relationship between bio-optical characteristics and

Aquatic botany

E L S E V I E R Aquatic Botany 59 (1997) 237-251

Relationship between bio-optical characteristics and photoinhibition of phytoplankton

F61ix L. Figueroa a,., Jesfis Mercado a, Carlos Jim6nez a, Soluna Salles a, Jos6 Aguilera a, Maria Pilar Sfinchez-Saavedra b,

Michael Lebert c, Donat-Peter H~ider c, Olimpio Montero d, Luis Lubifin d

a Departamento de Ecologfa, Facultad de Ciencias, Universidad de M{tlaga, Campus Universitario de Teatinos s / n, E-29071 M61aga, Spain

h Centro de lnvestigaci6n Cienfffica 3' de Educaci6n Superior de Ensenada (CICESE) Apdo Postal 2732 Ensenada, M£xico

c lnstitutfiir Botanik und Pharmazeutisehe Biologie, Friedrich-Alexander-Unitersit~t, Staudstr 5, Erlangen D-91058, Germany

Instituto de Ciencias Marinas de Andalucfa, C.S.L C, E-11510 Puerto Real, Cddiz, Spain

Accepted 3 July 1997

Abstract

The relationship between the bio-optical properties of different microalgae and photoinhibition after short-term exposure (15 and 30 min) to solar radiation was analyzed. Photoinhibition was determined as the decrease in oxygen production and in the in vivo-induced chlorophyll fluorescence. Microalgae with different chlorophyll concentrations, cell size and volume were used. Both photoinhibition and recovery of oxygen production and quantum yield were higher after 30 than after 15 min exposure to solar radiation. Photoinhibition was reduced when UV-A and UV-B radiations were eliminated from the solar radiation. The decrease of effective quantum yield and oxygen production was not dependent on cell size, biovolume or chlorophyll concentration in the algal cultures. It was, however, related to the bio-optical property of the cultures. manifested as the specific attenuation coefficient (Kc). As a general response, the inhibition of

Corresponding author. Tel.: +34 52 131672; fax: +34 52 132000; e-mail: [email protected]

0304-3770/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0304- 3 770(97)00065-X

238 F.L. Figueroa et al./ Aquatic Botany 59 (1997) 237-251

effective quantum yield slightly decreased with the increase in K c. Recovery of yield and oxygen production in darkness after exposure to solar radiation was also clearly related to K¢. The relation between the recovery of the yield and K c followed a parabolic function. It was also found that the recovery of the inhibition of the yield was higher in phytoplankton with active xanthophyll cycle but that it was not dependent on the concentration of total carotenoids. © 1997 Elsevier Science B.V.

Keywords: Bio-optical properties; Quantum yield; Microalgae; Photoinhibition; Pulse amplitude modulate fluorescence; Xanthophyll cycle

1. Introduction

Since the discovery of the ozone hole, (Farman et al., 1985) an increasing amount of work has been devoted to measuring the impact of the enhanced UV-B radiation on aquatic ecosystems (Weiler and Penhale, 1994; H~ider, 1996). Given that phytoplankton cells are in the base of the food chain and that they represent an important component in the biogeochemical cycles, the investigation on phytoplankton ecophysiology is relevant (see H~ider, 1994; Pr6zelin et al., 1994). The incorporation of carbon into organic material on our planet has been estimated to be about 2 × 101~ metric tons annually, half of which is due to phytoplankton (Houghton and Woodwell, 1989). Thus, an evaluation of the effect of UV-B on photosynthesis in aquatic ecosystems is crucial in order to estimate the flow of carbon in the oceans and to increase our knowledge of global climate change. Enhanced UV-B can decrease phytoplankton productivity by direct effects on carbon assimilation (Lesser et al., 1994; Schofield et al., 1995), nitrogen assimilation (Behrenfeld et al., 1995), DNA damage (Karentz et al., 1991; Buma et al., 1996) or due to the inhibition of motility (H~ider and Liu, 1990; Tirlapur et al., 1993; van Donk and Hessen, 1996).

