influence of the combined effect of phosphorus and small scaleturbulence on the ecophysiology of ...

8/3/2019 Influence of the Combined Effect of Phosphorus and Small ScaleTurbulence on the Ecophysiology of Dunaliella viridis

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The chlorophyteDunaliella viridis was cultured under

four conditions resulting from the combination of high

and low turbulence supplied through non-manipulated

air bubbling system, and high and low phosphorus.

Under such conditions eleven variables were analyzed,

cell density, carrying capacity, growth rate,

nephelometry, pH, pigment composition, package

effect, free-phosphate concentration, photosynthesis

and respiration rates and P:B ratios. The results

showed that both phosphorus concentration and

turbulence level affects culture ecophysiology at all

levels. High turbulence, thought its effect on the

boundary layer, favored synergistically the positive

effect of high phosphate conditions on the population

dynamics. Moreover phosphorus level had a decisiveeffect on system productivity, reflecting its role as an

essential nutrient. The analysis of the effect of

phosphorus limitation on chlorophyll metabolism,

which was expected to affect both concentration of

macromolecules and the specific activities of enzymes,

yielded positive results that support this hypothesis.

Statistical analysis carried out on photosynthesis and

respiration rate values yielded striking result, since

turbulence, given its effect on the boundary layer and

mixing, was expected to have a positive influence on

the photosynthesis rate, but nevertheless, the obtainedresults seemed to indicate otherwise. In regard to the

respiration rates, the results indicate that the energetic

of phosphorus incorporation strongly affect cell

metabolism, causing increased respiratory rate in low

phosphorus cultures, in which phosphorus

incorporation is more costly energetically. Likewise, it

was confirmed the usefulness of nephelometry and

chlorophyll A as cell density estimators.

Keywords Dunaliella viridis Phosphate Turbulence

Pigments Cell density Respiration Photosynthesis

Introduction

Microalgae have considerable phenotypic plasticity,

allowing acclimation to wide variations in irradiance,nutrient supply and temperature, and although uncoupling

of light harvesting, carbon dioxide fixation and nutrient

acquisition allows metabolic flexibility in dynamic

physical/chemical environments, a balance must be

achieved over time scales of hours to days

(Geider et al., 1998).

In spite of its abundant in nature, phosphate is

frequently the limiting nutrient in many environments

since it is mainly found in forms not readily available

such insoluble salts, being an essential nutrient both as a

part of several key plant structure compounds and as acatalysis in the conversion of numerous key biochemical

reactions in plants. Increase in cell size, shape and cell

wall thickness and disorganization in the internal structure

are the dominant features of phosphorus starvation. In

addition, Theodorou et al. found that Selenastrum

minutum cultured under conditions of phosphorous

limitation resulted in noticeable decrease in rate of

respiration, rate of photosynthesis and significant increase

in activities phosphoenol pyruvate (PEP) carboxylase and

PEP phosphates (Said, 2009). Phosphate uptake and

storage has been studied extensively in fresh water and inmarine algae, and whose deprivation was found to induce

in algae a remarkable enhancement of phosphorus uptake

as well as enhanced extracellular phosphatase activity

(Shimogawara et al., 1999). Uptake of inorganic

phosphate was found to depend on Na+

in microalgae as

Dunaliella sp. (Ullrich and Glaser, 1982), indicating a

Na+-coupled symport mechanism. Likewise, Wikoff et al.

(1999) showed that phosphate limitation triggers a

starvation response, which promotes enhanced utilization

of phosphate from external and internal origins.

The dynamics of planktonic communities is also

markedly driven by the physical properties of the aquatic

ecosystems. Physical forcing not only shapes the structure

Influence of the Combined Effect of Phosphorus and Small Scale

Turbulence on the Ecophysiology ofDunaliella viridis

Cristbal Gallardo Alba*

Departamento de Ecologa, Facultad de Ciencias, Universidad de Mlaga, Campus Teatinos, E-29071, Spain

*Corresponding author, e-mail: [email protected]


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of the pelagic environment but also affects biological

processes in many direct and indirect ways, since

autotrophic organisms, for example, experience changes

in inorganic nutrients and light regimes which influence

their physiological process. Turbulence, which results

from (entropic) dissipation of mechanical energy that

works in a fluid, may exert direct effect of potential

ecophysiological importance through interaction with the

transport of molecules in and out the cells (Berdalet and

Estrada, 2006). As any solid surface submerged in water,

phytoplankton cells generate around them a layer of

reduced fluid movement or boundary layer, due to the

stress or drag effect exerted by solid. Viscosity causes the

average speed of the flow and the size of the turbulent

eddies to decrease from their values in the open water to

zero at the boundary, creating problems for organism

which require the transport of nutrients and metabolites to

and from the cells, including gas exchange. At the small

scale domain of the phytoplankton cells ( 1 mm), those

fluxes are controlled by random motion of molecules and

is much slower than turbulent diffusion over long

distances. Thus, if nutrients cannot be appropriately

supplied by molecular diffusion, the possibility of

diffusion limitation of nutrient uptake increases. The

thickness of a boundary layer is reduced in proportion to

the speed of the water moving past it. Relative motion of

cell and fluid, either originated by active swimming or

sinking the organism, or by motion of the fluid, will have

an effect on renewing the depleted zone (Berdanet andEstrada, 2006).

Dunaliella sp. is motile, unicellular, rod to avoid

shaped (9-11 m), green algae (Chlorophyceae) which

are common in marine waters, being the most important

phytoplanktonic organisms in hypersaline environments.

Dunaliella cells are enclosed by a thin elastic plasma

membrane covered by a mucous surface coat which

permits rapid changes in cell volume in response to

extracellular changes; in addition, -carotene is

accumulated by some species. Solute concentrations in

this salt-adapted unicellular green algae are important inrelation to its capacity for osmoregulation, having been

found large quantities of phosphate by chemical analysis

in this organism, and since the metabolic function of this

phosphate must depend on its state in the organisms,

different studies have been carried out in order to

characterize the phosphorus pool, allowing to identify

glycerolphosphoryglycerol (GPG) as the major

component, which likely is involved in the maintenance

of the high glycerol content ofDunaliella cells under

hypersaline conditions (Ginzburg et al., 1987).

The aim of this work was to study the effect ofphosphorus and turbulence on the ecophysiology of

Dunaliella viridis cultures by examining the population

dynamics and changes in pigment composition,

photosynthetic and respiratory alterations, free

phosphorus concentration, light extinction and package

effect. In addition, it was also contrasted the usefulness of

nephelometry and chlorophyll A concentration as cell

density estimators. The results showed that both

phosphorus concentration and turbulence level affects

culture ecophysiology at all levels, which could have

wide application in commercial farms.

Materials al methods

Plant material and experimental design

D. viridis Teodoresco was provided by the Department of

Ecology of the University of Malaga, maintained in

culture in the laboratory at 1,5 M NaCl, a photoperiod of

12 hours light/12 hours dark and 25C. For

experimentation, from the stock culture were isolated

eight cultures of 450 mL in Erlenmeyerflasks, which, in

duplicate, were subjected to the treatments: high

phosphate (P+) (200 M) and high air bubbling (T+) (1L

min-1

), high phosphate and low air bubbling (T-) (0.02 L

min-1

), low phosphate (P-) (50 M) and high air bubbling,

and finally low phosphate and low air bubbling,

maintaining the same conditions that in the original

culture. During the experiment the cultures were

maintained at a constant photon fluence rate (PFR)provided by fluorescent lamps (OSRAM); the light

intensity that each culture received was measured by a

spherical sensor at 5 cm depth of culture. All cultures

started at a cell density of 0,35 106

cells mL-1

, and the

measurements of the different variables were carried out

on the third, sixth, eleventh and twelfth day of treatment.

