DIFFUSION BOUNDARY LAYER TRANSPORT IN GRACILARIA CONFERTA(RHODOPHYTA)1

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JOSE L. GARRIDO ET AL.

Nelson, J. R. & Wakeham, S.G. 1989.A phytol-substituted chlorophyll c from Emiliania huxleyi (Prymnesiophyceae).J . PhyC O ~ . 25:761-6. Rickettts, T. R. 1966.Magnesium 2,4-divinylphaeoporphyrina, monomethyl ester, a protochlorophyll-like pigment present in some unicellular flagellates. Phytochemistry 5:223-9. Stauber, J. L. &Jeffrey, S. W. 1988. Photosynthetic pigments in fifty-one species of marine diat0ms.J. Phycol. 24:158-72. Vesk, M. &Jeffrey, S. W. 1987. Ultrastructure and pigments of two strains of the picoplanktonic alga Pelagococcus subuiridis (Chrysophyceae).J. Phycol. 24:158-72.

Wright, S. W., Jeffrey, S. W., Mantoura, R. F. C., Llewellyn, C. A., Bjerland, T., Repeta, D. & Welschmeyer, N. 1991. Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar. Ecol. Prog. Ser. 77:183-96. Zapata, M. & Garrido, J. L. 1991. Influence of injection conditions in reversed phase high performance liquid chromatography of chlorophylls and carotenoids. Chromatographia 3 1~589-94.

J. Phycol. 31, 768-773 (1995)

DIFFUSION BOUNDARY LAYER TRANSPORT IN GRACILARIA CONFERTA (RHODOPHYTA)' Yael Gonen, Eitan KimmeP Agricultural Engineering, Technion IIT, Haifa 32000,Israel

and Michael Friedlander National Institute of Oceanography, P.O. Box 8030,Haifa, 31080,Israel ABSTRACT

A theoretical framework on the combined effect of water velocity and solute concentration on the photosynthetic performance of the red alga Gracilaria conferta (Rhodophyta) is developed. This is based on the balance between the rate oftransport through boundary layers and MichaelisMenten-type equations for carbon consumption and for production of oxygen and hydroxyl ions. By comparing the theoretical models with experimental data, we found that the mechanism of enhancing photosynthetic rates by increasing water velocity cannot be attributed to enhanced bicarbonate and CO, transport, nor to CO, as a sole source of carbon. Velocity-facilitated photosynthesis may be due to the enhanced removal of OH- ions, which inhibit photosynthesis when accumulated on the algal surface. Oxygen had no inhibitory effect on Gracilaria conferta.

Key index words: boundary layer; carbon; Gracilaria conferta; hydroxyl ions; photosynthesis; Rhodophyta; transport

Cultivation of seaweeds in ponds requires seawater mixing for supply of carbon and nutrients and the removal of excreted compounds. Enhanced photosynthetic and growth rates can be obtained with increased water motion and carbon concentration as observed for Macrocystis (Gerard 1982),Ulva (Parker 1981,Koch 1993),and Gracilaria conferta (Gonen et al. 1993). In contrast, photosynthetic rates Received 30 January 1995. Accepted 22 June 1995.

* Author for reprint requests.

in some species of algae are reduced by elevated oxygen concentrations (Weber et al. 1981, Raven 1991, Marcus et al. 1992).Increased pH of the seawater or elevated concentrations of OH- ions also reduce photosynthetic rates in Gracilaria (Weber et al. 1981, Lignell and Pedersen 1989, Friedlander and Ben-Amotz 1991, Friedlander 1992)or in other algae, respectively (Lucas 1983, Israel and Beer 1992). The transport of the preceding ingredients, which affect photosynthetic rates, toward and away from the alga takes place through a thin diffusion boundary layer. Solute transport to the alga can be enhanced by increasing the concentration difference across the boundary layer or by reducing the boundary layer thickness, e.g. through an increase in the relative water velocity around the algae (Wheeler 1980). Dissolved inorganic carbon (DIC) in seawater is one of the major factors controlling growth of seaweeds, especially in intensive cultivation systems (Friedlander and Ben-Amotz 1991). A shortage of DIC causes reduction of the photosynthetic rate (lsrael et al. 1991, Gonen et al. 1993).Diffusion rates of DIC in water are much lower than in air, and thus the DIC may be limiting to the plant. In natural seawater (pH 8), DIC mostly appears in the form of bicarbonate (HCOS-) with concentrations of about 2 mM. At this pH, another form of DIC, CO, appears in very low concentrations of about 10 HM (Raven et al. 1985).Bicarbonate has been suggested as the carbon form that Gracilaria uses (Israel et al. 1991). On the other hand, Lignell and Pedersen