Most of the studies on the effect of UV-B radiation on photosynthesis have been conducted in short-term experiments (Smith et al., 1992; Holm-Hansen et al., 1993; Pr6zelin et al., 1994) and with single species (Karentz et al., 1991). A few investigations have analyzed the long-term effects of UV-B on natural communities of phytoplankton either by experimental increase (Worrest et al., 1978) or selective exclusion of UV from natural sunlight (Bothwell et al., 1993; Helbling et al., 1994).

In this study, the effects of full solar radiation (PAR + UV-A + UV-B) and UV- filtered solar radiation (PAR) on quantum yield of fluorescence and photosynthesis in phytoplankton are investigated in short-term experiments. Cell cultures were exposed to solar radiation for 15 and 30 min and then transferred to darkness to investigate the possible recovery of yield and of oxygen production. The microalgal species were selected according to different bio-optical characteristics (i.e., pigmentation, cell size and specific attenuation coefficient). In addition, carotenoid composition of the cell cultures before the exposure to solar radiation was characterized by high performance liquid chromatography (HPLC) in order to relate the concentration of specific carotenoids and the action of the xanthophyll cycle to the depletion and recovery of yield and photosynthesis by solar radiation.

F.L Figueroa et al. /Aquatic Botany 59 (1997) 237-251 239

2. Material and methods

2. I. Microalgae

Algae from the algal collection of the Institute of Marine Sciences of Andalucla (Cfidiz, Spain) were used. The species were as follows: the Eustigmatophyceae Nan- nochloropsis gaditana Lubifin (strain B-3) and N. oculata (Droop) Hibberd (strain OCU 66), the Chlorophyceae Nannochloris maculata Butcher, the Cryptophyceae Rhodomonas baltica Karsten (strain USA) and the Prasinophyceae Tetraselmis suecica (Kylin) Butcher. The algae were cultivated in 21 beakers with F / 2 medium (Guillard and Rhyter, 1962) modified with double nitrate and phosphate concentration, mixed by means of air bubbling at 20°C and at irradiance of 75 /xmol m 2 S-1 (17 W m 2) of continuous light regime under white light provided by fluorescent lamps (Osram Day-light, 20 W).

2.2. Measurement of solar radiation

Solar radiation was measured in three wavelength bands (UV-B, 280-315 nm; UV-A, 315-400 nm; PAR, 400-700 nm) using a recently developed dosimeter (ELDONET, Real Time Computer, Erlangen, Germany). The instrument uses water- proof sensors (GriSbel, Ettlingen, Germany) and takes readings at 1 s intervals which are averaged over 1 min. After amplification and analog/digital conversion, the data are graphically displayed and stored in a portable computer. The incident irradiance during the exposure to solar radiation around noon was 480 W m -2 of PAR, 45 W m -2 of UV-A and 1.8 W m -2 of UV-B.

2.3. Light measurement in the algal cultures

Underwater spectral composition in the algal cultures was determined with a Licor spectroradiometer model Li-1800 UW provided with a 4p custom-made spherical diffuser (Dr. Garcfa-Pichel, Bremerhaven) connected at the end of an optic fiber (Licor 1800-10). The system was calibrated against the 27r sensor of the Li-1800 UW. The error of angular response was less than 5%.

Diffuse attenuation coefficient for phytoplankton ( K p ) f o r photosynthetic active radiation (PAR) was determined by linear regression following the equation (Kirk. 1994):

Kp = K v - K w in m - J

being K v the total attenuation coefficient in the cultures and calculated as [(ln l o - l n l J z ] where Io is the incident photon fluence rate at 5 cm deep from the surface determined with a 47r sensor as described above, I S was the photon fluence rate into the culture at the distance z from the surface of the culture vessel and K w the attenuation coefficient due to the water. Other potential absorbers are the galvinic and yellow

240 F.L. Figueroa et al. /Aquatic Botany 59 (1997) 237-251

substances but the contribution to the light attenuation (K G) in algal cultures is very low (Kirk, 1994), thus the value of K~ is considered zero. The specific attenuation coefficient was calculated as the ratio of Kp and the concentration of chlorophyll in mg m -3, being expressed in m 2 mg -1 Chla.

