CURRENT MICROBIOLOGY Vol. 51 (2005), pp. 379384 DOI: 10.

1007/s00284-005-0069-z

Current Microbiology
An International Journal
Springer Science+Business Media, Inc. 2005

Characterization of Exopolysaccharides Produced by Cyanobacteria Isolated from Polynesian Microbial Mats

Laurent Richert,1 Stjepko Golubic,2 Roland Le Guds,3 Jacqueline Ratiskol,4 Claude Payri,1 Jean Guezennec4
1 2

Laboratoire dEcologie Marine, Universit de la Polynsie Franaise, B.P. 6570, 98 702 Faaa, Tahiti, Polynsie Franaise Biological Science Center, Boston University, 5 Cummington St., Boston, MA 02215, USA 3 Laboratoire VP/PBA, Centre de Nantes, French Research Institute for Exploitation of the Sea, B.P. 21105, 44311 Nantes, France 4 Laboratoire VP/BMM, Centre de Brest, French Research Institute for Exploitation of the Sea, B.P. 70, Plouzane, France Received: 29 March 2005 / Accepted: 13 June 2005

Abstract. Six cyanobacterial isolates recovered from Polynesian microbial mats, called kopara, were cultured using laboratory-closed photobioreactors and were shown to produce exopolymers as released and capsular exopolysaccharides (EPS). These polymers have been chemically characterized using colorimetric and elemental assays, infrared spectrometry, and gas chromatography. Both capsular and released EPS consisted of 7 to 10 different monosaccharides with neutral sugars predominating. Interestingly, four isolates exhibited sulfate contents ranging from 6% to 19%. On the basis of preliminary data, cyanobacteria from this unusual ecosystem appear to be an important source of novel EPS of a great interest in terms of their biological activities.

Polysaccharides have found applications in many industrial sectors because of their many interesting physical and chemical properties, such as stabilizing, suspending, thickening, gelling, and water-retention capability. They are used in textiles, adhesives, paint, food, and beverage industries [45]; in pharmaceuticals [4, 20]; in oil and metal recovery from ore and industrial wastes [21]. Previous investigators have reported cyanobacterial exopolysaccharides (EPS) of great biotechnological interest such as EPS of Cyanospira capsulata [6] and Aphanothece halophytica GR02 [32], which display similar viscosity behavior as xanthan. The metal ion binding capacity of EPSs produced by Anabaena sp. [7] and Cyanothece spp. [42] may find useful applications in wastewater treatment, whereas the water-holding capacity of EPS produced by Nostoc muscorum [8] may be used as a soil conditioner. EPSs produced by Cyanospira capsulata strain Mag I 504 ATCC 43193 is of great interest for oil recovery [9].

Correspondence to: L. Richert at present address: CAIRAP, BP 1720, 98 713 Papeete, Tahiti, French polymesia; email: lrichert@cairap.pf

Cells of most cyanobacteria are surrounded by mucilaginous external layers essentially constituted of polysaccharidic material. These layers are classified into three types: the sheath, the capsule, and the slime [11]. In addition to these polysaccharidic layers more or less attached to the cell surface, a solubilized fraction of polysaccharides is often present in the culture medium. The protocols used for cyanobacterial EPS extraction distinguish generally three fractions of polymers: the sheath; the capsulated polysaccharides (CPS), which include the capsule layer and the part of the slime layer well attached to the cell surface; and the released polysaccharides (RPS), which include the solubilized fraction of the slime layer loosely attached to the cell surface [1, 5]. In French Polynesian atolls, microbial mats are developing in water ponds exposed to salinity fluctuation and high solar irradiation. These microbial mats, which are called kopara by the inhabitants of Tuamotu archipelago [13], are mainly composed of cyanobacteria and present high contents in polysaccharides [27, 41]. The aim of this study was to grow six cyanobacterial isolates recovered from such microbial mats using

380 particular culture procedures involving laboratoryclosed photobioreactors. Under unbalanced conditions, these six microorganisms were shown to secrete both capsular and released EPS, which were subsequently analyzed.

