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Journalof Applied Phycology 8: 131-137, 1996.

131
1996 Kluwer Academic Publishers. Printed in Belgium.

Effect of blue-green light on growth rate and chemical composition of three


diatoms
Maria del Pilar Sdnchez-Saavedral & Domenico Voltolina2
1Departamentode Acuicultura, Centro de Investigaci6n Cientifica y de Educaci6n Superiorde Ensenada,
ApartadoPostal 2732, Ensenada, 22800 Baja California, Mexico
2
Present address: UniversidadAuton6ma de Sinaloa, Facultadde Ciencias del Mar, Apartado Postal 610,
Mazatlan, 82000 Sinaloa, Mexico

Received 12 March 1996; revised 25 May 1996; accepted 28 May 1996

Key words: diatom cultures, light quality, irradiance, growth rate, chemical composition, Chaetoceros sp., Skelet-
onema costatum, Thalassiosirapseudonana

Abstract

The diatoms Chaetoceros sp., Skeletonema costatum and Thalassiosira pseudonana were grown with different
irradiances of white and of blue-green light, and with a mixture of blue-green plus 6.5 mol m -2 s- of white light.
Exponential growth rates were higher in mixed blue for the first two, while T pseudonanagrew faster in white light
but, in all cases, mean cell division rates did not differ with increasing irradiances. Harvesting in stationary, rather
than in late exponential growth phase, resulted in higher protein contents for Chaetoceros sp. and S. costatum, but
for T pseudonanathe highest value was in the exponential phase. The highest protein content was in blue-green
light for the three species and it increased with irradiance. As to other fractions, the three strains showed different
responses, related to light quality and quantity, as well as to culture ages.

Introduction However, only a few authors (Flaak & Epifanio,


1978; Snchez-Saavedra & Voltolina, 1994, 1995)
The culture of microalgae as food for the commercial have explored the possibility of obtaining live food
rearing of marine animals is of critical importance in of different qualities for filter feeders using large-scale
mariculture. Apart from the dietary requirements of cultures of one microalga under light with different
the organisms feeding on them, the nutritional value of spectral characteristics. However, the results obtained
microalgae depends on their biochemical composition in these previous works differed considerably, indicat-
(Brown et al., 1989), which may be manipulated by ing the possibility of a species-specific response to
modifying culture conditions. light quality. In the present paper we compare the
Light is the driving force of photosynthesis. As growth rates and the proximate composition of the
such, its quantity, its spectral composition and their same two diatoms (Thalassiosirapseudonana Hasle
fluctuations within the culture control biomass pro- and Heimdal and Chaetoceros sp., respectively) and
duction rates (Dubinsky et al., 1995) and may cause of a third one Skeletonema costatum (Greville) Cleve
variations in the photosynthetic responses, growth rates grown to their late exponential and early stationary
and cell metabolism of algae (Senger, 1987; Sukenik growth phases with different photon fluence rates of
et al., 1989). For instance, more amino acids and pro- white and of blue-green light.
tein may be found in algal biomass produced under
blue light than under equal levels of white light, while
red light has been shown to favour glycogenesis over
proteogenesis (Voskresenskaya, 1972).
132

Materials and methods 1.2 l i . .


l .
l . . .
I
l l . .
l .
l . . . . . . . . . . . . . .
...
I

1.0
The marine diatoms used for this work were the
clones CH-X-1 (Chaetoceros sp.), SK-C-2 (Skelet- 0.8
onema costatum = CCMP1332), and TH-P-1 (Thalas- r
siosirapseudonana = CCMP1335), of the Centro de Er 0.6
Investigaci6n Cientffica y de Educaci6n Superior de
Ensenada (C.I.C.E.S.E.) culture collection (Trujillo- 0.4
Valle, 1993). The first is a local strain, isolated in
coastal waters and used in several Mexican commer- 0.2
.. ..... ..............
cial hatcheries, the other two were obtained from -----
. . ------
.. ..... . ....::::.
Provasoli-Guillard Center for Culture Collection for V.LU .. . . . . . . .. . . . . .
I E.
I .
F .
I .
i .
I . . . I . . . . . .i-r I