2.4. Cut-off filters for UV radiation and light treatments

Samples from the algal cultures were transferred to Petri dishes with 3 cm deep which were covered with Ultraphan filters (Digefra GmbH, Mtinchen, Germany). In order to cut off both UV-A (315-400 nm) and UV-B (280-315 nm), the Petri dishes were covered with a filter opaque to radiation shorter than 400 nm (Ultrapahn 395), this treatment was denoted as PAR (only photosynthetic active radiation). The irradiance in PAR treatment is reduced about 10% with Ultraphan 395 filter and thus, in order to have the same irradiance of PAR in both treatments, Ultraphan 295 was used to reduce the irradiance at the same level as Ultraphan 395 produced.

2.5. Measurements of fluorescence induction

In vivo-induced chlorophyll fluorescence was determined with two portable pulse amplitude modulated fluorometers (PAM-2000, Waltz, Effeltrich, Germany). The method of quenching analysis is based on the measurement of the fluorescence parameters in response to saturating light in dark- or light-adapted specimens (Schreiber et al., 1995). In this work, the effective quantum yield of light-adapted algae was determined by using the expression ( F " - F t ) / F " = A F / F ' , where F m' is the maximal fluorescence of light-exposed algae which normally decreases with increasing irradiance compared to the maximal fluorescence in dark-adapted algae (Fm), and F t is the basal fluorescence after the saturating light pulse. The effective quantum yield was immediately determined after 15 or 30 min exposure to solar radiation from at least 8 replicates immersing the optic fiber in the algal cultures and covering the cultures with neutral density filters to reduce the irradiance until 75 /xmol m -2 s-1 (17 W m-E). Subsequently, the samples were transferred to darkness for 6 h, and recovery of the photosynthetic yield was determined at predefined intervals (1, 2, 3, 4 and 6 h).

2.6. Photosynthetic rate

Photosynthetic rate was determined by oxygen evolution at saturating light (800 /zmol m -2 s -1) in two CWI Hansatech oxygen measuring chambers supplied with Clark-type oxygen electrodes (model CBID Hansatech). The photosynthetic rate was determined after 30 min of exposition to PAR or PAR + UV-A + UV-B and expressed as fmol 02 cell- l h - 1. Recovery of oxygen production was determined after 2 and 6 h in darkness.

2.7. Pigment analysis

Pigments were extracted from a volume of 10 ml cells harvested by centrifugation, then sonicated in methanol or N,N-dimethylformamide (DMF) and kept overnight at

F.L. Figueroa et al. / Aquatic Botany 59 (1997) 237-251 241

4°C. The pellets were extracted again until colorless. Measurements were performed with a Lambda 5-Perkin Elmer spectrophotometer, and the trichromatic equations given by Porra et al. (1989) were used to calculate both Chl and carotenoid concentrations. In order to determine the contribution of each carotenoid to the total carotenoid content, methanolic and DMF extracts were analyzed by means of High Performance Liquid Chromatography in a Waters 600E multisolvent delivery system equipped with a RP-C18 column packed with spherisorb ODS-2. Samples were filtered through a nylon membrane of 0.2 /zm, and 20/~1 were injected. Elution was carried out in a two-solvent gradient system according to Minguez-Mosquera et al. (1992). A programmable photo- diode array detector (Waters type 991) was used for pigment detection by plotting each peak in the chromatogram and its maximum wavelength, since data were acquired three-dimensionally (absorbance-time-wavelength) over the wavelength range of 350 to 800 nm. The epoxidation state of xanthophyll cycle was calculated as (V + 0 . 5 A ) / ( V + A + Z) where V (violaxanthin), A (anteraxanthin) and Z (zeaxanthin) are expressed in fg per cell.

2.8. Statistics

PAM measurements for each treatment were repeated at least eight times and mean values and standard deviation were calculated. Oxygen determinations and pigment analysis were run in triplicate. Statistical significance of means were tested with a model 1 one-way ANOVA followed by a multirange test (Fisher's protected least significance difference.

