J. Phycol. 46, 477–483 (2010) Ó 2010 Phycological Society of America DOI: 10.1111/j.1529-8817.2010.00823.x CARBOHYDRATE RELEASE BY A SUBTROPICAL STRAIN OF SPONDYLOSIUM PYGMAEUM (ZYGNEMATOPHYCEAE): INFLUENCE OF NITRATE AVAILABILITY AND CULTURE AGING1 Fernanda Reinhardt Piedras Po´s-graduac¸a˜o em Oceanografia Biolo´gica, Instituto de Oceanografia, Universidade Federal de Rio Grande- FURG, Av. Italia, Km 8, Rio Grande, RS 96201-900, Brasil Paulo Roberto Martins Baisch, Maria Isabel Correˆa da Silva Machado Laborato´rio de Oceanografia Geolo´gica Instituto de Oceanografia, Universidade Federal de Rio Grande- FURG, Av. Italia, Km 8, Rio Grande, RS 96201-900, Brasil Armando Augusto Henriques Vieira Departamento de Botanica, Unversidade Federal de Sao Carlos, Via Washington Luis, Km 235, Sao Carlos, SP 13565-905, Brasil and Danilo Giroldo2 Laborato´rio de Botaˆnica Criptogaˆmica, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Rio Grande – FURG, Av. Italia, Km 8, Rio Grande, RS 96201-900, Brasil This paper describes the influence of nitrate avail- availability. EPS molecules >12 kDa were composed ability on growth and release of dissolved free and mainly of xylose, fucose, and galactose, as for other combined carbohydrates (DFCHOs and DCCHOs) desmids. However, a high N-acetyl-glucosamine con- produced by Spondylosium pygmaeum (Cooke) W. tent was found, uniquely among desmid EPSs. West (Zygnematophyceae). This strain was isolated Key index words: carbohydrate; desmid; growth; from a subtropical shallow pond, located at the nitrate; Spondylosium extreme south of Brazil (Rio Grande, RS). Experi- ments were carried out in batch culture, comparing Abbreviations: Ara, arabinose; DCCHO, dissolved two initial nitrate levels (10 ⁄ 100 lM) in the medium. combined carbohydrate; DFCHO, dissolved free Growth was monitored by direct microscopic cell carbohydrate; EPS, extracellular polysaccharide; counts and chl a content. Nitrate consumption was Fuc, fucose; Gal, galactose; GalA, galacturonic determined by ion chromatography, while the pro- acid; GalNAc, N-acetylgalactosamine; Glc, glu- duction of extracellular carbohydrates was moni- cose; GlcA, glucuronic acid; GlcNAc, N-acetylglu- tored by the phenol-sulfuric method. The cosamine; Man, mannose; Rha, rhamnose; monosaccharide compositions of DFCHOs and TDCHO, total dissolved carbohydrate; Xyl, xylose DCCHOs were determined in each growth phase by HPLC with pulse amperometric detection (HPLC- PAD). At the end of the experiment, the total EPSs are the main fraction of dissolved organic composition of extracellular polysaccharide (EPS) carbon released by freshwater phytoplankton (Paul- molecules >12 kDa was determined by gas chro- sen and Vieira 1994), and such production is a sig- matography. Nitrate availability had no influence on nificant source of carbohydrates in natural waters S. pygmaeum cell density at any phase. On the other (Biersmith and Benner 1998). However, few studies hand, chl a content decreased after a few days have attempted to characterize the composition of growth when the availability of nitrate was restricted, carbohydrates released by phytoplankton, comparing but continued to rise when nitrate was plentiful. to the large variety of species whose products have Also, nitrate depletion was faster at 10 lM nitrate. not been studied yet (Giroldo and Vieira 2005). No influence of the growth phase or nitrate avai- Production of EPSs is a conspicuous process lability on the total carbohydrates (TDCHOs) among desmids, because of the formation of muci- released per cell was observed. Only DCCHOs were laginous capsules, as well as the release of dissolved released by S. pygmaeum, and the composition varied polysaccharides (Domozych 2005). EPS release is between growth phases, especially at lower nitrate the result of the action of physiological processes on the allocation of photoassimilated carbon (Vieira 1Received 25 June 2009. Accepted 18 November 2009. and Myklestad 1986) and is associated with Golgi 2Author for correspondence: e-mail [email protected]. vesicle production (Domozych et al. 2007). Mucilage 477 478 FERNANDA REINHARDT PIEDRAS ET AL. capsules have been reported to result from diverse tions and occurrence of species in natural waters adaptative strategies, including buoyancy improve- (Reynolds et al. 2002, Salmaso and Padisak 2007). ment, grazing reduction, and selective permeability (Reynolds 2007). Although the formation of a cap- MATERIALS AND METHODS sule itself is not influenced by nutrient availability Organism and culture conditions. S. pygmaeum (Desmidiaceae) in Staurodesmus convergens (Gouveˆa et al. 2002), the is one of the smaller filamentous desmids (Croasdale et al. thickness and texture of the mucilage formed vary 1983). A subtropical strain (FURGZY006) was isolated from a with the environment (Reynolds 2007). small (surface area 1,000 m2), shallow (average depth 1.8 m) Dissolved polysaccharide release is also a process pond, located in the Carreiros campus of the Federal University described as depending on nutrient availability of Rio Grande in the extreme south of Brazil (32°04¢18¢¢ S and 52°09¢57¢¢ W). The cells are elliptical in side view and subellip- (Myklestad 1995). Nitrogen and phosphorus defi- tical in top view, with a smooth cell wall. The isolated cells ciency increases the EPS release rate in many spe- (N = 50) were 6.6 ± 0.9 lm long and 6.5 ± 0.3 lm broad with a cies (Lombardi et al. 1998, Giroldo and Vieira 3.6 ± 0.6 lm isthmus. Axenic cultures were obtained by micro- 2005). Excess light and nutrient depletion together manipulation and several washes under the microscope (mod. decrease cytokinesis while photosynthesis is still CX41F; Olympus, Tokyo, Japan). Cultures were maintained axenically in WC medium (Guillard and Lorenzen 1972) at pH active, leading to an excess of photoassimilated car- ) ) 7.0, in 12:12 light:dark (L:D) cycles with 100 lmol Æ m 2 Æ s 1 of bon (Fogg 1983), which is diverted into carbohy- irradiance and temperature controlled at 25°C±1°C. drate release. Under such stress conditions, excess Experimental design. Triplicate experiments were carried out electrons accumulating in the photosynthetic elec- with S. pygmaeum in batch culture, using media based on WC tron transport chain (PETC) may induce overpro- medium,withsodiumnitrateasthesolenitrogensource,reduced duction of reactive oxygen species, causing from the original 1,000 lM to 10 and 100 lM. A 150 mL culture of S. pygmaeum in WC medium, at the exponential growth phase photoinhibition and damage to membranes. The 7 )1 synthesis of carbohydrate, or triacylglycerols, con- (30d,10 cell Æ mL ),wascentrifuged(5 min,3,000g)inaSigma 2 K15 centrifuge (Osterode am Harz, Germany), washed, and sumes NADPH generated by the PETC, relaxing the resuspended in nitrogen-free WC medium (600 mL). This was overreduced PETC under stress conditions. More- kept under starvation for 7 d in the culture conditions described over, in such conditions, carbohydrate release acts above, to exhaust most of the intracellular nitrogen reserves. as a protection of the photosynthetic apparatus Aliquots of 30 mL of thestarved culture were inoculated insix 1 L (Smith and Underwood 2000) and also stimulates Erlenmeyer flasks with 600 mL of WC medium each, at each of bacterial growth associated with the dissolved the nitrate test levels. In parallel, 100 mL aliquots of the starved culture were inoculated in duplicate 4 L carboys with 3.5 L of WC organic matter (DOM) excreted by phytoplankton medium and the respective nitrate concentrations. The experi- (Giroldo et al. 2007). Bacterial growth on algal ment continued until stationary growth phases had been DOM as a substrate increases nutrient reminerali- observed at both nitrate levels. zation and, consequently, may promote phytoplank- An aliquot of 30 mL was sampled from each 1 L Erlenmeyer ton growth by the coevolution of complex plankton flask at intervals of 48–72 h to measure growth by direct communities (Wood and Van Valen 1990). microscopic cell counts and chl a content determination. Subsamples (4 mL) were fixed and stained with Lugol’s More recently, EPS production has been related solution before cells were counted in a hemocytometer to the formation of transparent exopolymer parti- improved Neubauer (Brand, Wertheim, Germany). An 11 mL cles, responsible for the formation of large aggre- aliquot was centrifuged for 20 min at 3,745g, and the super- gates in marine environments (Alldredge et al. natant used to determine total extracellular carbohydrate 1993, Passow 2002). Such particles play an impor- (Dubois et al. 1956). To determine chl a content, the cell pellet was extracted for 24 h in 10 mL methanol in the dark at tant part in diatom bloom coagulation, providing ) an adaptative function for the cells of a declining 4°C and centrifuged again for 10 min at 3,745g. The supernatants were read in a Q798-U spectrophotometer (Qui- bloom, which drop down to depths with better mis, Diadema, Brazil) at 665 and 750 nm, and chl a content was nutrient conditions than the depleted surface (Pas- calculated, using the coefficient proposed by Mackinney sow et al. 1994). Although this process was proposed (1951). In the periodic samples of culture supernatant, the to explain large aggregates and bloom dynamics in dissolved carbohydrates were estimated by HPLC with pulse the marine environment, the significance of such a amperometric detection (PAD), and the extracellular concen- process in freshwater has also been pointed out by tration of the nitrogen