Cell density, carrying capacity and growth rate

The cell density of each culture was measured by cellcounting using the Neubauer hemacytometer and a Leica

CME microscope operated at a magnification of 200x, for

which was taken 1 mL culture and a drop of lugol (to fix

the cells). If the cultures were too dense, they were diluted

with culture medium. Finally, the average value of cells in

each square was multiplied by 160.000 and by the dilution

factor to obtain the cell density (n cells mL-1

).

The carrying capacity of each replica was obtained

from the maximum value of cell density, while that the

growth rates were estimated from the linearization of the

logistic growth equation:

Influence of the combined effect of phosphorus and small scale turbulence on the ecophysiology of Dunaliella viridis


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where r is the growth rate, t is the time (days), K is the

carrying capacity, N is the cell density at time t, and c is

the cell density when t = 0.

Nephelometry

The measurements were done using 1 mL of culture

(making the white with the free-cell culture medium), by

means of a Thermo Spectronic spectrophotometer. At the

same time were also measured the absorvances at 668 nm.

pH measurement

For pH measurements were used Crison pH 25 electrodes,

previously calibrated. It was introduced in each culture,

and the values were taken when the measures were

stabilized while the electrode was slowly shaken.

Pigment composition

For the determination of the pigment concentrations,

3 mL of each culture were taken at the third, sixth and

eleventh day after the beginning of the experiment and

filtered using GF/F filters (retaining the supernatant, since

later it will be necessary) . The filters were then extracted

in 90% acetone in falcon tubes of 15 mL for 10 minutes inthe dark, after which they were centrifuged for 3 minutes;

finally, using the supernatant, the concentrations of

chlorophyll A, chlorophyll B and total carotenoids were

determined using the spectrophotometric method and

equations recommended by Lichtenhaler (1987).

Production: biomass ratio

The system productivity (or production-to-biomass ratio)

was estimated based on cell densities and not on actualmeasurements of the culture masses. It was calculated as:

where B is the difference between the final and the

initial cell density, t is the time interval between

measurements (12 days) and B0 is the initial cell density.

Phosphate measurement

Phosphate concentrations were estimated using the

supernatants which resulted of the photosynthetic

pigments filtration. It required a calibration curve that

provided a reliable way to estimate the concentration from

spectrophotometric data. Thus 0, 5 mL of supernatant

from the filtrates of each culture were mixed with 1 mL of

malachite green reagent (according with the method

proposed by Fernndez et al., 1985), making two replicas.

After 5 minutes of incubation, the absorvance at 660 nm

of each replica was measured in a Jenway 6500

spectrophotometer, using as white 0, 5 mL of deionized

water with 1 mL of malachite green reagent. With regard

to the calibration curve, it was done with the

concentrations 2, 5 M, 5 M, 10 M, 15 M and 25 M.

In this case, it was not necessary to make dilutions. The

phosphate incorporation rate (PIR) was estimated as:

Photosynthesis and respiration rates

Net photosynthesis rates (NPR) and respiration rates (RR)

were estimated as O2 evolution rates. For each replica,

two flasks of 100 mL filled to the brim (covering with

parafilm, preventing air bubbles),were incubated, one ofthem in light (for photosynthesis),and the other one in

dark (for respiration), measuring the initial and final

oxygen in each incubation, for which was used a VWR

oxygen electrode. NPR and RR were estimated according

with the formula:

where is the change in oxygen concentration and t

is the incubation time.

Extinction coefficient

Vertical light extinction coefficient (Kd) was estimated

according to Beers Law:

where I0 is the incident light (E m-2 s-l), Iz is the light at

deep z and kd

is the vertical light extinction coefficient

(m-1).



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The light extinction coefficient depends on both the

physical structure of cells as the pigments; in the case of

the pigments, the relation can be estimated as:

where kd is the light extinction coefficient (m-1

), kd max is

the maximum extinction coefficient value (m-1

) , Km is the

pigment concentration corresponding to the half

maximum extinction value and C is the pigment

concentration .This relation was analyzed taking

ChlA, total carotenoids and total pigment concentrations.

Package effect

Package effect was spetrophotometrically estimated by

measuring the optical density at 668 nm of cultures (using

as white the measurement at 668 nm of the free-cell

culture medium) and using the absorvance at 664 nm of

the acetone extract (subtracting both the absorvance date

at 750 nm of the culture). Thus, the package effect was

estimated as:

PE = 1- (DO668 culture)/(DO664 acetone extract)

Statistical analysis

Two-ways analysis of variance (ANOVA) test,

considering turbulence and phosphorus levels, were

performed to evaluate significant differences between

treatments for carrying capacity, growth rate, pigment

composition, P/B ratio, photosynthesis and respiration

rates and phosphate incorporation rate. In addition,

photosynthesis and respiration rates were analyzed by

three-way ANOVA. When significant differences were

found, the Tukey method for multiple comparisons among

means was applied in order to identify differencesbetween treatments. Carrying capacities and growth rates

were also subjected to Kruskall-Wallis tests, since

homoscedasticity tests were no overcome .With the aim

of analyzing their utility as estimators of cell density,

were performed two regression analyses comparing the

cell density value against the nephelometry value and the

amount of chlorophyll per cell, and to test whether the

correlation values obtained were significant were used the

t-tests, and the slopes were compared by using the test for

comparison of slopes. Finally, Mann Whitney sum rank

tests were conducted in order to analyze significative

differences between treatments in cell density and free

phosphorous concentration.

All the statistical tests, except Kruskall-Wallis and

Mann Whitney tests (Statgraphics Centurion XVI version

16.1) were performed using Sigmaplot for Windows

version 11.00. In all cases the significance level was 0.05.

Results

Cell density, carrying capacity and growth rate

Cell density increased in the cultures for the four

treatments according to a sigmoidal trend, being strongest

the increase in high phosphate cultures ; furthermore, both

P+T+ as P-T- showed a decrease in cell density from the

day 11 (Fig.1); in the latter case, the cell density remained

almost constant throughout the experiment. The

maximum values were recorded in the P+T+ cultures(mean 48 mill. cell mL

-1to the eleventh day), while that

the minimum values whereas the minimum values were

measured in the P-T- cultures (mean 4 mill. cell mL-1 to

the twelfth day).

To test if significative differences exist between the

P+T- and P-T- cultures, a Mann Whitney sum rank test

was conducted, whose results indicated that the difference

in the mean values was greater than would be expected by

chance, that is, there were significative differences in celldensities between treatments for = 0,05 (Table 1).

Group N Missing Median 25% 75%

P-T- 8 0 4,6 4,01 5,3

P+T- 8 0 18,55 12,74 23,1

Mann-Whitney U Statistic= 0,000 (P = 0,001)

0

10

20

30

40

50

0 2 4 6 8 10 12

Time (days)

Cell

density(106

cellmL-1)

P+T+

P-T+P+T-

P-T-

Figure 1. Evolution of the mean cell density values in cultures over

time in response to treatments. Standard deviation as bars (n = 2).

Table 1. Results of Mann Whitney test on the mean cell density values of

high phosphorus and low turbulence cultures and low phosphorus and

turbulence cultures.

Influence of the combined effect of phosphorus and small scale turbulence on the ecophysiology ofDunaliella viridis


5/24

With the aim of determining if the obtained carryingcapacity and growth rate values (Table 4) showed

significant differences between treatments, the results

were subjected to statistical analysis.

Carrying capacity and growth rate values were

subjected to tests to check for homoscedasticity and

normality, not exceeding the homoscedasticity tests in

either case, so that, given the limited number of replicates,

were subjected to both parametric and nonparametric tests.

The ANOVA results for the carrying capacity are

detailed in table 2. The results showed that the effect of

different levels of phosphorus on the carrying capacity

depends on what level of turbulence is present because

there is statistically significant interaction betweenphosphorus and turbulence (P = 0,007). Thus, since the

results showed significant values for interaction, a

Tukey's multiple comparison test was applied (Table 5).

The results showed significative values in all cases, except

for the interaction between low phosphate and turbulence.