3. Resul ts

Effective quantum yield of the algal cultures was drastically reduced after both 15 min (Fig. 1) and 30 min (Fig. 2) of exposure to full solar radiation. The decrease of the quantum yield after 15 rain exposure was more pronounced under full solar radiation (PAR + UV) than under PAR alone while after 30 min of exposure, it was similar in both treatments (Fig. 2). Decrease of quantum yield was significantly ( p < 0.05) more pronounced after 30 min than after 15 min of exposure in all species except N. maculata. When the algae were transferred to darkness, gradual recovery was found in most algae investigated; only N. aculata did not recover after exposure to either solar (PAR + UV) or to PAR. In addition, recovery after exposure for 15 min was similar in both full solar or solar without UV radiation (Fig. 1). After 6 h in darkness, in the 15 min exposure treatment, significant differences ( p < 0.05) between PAR and PAR + UV treatments were found in N. gaditana, R. baltica and T. suecica. However, recovery after 30 min of exposure was faster in PAR than PAR + UV (Fig. 2). In 30 min exposure treatment after 6 h in darkness significant differences ( p < 0.05) between PAR and PAR + UV were found on N. oculata, R. baltica and T. suecica. Recovery was significantly ( p < 0.05) higher after 30 min exposure than after 15 min except in N.

242 F.L Figueroa et al. /Aquatic Botany 59 (1997) 237-251

0.7-

0.6~ I

0.51 o.41

"o -~ 0.3 ~

0.2~

0.1

A N. oculata 0.7 B N. gaditana

1 • i!i!l 0.6

~ 0.5

0.3 :i:: ::.;::

~ 0.5 ':: :::::

o l ~i: ~;:: . . . . 0

l h D 15 rain L l h D 2h D 3h D 4h D 6h D l h D 15 mln L l h D 2h D 3h D 4 h D 6h D

Time Time

~ 0.5

~ 0.4

"~ 0.3

0.7.

"~ 0.5

~ 0.4

'~ 0.3

0.2

0.1

0

0] 0.6 C N. maculata

o llii!i ' 0.4 0.3 0.2 ~ *

l h D 1 5 m i n L l h D 2 h D 3 h D 4 h D 6 h D

Time

0.7 E T. suecica

0.6 . ' r ~

o l h D 1 5 m i a L l h D 2 h D 3 h D 4 h D 6 h D

Time

D R. baltica

l h D 1 5 m i n L 1 h D 2 h D 3 h D 4 h D 6 h D

Time

Fig. 1. Effective quantum yield (AF/F ' ) of different microalgae after 15 min of exposure to P A R + U V (closed bars) and only PAR (dotted bars) and in darkness for 6 h. The initial yield was determined after 1 h of incubation in darkness (1 h D). Significant differences at <0.05 level between PAR and P A R + U V treatments at each time are indicated by asterisks.

maculata in w h i c h n o s i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d . B o t h s p e c i e s o f Nan- nonochloropsis (N. oculata a n d N.gaditana) s h o w e d a b o u t 9 0 % o f r e c o v e r y o f t he i r

e f f e c t i v e q u a n t u m y i e l d a f t e r 6 h in d a r k n e s s , w h i l e in b o t h Rhodomonas a n d Te-

F.L. Figueroa et al. /Aquatic Botany 59 (1997) 237-251 243

traselmis, t h e r e c o v e r y w a s a b o u t 8 0 % ; t h e r e c o v e r y w a s v e r y l o w ( 1 0 - 3 0 % ) in N.

maculata.