CURRENT MICROBIOLOGY Vol. 51 (2005) 10C). The cell-free supernatant containing the released polysaccharide was sequentially filtered to a final filter-porosity of 0.7 lm, then passed through a tangential-flow filtration concentrator (100,000 or 30,000 Da cutoff; Millipore, Beford, MA) until the conductivity of the filtrate was stabilized at approximately 5 lScm1. The concentrated and desalted RPS solutions were freeze-dried and stored at ambient temperature. The pellet was resuspended in dH2O before freeze-drying. The dried cells were added to salted water [15 g/L NaCl] (100 mL salted water/10 g cells) and heated for 4 hours at 80C under agitation. The cells were then eliminated by centrifugation (20,000 g for 45 minutes at 10C), and the CPS solution was submitted to the same treatment as the RPS. The resulting desalted CPS was freeze-dried and stored at ambient temperature. Chemical analysis. Uronic acid contents were measured by the metahydroxyphenyl method [14], and neutral sugars were analyzed by the orcinol-sulfuric acid method [40]. Protein content was determined by the bicinchoninic acid method [43] (Pierce Eurochemie bv, Holland). Fourier transform infrared spectroscopy. For Fourier transform infrared spectroscopy (FT-IR), pellets were obtained by grinding a mixture of 2 mg EPS with 200 mg dry KBr and pressing the mixture into a 16-mm-diameter mold. FT-IR spectra were recorded on a Bruker Victor 22 instrument with a resolution of 4 cm1. Spectra were obtained in the 4000-400 cm1 region, and sulfate contents were calculated according to Lijour et al. [26]. Gas chromatography analysis. Monosaccharide analysis was performed by gas chromatography analysis. Methanolysis was performed in methanolic 2 M HCl at 100C for 4 hours, and the methylglycosides were converted to the corresponding trimethylsilyl derivatives as previously described by Montreuil et al. [30]. Gas chromatography was carried out on an HP-5890 system (Hewlett Packard Corporation, Palo Alto, California) equipped with an automatic sample loader, a flame ionization detector, and a CP-Sil5CB (Chrompack) column with helium as carrier gas. Temperature profile was programmed as follows: 50C for 1 minute, from 50 to 120C at 20Cmin)1, from 120C to 240C at 2Cmin)1, from 240C to 280C at 10Cmin)1, and 280C for 10 minutes.