On the other hand, the results of the Kruskal-Wallis test

are shown in the table 6; in this case, the outcome of the

test did not shown significative differences between the

treatments for the carrying capacity, considering = 0,05.

The ANOVA results for the growth rate are showed inthe table 3, and the calculations are detailed in the annex.

In this case the test indicated that there wassignificative interaction between treatments (P = 0, 05).

Given the positive interaction, we conducted a Tukeys

test (Table S1), whose results indicated that there were

significant interaction in all cases, highlighting the

interacton between high phosphorus and turbulence levels

(P = 0,009), and between high turbulence and phosphate

levels (P = 0,013), showing a difference of means of

0.556 in the first case, and 0,497 in the second. For the

Kruskall-Wallis tests there were not a statistically

significant differences amongst the medians at the 95,0%

confidence level since the value of H is smaller than thevalue of

23, 0, 95 (Table 7).

Comparisons for factor: turbulence within P+

Comparison Diff of Means p q P

T+ vs, T- 23,05 2 11,23 0,002*

Comparisons for factor: turbulence within P-


T+ vs, T- 1,8 2 0,877 0,569

Comparisons for factor: phosphorus within T+


P+ vs, P- 40,85 2 19,893


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Treatment Ranks Ri Ri2/2

P+T+ 7 8 15 112,5

60,524,54,5

P+T- 5 6 11P-T+ 3 4 7P-T- 1 2 3

H= 6, 66; 23, 0, 95 = 7, 81 Ri2/2 = 202

Treatment Ranks Ri Ri2/2

P+T+ 7 6 13 84,54,5

24,584,5

P+T- 2 1 3P-T+ 3 4 7P-T- 5 8 13

H= 6; 23, 0, 95 = 7, 81 Ri2/2 = 198

Nephelometry

In order to check if the nephelometry is a good estimator of

cell density, the relation between both variables was

analyzed (Figure 2). Thus, firstly was performed an

ANOVA of regression, in order to check statistically

whether the variability of nephelometry values could be

explained by cell density variability (Table 8). In addition,

to confirm the dependence between both variables, the

Pearsons correlation coefficients obtained were subjected

to Students t-tests, considering the correlation of both the

independent treatments and the data as a whole (Table 9).

The results showed that in all cases, except for low

phosphorus and low turbulence treatment, the correlation

coefficient values were significant, that is, there is linear

correlation for = 0,05.It is noteworthy that the maximum

correlation coefficient value was obtained when the total

data were consider.

Critical value of F = 3, 3059E-15

Regression coefficient () values were also deeply

analized. On one hand, it was contrasted if the regression

coefficient values obtained were significant, for which itwas used Students test (Table 10). As in the case of

correlation coefficients, in all cases, except for the

treatments of low phosphate and low turbulence (P-T-),

the obtained values for were significant. On the other

hand, to check whether there were differences between

regression coefficients, a comparison of slopes test was

conducted, according with Sokal & Rolf (Table 11).Only

two combinations of slopes were no significant, high

phosphorus and high turbulence against low phosphorus

and low turbulence, and low phosphorus and high

turbulence against high phosphorus and low turbulence.

Treatment SCI Sb ts t criticalP+T+ 0,028 0,198 0,000 183,458* 2,447P-T+ 0,067 0,017 0,004 17,001* 2,447P+T- 0,052 0,154 0,001 102,836* 2,447P-T- 0,012 0,013 0,010 1,247 2,447

Total 0,028 0,705 2,43E-8 1160,5 * 2,042

Combinations S2xy Fs Fcritical

P+T+ x P-T+ 0,016 25,007* 4,747

P+T+ x P+T- 0,002 43,053* 4,747

P+T+ x P-T- 0,016 2,099 4,747

P-T+ x P+T- -0,013 3,712 4,747

P-T+ x P-T- -0,001 101,278* 4,747

P+T- x P-T- 0,013 15,626* 4,747

DF SS MS FRegression 1 5,045 5,045 214,487*Residues 30 0,705 0,023

Total 31 5,750

Treatment r Sr Tr Tcritical

P+T+ 0,907 0,172 5,279* 2,477

P-T+ 0,884 0,191 4,626* 2,477

P+T- 0,921 0,159 5,801* 2,477

P-T- 0,229 0,397 0,577 2,477

Total 0,937 0,064 14,64* 2,042

Table 6. Results of Kruskall Wallis test performed on carrying capacity

values.

Table 7. Results of Kruskall Wallis test performed on growth rate values.

y = 0,0283x + 0,1664

R2

= 0,8773

0

0,4

0,8

1,2

1,6

0 10 20 30 40 50 60

Mill. cell mL-1

Ab

sorvance750nm

Figure 2. Plot of cell density, measured as millions of cells per

milliliter of culture, vs. absorbance at 750 nm.

Table 8. Results of ANOVA of regression test.

Table 9. Students t test results to contrast the significance of the

correlation coefficient values.

Table 10. Results of Students t test for regression coefficient values

of both individual treatments and the data as a whole. Significant

values are marked with an asterisk.

Table 11. Results of the comparisons test of regression coefficients.

S2xy is the weighted average of each two groups. Significant

values are marked with an asterisk.



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pH measurements

Figure 3 shows the data obtained from pH measurementsfor each treatment at different times, ranging from 8,91 to

9,88. It should be noted that, in all cases except for high

phosphate and high turbulence treatments, the pH values

showed an upward trend throughout the experiment. To test

the existence of significant differences in pH values on the

eleventh day between treatments, an ANOVA contrast was

performed (Table 12) .According with the analysis, the

difference in the mean values among the different levels of

turbulence was greater than would be expected by chance,

reporting statistically significant differences

(P = 0,006) ,while the difference in the mean values amongthe different levels of phosphorus were not great enough to

exclude the possibility that the difference is just due to

random sampling variability. Furthermore, the results also

indicated pH values recorded at different levels of

phosphorus did not depend on what level of turbulence is

present.

Since the removal of CO2 by photosynthesis reduces the

acidity of the water, increasing the pH, and the respiration

produces CO2, which dissolves in water as carbonic acid,

thereby lowering the pH, the correlations between pH

values, photosynthesis rates and respiration rates were

analyzed. The results showed a significative Pearsons

correlation coefficient, r = -0,411, between pH and

respiration rates. (P = 0, 0458).

Pigment composition

The evolution of the pigment composition over time in

response to different treatments is reflected in figure 4. In

the case of chlorophylls, both chlorophyll A (ChlA) and

chlorophyll B (ChlB) exhibited similar patterns

throughout the experiment, showing significative

differences between treatments and interaction, according

with ANOVA analysis (Table S2 and S3). Thus, in the

case of high phosphorus and high turbulence cultures,

chlorophylls increased over time. In the case of the

cultures of high turbulence and low phosphate, it was

detected a decrease in chlorophylls levels between the

third and sixth day, and an increase between the sixth and

the eleventh day.

8,00

8,50

9,00

9,50

10,00

10,50

P+T+ P-T+ P+T- P-T-

pH

0

5

10

15

1 2 3 4

ChlorophyllA(gmL-1)

0

2

4

6

8

P+T+ P-T+ P+T- P-T-

Chloroph

yllB(gmL-1)

0

0,7

1,4

2,1

2,8

3,5

P+T+ P-T+ P+T- P-T-

Carotenoids(gmL-1)

Source of Variation DF SS MS F P-value

Phosphorus 1 0,049 0,049 4,967 0,09

Turbulence 1 0,285 0,285 28,537 0,006*Phosphorus x Turbulence 1 0,067 0,066 6,67 0,06

Residual 4 0,040 0,009

Total 7 0,441 0,063

Figure 3. Mean values of pH recorded for the different treatments at

third day, sixth day and eleventh day. Symbols as in figure 1. Standard

deviation as bars (n = 2).

Table 12. Two-way ANOVA performed on pH values of cultures under different treatments. Significant values are marked with an asterisk (P < 0,05).