T h e p h o t o s y n t h e t i c o x y g e n p r o d u c t i o n d e c r e a s e d a f t e r 3 0 m i n o f e x p o s u r e in b o t h

A

0.6 - o . 6 ~ ~ .~.. _ ~

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o. , ..:-:.:, :--i :,,:; :~.~- ~- ::-- Oo:,

O.~o lh D 30 minL lh D 2h D 3h D 4h D 6h D

Time

0"71 C 0.6 N. maculata

0.5

0.4

0.2

:

lh D 30 rain L l h D 2h D 3h D 4h D 6h D

Time

lh D 30 minL lh D 2h D 3h D 4h D 6h D

Time

°'71~,~.,, D R. baltica

o 6 t l : i : t . . "

.otliiil, .|iiil liil,lilil !!i)l ! lh D 30 minL lh D 2h D 3h D 4h D 6h D

Time

° . 7 1 ~ ~ 7. s°ecic.

i!: t

0 ,, .--T_ J-'-"i-i : : : , lh D 30 minL lh D 2h D 3h D 4h D 6h D

Time

Fig. 2. Effective quantum yield (A F~ F m) of different microalgae after 30 min exposure to PAR + UV (closed bars) and only PAR (dotted bars) and in darkness for 6 h. The initial yield was determined after l h of incubation in darkness (1 h D). Significant differences at <0 .05 level between PAR and P A R + U V treatments at each time are indicated with asterisks.


O" o

1"

1

1

A N. oculata 1-

1

el 0 o

N. gaditana

-h

0

1.2

Initial

C

30 rain L 2 h D 6 h D Initial 30 min L 2 h D 6 h L

D

N. maculata

O" o

Initial 30 rain L 2 h D 6 h D Initial 30 rain L 2 h D 6 h D

E 600"

T. s u e c i c a

500.

400-

O" 300 ~ 1 i 200

100

G : ' : Initial 30 min L 2 h D 6 h D

Fig. 3. Photosynthetic rate (fmol 0 2 cell- l h - i ) of different microalgae from saturating white light submitted to 30 rain of exposure to P A R + U V - A + U V - B (closed bars) and PAR (open bars). After solar exposure, the algal cultures were transferred to darkness and then the 0 2 evolution was determined after 2 and 6 h. The initial yield was determined after l h of incubation in darkness (1 h D). Significant differences at < 0.05 level between PAR and PAR + UV treatments at each time are indicated with asterisks.

F.L Figueroa et al. / Aquatic Botany 59 (1997) 237-251 245

PAR and PAR + UV except in N. gaditana, where only a small reduction was found under full solar radiation (Fig. 3). In N. oculata and R. baltica, the oxygen production was similar in PAR and PAR + UV after 30 min exposure. However, in T. suecica, UV radiation provoked more pronounced decrease of the oxygen evolution (Fig. 3). N. maculata was the most affected alga after solar exposure, and after 30 min, no net photosynthesis was detected.

After the transfer to darkness, N. gaditana showed full recovery in 2 h and had a higher photosynthetic rate than the cultures under laboratory conditions (initial values). T. suecica partially recovered after 6 h in darkness (75% of the initial). In both N. oculata and R. baltica, recovery was significantly ( p < 0.05) higher after 2 h in darkness than after 6 h. Finally, N. maculata presented very small recovery after 6 h in darkness, both after PAR and PAR + UV treatments. For all, except N. maculata, the recovery was not significantly different in both treatments after 6 h.

The different response of the algal populations to the solar radiation could be related to the bio-optical properties of the cultures. Bio-optical properties can be defined by the capacity of light absorption, which depends on the pigment composition, cell structure and size. Some parameters related to bio-optical properties are presented in Table 1. The attenuation coefficient of the downward radiation ranged from 7.8 m-1 (T. suecica) to 12.6 m ~ (N. maculata), the last value corresponding to high cell densities. Chlorophyll a concentration ranged from 2.6 (R. baltica) to 7.3 mg 1-~ (N. maculata). According to the chlorophyll concentration, the specific attenuation coefficient ranged from 0.0017 in the cultures of N. maculata to 0.0035 in the R. baltica ones.

Algal species were grouped according to their cell volume (Table 1): (1) small cells (3.1 to 4.6 /xm 3 of cell volume) including the two Nannochloropsis species and N. maculata, and (2) large cells (204 to 339/xm 3 of cell volume) including R. baltica and T. suecica. Total biovolume, calculated as the product of averaged volume and the cell density, ranged from 0.59 in cultures of T. suecica to 0.16 mm 3 m1-1 in those of N. gaditana (Table 1). Chlorophyll and carotenoid content per unit culture volume was higher in cultures with small cells than in cultures with large cells (Table 1). Table 2 summarizes the concentration of the main carotenoids found in the microalgae used throughout this work.