300 400 500 600 700


Marine Phytoplankton (Andersen et al., 1991).
Wavelength
Non-axenic batch cultures of each strain were run
in triplicate, in 250 ml Erlenmeyer flasks with Guillard
2 . l . ll.. . *,I . . .. . . .rll l. ,,l ,, ,,,l
& Ryther's (1962) 'f' medium, prepared with sterile
I.
natural seawater (salinity = 33 1 g kg-'). Cultures
conditions were 23 1 C and pH between 7.5 and
1:3 o b
8.5. Stirring was manual, twice daily, and lighting was ' 0.1
continuous, provided by commercial fluorescent lamps
Sylvania Cool White F40CW or General Electric Blue- 0.
green F40B (peak of emission = 440 nm) with known 4
spectral characteristics (Figure 1) (Sdnchez-Saavedra TO. 4
& Voltolina, 1994). In the case of blue-green, one set of
cultures was isolated from ambient light and a second 0.;
one was allowed to receive 6.5 umol m- 2 s - I of the
1O _. . ., . .I . ,.. . I.........., . I
laboratory lighting, with the same spectral characterist- v
300 400 500 600 700
ics as the experimental white light. In this case, the dis-
Wavelength
tance of the cultures from the light sources was adjus-
ted so that the total irradiance, measured at the centre Figure 1. Spectral distribution (in relative units) of the three fluor-
of the culture vessel with a Biospherical Instrument escent light sources: --- blue light, - mixed blue light; ......
ambient light; b: white light.
QSL-100 quanta meter, was the same as those of white
and blue-green light. Growth experiments were run in
light gradients, from 100 to 547 /mol m - 2 s-l for both
types of blue-green, and from 100 to 718/ mol m-2 s- Proximate cell compositions were determined for
for white light, and were repeated at least three times each culture in late exponential and in stationary phase
for each value irradiance. of growth in three or four triplicate sets of samples for
The growth caracteristics obtained with these each type of analysis and determined independently
experiments for each strain and irradiance were used in each case: soluble protein content, using bovine
in separate sets of experiments, to obtain samples for albumin (98%) as standard, was determined according
proximate analysis during the late exponential and the to Lowry et al. (1951) and Malara & Charra (1972a)
stationary phases of growth, 24 h before and 24 h after after pretreatment for 10 min with 0.1 N NaOH at
reaching the maximum value of cell concentrations at 100 C for Chaetoceros sp. and at 80 C for S. cost-
100 and 400 iumol m- 2 s -1 of irradiance. atum and with 0.5 N NaOH at 80 C for 10 min for
Each set of cultures was started with inocula T pseudonana, adjusting the final pH value to <8.0
acclimated to the light condition tested, for at least (Firber-Lorda, 1986). Carbohydrates were measured
eight to ten generations. Increases in cell concentra- with the phenol-sulphuric method by Dubois et al.
tions were checked by daily measurements of optical (1956), using glucose (99%) as standard, as described
density at 550 nm and by occasional hemacytometer by Malara & Charra (1972b), pretreatment was as in
count. Mean cell division rates were calculated as in Whyte (1987). Lipids were extracted following Bligh
Fogg & Thake (1987), using log2 transformation. & Dyer (1959) and Chiaverini (1972) and quantified
133

