CHAPTER III: NITROGEN UPTAKE STUDIES

3.1 NITROGEN UPTAKE KINETICS

3.1.1 Introduction

The first investigation on the rate of nutrient uptake by a marine phytoplankton species was

conducted by Ketchum (1939) using cultures of Nitzschia ciosterium. Since then extensive

research on nutrient uptake kinetics has been carried out for phytoplankton (Collos, 1998;

Dortch, 1990; Harrison et al., 1989; Parslow et al., 1985; Wheeler et al., 1983; Goldman

and Gilbert, 1982; Glibert and Goldman, 1981; Eppley et al., 1969). Likewise the research

on the uptake of nutrients on macroalgae has also been widely carried out (D'Elia and De

Boer, 1978; Haines and Wheeler, 1978; Hanisak and Harlin, 1978; Wallentinus, 1984;

Williams and Fischer, 1985; Campbell, 1999; Torres et at., 2003; Phillips and Hurd, 2003;

Tarutani et al., 2004; Fong et al., 2004; Cohen and Fong, 2005).

Many studies have focused on the uptake physiology i. e. determination of kinetic

parameters, interactions between ammonium and nitrate uptake, etc. and other detailed

studies have also been reported (Haines and Wheeler, 1978; Hanisak and Harlin, 1978;

Topinka, 1978; Friedlander and Dawes, 1985; Thomas et al., 1987; Lobban and Harrison,

1997; Naldi and Wheeler, 2002).

N uptake rates by macroalgae are usually described as a saturating function of substrate

concentration. Studies conducted to determine the kinetic parameters- V and K s, have

reported that sometimes the uptake rates deviated from the saturating kinetic pattern to

non-saturable kinetics. Non-saturable kinetics has been shown for macroalgae such as

Gracilaria foliifera (D'Elia and De Boer, 1978), Gracilaria tikvahiae (Friedlander and Dawes,

1985) and Stictosiphonia arbuscula (Phillips and Hurd, 2004) and saturable kinetics has

been reported for many macroalgae (Hanisak and Harlin, 1978; Topinka, 1978; Harlin and

Craigie, 1978; Probyn and Chapman, 1982; Thomas et at, 1985; Wallentinus, 1984; Haines

and Wheeler, 1978).

As NH4+ is a reduced form of N it is usually taken up in preference to other N forms (Lobban

and Harrison, 1994) and the preferential uptake of ammonium has been often shown in

39

many marine algae (Caperon and Meyer, 1972; Caperon and Ziemann, 1976; Conway,

1977; Phillips and Hurd, 2004). Macroalgal species have exhibited saturable kinetics in

many cases such as in Ulva lactuca, Catenefia nipae (Runcie et a/., 2003), U. lactuca

(Pedersen, 1994), Enteromorpha prolifera (O'Brien and Wheeler, 1987), Polysiphonia

decipiens (Campbell, 1999) and Gracillaria follifera (D'Elia and DeBoer, 1978). However

different species exhibited different uptake patterns for example Codium fragile has shown

ammonium saturation kinetics, suggesting active transport at concentration above 5 IAM

ammonium (Hanisak and Harlin, 1978) and at the concentration of more than 30

ammonium, Fucus spiralis showed saturating uptake kinetics (Topinka, 1978). Rates of

ammonium uptake are a linear function of ammonium concentration in many cases such as

in Macrocystis pyrifera (Haines and Wheeler, 1978), Laminada groenlandica (Harrison et

al., 1986), Gracilada pacifica (Thomas et aL, 1987), G. tikvahiae (Friedlander and Dawes,

1985), Chondrus crispus (Amat and Braud, 1990), Enteromorpha sp. (Fujita, 1985), Ulva

sp. and Chaetomorpha sp. (Lavery and McComb, 1991) and Fucus distichus (Thomas et

a/., 1985).