No relation was found between cell size or biovolume and the percentage of decrease of the effective quantum yield. After 15 min exposition, the smallest cells, i.e., N. gaditana, were the most affected (about 90% of decrease in the yield) but the least affected were not the largest, i.e., T. suecica (339/xm 3) but R. baltica (204/xm3). After 30 rain exposure, the decrease of the yield was more pronounced in N. maculata than in N. gadimna. However, recovery of the yield after 6 h in darkness was clearly dependent, in a parabolic way, on the bio-optical properties of the algal cultures, i.e., the specific attenuation coefficient (K c) (Fig. 4). The recovery of yield increased until Kc values of 0.0025 m 2 mg ~ Chla corresponding to cultures of N. gaditana and then slightly decreased for higher K c values corresponding to cells of both smaller (N. oculata) and bigger size (R. baltica). Thus, the cultures of N. gaditana were most affected by solar radiation (highest decrease of yield) but they also showed the highest recovery capacity. Correspondingly, N. gaditana showed the lowest inhibition of oxygen production (Fig. 5). The percentage of inhibition of photosynthesis versus K c followed a negative

246 F.L. Figueroa et al. /Aquat ic Botany 59 (1997) 237-251

Table 1 Bio-optical characterization of the algal populations used in the experiments

Algal species Kp K c (m 2 Number Cell Total Bio- Chl a Total ( m - J ) m g - l of cells volume volume (mg 1 - i ) carotenoids

Chl a (10 6 ml - i) (l~m 3) (mm 3 ml - t) (mg 1-1)

N. oculata 9.8 0.0032 67.2 3.6 0.25 3.0 1.5 N. gaditana 10.5 0.0025 53.4 3.1 0.16 4.2 2.4 N. maculata 12.6 0.0017 66.6 4.6 0.31 7.3 3.7 R. baltica 9.1 0.0035 1.1 204.5 0.24 2.6 0.3 T. suecica 7.8 0.0021 1.7 339.0 0.59 3.7 0.8

Kp = attenuation coefficient due to phytoplankton ( m - t ). K c = specific attenuation coefficient (m 2 m g - 1 Chl a). For all data, SD was between 10-12%.

parabolic function, with high inhibition of photosynthesis at low K c and low inhibition at intermediate Kc values. The percentage of recovery of the oxygen evolution increased drastically with Kc and then decreased (Fig. 5), showing a similar pattern to that of recovery of yield (Fig. 4).

The decrease of yield and oxygen evolution was not dependent on the concentration of carotenoids (Table 1, 2). However, a relation between the existence of the xanthophyll cycle (Table 2) and the decrease of the effective quantum yield and its recovery was found. Nannochloropsis species, with zeaxanthin and epoxidation state of 0.91-0.95, showed higher recovery than algal cultures without zeaxanthin or violaxanthin cycle (Figs. 4 and 5, Table 2).

Table 2 Concentration of the carotenoids, in fg cel l- t with role in photoprotection, i.e., /3-carotene and of those involved in the xanthophyll cycle, violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z) in various microalgae

Algal species Violaxanthin Anteraxanthin Zeaxanthin Epoxidation (fg cell - t ) (fg cell- i ) (fg cell - i ) state

V+0 .5 A

/ V + A + Z

13-carotene Major carotenoids (fg cell- i ) (%)

N. oculata 14.31 0.43 0.45 0.95 - violaxanthin (64.6%); vaucheriaxanthin (15.8%)

N. gaditana 20.0 0.67 1.48 0.91 1.5 violaxanthin (43.4%); vaucheriaxanthin (17.8%)

N. maculata 5.33 1.17 - - 1.7 lutein (48.0%); cantanxanthin (12.9%)

R. baltica . . . . . alloxanthin (80.3%); chrocoxanthin (16.5%)

T. suecica 128.1 - - - 19.8 violaxanthin (32.3%); lutein (31.5%); siphoxanthin (16.5 %)

The epoxidation state of the xanthophyll cycle is also represented and calculated as V + 0 . 5 A / V + A + Z.