with the colorimetric method by Pande et al. (1963) carbohydrates remained practically unchanged. The
using tripalmitin (99%) as standard. All calibrations same tendency was evident for S. costatum in the
curves were routinely checked once weekly with new case of lipids, although differences never reached the
standards. level of significance. On the other hand, carbohydrates
A two-way analysis of variance and the aposteriori decreased with irradiance in the case of cultures grow-
Student Newman Keuls (SNK) test (Sokal & Rohlf, ing exponentially in blue-green and mixed blue-green
1979), all for a = 0.05, were used to compare the light, but had an inverse tendency in white light.
effect of each variable. In the case of T pseudonana,lipids increased with
irradiance in white and mixed blue-green light for cul-
tures in exponential growth, but not in blue-green light
Results only. In the stationary phase there was a tendency to
lower carbohydrates with high light, although the dif-
In all cases, mean cell division rates, from inoculum to ference was not significant in most of the cases.
the end of exponential growth, did not differ signific- The response to light quality is complicated by the
antly with increasing irradiance. However, the three responses to age and irradiance already described. In
species showed different responses to light quality. general terms, short-wave radiation causes an increase
Chaetoceros sp. and S. costatum grew faster in mixed in proteins, but the presence of a small percentage of
blue-green than in blue-green light only, and the slow- white light seems to have an effect which is not appar-
est growth was in white light. This was, on the other ently commensurate to such a small amount of con-
end, the best growth-promoting type of light, followed tamination by other wavelengths. In all cases, cultures
by mixed blue, for T pseudonana.As to cell numbers, grown in mixed blue-green have a higher protein con-
Chaetocerossp. and T pseudonanahad the highest cell tent than those grown in other types of light of equal
densities in white light, followed by mixed blue-green, photon fluence rate, those in blue-green are second
while S. costatum showed an inverse trend, with the and white light-grown cultures have the lowest protein
highest and lowest cell concentrations in blue-green content.
and in white light, respectively (Tables 1 and 2). An even more striking effect of light type is the low
As to proximate composition, responses to light amount of carbohydrates of mixed blue-green cultures
quality and quantity, as well as to culture age, differed of Chaetoceros, which was between one third and 50%
for each species so that, for the sake of simplicity, the of the levels found with the other types of light.
effect of each variable, summarized in Tables 3 and 4,
will be described separately.
The effects of culture age on Chaetoceros sp. and Discussion
S. costatum was a consistent tendency, independent
from light quality and quantity, to higher protein and Our results seem to disagree on a number of points
lipid contents during the early stationary than in the from some of the common generalizations on microal-
exponential growth phase. Carbohydrates, as well as gae. For instance, the protein content is expected to
the ash content had an inverse trend. be higher during exponential, rather than in station-
On the other hand, upon reaching the stationary ary phase of growth. Two out of the three species we
phase, T: pseudonana showed a sharp decrease in tested did not fulfil this expectation, possibly due ot the
proteins, paralleled by equally sharp increases in all late conversion of their nitrogen products into soluble
other fractions in blue and mixed blue-green light. In protein (Fernandez-Refriz et al., 1989).
white light, only the ash content increased while car- Another common assumption is that, if the growth
bohydrates and lipids remained practically unchanged. rate of one microalga is not dependent from the exper-
As a general response to irradiance, protein con- imental variable, the biomass harvested will have a
tents increased with increasing photon flux density, but similar composition. Even if the yields and the growth
differences were significant only during exponential rates of our strains were found to be independent from
growth and simply indicative of this trend in the sta- irradiance for each type of light, we found signific-
tionary phase. As to other organic fractions, the three ant irradiance-related differences in chemical compos-
strains showed different responses, in some cases also ition, during both phases of growth.
related to light quality: Chaetocerossp., for instance, Evidently, this is due to changes in biochemical
showed an irradiance-related decrease in lipids while pathways induced by quantitative changes of the light
134
Table 1. Mean cell division rate () and standard deviation (in brackets) during exponential growth of three
diatoms cultured with different photon fluence rates of blue-green (A), blue-green plus 6.5 timol m- 2 s-l
of white (B) and white light (C). Equal letters indicate lack of significant differences (Two-way ANOVA and
Student-Newman-Keuls a posteriori test, a = 0.05): a<b<c.

Species Light Photon fluence rates (mol s-l m- 2 )


100 210 362 547 718

Chaetoceros sp. A 1.39(0.08)b 1.39(0.03)b 1.38(0.07)b 1.40(0.03)b -


B 2.17(0.02)c 2.15(0.04)c 1.96(0.05)c 2.63(0.07)c -
C 0.73(0.04)a 0.75(0.07)a 0.70(0.05)a 0.71(0.07)a 0.74(0.52)a

S. costatum A 1.02(0.01)b 1.03(0.01)b 1.03(0.01)b 1.03(0.01)b -


B 2.59(0.04)c 2.65(0.02)c 2.59(0.02)c 2.61(0.01)c -
C 0.95(0.03)a 1.15(0.06)b 0.97(0.06)b 0.97(0.04)a 1.07(0.04)b

T pseudonana A 1.95(0.04)b 1.83(0.03)a 1.94(0.03)b 1.96(0.03)b -


B 2.53(0.02)d 2.35(0.04)c 2.03(0.04)ab 2.27(0.04)c -
C 2.74(0.06)e 2.97(0.05)f 2.76(0.03)e 3.23(0.09)9 2.88(0.06)f

Table 2. Mean cell concentrations (cell 103 ml- 1) and standard deviation (in brackets) during exponential
growth of three diatoms cultured with different photon fluence rates of blue-green (A), blue-green plus
6.5 mol m- 2 s- of white (B) and white light (C). Equal letters indicate lack of significant differences
(Two-way ANOVA and Student-Newman-Keuls a posteriori test, a = 0.05): a<b<c.