Figure 4. Average of pigment concentrations recorded over timein different treatments. Figure 4A shows the chlorophyll A

concentrations, figure 4B the chlorophyll B concentrations, and

figure 4C the total carotenoids concentrations. The colors

correspond to the third day (black), sixth day (white) and

eleventh day (gray). Standard deviation as bars (n=2).

DF: degree of freedom SS: sum of squares MS: mean square F: F static


A

B

C

8,80

9,00

9,20

9,40

9,60

9,80

10,00

3 5 7 9 11

Time (days)

pH


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Finally, the two remaining cultures showed a similar

trend, recording the highest values on the sixth day. For the

carotenoids, all treatments showed a similar pattern,

increasing the concentrations over time, although less

intensely in low phosphorus and low turbulence cultures.

ANOVA analysis indicated significant differences between

phosphate levels (P = 0, 03).

To test the existence of significant differences between

the quantities of each pigment per cell, five two-way

ANOVA tests were performed for different times, three ofthem for chlorophyll A (Tables 13-14 and Table S4), and

the remaining two for carotenoids (Table 15 and Table S5).

Since the concentrations of chlorophyll A were practically

identical to chlorophyll B, the obtained results could be

applied to both. ANOVA tests for chlorophyll indicated

significant differences between the phosphorous levels on

the third day (P = 0,017), being on average 53% higher in

high phosphate cultures, and between the turbulence levels

on the sixth day (P = 0,008), being on average 70% higher

in high turbulence cultures. In the case of total

carotenoids per cell, ANOVA analysis

showed significant differences between phosphorous

levels in all cases, being approximately 10 times higher in

low phosphate cultures. It was also analyzed the possible

correlation between the pigments levels with other

variables through multivariable correlation analysis

(Table 16). The results showed significant positive

correlation both between chlorophylls with the

phosphorus concentration of the medium and with

photosynthesis rates. Moreover, carotenoids showed

significative correlation with cell density and respiration.

In addition, to test if the correlation between the

carotenoids and cell density kept been significant in the

treatments separately, correlation analysis were performed

for each of them, although in this case any of the obtained

values was significant (Table 17).

Another objective was to analyze the potential

usefulness of chlorophyll A as estimator of biomass, for

which we studied the correlation between chlorophyll A

concentration (g. mL-1

) and cell density, obtaining a

Pearson correlation coefficient of 0,9093 (Figure 5),

which turned out to be significant after being subjected to

a Students t test (P = 7,3E-10).

Source of Variation DF SS MS F P

Phosphorus 1 0,068 0,0,069 15,671 0,017*Turbulence 1 0,008 0,007 1,768 0,254Phosphorus x Turbulence 1 0,0173 0,0173 3,929 0,119Residual 4 0,0176 0,0044

Total 7 0,111 0,0159


Phosphorus 1 0,0122 0,0122 4,287 0,107Turbulence 1 0,0570 0,0570 20,012 0,011*

Phosphorus x Turbulence 1 0,00012 0,00012 0,0433 0,845Residual 4 0,00285 0,00285Total 7 0,0807 0,0115


Phosphorus 1 0,018 0,018 26,481 0,007*Turbulence 1 0,000399 0,000399 0,586 0,487

Phosphorus x Turbulence 1 0,00515 0,00515 7,56 0,051Residual 4 0,00272 0,000681Total 7 0,0263 0,00376

Table 13. Results of two-way ANOVA performed on concentrations of chlorophyll A per cell values of cultures under different treatments on

the third da . Si nificant values are marked with an asterisk.


Table 14. Results of two-way ANOVA performed on concentrations of chlorophyll A per cell values of cultures under different treatments on

the sixth da . Si nificant values are marked with an asterisk.


Table 15. Results of two-way ANOVA performed on concentrations of total carotenoids per cell values of cultures under different treatments

on the eleventh day. Significant values are marked with an asterisk.




9/24

ANOVA of regression, conducted in order to contrast

its significance (Table 18), resulted to be significant for

= 0, 05, that is, the variability of chlorophyll A values

could be explained by cell density variability. In addition,

it was also analyzed the regression coefficient obtained,

( = 3,383), which proved to be significant, obtaining a

ts value of 9832, 2 (tcritical = 2,073). On the other hand it

was also studied the relation between accessory pigments

and chlorophyll A (Figure 6).

For all the treatments, except for cultures of high

phosphorus and high turbulence, the ratio betweenaccessory pigments and chlorophyll A showed an

increasing trend, being more pronounced in the low

phosphorus and high turbulence cultures, while that in the

low phosphorus and low turbulence the increase was

practically constant. However, the cultures of high

phosphorus and high turbulence displayed an increase

between the third and the sixth day, and a decrease

between the sixth and the eleventh day (Figure 6). To test

whether there were significant differences on the eleventh

day, an ANOVA was conducted (Table 19), whose results

showed that there were interactions between factors, thatis, the effect of different levels of phosphorus depended

on what level of turbulence was present. Finally, to find

which means are significantly different from one another,

a Tukeys test was conducted, whose results, detailed in

table 21 ,indicated that no interaction were detected only

for low turbulence.

Production: biomass ratio

The P/B ratio results, ranged from 0,869 to 8,678 d -1,

showed higher values in the cultures exposed to high

phosphorus treatments; likewise, those cultures exposed

y = 3,383x - 0,9832

R = 0,9093

0

20

40

60

0 5 10 15

Millions of cells mL-1

ChlorophyllA(gmL-1 )

Photos. rate Resp. rate Cell density Free phosphorus

Chlorophyll A r = 0,7019 r = -0,1396 r = -0,244 r = 0,7439

(g. mL-1) N = 24 N = 24 N = 24 N = 24P = 0,0001* P = 0,5152 P = 0,2505 P = 0,000*

Chlorophyll B r = 0,4646 r = -0,2694 r = 0,0555 r = 0,6861

(g. mL-1

) N = 24 N = 24 N = 24 N = 24P = 0,0222* P = 0,2031 P = 0,7966 P = 0,0002*

Carotenoids r = 0,1571 r = 0,4681 r = -0,8573 r = -0,1915

(g. mL-1

) N = 24 N = 24 N = 24 N = 24P = 0,4634 P = 0,0211* P = 0,000* P = 0,37

Treatment r P-value

P+T+ 0,256 0,744P-T+ 0,137 0,863P+T- -0,889 0,111P-T- -0,132 0,868

DF SS MS F

Regression 1 286,46 286,46 105,65*

Residues 22 59,65 2,71

Total 23 346,11

Table 16. Results of multivariable correlation analysis conducted on chlorophylls and total carotenoids concentrations, photosynthesis and

respiration rates, cell densities and free phosphate concentration. Significant values are marked with an asterisk (P < 0, 05).

Table 17. . Results of the correlation analysis conducted between the

total carotenoids concentrations and the respiration rates for eachtreatment.

Figure 5. Regression line between the absorbance at Chlorophyll A

concentration and cell density, measured as millions of cells per

milliliter of culture.

r: Pearsons correlation coefficient

Table 18. . Results of the ANOVA of regression conducted on variables

cell density (independent) and chlorophyll concentration (dependent).Significant value is marked with an asterisk.

Critical value of F = 7.3E-24



10/24

to high turbulence showed higher values than the low

turbulence cultures (Figure 6).To test whether the

observed differences were statistically significant were

analyzed by ADEVA (Table 20). According with the

test, main effects cannot be properly interpreted because

significant interaction is determined, that is, the effect of

different levels of phosphorus depends on what level of

turbulence is present. Since there is a statistically

significant interaction between phosphorus and

turbulence (P = 0,048), a Tukeys test was conducted

(Table 23).