769

BOUNDARY LAYER TRANSPORT

(1989) associated the carbonic anhydrase activity near the surface of G. secundata with CO, consumption. Some of the excreted products of the photosynthetic process in algae can inhibit photosynthesis. Oxygen inhibition of photosynthesis is ubiquitous in CJ terrestrial plants at present atmospheric CO, levels and in some species of seaweeds (Newman and Cattolico 1987, Raven 1991). High internal O2 accumulation in Hydrodictyon and Chara inhibited photosynthesis at low water velocities (Weber et al. 198 1). In some other species of seaweeds, however, oxygen had no inhibitory effect (Reiskind et al. 1989, Johnston 1990, Israel et al. 1991). When the pH level at the boundary layer rises during DIC consumption (Israel and Beer 1992), Gracilaria may be stressed due to a drop in available DIC (Lignell and Pedersen 1989) or to inhibition of enzymatic processes (Frost-Christensen and SandJensen 1990). Use of HC03- by green algae results in an increase in the pH level and consequently a reduction in HC0,- concentration in the boundary layer (Axelsson and Uusitalo 1988). This creates a steep concentration gradient in the water surrounding the plant. Bicarbonate utilization is followed by COs assimilation and the excretion of OH- ion (antiport) or the uptake of protons (symport). Both mechanisms induce an increase of the pH level (Prins and Elzenga 1989) at which only HCOS- utilization can occur. Weber et al. (198 1) and Friedlander (1992) have shown a massive decline in photosynthetic rate of Gracilaria as the water pH increased. Growth rate declined dramatically at pHs higher than 8.6 and decreased slower for pHs lower than 8 (Lignell and Pedersen 1989, Friedlander and Ben-Amotz 199 1). In many marine algae, photosynthesis increases with relative water velocity up to a certain “saturation” velocity where it reaches a maximal photosynthetic rate (Wheeler 1980). Any further increase of the velocity beyond the saturation velocity will not improve the photosynthetic rate (Wheeler 1980, Koch 1993). This phenomenon suggests a possible combined action of two “resistances” in series: 1) a boundary layer resistance to DIC and nutrient transport from the bulk medium, which explains the increase in photosynthesis as a function of velocity (Wheeler 1980), and 2) a limiting enzymatical rate at the plant surface, such as carbonic anhydrase activity or carrier activity (Wheeler 1980,Jenkins and Proctor 1985, Kerby and Raven 1985, Koch 1993), which explains the saturation phenomenon. T h e concentrations on the alga surface of, e.g. carbon, oxygen, and OH- ions, influence the effectiveness of each of the preceding resistances and seems to be a key factor in resolving the question of why the photosynthetic rate in Gracilaria conferta is increased by more powerful mixing. T h e present paper is aimed at identifying which of the following ingredients-carbon, oxygen or OH- ions-affect the photosynthetic rate of Gracilaria conferta when

Bulk of water (w)

Carbon

Qc, in

photosynthetic (P) consumption (Qc, in) of inorganic carbon FIG. 1. Schematic model of physical and biological processes involved in carbon transport through the boundary layer and in photosynthetic carbon consumption of a Gracilaria thallus.

its flux through the seaweed boundary layer is increased by more powerful mixing. MATERIALS A N D METHODS

Theoretical background for boundaly layer transport. The sequential processes of external (ex) transport and internal (in) enzymatic processes are shown clockwise in Figure 1. The boundary layer around each of the Gracilaria branches is considered for simplicity as the boundary layer formed over a uniform cylinder exposed to a cross flow. In this case, mass transport across this boundary layer is characterized by the expression (1) Sh = cRenSc”, where the nondimensional Sherwood number Sh, Reynolds number Re, and Schmidt number Sc, are defined by

where U is the relative water velocity, D is the molecular diffusion coefficient, d is the cylinder diameter, H denotes mass transport coefficient, and v is the kinematic viscosity. The constants c and n depend on the flow regime (Holman 1986:292). For simplicity, the diffusion coefficients for bicarbonate and all other tested compounds were assumed equal to 4 0 1 O - ~cm*.s-’. Based on Jenkins and Proctor (1985) and using Eq. 2, the rate of carbon flux per gram alga, from the bulk of water to the algal surface (see Fig. 1) is given by

Q.,..= H.S(C,

-

C,)

1

= -Sh.S.D(C,

d

- CJ,

(3)

where C. and C, are the carbon concentrations over the algal surface and in the bulk of water, respectively, and S denotes the branch surface area per gram alga. Because the variables U, H, d, and C. may vary within the thallus, their mean values have been applied to simplify the analysis. Variations in C, are negligible because the reduction in carbon concentration due to algal uptake is much smaller than the rate of supply of carbon by water mixing from the bulk of water.