It has been reported by Peckol et al. (1994) that under N-limiting conditions ammonium

uptake of G. tikvahiae was faster. Earlier Haines and Wheeler (1978) had also reported

faster uptake rates by Hypnea musciformis compared to Macrocystis pyrifera, which was an

adaptation to N- poor environment. Uptake of ammonium is of interest because many

species take up ammonium at rates that greatly exceed growth rates (references listed in

Hanisak, 1983) and are able to grow rapidly for days using stored N when N availability is

low (Hanisak, 1983; Fujita, 1985). Ammonium uptake by N sufficient algae should be

saturable and less rapid than uptake by N deficient algae if internal N pools regulate N

uptake (Wheeler, 1983). In addition, ammonium uptake rates generally increase with

decreasing tissue N content, an indication of increasing N starvation (D'Elia and DeBoer,

1978; Hanisak, 1983; Fujita, 1985). Thus the macroalgae living in N deplete environment

could derive N from transient pulses of ammonium (D'Elia and DeBoer, 1978; Rosenberg et

a/., 1984; Fujita, 1985)

Nitrate is the most thermodynamically stable form of DIN (dissolved inorganic nitrogen) in

aquatic environments, and hence it is the predominant form of fixed N in most of the aquatic

systems, but not necessarily the most readily available form (Falkowski and Raven, 1997).

Macroalgae can take up nitrate at extremely low substrate concentrations, suggesting an

40

active transport mechanism that is usually described by a Michaelis Menten hyperbola

(Lobban and Harrison 1997). Nitrate uptake typically shows saturation kinetics for

Laminaria longicruris (Harlin and Craigie, 1978), Fucus spiralis (Topinka, 1978),

Macrocystis pyrifera and Hypnea musciformis (Haines and Wheeler 1978) indicating active

transport, while linear increase in the rate of nitrate uptake has also been reported for

Chaetomorpha linum (Lavery and Mc Comb, 1991), Laminaria groenlandica (Harrison et al.,

1986) and Gracilaria pacifica (Thomas et al., 1987).

Although not given much consideration for the nitrite uptake in macroalgae, experiments

have shown that nitrite uptake exceeds nitrate uptake in several marine phytoplankton

species in culture and natural populations (Collos, 1998). High nitrite supply is caused by

processes such as summer upwelling events (Valiela, 1995), phytoplankton excretion

(Collos, 1998) and sewage inputs to coastal areas (Brinkhuis et at, 1989). However, the

detailed description of nitrite uptake kinetics has been largely ignored in macroalgae and

restricted to two studies (Hanisak and Harlin, 1978; Topinka, 1978). Topinka (1978) in his

study demonstrated that macroalga Fucus spiralis exhibited saturation type nitrite uptake

kinetics but the nitrite uptake during dark conditions was clearly a linear relationship.

In considering, the role of macroalgae in regulating DON (dissolved organic nitrogen) fluxes

and transformation in coastal waters, it is necessary to quantify the uptake process of DON

especially urea. Urea is said to be the excretory product of marine zooplankton (McCarthy,

1971), mixed assemblages of oceanic microzooplankton (Eppley et al., 1973), bivalve

molluscs (Allen and Garrett, 1971) and blue sharks (McCarthy and Kamykowski, 1972). In

one of the studies on the decomposition of zooplankton, it was found that zooplankton

contributes•to seawater urea (Mitamura and Saijo, 1980).

The first extensive study of the mechanisms involved in urea uptake was on the green alga

Chlamydomonas reinhattlii Dangeard (Hodson et al., 1975; Williams and Hodson, 1977).

These authors found that urea transport at concentrations of < 70 1AM was mediated by a

saturable transport system with the K, of 5.11.1M in acetate grown cells deprived of

ammonium for more than 2 h. At concentration of > 70 p.M urea, transport occurred by

passive diffusion. The same two pathways, active uptake at low urea concentrations and

passive diffusion at higher concentrations, were also found in the case of yeast (Cooper

and Sumrada, 1975).

41

Saturable uptake kinetics of dissolved organic nitrogen in the form of urea has been

reported for the first time for macroalgae Chordaria flageffiformis by Probyn and Chapman

(1982). In yet another study by Tarutani et al. (2004) a short term uptake of dissolved

organic nitrogen by an axenic strain of U/va pertusa (chlorophyceae) using 15N isotope

measurements showed that Ulva pertusa had the maximum uptake rate and half saturation

constant as 7.92 ± 1.10 1.1M N (g dry wt) -1 h-1 and 25.5 ± 7.0 jiM respectively which when

compared with ammonium or nitrate measured during the same study implied that U.

pertusa had a poor ability to use urea.