0

2

4

6

8

10

P+T+ P-T+ P+T- P-T-

P/Br

atio

(day

-1)


Phosphorus 1 0,122 0,122 51,1 0,002*

Turbulence 1 0,00468 0,00468 1,952 0,235Phosphorus x turbulence 1 0,174 0,174 72,866 0,001*Residual 4 0,00958 0,00239Total 7 0,311 0,0444


Phosphorus 1 72,573 72,573 269,938


11/24

Phosphate measurement

Phosphorus levels recorded at different times are detailed

in table 24. The results indicated a drastically decrease

between the days 0 and 3, remaining almost constant

during the rest of the experiment (except for the P+T-

treatment). In order to analyze whether the turbulence

level significantly affected the initial decrease of the

phosphate from the medium, these differences were

contrasted by Mann-Whitney Rank Sum test. (Table 25, A

and B). Neither in the case of high phosphate nor low

phosphorus cultures the differences in the mean values

between the two groups were not great enough to exclude

the possibility that the difference were due

to random sampling variability, namely, there are not

statistically significant differences.

On the other hand, phosphorous incorporation rates

(PIR) were estimated from the average between the final

and initial cell density values (Figure 8). The highest

incorporation rates corresponded to P+T- cultures (mean

= 13.3), while at the opposite circumstance was the P-T+

cultures (mean = 8, 12). To test whether the differences

were significant was conducted a Kruskal-Wallis test,

since was not passed the normality test. (Table 26). The

results indicated not significant differences between

treatments (P = 0,08331).

Comparisons for factor: Turbulence within P+


T+ vs. T- 2,821 2 7,695 0,006*

Comparisons for factor: Turbulence within P-


T+ vs. T- 0,75 2 2,046 0,222

Comparisons for factor: Phosphorus within T+

Comparison Diff of Means p q PP+ vs. P- 7,06 2 19,255


12/24

Treatment Sample Size Average Rank

P+T+ 2 3,5

P-T+ 2 1,5

P+T- 2 7,5

P-T- 2 5,5

Test statistic =6,666 P-Value = 0,083314

Photosynthesis and respiration rates

Photosynthesis and respiration rates (measured as O2

evolution) showed very different patterns. In the case of

the photosynthesis rate (Fig.8), the highest values were

reached on the third day in high phosphorus cultures,

with 0,049 fmol cell-1

h-1

for the high turbulence

cultures and 0,066 fmol cell-1 h-1 for the low turbulence

cultures. It should also be noted that both high

phosphate and high turbulence cultures such as low

phosphate and high turbulence cultures showed a

downward pattern throughout the experiment. In the

other treatments, photosynthesis rate showed an inverse

behavior to each other over time, reaching on the sixth

day the minimum value in the high phosphorus and low

turbulence cultures, and the maximum value in the low

phosphorus and low turbulence cultures. To test whether

the observed differences were significant, a three way

ANOVA was conducted, although this did not pass the

normality and homoscedasticity tests. (Table 27).

The results showed statistically significant

differences in the mean values among the different

levels of turbulence (P = 0,014), and time (P = 0,002).

Likewise, it also indicate that the effect of different

levels of time depends on what level of phosphorus is

present, i.e. there is a statistically significant interaction

between time and phosphorus (P = 0,019). The results

of Tukeys test are shown in table 28. We also

conducted a multiple correlation study with the aim to

find possible correlations between the photosynthesis

rate and other variables (Table 29). Two significant

correlations were found: with the phosphorus

concentration of the medium and with the amount of

chlorophyll per cell.

Figure 10 represents the respiration rate results

obtained for the different treatments. As can be seen, the

low phosphorus and high turbulence cultures showed

respiration rates higher than the other treatments, reaching

the maximum on the third day, while it remained almost

constant between the sixth and the eleventh day. The high

phosphorus and high turbulence cultures were those that

showed lower values, decreasing along the experiment. A

similar trend showed the low phosphorus and low

cultures, while in the case of high phosphorus and low

turbulence cultures, the highest values were observed on

the third day. With the aim of determining if there are

differences between treatments was performed a three

way ANOVA, but it did not passed the normality and

homoscedasticity tests (Table 30). In addition, two two-

way ANOVA were performed on the values of the thirdand eleventh day (Table S6S8).

0

2

4

6

8

10

12

14

P+T+ P-T+ P+T- P-T-

PIR

104(pmold-1

cell-1)

0

0,02

0,04

0,06

0,08

0,1

P+T+ P-T+ P+T- P-T-

Photosynthesisrate

(fmolcell-1h

-1)


P+T+ 2 0 5,812 1,88 9,75P+T- 2 0 19,332 18,01 20,65



P-T+ 2 0 0,83 0,83 0,83

P-T- 2 0 0,48 0,39 0,58


Table 25. Results Mann-Whitney Rank Sum test performed on phosphorous concentration values on the third day.

A) High phosphorus comparisons B) Low phosphorus comparisons

Figure 8. Phosphorous incorporation rates (PIR) of the different

treatments. The values are displayed multiplied by 103.

Table 26. Results of Kruskall Wallis test performed on phosphorous

incorporation rates.

Figure 9. Mean values of net photosynthesis rates of the different

treatments recorded at third day, sixth day and eleventh day. Symbols

as in figure 1. Standard deviation as bars (n = 2).


0

0,02

0,04

0,06

0,08

3 5 7 9 11

Time (days)

Photosynthesisrate

(fmolcell-1

h-1)

0

2

4

6

8

10

12

14

16

P+T+ P-T+ P+T- P-T-

PIR

103(pmold-1

cell-1)


13/24


Time 2 0,00319 0,0016 11,484 0,002*Phosphorus 1 0,000438 0,000438 3,15 0,101

Turbulence 1 0,00114 0,00114 8,192 0,014*

Day x Phosphorus 2 0,00155 0,000774 5,571 0,019*Day x Turbulence 2 0,000107 0,0000534 0,384 0,689Phosphorus x Turbulence 1 0,000425 0,000425 3,059 0,106

Day x Phosphorus x Turbulence 2 0,000412 0,000206 1,483 0,266Residual 12 0,00167 0,000139Total 23 0,00892 0,000388

A) Comparisons for factor: day within P+

Comparison Diff of Means t Unadjusted P Critical Level Significant?3 vs. 6 0,0426 5,116


14/24

y = 0,076x + 0,0138

0

0,03

0,06

0,09

0,12

0 0,2 0,4 0,6 0,8 1

1/Chlorophyll A (g

-1

mL)

1/kd(cm)

For the three-way ANOVA, the results showed that

in all case, except for the triple interaction between

phosphorus, turbulence and time, the differences were

significant. Meanwhile, the two-way ANOVA for the

third day values indicated the existence of statistically

significant differences for both phosphorus (obtaining a

mean value 7,8 times higher in low phosphorus

treatments) and turbulence jjjjjjjjjjjjjjjjjjjjjjjjk

kkk

0 5 10 150

20

40

60

Chlorophyll A ( gmL-1

)

kd(cm-1)

A

levels (being the mean value 2,75 times higher in high

turbulence treatments), while the eleventh day values

showed significant interaction between treatments.

Finally, it was also analyzed the correlation between

respiration rate, net photosynthesis rate, phosphorous

concentration of the medium, chlorophyll A concentration

and cell density (Table 26). Respiration rate showed a

significant Persons correlation value with cell density

and amount of chlorophyll A per cell.

Extinction coefficient

kd values estimated from the Lamber-Beer Law ranged

from 9,08 to 56,60, registering the lower in low

phosphorus and high turbulence cultures, and the higher

values in high phosphorus and high turbulence cultures.

As expected, kd and chlorophyll A concentration

presented an asymptotic relationship (Figure 9A). It was

also analyzed the relationship between kd and total

pigment concentration, since accessory pigments also

participate in the absorption of light, showing the same

pattern (Figure 9B).

0 5 10 15 20 250

20

40

60

Total pigments ( gmL-1

)

kd(cm-1)

B

0

0,03

0,06

0,09

0,12

P+T- P-T+ P+T- P-T-

Respirationrate

(fmolcell

-1h

-1)

Photosynthesis Cell density Phosphorous pg. ChlA cell

Respiration r = 0,0191 r = -0,4581 r = -0,2887 r = -0,1396N = 24 N = 24 N = 24 N = 24P = 0,9295 P = 0,0244 P = 0,1712 P = 0,5152

Figure 10. Mean values of respiration rates of the different

treatments recorded at third day, sixth day and eleventh day.