F.L. Figueroa et al./Aquatic Botany 59 (1997) 237 251 247

o~ 120

100 t~

80

60

40 // e

8 20

0 0 . 0 0 1 5

/ /

I I I I

0.002 0.0025 0.003 0.0035 0.004

K (m 2 m g l C h l a )

Fig. 4. Relation between the recovery of the effective quantum yield after 30 min exposure to P A R + U V

(circles) and PAR (dots) followed by 6 h in darkness, and the specific attenuation coefficient (m 2 mg i Chl

a) in the algal cultures.

100 I

o

N 8o

~ 60 O

~ 4O

• - 20

0 0.0015

• # / "\~ //.

0.002 0. 25 0.003 0.0035 I

0.004

_= o

O"

160~ B

140

120-

100 ~

80

60- / /

4O

20 ~

o 0.0015

m ,\

I I I I I

0.002 0.0025 0.003 0.0035 0.004

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Fig. 5. Relation between the inhibition of oxygen production (a) and recovery of oxygen production (b) alter 30 rain exposure to P A R + U V (circles) and PAR (dots) followed by 6 h in darkness, as a function of the specific attenuation coefficient of the algal cultures.


4. Discussion

In this work, the relationship between recovery of the effective quantum yield and photosynthetic capacity, after solar exposure followed by dark incubation, was closely related to the specific attenuation coefficient of the algal populations. By contrast, no relationships were found among the decrease of yield and cell size, biovolume or Chl concentration. The specific attenuation coefficient is a parameter which integrates the optical properties of the algae in the culture media since it is the ratio between diffuse attenuation coefficient and chlorophyll concentration. The attenuation coefficient depends on the absorption and scattering coefficients in the cultures which are inherent optical properties (Kirk, 1994). The fact that the recovery is more related to K c than the depletion of yield can be interpreted as follows: the cells are submitted to drastic changes in the light field, in the short term, since they are transferred from WL of 17 W m -2 without UV to full solar radiation of 480 W m -2 of PAR, 45 W m -2 of UV-A and 1.8 W m -2 of UV-B. Hence, the cells suffer a drastic decrease of yield after 15 min (72-90% under PAR and 84-93% under PAR + UV) and 30 min (86-95% under PAR and 95-100% under PAR + UV). The effect is so drastic that just after the exposure, it is not possible to detect if the decrease of yield is due to photodamage or reversible photoinhibition. In the following period in darkness, when the repair mechanisms operate, it is possible to determine from the extent of the recovery whether the effect of solar radiation on the effective quantum yield is due to reversible photoinhibition or to photodamage. The fact that the recovery was higher after 30 than after 15 min exposure is interpreted in terms of a dose dependence of the repair mechanisms. Although a 30 min exposure produces higher photoinhibition, it also seems to induce repair mechanisms.

Those algae with low specific attenuation coefficient showed higher photodamage than the algae with high K c values. Populations with low K c showed a lower efficiency of light absorption due to the increase of the package effect of the pigments (Berner et al., 1989; Mercado et al., 1996). Kc values decreased with Chl concentration, a higher total absorption is produced and consequently larger total photochemical effects are found. Irradiation of a 'strong UV absorber' (large cells) will cause larger total photochemical effects than irradiation of a ' weak absorber' (small cells) (Garda-Pichel, 1996). However, if the effects are normalized to the amount (i.e., mass, number, Chl, etc.) of the absorber, the specific photochemical effect will actually be much lower in a strong than in a weak absorber (Garcfa-Pichel, 1996). For example, in cells of Synechococcus sp. (1 /zm diameter), the number of thymidine dimers per gram of DNA caused by the same UV dose would be around 43% larger than in cells of a dinoflagellate of larger cell size (40 k~m diameter) (Garcla-Pichel, 1996). In this work, the recovery of photosynthesis and effective quantum yield was not dependent on the optical properties of the individual cells but on the optical properties of the algal populations as characterized by the K c values.