Materials and Methods

Cyanobacterial strains. Six cyanobacterial strains were isolated from kopara that developed in shallow brackish to marine ponds of Rangiroa atoll, Tuamotu archipelago, French Polynesia. They were identied using a polyphasic approach combining morphologic and phylogenetic analysis based on 16S rRNA sequence [39]. Half of them were filamentous species: Geitlerinema (Oscillatoria) sp. strain FE, Plectonema (Leptolyngbya) cf. golenkinianum Gomont strain FF, and Plectonema (Leptolyngbya) cf. battersii Gomont strain GF, whereas the others were unicellular: Chroococcus submarinus (Hansgirg) Kovacik strain BM, Johannesbaptistia pellucida (Dickie) Taylor and Drouet strain GC, and Rhabdoderma cf. rubrum (Alvik) Komarek and Anagnostidis strain CH. The six nonaxenic isolates, each with a single species of cyanobacteria, were maintained by regular transfers into fresh liquid medium at 22C under cool-white fluorescent light (20 lmol.s1.m2) with a light to dark cycle of 12:12 hours. The culture medium used was the Conway medium, which is a modified version of the Walne medium [50]. In 1 L natural sea water: 100.00 mg NaNO3; 45.00 mg Na2EDTA; 33.60 mg H3BO3; 26.00 mg NaH2PO4-2H2O; 1.28 mg FeCl3-6H2O; 0.36 mg MnCl2-4H2O; 1 lL trace metal solution; 50 lL vitamin solution. Trace metal solution was composed as follows: 1.05 gZnCl2; 1.00 g CoCl2-6H2O; 0.45 g (NH4)6Mo7O24-4H2O;1.00 g CuSO4-5H2O; and 50 mL dH2O. Vitamin solution was composed as follows: 400 mg vitamin B1; 20 mg vitamin B12; and 100 mL dH2O. Culturing procedures. All culturing procedures were performed aseptically. Stock culture, 10 mL, was used to inoculate an Erlenmeyer flask containing 150 mL double-spiked (2 concentrated) Conway medium and incubated for approximately 10 days under permanent cool-white fluorescent light (150 lmols1m2) at 30C. Then, 100 mL of the Erlenmeyer flask contents were transferred into a flat-bottom-balloon containing 1 L double-spiked Conway medium and 500 mL 4-mm diameter glass beads and incubated as before for 6 days. This culture, 200 mL, was used to inoculate a photobioreactor (Rhodella type, New Brunswick Scientific, USA, see [39] for details) containing 1.8 L 5 concentrated Conway medium. The pH was adjusted manually to approximately 8.0 just before inoculation with 1 M sterile NaOH. The culture was run for approximately 4 to 5 weeks at 32C under permanent lighting (300 lmols1m2) with an aeration flow of 0.125 v/v/min and mechanically agitated (250 rpm). The aeration was performed with filter-sterilized air supplemented with carbon dioxide to keep the pH at approximately 8.35. After 5 days of culture, the medium was re-enriched with Conway concentrate to obtain a 5 concentration. Samples, 2 mL, were taken daily to follow ionic concentrations using a Dionex 500 chromatograph (Dionex, Sunnyvale). The system was equipped with a precolumn AG9-HC associated to a column AS9HC for anions and a pre-column CG12-A associated to a column CS12-A for cations. Cultures were stopped 15 days after the disappearance of nitrates and phosphates in the culture medium. Exopolysaccharide extraction. Cells were separated from the culture medium using high-speed centrifugation (20,000 g for 45 minutes at

Results
Culturing of EPS-producing strains. For all strains, biomass and EPS were harvested after a fed-batch culture ran approximately 25 to 35 days. During the culturing phase of this study, cyanobacterial filaments showed the tendency to adhere to glass walls, which posed a problem because of decreased active cell surfaces. This problem was anticipated because of the nature of the isolated strains stemming from well-structured benthic microbial communities [39]. For the purposes of culturing and optimization of polysaccharide harvesting, a modification of the culture settings was required and carried out successfully. Glass beads were added to the culture medium at the balloon step, which increased considerably the surface to which the filaments adhered and promoted their growth. During culturing in the photobioreactor, the adherence of filaments on glass walls was decreased by strong mechanical agitation.

L. Richert et al: Exopolysaccharides Produced by Cyanobacteria Table 1. Yields of EPS production and colorimetric analysis Strain-EPS FE-CPS FE-RPS FF-CPS FF-RPS GF-CPS GF-RPS BM-CPS BM-RPS CH-CPS CH-RPS GC-CPS GC-RPS Neutral sugars (%) 77b 84 80 69 67 47 45 42 76 57 65 66 Uronic acids (%) 7 6 5 11 11 7 6 10 2 2 6 5 Proteins (%) 15 8 14 19 10 11 24 14 7 7 10 10 Sulfatesa (%) 0 0 0 0 11 16 6 14 7 13 17 19

381

Yield of EPS production (mg/g biomass) 118.7 110.2 97.0 27.1 81.4 7.7 50.9 23.6 50.4 56.2 124.0 34.5

a Sulfate contents are calculated by the method of Lijour et al. [26] using FT-IR spectra; bValues are w/w percentages of polymer dry weight. FT-IR, Fourier transforminfrared spectrometry; CPS, capsulated polysaccharides; RPS, released polysaccharides.