770

YAEL GONEN ET AL.

The rate of carbon consumption, Q,,",is presumably controlled by enzymatic processes that can be described by an expression such as

which follows the kinetics of Michaelis-Menten in the absence of inhibitors. In Eq. 4, Q,- is the maximal carbon consumption due to photosynthetic activity, and K, is the half-saturation constant. Under steady-state conditions, one can assume the equivalence Qcm

=

(5)

Qi.

because no dissolved carbon is accumulated in the cells of Gracilaria (Israel et al. 1991). The presence of OH- ions, CO,*- (Helder 1985) ions, or oxygen, on the seaweed surface, 0,,might affect photosynthesis as a competitive inhibitor, modifying Eq. 4 to Q,n

=

Q.m..

, C.-,

J

K, 1 + 2

?

(6)

+C.

where 0, denotes the concentration of the inhibitor on the alga surface. A similar way of introducing the influence of internal oxygen on the photosynthetic rate was suggested by Weber et al. (1981). Analogous to Eq. 3, the rate of removal (Qo,,) of the inhibitor is determined by 1 Q,.= . dSh.S.D(O.

- Ow),

(7)

where 0, denotes the inhibitor concentration in the bulk of water. Under steady-state conditions, the rate of the inhibitor production, which can be assumed equal to the carbon consumption rate, equals the rate of the inhibitor removal, Q,:

Q., = Q ; m = Q,in(8) Experimental procedure. Gracilaria conferta was cultured in indoor tanks (36 L) at 22'44" C during the spring of 1992. Water velocities around the algal surface were calculated from the dissolution rate of benzoic acid sticks (BASs), following the method of MacFarlane and Raven (1989). The sticks were attached to the algae using a method described previously by Gonen et al. (1993).

Net photosynthesis variations of cultured Gracilaria conferta with water velocity and carbon concentrations were measured in a closed Clark-type oxygen-electrode system (Beer and Israel 1990). Different water velocities between 0 and 8 cm.s-' (Gonen et al. 1993) were obtained in the photosynthesis chamber by a magnetic stirrer after calibration with BASs. The pH was kept at 8.2 by Tris-HCI buffer, the temperature was 25" C, and irradiation was 200 pmol photons.m-*.s-'. The DIC concentrations (0.5-4.0 mM) were obtained by adding NaHCO, to 7 mL of seawater. The influence of oxygen concentration on the photosynthetic rate was tested by the Winkler method (Dawes 1981). A wide range of oxygen concentrations in seawater was obtained by oxygen or nitrogen bubbling for different time periods prior to the experiment. Then, plants were placed for 30 min in BOD bottles with the prepared seawater and an irradiation of 200 pmol photons.m-**s-l. Photosynthetic rates were estimated from the rate of accumulation of oxygen in each bottle. T o show the relationship between the OH- mass transport and the flow field, 0, and Sh numbers for the OH- ions were estimated using the measured 0, and the water velocities that had been measured by the BAS method. RESULTS AND DISCUSSION

Transport parameters. A reference value of total carbon consumption rate = 1.18. lo-' g C -

