AQUATIC MICROBIAL ECOLOGY Published April 28 Aquat microb Ecol

Effects of light on pigments and photosynthetic activity in a phycoerythrin-rich strain of Spirulina subsalsa

L. Tomaselli, M. C. Margheri, A. Sacchi

Centro di Studio dei Microrganismi Autotrofi del CNR. P. le delle Cascine, 1-27-50144 Firenze, Italy

ABSTRACT: Data on acclimation to 2 photon flux densities (15 and 100 pm01 photons m-2 S-') in Spir- ulina subsalsa strain 3F, a highly fluorescent phycoerythrin-rich cyanobacterium isolated from the brackish Lake Faro (Messina, Italy), indicated plasticity of the photosynthetlc apparatus of this organ- Ism. High-irradiance grown cells showed the greatest photosynthetlc capacity even though they had a lower and content. decreased to a lesser extent but their com- position changed. b- decreased, while the amount of myxoxanthophyll more than doubled. The stability of both C-phycoerythrin and C- ratios in cells grown under different light quality (green and red) demonstrated the lack of complementary chromatic adaptation in S. subsalsa. This factor, combined with the efficient utilization of low-wavelength light, indicates the strong adap- tation of this strain to its habitat.

KEYWORDS: Pigments . Oxygen evolution . Photoacclimation . Spirulina subsalsa .

INTRODUCTION lar strain. Our results, besides contributing to the understanding of photoadaption in S. subsalsa, could Cyanobacteria of the genus Spirulina Turpin are fre- provide useful information for the possible exploitation quently found in thermal springs and in brackish or of this strain as a source of natural fluorescent dye in marine waters, mostly eutrophic, where they can form immunofluorescent assays (Strier et al. 1985). dense populations, and make major contributions to primary productivity (Anagnostidis & Golubic 1966, Tomaselli Feroci & Balloni 1976, Castenholz 1977, MATERIALS AND METHODS Bazzichelli et al. 1978). The common species S. laby- rinthiformis Gomont, S. major Kuetzing and S. sub- Organism and growth conditions. Spirulina sub- salsa Oersted are characterized by a blue-green pig- salsa strain 3F was isolated from a water sample col- mentation due to the presence of C-phycocyanin, the lected at a depth of 10 m in Lake Faro. Stock cultures main light-harvesting pigment of most cyanobacteria, were maintained under 25 pm01 photons m-2 S-' of The present study concerns a strain of Spirulina sub- continuous PAR, at 26OC on MN medium (Rippka et al. salsa, isolated from the brackish meromictic Lake Faro 1979). For the experiments on photoacclimation, cells near Messina (Italy), which shows an unusual red pig- were cultured in a Gallenkamp orbital incubator at mentation, due to phycoerythrin-rich 26"C, in an atmosphere of CO2-enriched air (5 %). Con- (Tomaselli et al. 1990). The phycoerythrin in this strain tinuous irradiation of 15 pm01 photons m-2 s-' (LI) and possesses a high degree of autofluorescence which 100 vmol photons m-2 S-' (HI) was provided by cool- increases with exposure to increases in photosynthetic white fluorescent lamps. Cultures were allowed to active radiation (PAR) (Tomaselli et al. 1993). acclimate to irradiance levels by diluting them every We have examined the effects of distinct photon flux week with fresh medium. In this way, cells had been density (PFD) and spectral quality on growth, pigment acclimated for at least 6 generations before biochemi- composition and photosynthetic activity of this particu- cal and physiological determinations were made. Sim-