Species Light Photon fluence rates (mol s-l m-2 )


100 210 362 547 718

Chaetocerossp. A 988(45)a 970(36)a 943(35)a 980(52)a -


B 1027(43)b 1129(26)b 1140(90)b 1040(52)b -
C 1340(29)c 1293(48)c 1322(53)c 1293(96)c 1323(90)c

S. costatum A 940(26)c 923(18)c 946(30)c 960(21)c -


B 790(45)b 810(25)b 797(36)b 820(42)b -
C 520(17)a 536(19)a 517(23)a 508(29)a 523(25)a

T pseudonana A 1248(96)a 1198(87)a 1240(83)a 1190(73)a -


B 1325(81)b 1420(76)b 1420(76)b 1340(36)b -
C 1536(99)c 1620(80)c 1551(67)c 1730(91)c 1630(86)c

environment, not unlike the one described by Helle- are due only to light quality, and that none was limited
bust (1970) for two marine coccolithophorids, whose in these aspects by the quantity of light. In two spe-
growth was saturated at light intensities far below those cies (Chaetoceros sp. and S. costatum) the response
necessary for maximum photosynthesis. was close to the expected since, as described by oth-
As to light quality, several reports in the literat- er authors (Hauschild et al., 1991; Ojala, 1993), blue
ure suggest that the response of photosynthetic organ- light caused an increase in growth rate but, in both
isms to blue light are by no means homogeneous and cases, only if this was enriched by a small amount of
that the expected higher growth rates, higher protein white. We found a different response to blue light for
and chlorophyll contents and increase of tricarboxilic T pseudonana, similar to that described by Flaak &
acid synthesis independently from the photosynthetic Epifanio (1978) for the same species, since its growth
activity is a composite picture, from which individual rate was higher in white than in blue light.
species may deviate in one or more aspects (Wallen & As to biochemical composition, blue light had the
Geen, 1971; Gostan et al., 1986). expected effect of higher protein contents for the three
In this work we found that the differences measured species, again made more evident by the mixture with
in growth rate and cell numbers for the three species white light and by an increase with increased irradiance
135

Table 3. Mean proximate composition (in percentage of the total dry weight) and standard deviation (in brackets)
during exponential growth of three diatoms cultured with different photon fluence rates of blue-green (A), blue-green
-2 -
plus 6.5 mol m s of white (B) and white light (C). Equal letters indicate lack of significant differences (Two-way
ANOVA and Student-Newman-Keuls aposterioHtest, a = 0.05): a<b<c.

Species Light /mol s- m- 2 Protein Lipids Carbohydrates Ash content

Chaetocerossp. A 100 33.26(1.49)b 20.12(2.16)d 15.26(0.56)c 26.13(2.01)a


400 42.26(1.16)c 10.15(0.41)a 16.26(0.76)c 23.16(2.01)a
B 100 38.12(1.29)b 24.12(0.87)f 7.23(0.89)a 30.41(3.06)a
400 51.26(2.15)d 16.44(0.37)c 6.43(0.39)a 26.33(1.22)a
C 100 30.15(1.46)a 22.41(0.49)e 12.23(0.76)b 26.84(1.36)a
400 36.18(1.37)b 12.44(0.62)b 13.29(0.47)b 23.22(1.44)a

S. costatum A 100 39.16(0.97)a 6.49(0.83)ab 19.21(0.77)d 39.36(1.67)a


400 43.16(0.87)c 5.43(0.46)a 17.21(0.69)c 38.44(1.39)a
B 100 42.17(0.94)c 7.43(0.92)ab 15.23(0.33)b 37.43(1.76)a
400 47.15(1.32)d 5.20(0.72)a 13.26(0.23)a 34.17(0.47)a
C 100 37.23(1.46)a 6.62(0.46)ab 21.73(0.49)e 38.43(1.69)a
400 39.26(0.87)b 4.17(0.37)a 24.33(0.71)f 36.11(1.72)a