Symbols as in figure 1. Standard deviation as bars (n = 2).

Table 31. Results of multivariable correlation analysis conducted on net photosynthesis rate, respiration rate, cell density, free phosphorous

concentration and amount of chlorophyll per cell (pg. cell -1 ).

y = 0,1436x + 0,0131

0

0,03

0,06

0,09

0,12

0 0,1 0,2 0,3 0,4 0,5 0,6

1/Total pigments (g-1

mL)

1/kd(cm)

C D

Figure 11. (A) kd (m-1

) values against chlorophyll A concentration ( g mL-1

) obtained during the experiment in the different treatments. (B) kd

(m-1

) values against total pigment concentration ( g mL-1

) obtained during the experiment in the different treatments. (C) and (D) figures


0

0,02

0,04

0,06

0,08

0,1

3 5 7 9 11

Time (days)

Respirationrate

(fmolcell-

1

h-1)

(m-1)

(m-1)

(m)

(

)


15/24

In order to estimate the parameters kdmax and km, we

carried out a Lineweaver-Burk double reciprocal plot,

reorganizing the original equation (Figure 9 C and D). For

the km, the values were 5,507 in the case of chlorophyll A,

and 10.96 in the case of total pigments. On the other hand,

the kdmax

values were 72,464 for chlorophyll and 76,33 in

the case of total pigments. Finally, with the aim to analyze

the influence of carotenoids on the light absorption, we

estimated the value of kdmax from the relation between the

total carotenoids concentrations and kd values, obtaining a

value of 169,49 (data not shown).

Package effect

Figure 12 shows the package effect values estimated

during the experiments in the different treatments. It

should be noted that only low turbulence cultures showed

a common trend between the treatments, recording the

maximum value on the sixth day. Meanwhile, the low

phosphorus and high turbulence cultures showed an

inverse trend, in which the lower value corresponds to the

sixth day. It was also examined through multivariable

correlation analysis the possible connection with other

variables, whose results indicated a significant negative

correlation of r = -0,5128 (P = 0,0104) with cell density.

Discussion

It has long been analyzed the influence that phosphorus

and turbulence levels have over physiology and ecology

of algae communities, and concretely on the halophilic

algaDunaliella viridis, although in all cases, both factors

have been independently analyzed. In this study we have

analyzed the effect of both variables together on theecophysiology ofD.viridis. The growth dynamics of algal

cultures showed similar patterns throughout the

experiment, at least until the eleventh day, increasing cell

density over time according to a sigmoidal trend, with the

strongest increase in the high phosphate cultures.

However, while that from the eleventh day the P+T+

cultures and P-T- cultures entered in the death or crash

phase, result of the deteriorate in water quality and

consumption of nutrients to levels incapable of sustaining

growth, the P-T+ and P+T- cultures showed a slight

increase. These results indicate that the high turbulence,

through its effect on the boundary layer, was able to

increase the available phosphorus pool in the medium to

be incorporated by cells, which determined that the P-T+

and P+T-showed a very similar trend over time despite

differences in initial phosphorus concentration. With

respect to phosphorus , its importance as limiting nutrient

seems to be confirmed by the significative differences

obtained when we analyzed through the Mann Whitneys

test the cell density values recorded in the P+T- and P-T-

treatments (Table 1).

The fact that phytoplankton growth may be limited by

one of several nutrients in a manner that has been related

to cellular stoichiometric ratios (Hecky and Kilham,

1988), although in the concrete case of freshwater

systems, the increase in green algae biomass with total

phosphorus is slow, which suggest that other factors may

be limiting their biomass (Watson et al., 1997). These

data support the obtained results for cell densities in

cultures subjected to different turbulence levels, which,although showed a sigmoidal increase along the time,

analysis revealed that the effect of different levels of

phosphorus on carrying capacity depended on what level

of turbulence was present. From these results we can infer

that, although the phosphorus was the factor which had

more influence, since that in low phosphorus cultures did

not exist significant differences between turbulence

levels, the latter had great influence on cell density. These

results fits with the findings of Aguilera et al. (1994),

although were not supported by Kruskal-Wallis test

(Table 6). The effect of the small-scale turbulence isbecause it allows overcome diffusible transport

limitations, since, at the small scale domain of the

Dunaliella cells (< 1 mm), nutrient and gas fluxes are

controlled by molecular diffusion. Thus, in absence of

turbulence, metabolites cannot be appropriately

transported to and from the cells by molecular diffusion,

so that, aeration, below certain threshold, improves gas

exchange through the water-air interference (involving pH

effects), nutrient transport regimes and light availability

(Aguilera et al., 1994, Jimenez et al., 1996). However,

inhibition has also been observed in different species atdiverse turbulence levels (Garca-Gonzlez et al., 2003).

0

0,4

0,8

1,2

1,6

2

P+T+ P-T+ P+T- P-T-

Packageeffect


0

0,3

0,6

0,9

1,2

1,5

1,8

3 4 5 6 7 8 9 10 11

Time (days)

Package

effect

Figure 12. Mean values of package effect values of the different

treatments throughout the experiment. Symbols as in figure 1.

Standard deviation as bars (n = 2).


16/24

The significant interaction identified between the

treatments on the growth rate by the ANOVA test

(Table 3) highlighted the synergic influence exerted by

the high turbulence over the positive effect of higher

availability of phosphorus. According with Gordillo et al.

(2001) this interaction could be partially determined by

the effect of a better mix of cultures, allowing more

efficient use of light, being supported this hypothesis by

the significant differences found between the turbulence

levels both for high and low phosphate in the Tukeys

test. In addition, another factor that may be involved in

the interaction is the effect of turbulence on the boundary

layer. Either way, the results seemed to support the

synergic positive effect that the high turbulence exerts on

the phosphorus availability, at least in the high

phosphorus cultures. However, further studies are needed

in order to confirm the role of the interaction between

phosphorus and turbulence on the growth rate, since the

results indicated that turbulence had a negative influence

on the low phosphorus cultures. Furthermore, the

performed Kruskall-Wallis test indicated that there were

not statistically significant differences between

treatments.

The results of statistical test yielded very promising

results to confirm the usefulness of nephelometry as cell

density estimator for both individuals and overall

treatments, except for the low phosphorus and low

turbulence cultures, in which there were not statisticallysignificance neither in the case of the correlation

coefficient nor regression coefficient. Therefore, the data

suggest that in very oligotrophic environments

nephelometry is not the most appropriate cell density

estimator, so that in these circumstances should be

resorted to other methods such as extract-fluorometry.

Likewise, the significant differences found in the

comparisons test of regression coefficients between

P+T+, P+T- and P-T+ seem indicate that the estimates

obtained by nephelometry should be contrasted with other

methods, because it may sometimes overestimate orunderestimate cell densities.

.

In a closed system, changes in pH and alkalinity are

the results of net fluxes of H+

across the plasma

membrane as well as influx of different dissociation states

of weak electrolytes such as NH4+/NH3. Thus, pH

dynamics is affected by many factor; for example, the fact

that cell density increases over time would imply that the

medium becomes more alkaline because there is a net

increase in CO2 consumption as a result of its fixation in

photosynthesis, resulting in a shift in carbonic carbonatebalance, and indeed, this was the observed behavior in

three of the treatments. This trend was most evident in the

low phosphorus cultures, which may be due to cell

density in the high phosphorus cultures reaches values so

high that the light that excites the photosynthetic

apparatus of most cells is insufficient, affecting carbon

fixation, since both processes are tightly coupled, which

could contribute to decrease pH in P+T+ cultures and to

stabilize in P+T-cultures. The differences in respiration

rates between the treatments during the experiment also

appear to be directly related to the observed variation in

pH levels, as a result of the release of CO2, according

with the correlation analysis. On the other hand, the

ANOVA results (Table 12) indicated that there were

significant differences between the turbulence levels,

which fit with previous data indicating that higher

aeration tends to decrease the pH through the buffer

system H2CO3-CO2 (Fbregas et al., 1993), which could

partially explain that high turbulence cultures showed

lower final pH values. The effect of turbulence may also

be influenced by the combined effect of the positive

influence of more intense mixing on the light received,

and the increase in the gas exchange.