Imbalanced nutrient conditions were introduced to cultures using a fed-batch technique: two medium enrichments (at the beginning and at day 5) were prepared to obtain a sufficient amount of biomass before the starvation period (15 days) toward nitrates and phosphates. Nutrient concentrations of culture medium were monitored every day. Nitrates and phosphates disappeared from the culture medium after 5 days after inoculation and a change in the color of cells from blue-green to yellow-brown could be noticed, indicating attainment of the stationary phase (with the exception of strain BM). Conway concentrate was then added to the medium to stimulate another phase of growth. The culture was then stopped after 15 days, corresponding to the disappearance of nitrates and phosphates in the culture medium. For strain BM, nitrates and phosphates were still present in the culture medium, and a water-soluble brown pigment could be observed after 5 days. Our culturing procedures have been successful by introducing several modifications, which to our knowledge are being applied for the first time or are unusual in culturing cyanobacteria for polysaccharide production. These include the application of Conway medium, which is usually used for eucaryotic microalgae, the fedbatch technique, and the use of glass beads as substratum for filamentous cyanobacteria. The Rhodella-type photobioreactor was constructed especially in very small quantities (< 5 U). Production and chemical analysis of EPS. The yields of different polysaccharides obtained (Table 1) were not correlated to those of biomass (data not shown). Indeed, total EPS (CPS + RPS) accounted for 9% of biomass dry weight for strain GF with the highest biomass (12.1 g/L)

compared with 23 % for strain FE, which exhibited the smallest biomass (3.0 g/L). Strains FF, BM, GC, and GF produced much more CPS than RPS, whereas strains CH and FE produced equal amounts of CPS and RPS. Colorimetric analyses showed a predominance of neutral sugars for all EPS, but uronic acids contents were also present in both RPS and CPS of strains FF, GF, and BM (Table 1). Protein contents ranged from 7% to 24% (w/w). FT-IR spectra were characterized by three intense absorption bands (Fig. 1): a broad band >3000 cm)1, resulting from O-H and C-H stretching bands at 3420 and 2900 cm)1, respectively; an intense absorption band at 16301650 cm)1 because of hydroxylic and carboxylic bonds; and a third characteristic band at 1050 cm)1 corresponding to the sugar skeletons. For both EPSs of strains GF, CH, GC, and BM, a doublet was observed at 12501230 cm)1 because of sulfate groups, indicating sulfate contents ranging from 6% to 19% (w/w) (Table 1), in agreement with elemental microanalysis (data not shown). Gas chromatography analyses of monosaccharides as per-O-trimethylsilyl methylglycosides derivatives are listed in Table 2. The 12 EPSs were composed of 7 to 10 monosaccharides. Fucose, xylose, and glucose were present in all of the EPSs. Uronic acids were absent from EPSs from strain FE. In addition, EPSs from strain FE mainly consisted of glucose with traces of other neutral sugars. Glucosamine residues were found in the RPS produced by strain CH and, to a lesser extent, in both EPSs from strain BM. Molar ratios of monosaccharides were similar between CPS and RPS for strains FE, GF, GC, and BM. For strains FF and CH, these ratios slightly differed between the CPS and their corresponding RPS.

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Fig. 1. FT-IR spectrum of the RPS of strain CH (Rhabdoderma cf. rubrum (lvik) Komrek and Anagnostidis). FT-IR, Fourier transforminfrared spectroscopy. Table 2. Monosaccharide molar ratios of the EPSs Strain-EPS FE-CPS FE-RPS FF-CPS FF-RPS GF-CPS GF-RPS BM-CPS BM-RPS CH-CPS CH-RPS GC-CPS GC-RPS Ara 1 1 1 1 1 Rha Tr Tr 1 1 2 2 3 0.2 Fuc Tr Tr 0.2 0.6 1 2 3 3 0.5 2 0.2 0.2 Xyl Tr Tr 1 2 1 2 5 5 1 5 0.4 0.2 Glc 1 1 3 2 4 4 4 3 2 3 1 1 Gal 0.4 1 2 3 2 2 Tr 1 1 1 Man 1 0.5 2 3 1 1 Tr 1 1 1 GlcA 0.2 0.5 1 1 1 1 Tr Tr 0.4 0.2 GalA Tr Tr 1 1 1 1 Tr Tr 0.2 0.2 N-Glc 1 1 Tr 10

Ara: arabinose; CPS, capsulated polysaccharides; EPS, exopolysaccharides; Fuc: fucose; GalA: galacturonic acid; Gal: galactose; Glc: glucose; GlcA: glucuronic acid; Man: mannose; N-Glc: glucosamine; Rha: rhamnose; RPS, released polysaccharides; Xyl: xylose.