0 lnter-Research 1995 Resale of full article not permitted 28 Aquat microb Ecol9: 27-31. 1995

ilarly, to determine the effects of spectral quality on growing at low irradiance (below 40 1.1mol photons m-' cell pigment content, S. subsalsa cultures were incu- S-') CPE cell content, which represented about 20 % of bated for a month in white (25 pm01 photons m-' S-' ), total cell protein, was more than 10 times greater than red (30 pm01 photons m-2 S-') and green light (28 pm01 that of chl a. It was also about 4 times greater than the photons m-* S-'). Chromatic illumination was obtained sum of the contents of C-phycocyanin (CPC) plus allo- by interposition of appropriate colored plastic filters phycocyanin (APC). Eight distint carotenoids were between the cultures and the light source (400 W revealed by thin layer chromatography, 4 of which incandescent lamp). were present in significant amounts: p-carotene, Biomass and pigment analyses. Growth was deter- myxoxanthophyll, zeaxanthin and a having mined by cell dry weight; trichomes were collected by spectral characteristics and Rf values similar to p- filtration through a membrane filter of 3 pm pore size, cryptoxanthin. washed and dried at 70°C to constant weight. Protein, carbohydrate and lipid cell contents were determined as previously reported (Tredici et al. 1988). Chloro- Effect of PFDs and spectral quality on phyll a (chl a) and phycobiliprotein (PBP) contents pigment content were determined spectrophotometrically using the methods of Vonshak et al. (1985), and of Bennet & Spirulina subsalsa cultures exposed to increased Bogorad (1973), respectively. Total carotenoid content PFD showed a general reduction in pigment cell con- was estimated spectrophotometrically as reported by tent. PBPs content decreased more markedly than chl a Paoletti (1969). Specific carotenoids were isolated by content (about 70% and 57%, respectively) (Table 1). thin layer chromatography (Kieselgel 60, Merck) and Therefore, with increasing PFD, there was a reduction identified spectrophotometrically in different solvents, in the CPE:chl a and CPC: chl a ratios. Total caro- following Davies (1965). tenoid content was reduced with increasing PFD, but Photosynthetic activity determination. Rates of pho- to a lesser extent than chl a content, resulting in an in- tosynthetic oxygen evolution and respiratory oxygen crease of the total carotenoids : chl a ratio. Decreases in uptake were measured on Spirulina subsalsa cultures, the relative abundance of p-carotene in the carotenoid diluted to a final concentration of about 2.5 mg chl a l-' pigments coincided with increasing PFD, while myxo- with fresh medium containing 1 g 1-' NaHC03, using a increased noticeably. Due to the reduction Clark-type O2 electrode, at 26°C under PFDs ranging of p-carotene, zeaxanthin was the prevalent caro- from 25 to 760 pm01 photons m-2 S-' (PAR). Red and tenoid at higher irradiance (Fig. l). green light was obtained as described above. Cell pigment composition of Spirulina subsalsa was Statistical analyses. Effects of PFD and spectral qual- altered little by spectral quality (Table 1). The ratio of ity on pigment content and photosynthetic activity phycoerythrin contents after growth in red and green were compared by an analysis of variance (ANOVA). light [CPE (red):CPE (green)] and the one of phyco- The significance between pairs of variable means was cyanin contents under the same conditions [CPC analyzed by a least significant difference (LSD) analy- (red):CPC (green)] did not vary by more than 15%, sis. indicating a lack of chromatic adaptation in this strain.

RESULTS Photosynthetic activity of L1 and HI cultures

Laboratory cultures of Spirulina subsalsa strain 3F The curves of photosynthetic activity to increasing showed the prevalence of the C-phycoerythrin (CPE) PFD of Spirulina subsalsa 3F cultures grown at LT or HI over the other photosynthetic pigments. In cultures showed that the strain underwent some degree of

Table 1 Effects of photon flux density (PFD)and spectral quality on pigment composition (mg g-' dry weight) of Spirulina sub- salsa strain 3F. Data are means * standard deviation (n = 4). For each experiment, variable means with the same letter are not significantly different (p > 0.05)