Usually spectrophotometric determination of changes in nutrient concentration in the

incubating medium over a certain time interval was the standard method used to estimate

nutrient uptake by macroalgae. This study, however, was carried out using stable isotope

15N. Earlier the isotopic methods, which provided precision and sensitivity, were used only

to study the phytoplankton uptake rates. The knowledge of the nitrogen cycle in marine and

fresh waters has advanced tremendously by application of tracer techniques, especially

those employing 15N, but until recently, Williams and Fisher (1985), for the first time applied

stable isotopic techniques to study the kinetics of nutrient uptake in macroalgae wherein

they measured both concentration changes and the incorporation of 15 N into the algal

tissue. Recently Naldi and Wheeler (2002) determined ammonium and nitrate uptake rates

in U/va fenestrate and Gracilatia pacifica by 15N accumulation in the algal tissue and by

disappearance of nutrient from the medium in long-term incubations.

The studies on the marine algae of the Indian Ocean were initiated in 17 th century by Dr.

Herman (Srinivasan, 1966). Since then several studies have been carried out on marine

macroalgae of Indian Ocean, but not a single study was carried out to study the uptake of

all the four N nutrients together, for example Datta and Datta (1999) measured under

laboratory conditions the dessication induced nitrate and ammonium uptake in the red alga

Catene//a repens from the intertidal mangrove forests of Sunderban, India. However this is

the first study to report the uptake kinetics of intertidal macroalgae from a eutrophic and

oligotrophic system for all the major nitrogen nutrients by 15N technique from India.

From the literature (see above), it has been observed that biphasic or non saturable rapid N

uptake is a characteristic of macrolagae growing under conditions of low N availability and

that saturable N uptake is the characteristic of the macroalgae growing under condition of

42

high N availability. Therefore it is expected that biphasic or linear uptake would be exhibited

by the macrolagae growing in the waters of Lakshadweep (oligotrophic) and that

macroalgae from Anjuna (eutrophic) would show saturable N uptake. With this in mind the

following investigations were carried out.

1) To determine N1-14+, NO3, NO2 and urea uptake rates for Dictyota dichotoma,

Sargassum tenerrimum, Stochoespermum marginatum, U/va lactuca, Acanthophora

specifera, Soliera robusta, Caulerpa sertulariodes and Padina tetrastomatica.

2) To determine Vmax , K and VF-flax/Ks (a) of nitrate, nitrite, ammonium and urea uptake in

short term incubations.

3) To compare the nutrient uptake from two different environmental conditions (eutrophic

and oligotrophic).

4) To determine the safety factor (safety factor is the ratio of the maximum nutrient uptake

rate to maximum ambient concentration of nutrient) for different macroalgae from two

different environments.

43

3.1.2 Methodology

Soon after the collection the algae were brought to the laboratory and were cleaned with

filtered seawater to remove the epiphytes and sediments attached to the thallus of the

algae. They were then held in filtered seawater with aeration for 1-2 h before

experimentation.

Uptake measurements were carried out with approximately 0.5 g of algal thallus placed in

beakers with 250 cc of seawater to which ammonium, nitrate, nitrite and urea was added at

concentrations 2, 4, 6, 8, 10, 20, 30, 40, 50 and 60 µM N. The spike in each case was

prepared by mixing 9 parts of unlabelled N compound with 1 part of 15N—labelled N

compound (99 atom % excess). For each set of experiments with a given concentration,

incubations with algal tissues were prepared and spiked with the N nutrient in the form of

NH4CI, KNO3, NaNO2 and urea. The beakers were shaken periodically to prevent localized

depletion of N nutrient in the incubation medium.

Incubations were carried out at a light intensity of 800 ttE m -2 s-1 (incubations in dark were

carried out in the samples collected from Goa only) and ambient temperature of 27 - 28°C.