s-l SgFW-l was estimated according to previous experimental data (Friedlander et al. 1987). Gracilaria conferta has two morphological types: a compact type (A) has about 300 branchlets-g-' alga, and a large type (B) has about 80 branchlets-g-'. T h e average diameter of a type A branchlet is 0.07 cm, and its average length is 1.O cm. T h e branchlet of type B is 0.09 cm in diameter and 2.0 cm in length. T h e estimated surface area (S) of the branchlets was estimated as 66.0 cm*-g-' for A and 45.2 cmP-g-' for B. Model simulations. Equations 1-5 can be used to derive the carbon concentration ratio across the boundary layer (C,/C,) versus the relative water velocity (U). This relationship is based on photosynthetic responses and is plotted for various carbon concentrations in the bulk of water (C,), given the preceding values of and S (Fig. 2). For typical water velocities (i.e. greater than about 1 cmms-l) and for common carbon concentrations, boundary layer effects induced differences of no more than 10% between C, and C,. It is the value of C, that determines the photosynthetic rate (Eq. 4), and while variations in C, mirror those in C,, boundary layer effects cannot affect C, that much at high U. For instance, for a two-fold rise in C,, C, has to increase by about the same factor. A similar rise in C,, while keeping C, constant (i.e. to double the ratio C,/C,) can be obtained by a velocity change only for velocities less than 0.01 cmas-l in Gracilaria of type A and less than 0.1 cmms-' in type B (see Fig. 2). Such extremely low values of relative water velocities are much smaller than the velocities that influence growth rates in vivo in aerated ponds or photosynthesis rates in vitro (Parker 1982, Gonen et al. 1993). It is most likely that the very high concentrations of bicarbonate (about 2000 rM) in the ambient seawater yield an excessive rate of supply from the bulk of the water under regular mixing conditions, in comparison to a relatively low rate of bicarbonate removal from the boundary layer due to algal consumption. T h e relevance of the preceding model (Eqs. 1-5) to bicarbonate can also be tested by considering the photosynthetic carbon consumption rate Qin) as an enzymatic process that depends directly and exclusively on the surface bicarbonate concentration C,. In this case, when the results of are plotted versus Q,in/C,, they are expected to cluster along a straight line with a negative slope (see the second form of Eq. 4 and Baily and Ollis 1986:128). Values of C,, were estimated using Eqs. 3-5 and the following measured data: photosynthetic rates in Gracilaria conferta (Gonen et al. 1993) as rates of carbon consumption and values of the Sh number for carbon transport, measured by the BAS technique. T h e experimental results, processed as already explained, do not follow the expected pattern (Fig. 3); therefore, the model presented by Eqs. 1-5 does not hold for bicarbonate exclusively.

77 1

BOUNDARY LAYER TRANSPORT 70

at

8.16 0

0.54

. 1.22 -x-

0

" 8.10 /

1.54 A A

2ov* lot

o.oO01

0.01

1

100

01

12

8 0.54

8.A6

I-

0 14

I

I

I

I

I

16

18

20

22

24

u, (cm/s) 1 .oo

FIG. 3. The rate of carbon consumption (Q,,,) as a function of the ratio QJC. for DIC concentrations (C,) of 0.5 1 (O), 2 (O), 2.3 (A), and 4 (0).Water velocities in cm.s-' are shown along each data point (n = 5-10).

m,

b

0.75

3

qrn0.50 u 0.25

0 0.001

0.1

10

loo0

u, ( c d s ) FIG. 2. Calculated values of carbon concentration ratio (C,/ C,) versus relative water velocity (U)for different carbon concentrations in the water (C,) and surface (C.) for Crudaria of a) type A and b) type B.

Th e possibility that Gracilaria depends on COPas the only inorganic carbon source can be tested by a procedure similar to the one that provided the simulations in Figure 2. Solving Eqs. 1-5 for CO, concentrations of 2- 12 pM, and the preceding values of and S, yields velocities for COP transport that are on the order of 1000 cm.s-'. Such extremely high values of relative water velocities are much higher than velocities that influence growth rates in uiuo in aerated ponds or photosynthesis rates in vitro. Therefore, C 0 2 cannot serve as the only carbon source for Gracilaria. This conclusion agrees with an earlier analysis by Raven (199 1) for macroalgae with a low surface area-to-volume ratio. To explain the mechanism for velocity-enhanced photosynthesis, we need to identify another component with 1) a rate of production or consumption that is comparable to the photosynthesis rate and 2) a lower concentration than bicarbonate (about 2000 pM) and higher concentration than COP(about 10 pM). Possible candidates are the OH- ions and oxygen with typical concentrations in seawater of less

than 100 and 200 pM, respectively. In the same way that water velocity can affect the carbon supply for photosynthesis, it can also contribute to photosynthesis by removing inhibitory solutes such as oxygen or OH- ions from the algal surface. Oxygen concentrations were found to have no inhibitory effect on the photosynthetic rate except for very high levels (above 800 pM). Therefore, the increase of water velocity does not effect photosynthesis through removal of 0, inhibition. T h e enhanced removal of carbonate ions from the plant surface, which appears to have no influence on photosynthetic rate in Ulua lactuca (Maberly 1992), contributes to increased photosynthetic rates in C02-consuming plants (Helder 1985). In the socalled bicarbonate-utilizing plants, an effect that may inhibit photosynthetic rates is the elevation of pH values in the boundary layer and the insufficient removal rates of OH- ions from the plant surface (Helder 1985). T h e increased pH and the accumulation of OHon the algal surface (O,),due to low removal rates of OH- ions, may be an inhibitor to photosynthesis (Lucas 1983, Israel and Beer 1992). T h e effect of OH- is analyzed by means of Eqs. 6-8, from which the OH- concentrations in the bulk of water Ow, and at the algal surface 0,,are derived. Rates of OH- production are presumably similar to carbon consumption rates during the photosynthetic process (Lucas 1983), and the concentration of OHions varies roughly between 1 and 100 pM. T he resulting O,/Ow ratio and its dependence on the Sh number is compared with the C,/Cw ratio (Fig. 4). As shown, the effect of Sh number on the bicarbonate ratio is minor (less than 10%)while variations in the OH- concentration ratio are much larger (from approximately 10 to approximately 150), sug-

772

YAEL GONEN ET AL.