Light conditions Chl a CPE CPC APC Carotenoids

White light (100 pm01 photons m-' S-') 4.02 * 0.18 a 29.32 r 1.94 a 6.09 * 0.68 a 0.88 * 0.15 a 0.97 * 0.10 a White light (25 pm01 photons m-2 S-') 9.42 * 0.32 b 96.50 t 2.49 b 22.90 * 1.35 b 1.58 + 0.12 b 1.39 * 0.15 b Red light (30 pm01 photons m-' S-') 8.87k0.26~ 88.55k2.84~ 22.4250.72bc 1.32+0.11c Green light (28 pm01 photons m-2 S-') 9 73 i 0.51 b 100.62 * 2.63 d 21.40 * 0.71 c 1.46 rt 0.15 bc - Tomaselli et al.: Spirulina photoacclimation

hydrates) to a prevalence of carbohydrates L I (43.6 % compared to 36.8% of proteins). @-carotene - Zeaxanthin Lipids remained unchanged (7.1% and @-cryptoxanthln 6.8% under low and high irradiance, ---- My*oxanthoph$l respectively)

Effect of spectral quality on photosynthetic activity

The photosynthetic activities of Spirulina subsalsa cultures exposed to white, red and green light at similar PFDs were compared (Table 2). Similar values for photosynthetic activity were observed under white and green light, whereas under red light the value was about 20 % lower.

DISCUSSION AND CONCLUSIONS Wavelength ( nm ) Wavelength ( nrn ) Spirulina subsalsa strain 3F efficiently Fig. 1. Absorption spectra of the prevailing carotenoids from SpiruLina regulated pigment content in response to subsalsa 3F cultures grown under 15 pm01 photons m-2 S-' (LI) and PFD changes. It followed classical photo- 100 pm01 photons m-2 s-' (HI) of continuous PAR. Note that the absorb- acclimation mechanisms of cyanobacteria, ance scales are different which are decreases in cell content of both chl a and PBPs, following a shift from lower acclimation (Fig. 2). In fact, at PFDs greater than to higher growth irradiance (Post 1986, Wyman & Fay 150 pm01 photons m-2 S-' the photosynthetic activity of 1987, Millie et al. 1990, Falkowski & LaRoche 1991). the HI cultures was always higher than that of L1 cul- The pigment required to sustain the energy needs of tures. The maximum photosynthetic activity (P,,,) in cells at their division rate decreases (Van Liere & the former was more than 20% greater than in the Walsby 1982). The reduction in pigment content, latter. As expected, dark respiration was also greater in accompanied by the decline of light-harvesting appa- HI cultures (79 pm01 O2 mg-l chl a h-') than in L1 cultures (39 pm01 O2 mg-I chl a h-'). The 200 - greater photosynthetic activity observed at PFDs less than 150 pm01 photons m-2 S-' in cul- - tures acclimated to L1 (Fig. 2) is explained by h -i zC150 - P----- the great capacity of this strain to alter light- Q 2 harvesting through a considerable increase of : U pigment cell content, especially CPE (Table 1). 2'; - -1 ; ; l00 R PFD Effect of on growth and cell composition 2-g O Growth rate increased with the increase of PFD levels and was enhanced up to 100 pm01 photons m-' S-'. Cultures growing at high irra- diance showed a mean generation time of 27 h, I 100 200 300 400 500 600 700 800 whereas it took 36 h in the cultures growing at 25 pm01 photons m-' S-'. The biochemical com- Light irradiance (pm01 photons m-2 S-l, PAR) position of Spirulina subsalsa, expressed as % Fig. 2. Photosynthetic activity of SpiruLina subsalsa 3F cultures accli- of dry changed from low high growth mated at 15 pm01 photons m-2 S-' (.) and 100 pm01 photons m-2S-' (0) irradiance, shifting from a prevalence of pro- of continuous PAR. Values are means and error bars are *standard teins (47.7% compared to 33.2% of carbo- deviations (n = 4) 30 Aquat microb Ecol 9: 27-31. 1995