Discussion
Culturing cyanobacteria or microalgae-producing EPS may encounter the undesirable phenomenon of cell adhesion to glass walls of culture vessels as previously described for several marine strains of Oscillatoria and Phormidium [19], for Nostoc [15] and even for the coccoid cyanobacterium Anacystis nidulans [24]. In our study, this problem only occurred with filamentous strains. It has been hypothesized that the cyanobacterial strains would optimally synthesize EPSs under imbalanced conditions of C/N and C/P ratios [10, 31]. Our strains were isolated from an oligotrophic environment,

which is subject to large salinity fluctuations [39] and thus likely to be nutrient limited most of the time. In addition, previous studies have documented the presence of high amounts of extracellular polymers within the microbial mats [27, 41]. With the exception of strain BM, the onset of the starvation period was visualized by the change in cell color from blue-green to yellowbrown as previously reported by Lehmann and Wber [24] and Reddy et al. [38]. Moreover, the nitrates and phosphates decreases in the culture medium were not related to an increase in intracellular reserves because the color changed again from yellow-brown to bluegreen after the re-enrichment of the culture medium with the Conway concentrate. The yields of polysaccharides

L. Richert et al: Exopolysaccharides Produced by Cyanobacteria

383 macromolecules. In this regard, the kopara microbial mats may be considered a great source of yet unexplored cyanobacterial EPS-producers.
ACKNOWLEDGMENTS The authors thank the CAIRAP SA (Arue, Tahiti) for financial support. International contacts and collaboration were supported by the Boston University Marine Program, Hanse Institute for Advanced Studies, Alexander von Humboldt Foundation and the Visiting Professor Program of the University of Marseille.

were in agreement with other values reported for cyanobacteria [11, 19, 34]. The sugar composition of both CPS and RPS lies within the range of variability of other cyanobacteria. The predominance of glucose in EPSs of strain FE is noteworthy and is comparable with homoglucans from Phormidium uncinatum [22] and Scytonema hofmanni [34]. Such high content of glucose in EPSs was suspected to be related to a too-drastic extraction procedure leading to a contamination by intracellular glucose reserves [33]. However, in our case, the same extraction protocol was applied to all samples of CPS, whereas RPS were obtained without extraction. Nevertheless, only polysaccharides (both CPS and RPS) from strain FE exhibited this characteristic. Protein contents showed generally too-high values (7% to 24%), indicating the need for a further purification treatment such as enzymatic treatment [18]. Sulfate contents of EPSs of strains GF, CH, GC, and BM were relatively high compared with values previously reported [3, 5, 12, 37, 44, 46]. This suggests that these sulfated polysaccharides should be further evaluated for their biological activities. Indeed, for several years there has been a growing interest in the recognition of biological activities of microbial polysaccharides, and it was found that such activities are often associated with sulfate content of polymers [51]. Such biological activities could include anticoagulant [35, 49], antitumor [17, 29] and immunostimulatory activities [28, 36] as well as antiviral effects [23], including antihuman immunodeficiency virus activity [2, 47]. Molar ratios of monosaccharides were similar between CPS and RPS for strains FE, GF, GC, and BM, which agrees with the studies of Li et al. [25], Vincenzini et al. [48], and De Philippis and Vincenzini [11], who supported the hypothesis that the RPS is a solubilized fraction of the CPS. But for strains FF and CH, these ratios differed slightly between the CPS and their corresponding RPS, suggesting that they are two distinct polysaccharidic fractions as previously observed by Gloaguen et al. [19], Forni et al. [16], and Nicolaus et al. [34].

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