Concentration of macromolecules and the specific

activities of enzymes and ribosomes are affected by

phosphorus limitation, although, by comparison with

other nutrients as nitrogen, there is relatively little

information on the response of microalgae photosynthetic

apparatus to phosphorus limitation (Geider et al., 1998).

These data appear to be supported by observations, since,although initially the expected results were that there were

a directly proportional relation between the chlorophyll

concentration and cellular density, at least until the

eleventh day, only the P+T+ cultures showed this trend,

which may be result of phosphorus starvation conditions,

being chlorosis a common response to nutrient limitation

and nutrient starvation in algae (Lapointe and Duke,

1984), although this decline do not appear require

chlorophyll degradation, since may simply arise from

dilution of the chlorophyll poll by cell division in absence

of synthesis (Colliner and Grossman, 1992). Thehypothesis of the effect of phosphorous limitation on

recorded chlorophyll levels is supported by the ANOVA

test results (Table 13), which also indicated a positive

synergistic influence of turbulence on the concentration of

available phosphorous. In the case of the low phosphate

and high turbulence cultures, the fact that chlorophyll

concentration kept almost constant may indicate that the

synthesis and dilution of chlorophyll were similar.

Finally, the decrease in chlorophyll concentration

between the sixth and the eleventh day in the high

phosphorus and low turbulence cultures, which reflect adecrease in the carbon to chlorophyll ratio (C:Chl),

probably was associated with the phenomenon of



17/24

photoaclimatation, since it is well known that the

phytoplankton C:Chl ratio decrease from high to low light

under nutrient-replete conditions (Geiger et al., 1996).

The hypothesis that phosphorus availability could

affect the chlorophyll synthesiswas also supported by the

analysis of the cellular quota of chlorophylls, showing

significant differences on the third day, according with

Geiger et al. (1996). Moreover, the analysis of data

recorded on the sixth day, when phosphorous levels had

reached similar levels in all cultures, showed the

influence of turbulence on the mineral nutrition through

its effect on the boundary layer, which would explain the

significant differences between the amounts of

chlorophyll per cell (Table 14). Probably, if we had

recorded chlorophyll concentrations in shorter time

intervals, we would have registered interaction between

phosphorous and turbulence, but further experiments are

needed to confirm this hypothesis. As expected, the

phosphorous concentration on the eleventh day reached

such low values that turbulence had no effect on its

availability (Table S4). The relation between the

phosphorus availability and the chlorophyll synthesis

seems to be also supported by the significant positive

correlation found between concentration of free phosphate

in the medium and chlorophyll concentration (Table 16).

The cellular quota of carotenoids showed significative

differences between the phosphorus levels, being higher

in the low phosphate cultures (Table 15). Those results

are in accordance with the fact that the carotenoidsaccumulation is triggered by suboptimal conditions, as for

example nutrient limitation (Fazelli et al., 2006). Among

the different turbulence levels, however, did not show

significant differences in the carotenoids concentration,

which disagree with the findings of Aguilera et al. (1994).

This may be due to that differences in bubbling were not

enough. On the other hand , the results obtained from the

correlation analysis, which seemed to indicate an inverse

relation between the amount of carotenoids per cell and

the cell density, were probably only an artifact resulting

from the dilution effect, since the correlation coefficientsbetween the separate treatments did not even showed

negative values.

With regard to the usefulness of chlorophyll A as cell

density estimator, the results indicated that this is a good

way to know the approximate culture density, since the

regression ANOVA, the Pearsons correlation coefficient

and the correlation coefficient revealed significant values,

according with Butterwick et al. (1982).

On the other hand, the relation between the accessory

and the main pigments gave an idea of how the algae

adapted to the lack of light produced by the increase incell density, and according with the statistic tests

performed on data collected the eleventh day, the

turbulence was the critical factor which determines such

adaptations, because, irrespective of the phosphorous

level, there were significant differences between the

turbulence levels, and, however, in the case of low

turbulence cultures, there were no significant differences

between the cultures of high and low phosphorus. Thus,

in low turbulence cultures, in which the relation between

the accessory and main pigment showed a similar

increasing patter throughout the experiment, their

response to the decline of light were to increase the

photosystem size. With respect to high turbulence

cultures, those in which are linked to low phosphorus also

responded to the decrease of light throughout the

experiment by increasing the photosystems size. Both

results are fit to the findings of other authors, which

indicate that a common structural adaptation to lower

light in Dunaliella is the increase in antenna size, which

allows the maintenance and rapid recovery of Pmax (Melis

et al., 2009; Gordillo et al., 2001). Meanwhile, the high

phosphorus and high turbulence cultures responded by

increasing the number of photosystems. The differences

between the behaviors of different treatments should be

caused by differences in the degree of light extinction due

to different cell densities between treatments, although

additional experiments are needed in order to determinate

the specific factors which govern such different

behaviors.

There is a well-established, positive relationshipbetween nutrient loading and productivity (Schindler,

1978), which is most readily apparent as increasing

phytoplankton biomass with eutrophication (Nicholls and

Dillon, 1978). The system productivity (or production-to-

biomass ratio ) although were only estimates because the

calculations were based on cell densities and not on actual

measurements of the culture masses, showed significative

differences between the treatments according with the

statistical tests. Thus, the results showed that phosphorus

level had a decisive effect on the P:B rate, reflecting its

role as an essential nutrient, whose deficiency has beenreported to decrease the rate of CO2 assimilation, and

having been demonstrated a significant direct correlation

between the phosphorus level and the Rubisco activity

(Brooks et al., 1987). In the case of Dunaliella,

phosphorus seems to have an additional role as

constituent of glycerophosphorylglycerols (GPGs), which

are a major component in the Dunaliella phosphorus

spectrum, and that seems to be involved in the

maintenance of the high glycerol content, necessary for

the osmotic control (Ginzbulrg et al., 1988). As the results

of the Tukeys test showed (Table 23), turbulence,through its effect on the boundary layer and the

availability of light by cells, acts by enhancing the



18/24

positive effect of phosphate on growth. Such results

conform to expectations, since high phosphate cultures

have more eutrophic character, characterized by higher

P:B ratio, while the low phosphate cultures have more

oligotrophic character (Rodriguez J., 1999).

With reference to the evolution of phosphate in the

medium, the results showed that there was not a

statistically significant difference between the treatments

analyzed separately (since they started from different

initial concentrations); the steep decline between the day

0 to 3 should be due to going from the stock solution,

poor in phosphorous, to fresh media, rich in phosphorus,

Dunaliella began to incorporate it in large quantities,

because the total phosphorus content ofDunaliella is high

in comparison with other algal species, which is stored in

diverse forms as phosphomonoesters, including glycerol

phosphate and sugar phosphates, and polyphosphate

chains (Benthal et al., 1988). Calculations show that

within less that 1 hour,D.viridis may accumulate 500mM

equivalents of Pi from media containing mol/L Pi

concentrations (Li et al., 2006). In previous studies have

been shown thatDunaliella accumulates large amounts of

polyphosphates within acidic vacuoles, and the stored

polyphosphates may be utilized to resist alkaline stress

(Pick and Weiss, 1991). Thus, when the cultures, which

were from aged stock cultures, were exposed to

concentrations of 50 and 250 M quickly began to store

phosphorus, and although apparently there are differencesbetween high and low turbulence cultures, the statistical

evidences does not support this hypothesis. With regard to

the results pertaining to the phosphorus incorporation

rates (Figure 8), according with the statistical analysis no

significant differences existed between the treatments.