Duplicate sets of algal tissues were withdrawn at the end of 1 h.

Algal material removed from the incubation medium was briefly rinsed with deionized water

to remove the traces of N adsorbed on the thallus and dried to a constant weight at 70°C.

Dried samples were homogenized by grinding with mortar and pestle for the estimation of

particulate organic nitrogen (PON). PON content of the algal tissues was determined by

Kjeldahl digestion, steam distillation and titration with a normal solution of NaOH (precision

0.15 AM N). At the end of the titration, the ammonium was trapped by acidification with a

few drops of 0.05 N HCI, concentrated to a smaller volume and taken for measurements of

14N: 15N isotopic ratios in a Jasco N-151 Heavy Nitrogen analyzer (refer chapter II). PON

concentrations and isotopic ratios thus were obtained from the same sample and uptake

rates were calculated with the equation (Dugdale and Wilkerson, 1986) where PON

concentration at the end of the incubation is used. Control algal samples were used for the

determination of natural abundance of 15N.

44

Safety factor was calculated for different species of algae. It is the ratio of the V max for

nutrient to the rate of nutrient uptake at maximum ambient concentration of nutrient. It

provides a simple estimate of the amount of surplus capacity of a nutrient uptake system. I

calculated safety factors based on maximum nitrate, nitrite, ammonium and urea

concentrations in the coastal waters of Goa and Lakshadweep. The maximum N

concentrations are given in table 3.1.

Table 3.1: Maximum concentration (RM) of nitrate, nitrite, ammonium and urea in seawt

from Anjuna (Goa) and Kavaratti (Lakshadweep).

Study area NO3- NO2- NI-14+ Urea

Goa 17 0.5 6 2.45

Kavaratti 1.09 0.23 1.44 3.35

Statistical analysis

Analysis of covariance (ANCOVA) was employed to examine the difference in uptake rates

between different species and also the differences in light and dark. The significance level

used was P < 0.05. 95 % confidence limit was used to see how the data is statistically valid.

45

3.1.3 Results

3.1.3.1 N uptake kinetics for macroalgae in an eutrophic environment

3.1.3.1.1 Ambient N concentrations

The minimum and maximum ambient concentration of the nitrite, nitrate, ammonium and

urea measured during the experimental period ranged between 0.21 and 1.71 (average

0.79 t 0.44 1.1N1 N, n=10), 0.98 to 4.4 (average 2.94 t 1.19 RM N, n=10), 0.07 to 1.53

(average 0.54 t 0.45 11M N, n=10) and 0.05 to 2.45 (average 0.73 ± 0.72. !AM N, n=10)

respectively.

3.1.3.1.2 Uptake kinetics

The six intertidal macroalgal species namely Stoechospermum marginatum, Sargassum

tenerrimum, Ulva lactuca, Dictyota dichotoma, Caulerpa sertularfodes and Padina

tetrastomatica were collected from Goa to study the uptake kinetics for ammonium, nitrate,

nitrite and urea in light and dark. The uptake results showed saturation for all the N species

and thus could be fitted to the Michaelis Menten relationships.

The Vr„,„ values reported for the algae for different species of N were almost similar in tight

and dark (Table 3.2). The highest Vmax value for NO3" in light and dark was observed in D.

dichotoma (Table 3.2). The highest Vmax value for NO2, NH4+ and urea in light was

observed for C. sertulariodes, D. dichotoma and S. marginatum respectively, whereas in

dark U. lactuca showed highest Wax. The uptake rates of ammonium, nitrate and urea for

C. settulariodes were not measured in light and dark, nitrite was measured in light only. The

N uptake rates for S. marginatum were not measured in dark.

S. tenerrimum showed highest affinity (a) for NO3 uptake in both light and dark. Highest

affinity for nitrite was seen in C. sertulariodes in light and D. dichotoma in dark, whereas for

NH4+, U. lactuca exhibited highest affinity in light and S. tenerrimum in dark. P.

tetrastomatica and U. lactuca exhibited maximum affinity for urea uptake in light and dark

respectively. The preference (affinity) to take up N species for S. marginatum, S.

