; Marshall et al. 2006; Rao ated bacteria include phytohormone produc- et al. 2006). Goecke et al. (2010) found out that tion, morphogenesis of macroalgae triggered by the beneficial relationship is based on the algal bacterial metabolites, specific antibiotic activi- activity to produce organic compounds and ox- ties affecting epibionts and elicitation of oxida- ygen which will be utilized by bacteria. Shiba tive burst mechanisms (Goecke et al. 2010). On and Taga (1981) reported that the bacterial flora the other hand, negative interactions induce or attached on a macroalga changes during the generate algal diseases (Goecke et al. 2010). repeatedly transferred batch culture of algae in Beneficial associations between macroalgae and a sterile medium. They pointed out that the 1 Health Research Institute, AIST Shikoku, National Institute of Advanced Industrial Science and Technology, Hayashi, Takamatsu, Kagawa 761-0395, Japan ＊Corresponding author : Tel: +81-878-693561, fax: +81-878-693553, e-mail: h-kakita@aist. go.jp 95 Hirotaka KAKITA, Hideki OBIKA and Hiroshi KAMISHIMA growth-stimulated bacteria become predominant Therefore, to characterize the cultivable epi- in the repeatedly transferred batch culture of phytic bacteria derived from Japanese Gracilar- algae in a sterile medium and the growth-in- iopsis chorda (Holmes) Ohmi, we investigated the hibited bacteria are washed out, because algal population and the generic composition of the exudates are the sole organic carbon sources in cultivable epiphytic bacteria derived from the batch culture of algae in a sterile medium. Japanese G. chorda using the repeatedly trans- Goecke et al. (2010) also reported that bacteria ferred batch culture of algae in a sterile medi- mineralize the organic substrates, supplying the um. In this paper, we conducted phenotypic and algae with carbon dioxide, minerals and‘growth generic characterization as for the epiphytic factors'. Several researchers have reported that bacterial isolates obtained from the unialgal bacteria supply macroalgae with growth facters culturestrainoftheredalgaG. chorda collect- relating to the production and turnover of var- ed from the estuary of Katsuura River in ious phytohormones and biostimulators of cell Tokushima Prefecture in southwest Japan. growth and development (Berland et al. 1972, Bolinches et al. 1988, Meusnier et al. 2001). Materials and Methods Dimitrieva et al. (2006) reported that a favora- ble growth-promoting effect by the bacterium Collection of materials Pseudoalteromonas porphyrae was observed on The algal specimens of the red alga Gracilar- Laminaria japonica. Freis (1975) clarified the iopsis chorda (Holmes) Ohmi were collected from ability of bacteria living on Enteromorpha spp. the estuary of Katsuura River [latitude 34.04 N to convert tryptophan into the phytohormone and longitude 134.58 E, the World Geodetic Sys- indole-3-acetic acid. tem 1984 (WGS 84)] in Tokushima Prefecture in Furthermore, an increased growth rate of an southwest Japan in June 2007. algal strain leads to an increase in its total production. The Japanese species of the red Unialgal culture strain algal family Gracilariaceae are important indus- The algal specimens were washed with ster- trial macroalgae, because they have been har- ile seawater and cultured in a sterile medium vested in Japan as commercial sources of agar for eight days, as described previously (Kakita and for food additives (Tokuda et al. 1987). et al. 2013). Thirty Erlenmeyer flasks were used However, no trials of the growth stimulation of for the repeatedly transferred batch culture of Japanese Gracilariaceae using epiphytic bacteria algae in a sterile medium. Three hundred mil- have ever been reported. In general, cultivable ligrams in wet weight of the washed alga were bacteria are easy to isolate, to increase, and to inoculated under a clean booth into each Er- store in comparison with unculturable bacteria. lenmeyer flask containing 400 mL of the sterile Therefore some kind of cultivable bacteria may medium and were incubated with aeration at be more applicable to algal growth stimulation 18℃, in a 14 hL:10 hD regime and at photon than unculturable bacteria. To utilize beneficial irradiances of 60 mol photons m-2s-1 under cool- bacteria for stimulating algal growth, the culti- white lamps. After the repeatedly transferred vable epiphytic bacteria derived from the red batch culture of algae in a sterile medium for algal family Gracilariaceae have to be isolated, eight days, the macroalga obtained was collect- characterized, and identified at the species level. ed and used for further study as a sample of a Sucha bacterial generic classification is a neces- cultured alga. sary prerequisite for the algal growth stimula- The unialgal stock culture of the red alga G. tion by using cultivable epiphytic bacteria. How- chorda was started from tetraspores released by ever the details of the generic composition, matured tetrasporophytes among the cultured which is based on the 16S ribosomal RNA gene algae. The establishment of a unialgal culture sequences of the cultivable epiphytic bacteria strain followed the methods of Yamamoto and derived from the Japanese Gracilariaceae, have Sasaki (1987). Spore inoculation, medium re- never been reported. newal, and upright body inoculation were car- 96 Cultivable bacteria from the culture strain of Gracilariopsis chorda collected in Katsuura River ried out under a clean booth. Ten spores were per one gram of a macroalga was calculated. inoculated into each 50 mL-volume screw tube Colonies with the same characteristics [i.e. containing 30 mL of the sterile medium and shape, color, size, the presence or absence of a were incubated without aeration at 18℃,ina14 hollow (a circular transparent zone)] were re- hL:10 hD regime, in salinity of about 33 ‰ and garded as equivalent colony types. Eight strains at a photon irradiance of 40 mol photons m-2s-1. in total were isolated from different types of In the static culture, the sterile medium was predominant colonies. The eight bacterial iso- replaced with a fresh one once every 3 weeks. lates were taken for morphological and pheno- Upright bodies obtained (about 10 mm length) typic classification and genetic analysis. were taken from the bottom of the screw tube with tweezers and inoculated into a flask to Morphological and phenotypic classification of perform the culturing of the upright bodies. The bacterial isolates unialgal stock culture of upright bodies was in- Gram stain was carried out using Hucker's cubated in 1000 mL round-shaped flasks con- modification (Conn et al. 1957). Motility, cell taining 800 mL of the sterile medium with shape, flagella staining, pigment production, aeration at 18 ℃, in a 14 hL:10 hD regime and oxidase activity, gelatin hydrolysis and DNA at a photon irradiance of 40 mol photons m-2s-1 hydrolysis were tested as described in Cowan under cool-white lamps. The macroalga was and Steel's Manual (Barrow and Feltham 1993). taken from each flask with a pair of tweezers Oxidation-fermentation on glucose was investi- and inoculated into new flasks containing a new gated by Leifson's method (1963). A salt re- sterile medium bi-weekly. After the unialgal quirement examination was carried out with stock culture in a sterile medium, the macroal- Hidaka and Sakai's basal medium (0.05 % of ga obtained was collected under a clean booth, polypeptone and 0.01 % of yeast extract) (Hida- referred to as unialgal culture strain UGC1, and ka and Sakai 1965). Medium A (0.05 % of polypep- used for bacterial experiments. tone and 0.01 % of yeast extract, pH7.6), medi- um B (the medium A containing 1 % NaCl), and Enumeration of bacteria medium C (the medium A dissolved in sterile Five grams in wet weight of the unialgal cul- seawater) were used. The bacteria grown in the ture strain UGC1 and 45 mL of a sterile solu- medium A were referred to as terrestrial type.