T pseudonana A 100 44.12(0.69)c 15.33(0.72)b 12.33(0.43)c 22.47(1.99)a


400 48.27(0.33)e 17.27(0.69)d 11.28(0.23)b 24.72(2.06)a
B 100 46.47(0.47)d 13.23(0.47)a 10.11(0.62)a 26.41(2.31)a
400 52.21(1.69)f 16.44(0.33)c 12.44(0.39)c 23.12(1.76)a
C 100 31.27(0.42)a 20.12(0.12)e 13.62(0.42)d 21.33(1.49)a
400 36.47(1.33)b 20.41(0.36)e 15.23(0.26)e 20.88(1.20)a

Table 4. Mean proximate composition (in percentage of the total dry weight) and standard deviation (in brackets)
during stationary phase of growth of three diatoms cultured with different photon fluence rates of blue-green (A),
-2
blue-green plus 6.5 /mol m s- of white (B) and white light (C). Equal letters indicate lack of significant differences
(Two-way ANOVA and Student-Newman-Keuls aposterioritest, a = 0.05): a<b<c.

Species Light pmol s- m-2 Protein Lipids Carbohydrates Ash content

Chaetocerossp. A 100 37.13(1.22)b 21.46(1.16)d 12.29(0.55)c 21.12(1.41)a


400 45.26(0.96)c 12.13(1.11)a 13.33(0.67)c 19.47(2.47)a
B 100 41.33(0.99)b 25.37(0.96)f 5.33(0.96)a 26.11(1.13)a
400 53.46(1.21)d 17.61(0.68)c 4.37(0.93)a 22.17(1.72)a
C 100 33.29(0.97)a 23.17(0.99)e 10.29(0.62)b 24.29(1.62)a
400 39.18(0.86)b 14.14(0.84)b 11.43(0.72)b 20.17(1.17)a

S. costatum A 100 42.17(1.12)b 8.45(0.62)a 14.33(0.77)b 36.21(0.89)b


400 45.23(1.43)b 7.36(0.72)a 12.11(0.89)b 35.43(0.72)b
B 100 44.27(1.29)b 9.36(0.61)a 12.21(0.92)a 35.21(0.99)b
400 49.86(0.43)c 7.17(0.66)a 10.40(0.96)a 32.11(0.69)a
C 100 39.21(0.26)a 8.23(1.43)a 17.11(0.87)c 35.11(0.77)b
400 41.53(0.23)b 6.14(1.17)a 18.24(0.67)c 32.11(0.74)a

T pseudonana A 100 20.16(0.44)a 22.33(0.79)b 26.18(0.74)c 36.14(0.72)a


400 24.45(0.47)b 21.46(0.87)b 24.12(0.89)b 35.26(0.72)a
B 100 37.93(0.72)c 19.36(0.63)a 17.33(0.99)a 39.12(0.74)b
400 38.43(0.86)c 18.73(0.49)a 16.44(0.68)a 38.41 (0.82)b
C 100 18.61(1.12)a 20.12(0.72)b 16.93(0.77)a 38.10(0.96)b
400 19.61(1.17)a 22.21(0.86)b 15.76(0.74)a 38.44(0.94)b
136

levels. From these discrepancies between our results Dubinsky Z, Matsukawa K, Karube 1(1995) Photobiological aspects
and those of the general picture, it is clear that the blue of algal mass culture. J. Mar. Biotech. (in press).
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cobiliprotein/chlorophyll a fluorescence quotient of some species
of freshwater algae culture. Phycologia 32: 22-28.
To Centro de Investigaci6n Cientifica y de Educaci6n Pande SV, Khan RP, Venkitasubramanian TA (1963) Microdetermin-
Superior de Ensenada (C.I.C.E.S.E.), and Consejo ation of lipids and serum total fatty acid. Analyt. Biochem. 6:
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Nacional de Ciencia y Tecnologfa (Co.Na.C. y T.) for
Sanchez-Saavedra MP, Voltolina D (1994) The chemical composi-
financial support for this investigation. tion of Chaetocerossp. (Bacillariophyceae) under different light
conditions. Comp. Biochem. Physiol. 107B: 39-44.
Sinchez-Saavedra MP, Voltolina D (1995) The effect of different
light quality on the food value of the diatom Chaetoceros sp. for
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