46

tenerrimum, U. lactuca, D. dichotoma and P. tetrastomatica in light are NO2> NO3> NW>

urea, NO3- > urea > NH4+ > NO2, NH4+ > NO2 > NO3- > urea, NO2 > NO3- > urea > NH4+ and

urea > NO2 > NO3> NH4+ respectively. In dark the preference order for S. tenerrimum, U.

lactuca, D. dichotoma and P. tetrastomatica are NI-14+ > NO3 > NO2- > urea, NO2- > NI-14+ >

urea > NO3- , NO2> > NO3- > urea and NH 4+ > NO2> NO3> urea respectively.

The nitrate was preferred by the different algal species in the following order. S. tenerrimum

> D. dichotoma > U. lactuca > P. tetrastomatica > S. marginatum and S. tenerrimum > P.

tetrastomatica > U. lactuca > D. dichotoma in light and dark respectively, whereas the

preference for ammonium was shown in the following sequence: U. lactuca > S.

tenerrimum > D. dichotoma > S. marginatum = P. tetrastomatica and S. tenerrimum > P.

tetrastomatica > U. lactuca > D. dichotoma in light and dark respectively. The preference for

urea was shown by P. tetrastomatica > S. tenerrimum > D. dichotoma > U. lactuca and U.

lactuca > S. tenerrimum = D. dichotoma > P. tetrastomatica in light and dark respectively

and for nitrite the preference order is C. sertulariodes > D. dichotoma > U. lactuca > P.

tetrastomatica > S. tenerrimum > S. marginatum and D. dichotoma > P. tetrastomatica > U.

lactuca > S. marginatum in light and dark respectively. Thus except for the preference order

of S. tenerrimum, P. tetrastomatica, U. lactuca and D. dichotoma for nitrate and ammonium

in dark, the others do not show any pattern to take up a particular N species.

Compared with the uptake rates of different N species the uptake rates of urea are much

lower and in most of the species the ammonium uptake rates are higher in both light and

dark (Figs 3.1-3.6). The data points used for fitting Michaelis Menten kinetics for different

species of N for the different algae are within 95% confidence limits (Figs 3.7-3.12). The

significance of the differences in the responses in uptake for different species of N to those

of particular algal species are given in table 3.3 and 3.4 and the significance in uptake for

particular N species to that of different algae is given in table 3.6 and 3.7. The variance

ratios computed for differences in uptake of N compound in light and dark by an algal

species is given in table 3.5.

The highest safety factor was calculated for nitrite uptake for S. marginatum and S.

tenerrimum in light (Table 3.8). Safety factor for urea in S. marginatum in light shows

highest values compared to the other species of algae. The range for safety factor for

nitrate is 2 to 6, ammonium 1 to 6, nitrite 3 to 22 and urea 1 to 9.

47

Stoechospennum marginatum

• Uptake of all four N nutrients for this species followed substrate-saturable kinetics (Fig. 3.1

a-d), with the highest Vmax for ammonium, followed by those for nitrite, urea and nitrate

(Table 3.2). Analysis of covariance showed that the differences in the velocity of uptake of

ammonium in light were significantly higher than for other three nutrients (Table 3.3).

However, differences between uptake rates of nitrate, nitrite and urea in light and dark were

insignificant or not as pronounced as with ammonium. The affinity indexlk s) varied

within a narrow range (2.2 to 4.5) between the four nutrients (Table 3.2). The analysis of

covariance showed that the differences in the rate of uptake of ammonium and nitrate (in

light) when compared was (F = 54.78, d. f. =1, 20, p < 0.05) highly significant, whereas, the

differences in the uptake rates of nitrite and urea was not significant (Table 3.3). Amongst

the different species studied, the highest safety factor was calculated for this species for

nitrite (22.27) and urea (9.68) in light (Table 3.8).