However, further experiments are needed in order to

confirm these results, since the small number of replicas..

Statistical analysis carried out on photosynthesis and

respiration rate values yielded remarkable results. In the

case of photosynthesis rate, turbulence, given its effect onthe boundary layer, facilitating gas and metabolites

exchange and allowing most of the cells to receive

sufficient light through a better mix, was expected to have

a positive influence on the photosynthesis rate, according

with the findings of Jimenez et al.(1996), but

nevertheless, the obtained results indicated otherwise. On

the other hand, the results indicated significative

differences in high phosphate cultures between the third

and the sixth day, probably as consequence of cell density

reached values high enough to interfere with the

photosynthetic process, preventing cultures receiveenough light. Another possible complementary

explanation for both results is that the high concentration

of dissolved oxygen produced by photosynthetic activity

could be toxic for cells thus resulting in a decrease in

photosynthetic activity (Fbregas et al., 1993). The

decrease in the photosynthetic rate throughout the

experiment should also be directly related, especially in

low phosphate cultures, with the nutrient deficiency;

phosphate availability has been implicated in reduction in

the efficiency of photosynthetic electron transfer and

photosynthesis rates, by repressing the amount and

activity of Rubisco, the ribulose 1,5-bisphosphate

regeneration and the ATP synthesis (Evans, 1996; Fazely

et al., 2006, Pieters et al., 2001). This latter hypothesis

seems to be supported by the significative positive

correlation found between the photosynthesis rate and the

phosphorus concentration of the medium, although further

experiments are needed to confirm such relation.

In regard to the respiration rates, the previous data had

indicated that the phosphorus limitation of Dunaliella

resulted in a significant decline in respiration rates (Said,

2009, Theodorou et al., 1991). However, neither the

ANOVA tests nor the correlation analysis fit with these

data. Thus, on one hand, both the ANOVA results

performed on the respiration rate values registered on the

third and eleventh day indicated that the higher respiration

rates were reached on low phosphorus treatments (Table

S6-S8). Specifically in the case of the results for the

eleventh day, the Tukeys test showed significant

differences between the P+T+ and P-T+ treatments, being

higher the mean value of the P-T+ treatment (P = 0,031).These results seems to be also supported by the

correlation analysis values, which although were not

statistically significant, indicated a negative correlation

between respiration and free phosphorus level. One

possible explanation that could explain these results is

that the increase in respiration rates was a direct

consequence of the high phosphate incorporation rate in

Dunaliella, since its incorporation occurs against

electrochemical gradient, that is, with energy

consumption. For this reason, given that the energy

required by low phosphate cultures for incorporatingphosphate is higher, they require a higher respiration rate

in order to provide the necessary energy. On the other

hand, the obtained results for interaction between the low

phosphorous and turbulence levels disagree with the

findings of Jimnez et al. (1996), since it was expected to

record higher respiration rates in low turbulence cultures,

as a result of the higher concentration of reached

dissolved O2, although additional experiment are needed

to confirm it. The fact that no differences had been found

between turbulence levels, despite of the findings of

Jimnez et al.(1996), seems to indicate that air bubblingof 0, 02 L min-1

was enough to overcome, at least

partially, the deleterious effect of photoinhibition as a



19/24

result of the high concentration of O2. We decided not to

interpret the three way ANOVA results in this paper,

since it was very complex as a result of the large number

of significant interactions.

With regard to the ligh attenuation, as expected, whenthe vertical attenuation coefficients (kd) recorded in all

cultures throughout the time were plotted against the

chlorophyll A and total pigment concentrations,

hyperbolic relations were obtained for both cases,

according with Mercado et al. (1996). Such results

reflected the range of tropic states of cultures exposed to

different treatments, since D.viridis, being pigmented

organisms, contribute both to absorption and the

scattering of light. Chlorophyll A is the main pigment

responsible of absorbs light for use in photosynthesis, and

whose absorption peaks are between 400 and 475 nm and

around 665 nm. The observed relation is due to that, with

increasing the pigment concentration, , the extinction

coefficient increases linearly up to a point where the

concentration is such that the extinction coefficient does

not change although increase the pigment concentrations,

since all the corresponding light to the wavelength at

which chlorophyll and carotenoids absorb has already

been absorbed. The fact that the obtained kdmax values for

both chlorophyll A and total pigments have been so

similar (72.464 for chlorophyll A and 76,33 in the case of

total pigments), and in turn so different from the value

obtained for carotenoids (169,49), highlight the central

role of chlorophyll A in the absorption of light. On the

other hand, the obtained km values revealed that, for

chlorophyll A, concentrations above 5,507 g mL-1

imply

a decrease in the efficiency of energy absorption, since it

is necessary to synthesize a greater amount of chlorophyll

to absorb similar amounts of light. A similar conclusion

is possible to extract for total pigment concentrations

above 10,96 g mL-1

.

The package effect has been examined intensively

using theoretical formulae during the last decade, and it isnow clear that this effect depends both on the cell size and

pigment content of the cell for a given pigment

composition (Kirk, 1994), increasing when either the

average cell size or the absorption coefficient of the

cellular material increases. Thus, decreasing trends were

expected in absorbances, as a result of the increased

package effect, since the cell densities were increasing,

according with Bricaud et al. (1995). This hypothesis was

supported by the significant negative correlation found

between the package effect estimator and the cell density.

However, such trend was not apparent according to thedata shown in figure 11, which seems to indicate the

existence of a threshold above which are triggered the

mechanisms associated with the package effect, such as

changes in cell size, shape or morphology, changes in

chloroplast size, shape, number, morphology and

distribution within the cell; changes in the degree of

stacking of thylakoid membranes within the chloroplast

and changes in the optical properties of the thylakoid

membranes (e.g. the transparency of the membrane).

Further researches are needed in order to confirm the

existence of this physiologic threshold.

As concluding remarks, the influence of the

interaction between phosphorus availability and

turbulence on the ecophysiology ofDunaliella viridis has

been confirmed in this research. The effect of turbulence

is manifested most strongly in conditions in which the

phosphorus level begins to drop below optimum levels,

exerting its influence apparently by increasing the

available phosphorus as a result of the decrease in the

boundary layer, although other factors such as better

mixing can be also involved. In order to identify these

processes and the extent to which they contribute, further

researches are needed. Finally, the usefulness of

nephelometry and chlorophyll A as cell density estimators

were confirmed.

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21

Supporting information

Growth rate ANOVA step by step

P+ P- P+ P-

T+ 1,07421,106

0,54070,5281

3,249 T+ 1,15390564 0,29235649 2,94838774

1,223236 0,27888961

T- 0,54830,6376

0,80451,1192

3,1096 T- 0,30063289 0,64722025 2,60699554

0,40653376 1,25260864 3,3661 2,9925 6,3586 3,08430829 2,47107499 5,555

1 = 3,249 + 3,1096 = 6,3586

2. = 2,9483 + 2,6069 = 5,555

3.

= (4,753 + 1,4063 + 1,1423 + 3,70)/2 = 5,501

4.

= (11,33+ 8,955)/(22) = 5,071

5.

= (10,556 + 9,669)/(2 2) = 5, 056

6.

= (6,35862)/(222) = 5,05397

7. (

= 5,5555,05397 = 0.5014 = SCTotal

8. (

(

= 5,5015,05397 = 0.44732 = SC subgroup

9. (

)(

5,0715,05397 = 0,002429 = SCA

10. (

)

5, 0565,05397 = 0.0021 = SCB

11. SC subgroup SCASCB = 0,42744

12. SCTotal

SC subgroup = 0,05409

F.U. Gl SC MC Fobs Fcrit

A 1 0,01744712 0,01744712 1,29022196 7,7

B 1 0,002429045 0,00242904 0,17962891 7,7

A*B 1 0,42744258 0,42744258 31,609561 7,7

Error 4 0,05409029 0,01352257

Influence of the combine

influence of the combined effect of phosphorus and small scaleturbulence on the ecophysiology of ...

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