Table 2. Effects of light spectral quality on photosynthetic The ability of Spirulina subsalsa to increase the P- activity of Spiruljna subsalsa 3F cultures. Data are means * carotene cell content may be of ecological relevance in standard deviation (n = 3). Variable means with the same the eutrophic, sulphide-rich and scarcely transparent letter are not significantly different (p > 0.05) waters of Lake Faro, where the most penetrating com- ponent of the downwelling radiation is the low-wave- Light conditions Photosynthet~cactivity length blue and green light absorbed specifically by (pm01 O2mg-' chl a h-') p-carotene and phycoerythrin (Skulberg 1978). The White light 115.00 + 3.69 a efficient utilization of low-wavelength light shown by (250 pm01 photons m-' S-'l this strain also resulted from the greater photosyn- Red light 93.02 + 3.17 b thetic activities measured under green light in respect (260 pm01 photons m-' S'.j Green light 117.45 & 2.13 a to red light of similar quantum flux. (240 pm01 photons m-2 S-') Therefore, Spirulina subsalsa strain 3F appears to be well adapted to the environment where it lives with its specific equipment of antenna pigments, the preva- ratus, leads to considerable conservation of energy that lence of CPE, and the capability of extensive accumu- may be directed toward the biosynthesis of other cell lation of this pigment under low irradiance. Despite the constituents (Wyman & Fay 1987). Indeed, in our fact that it grows in a relatively narrow range of low experiments, when compared with cultures growing irradiance, this organism shows a certain degree of under low irradiance, those growing under high irradi- photoacclimation to high PFD. This is due not only to ance showed a reduction in cell contents of chl a and pigment regulation, but also to its capability to dissi- even more of PBPs, accompanied by a decreased mean pate a greater fraction of the light energy absorbed by generation time (27 instead of 36 h). The photosyn- CPE as autofluorescence (Tomaselli et al. 1993). The thetic activities of HI grown cultures, measured at inci- latter phenomenon, previously observed in a marine dent irradiances greater than 150 pm01 photons m-2 phycoerythrin-rich strain, is consid- S-', were also considerably higher than those of L1 ered to be of strategical importance in preventing grown cultures. This may be a result of a lower inci- photoinhibition (Wyman et al. 1985). dence of the reciprocal shading of individual pigment The behavior of Spirulina subsalsa 3F in the photo- molecules, which leads to a greater capacity of light- acclimation response to high growth irradiance. harvesting by each pigment molecule (Falkowski & together with the lack of chromatic adaptation, could LaRoche 1991). be a good basis for the exploitation of this organism as During the acclimation to high irradiance, the ob- a source of phycofluors. served decrease in the cell chl a:carotenoids ratio, specifically affecting the synthesis of chlorophyll, Acknowledgements. Research supported by the National Re- suggests a photoprotective role for carotenoid pig- search Council of Italy, Special Project RAISA, Sub-project 4, ments in Spirulina subsalsa 3F, as has been well docu- paper no. 1769. The authors are grateful to Dr M. Bindi of the mented in other cyanobacteria (Kellar & Paerl 1980). Ce.S.l.A.,Accademia dei Georgofili of Florence, for carrylng Also, the increase in the relative abundance of myxox- out the statistical data analyses, and M. D. Mannelli of the DISTAM at the Univers~tyof Florence, for the drawings. anthophyll and zeaxanthin found in S. subsalsa 3F, similar to what Millie et al. (1990) observed in Oscilla- toria agardhii, may be considered an adaptive LITERATURE CITED response for protection against photooxidation. On the other hand, the greater carotenoid pigment content, Anagnostidis K, Golubic S (1966) iJber die Okologle einiger observed in L1 grown cultures of S. subsalsa 3F, seems SpiruUna-Arten. Nova Hedwigia 11:309-335 Bazzichelli G, Abdelahad N, Florenzano G,Tomaselli L (1978) to indicate also a considerable contribution of these Contributo alla conoscenza delle Terme di Saturnia (Tos- lipophilic pigments to light-harvesting, as already pro- canal Ann Bot 37:203-232 posed for bloom-forming cyanobacteria (Paerl et al. 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