Sargassum tener► imum

Substrate saturable kinetics has been observed for all the four N species in light (Fig. 3.2 a-

d) and dark (Fig. 3.2 e-h) incubations. The highest Vmax was observed for ammonium in

light followed by almost similar Vmax for nitrate and nitrite. The V rn,, for urea was four times

less than the Vm calculated for ammonium in light and dark. Even though the Vmax for

ammonium (84 liM N (g dry wt) -'h -1) was much higher than for nitrite in light it was

interesting to note that the Vmax in dark incubations for both are almost similar (50 µM N (g

dry wt) -1 11" 1 ). The analysis of covariance showed that the differences in the uptake rate of

nitrate and ammonium when compared was significant in both light (Table 3.3) and dark

(Table 3.4). It is also seen that the differences in the velocity of nitrite, ammonium and urea

uptake when compared in light and dark were significant whereas the nitrate uptake was

insignificant (Table 3.5). Similar to S. marginatum, highest safety factor was calculated for

nitrite (22.47) in light for this species (Table 3.8).

Ulva lactuca

Substrate saturable kinetics has been observed for all the N species in light and dark (Fig.

3.3 a-d and Fig. 3.3 e-h respectively). It is interesting to note that the Vmax for ammonium in

48

dark (105 µM N (g dry wt)'h') was almost thrice higher than in light (38 µM N (g dry wt)'h

1 ) but the affinity for ammonium in tight was comparatively higher than in dark. Similar Vmax

(84 µM N (g dry wt) "'h"') values for nitrate in light and dark and a = 8 and 11 respectively for

nitrate in light and dark were observed. The affinity for nitrite (a = 19) and urea (a = 9) was

similar in light and dark but the affinity for nitrite in both light and dark was twice higher than

that for urea. Except for the significant differences in the ammonium uptake rates (F= 47.5,

d. f. = 1, 16, p <0.05) in light and dark (Table 3.5), the differences in the nitrite, nitrate and

urea uptake rates in light and dark were insignificant (Table 3.5). The safety factor

calculated for nitrite in both light (14.38) and dark (10.69) was highest compared to the

safety factors calculated for other N nutrients (Table 3.8).

Dictyota dichotoma

Uptake of all four N nutrients followed substrate-saturable kinetics (Fig. 3.4 a-d and Fig. 3.4

e-h for light and dark respectively). The Vmax values for light and dark incubations were

almost similar for all the N species (Vmax for NO3 = 105 and 102; NOZ = 54 and 56; urea =

20 and 22 p.M N (g dry wt) -'h' respectively in light and dark) except for ammonium wherein

the Vmax in light was thrice higher than in dark (Vmax for NH4 += 162 and 54 µM N (g dry wt)"

'ft' in light and dark respectively). The affinity for nitrite in light and dark was similar (a = 32)

and was the highest among ail, which implies that this alga can effectively acquire nitrite at

low concentrations. The analysis of covariance showed that the differences in the uptake

velocities of two different N species when compared in light (Table 3.3) and dark (Table 3.4)

were significant except for ammonium and urea in light and nitrite and urea in dark. The

analysis of covariance also showed that the differences in the uptake rates of nitrate and

nitrite (Table 3.4) when compared in light and dark were insignificant but the differences in

the ammonium uptake rates was highly significant (F = 49.5, d. f. = 1, 16, p <0.05). The

differences in the uptake of urea when compared in light and dark were also significant

(Table 3.5). The safety factor calculated for NO3 and NO2 showed similar values in light

and dark respectively whereas for NH4 + and urea the safety factor values in light were

double than that of dark (Table 3.8).

49

Padina tetrastomatica

All the four N species followed substrate saturable kinetics (Fig. 3.5 a-d and Fig. 3.5 e-h for

light and dark respectively). The V r„,, of nitrate and nitrite in light and dark showed similar

values ranging from 27 to 3411M N (g dry wty l h-1 ) but the affinity in light was comparatively

less than that of in dark. The analysis of covariance showed that the differences in the

uptake rates of nitrate in light and dark was insignificant whereas of ammonium, nitrite and

urea were significant (F = 17.5, d. f. = 1, 20, p < 0.05; F = 4.91, d. f. = 1, 20, p < 0.05 and F

= 21.3, d. f. = 1, 20, p < 0.05 for NH 4 ', NO2 and urea respectively). The safety factor

calculated for all the N nutrients showed that the safety factor for nitrate is double in light

than that of dark uptake whereas for nitrite the light uptake is double than that in dark

uptake (Table 3.8).