I

160

a 0

A

o

AA

['21

4

A

9.8

0

A

d::%

AA

801

A

0

0 A

0

I

0

40

0 A

c =

0

9.0

0

I

I I

I

5

I

I

I

15

I

I

45

35

25 Sh number

I

8.81 5

I

I

I

15

I

I

I

5

35

25

Sh number

FIG.5. pH at the surface of Gracilaria conferta (pH.) versus Sh number, calculated for water velocities between 0 and 8 cm.s-I for different carbon concentrations in the water ((2,): 0.5 0,1 (0), 2 (O),2.3 (A), and 4 mM (0)(n = 5-10). 0.97

0

0.931

CONCLUSIONS

*

0.911

t n

0.891

5

1

I

15

I

I

25

I

I

35

I

II

45

Sh number FIG.4. Concentration ratios versus Sh number. a) Hydroxyl

Our investigation focuses on the influence of water velocity on the photosynthetic performance of the seaweed Gracilaria conferla. For solutes such as DIC, oxygen, or OH- ions to be a factor in velocityfacilitated photosynthesis, it must have an effect on the photosynthetic rate on the one hand, and it must exhibit significantly different surface concentrations throughout the common range of relative water velocities on the other hand. Through a combination of theoretical modeling and experimental measurements, we propose that the mechanism relating increased photosynthetic rate in Gracilaria conferta to increased water velocity is the enhanced removal of OH- ions from the algal surface.

concentration ratio OJO, and b) carbon concentration ratio C,/ C, calculated for water velocities between 0 and 8 cm.s-' for different carbon concentrations in the water (CJ: 0.5 0,1 (0), We thank Dr. A. Sukenik for useful discussions and Prof. S. Beer 2 (O), 2.3 (A), and 4 mM (0)(n = 5-10). for reviewing the manuscript. The technical assistance of S . Hershler and his team is highly appreciated. This work was supported in part by a grant from the Fund for Collaborative Re-

gesting a greater gradient across the boundary layer for the OH- ions in comparison to bicarbonate, due to a more substantial emptying rate in the boundary layer, especially for high Sh numbers. These gradients are illustrated as values of pH, over the alga surface (Fig. 5 ) , which are much different from the pH, = 8 of the bulk of water. By rearranging Eq. 6, 0, is linearly proportional to the reciprocal value of photosynthetic rate, Qjn-', for a given C,. Due to this (theoretical) result, measured photosynthetic rates are described versus the calculated 0,values (Fig. 6). As before, the different symbols represent different values of C,. For a given C,, a significant inhibition of photosynthetic rate is associated with an increase in 0, (Fig. 6). These results lead us to hypothesize that as the relative water velocity increases and the boundary layer thickness decreases, the OH- ions would be swept away from the algal surface and the photosynthetic rate would increase.

70

I

8.16

"1

y6 =

0.54 0.61

0

0.54

A

0

044 0 0

"18.16 10

A

8.16

401

0 I

01

0

I

I

I

I

I

20

40

60

80

100

I

120

I

140

1

0 s Qm)

FIG.6. Photosynthetic rate of Gracilaria conferta expressed as hydroxyl ions production rate (QJ versus surface hydroxyl concentration (0,) at different carbon concentrations in the water ((2"): 0.5 1 (O), 2 (O), 2.3 (A), and 4 mM (0).Water velocities In cm.s-' indicate each data point (n = 5-10).

m,

BOUNDARY LAYER TRANSPORT

773

search between the Faculty of Agriculture of the Hebrew University, Rehovot, and the Faculty of Agricultural Engineering, Technion, Haifa.

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Lignell, A. & Pedersen, M. 1989. Effects of pH and inorganic carbon concentration on growth of Gracilaria secundata. Br. Phycol. J . 24:83-9. Lucas, W. J. 1983. Photosynthetic assimilation of exogenous HC0,- by aquatic plants. Annu. Rev. Plant Physiol. 34:71-

324-30.

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