The relation between cell size and the damage by UV is still confusing in the literature. Karentz et al. (1991), Bothwell et al. (1993) and Garcfa-Pichel (1996) argued that small algae are bound to be more sensitive to UV than larger ones. However, Helbling et al. (1994) found that smaller algal cells were less affected by UV-B than


larger cells. W~ingberg et al. (1996) did not find any clear relation between cell size and the sensitivity to UV-B irradiation. In this work, after 15 min of exposure to full solar radiation, the smallest species, N. gaditana, showed the most pronounced decrease of quantum yield, however, after 30 min the inhibition was higher in the largest species. The drastic decrease of yield and rapid recovery in Nannochloropsis species can be explained in terms of photoacclimation to the natural light field. These algae grow naturally in ponds exposed to very high light and temperature stress (Lubifin and Establier, 1979). In Nannochloropsis species, there must be mechanisms for dissipation of the excess of light. One of the possible mechanisms is the production of zeaxanthin, a quencher of excess of absorbed energy (see Table 2). The violaxanthin cycle consists of the formation of zeaxanthin (no-epoxy groups) from violaxanthin (two-epoxy groups) under high irradiance through the intermediate antheraxanthin (one epoxy group) in a series of enzyme-mediated reactions of de-epoxidation, whereas low light and darkness revered the reaction to replenish the violaxanthin content (Pfiindel and Bilger, 1994). The rise of non-radiative energy dissipation associated with the increase of zeaxanthin content has been related to (1) a direct quenching of the excited singlet-state of the chlorophyll a by energy transfer from chlorophyll to zeaxanthin (Demmig-Adams, 1990) and (2) alteration of the thylakoid membrane properties by promoting aggregation of the major light-harvesting complex (Noctor et al., 1991) or changing the fluidity of the thylakoid membrane (Horton and Ruban, 1992). In this case, the violaxanthin cycle would act as amplifier rather than as a direct quencher (Thiele and Krause, 1994). Nannochloropsis species have the violaxanthin cycle which could account for the low depletion of yield after 30 rain of exposure and rapid recovery in darkness.

In this work, the bio-optical properties of the algal populations are considered for the study of photoinhibition of microalgal cultures instead of the absorption characteristics of individual cells. This approach is closer to which occurs in the natural environment, in which the photochemical effects are dependent not only on the optical properties of the individual cells but on the optical properties of the whole system including phytoplankton and water (Kirk, 1994). Dubinsky et al. (1995) established that the light field characteristics in algal mass cultures and consequently in natural environments with high cell densities depended on: (1) optical density of the cultures, biomass concentration, pigmentation, and cell geometry, (2) path length of light within the culture, which depends on the light source and vessel configuration, or on latitude, season and depth in ponds and (3) mixing regime, which also affects light delivery to individual cells.

In summary, it has been illustrated that the effect of solar radiation in algal cultures is not dependent on the individual absorption properties, but on the bio-optical properties of the cell populations determined by the specific attenuation coefficient of the cultures. The drastic change of the light field induced by the transfer of cells from the laboratory to solar radiation provoked a general and drastic decrease of yield in all cell populations. The capacity for photoprotection and repair can be detected and related to K~. only during the recovery in darkness. This is due to the different kinetics of photoinhibition and photodamage compared to photorepair processes (Osmond, 1994), Photoinhibition operates more rapidly than repair mechanisms (Osmond, 1994). In the phytoplankton species analyzed, recovery of yield and oxygen production occurred in darkness in


contrast to higher plants, which require at least low irradiance with white light (Critchley, 1994). In order to explain the effect of UV radiation on photosynthesis, one must consider the optical properties and self-shading effects as indicated previously by Garcla-Pichel (1994).

Acknowledgements

This work was financed by the Spanish Ministry of Education and Science project AMB 94-0684 CO2 to F.L.F. and L.M.L, by the Acci6n Integrada Hispano-Alemana 133-B to F.L.F. and D.-P.H., by DADD (322-AI-e-dr) to D.P.-H. and by the European Union (Environment programme, ENV-5V-CT94-0425; DG XII) to D.-P.H.

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