Tables 3.6 and 3.7 shows the data of the velocity of uptake of different N species with

different species of alga when analysis of covariance was applied. The analysis of

covariance showed that the differences in the uptake rates for U. lactuca and S. tenenimum

and U. lactuca and P. tetrastomatica for all the N species when compared in light and dark

were significant (Table 3.6 and 3.7).

p (II

M N

(g

dry

wt)-

1 Pi)

(a)

100 50 intivr:4 8 0

0 20 40 60

(b)

50 • • ■

20 40 60

(C)

50

;?

...T, ■

• a

0 20 40 60

(d)

40 - ■

20 0 I I

20 40 60

Concentration (04)

Figure 3.1: Uptake rate as a function of substrate concentration (NH 4+ (a), NO3 (b), NO2- (c)

and urea (d)) for S. marginatum in light. Curves are fitted by using Michaelis Menten

equation

50

6040 --

20 0

(h)

sp—r1"—.■

• p r

(e)

■ 100

50

(f)

100

50

0

(9)

• 11

0 20 40 60

0

0 20 40 60 0 20 40 60 0 20 40 60

200

100

0

p ON

N (g

dry

`nit)

-' h

it)

(a)

(b)

15°°0

0 t/11 0 20 40

100 •

50 ,111-5-1-1 0

0 20 40

(e)

100

50

0

0 20 40

(c)

100 iiieriTt. 50

0

30 20 10 0

(d)

0 20 40 0 20 40

(g) (h)

100 ao 50 20 ■

0 0

0 20 40 0 20 40

Concentration (RM)

Figure 3.2: Uptake rate as a function of substrate concentration (N1-14 + (a, e), NO3 (b, f),

NO2 (c, g) and urea (d, h) in light and dark respectively) for S. tenerrimum. Curves are fitted

by using Michaelis Menten equation

p (p

.A4 N

(g d

ry W

t)-1 h

-1)

(a)

200

100

0 I • I 101 0 20 40 60

(b)

100 - ■ 50

0

0 20 40 60

Concentration (11M)

Figure 3.3: Uptake rate as a function of substrate concentration (NH4 + (a, e), NO3 (b,

NO2 (c, g) and urea (d, h)) in light and dark respectively) for U. lactuca. Curves are fitted by

using Michaelis Menten equation

51

p (4

M N

(g

dry

wti

l h-)

(a)

200 100 irirsLi

■

_■

_nr: 150 100 50 0

(b)

pe—O—ir-11

100

50

(c)

0 50 0 50 so

60 40 20

0

(e)

150 100 50

0

(f)

iota_ ■ 100

50

spr

(g)

• 0 50 0 50 0 50

(d)

30 20 10 tii—a—a—•7 0

0

50

(h)

30 20 jerj—e—P—, 10 • •

0 0 50

p (.

1.M N

(g

dry

wt)

-' 11

1)

Concentration (JIM)

Figure 3.4: Uptake rate as a function of substrate concentration (NH 4+ (a, e), NO3 (b, f),

NO2 (c, g) and urea (d, h) in light and dark respectively) for D. dichotoma. Curves are fitted

by using Michaelis Menten equation

Concentration (ti,M)

Figure 3.5: Uptake rate as a function of substrate concentration (NO2 (a) in light) for C.

sertulariodes. Curves are fitted by using Michaelis Menten equation

52

60 ao 20 0

(a)

■

■

(b)

ao 20 ir:171

0 0 20 40 60

60 40 20

(c)

■

0 20 40 60 0 20 40 60

(f)

ao 20

0 0 20 40 60

(g)

20

0 0 20 40 60

(h)

40

20

rrr" 0 0 20 40 60

p GL

M N

(g d

ry w

til W

1)

Concentration (RM)

Figure 3.6: Uptake rate as a function of substrate concentration (NH 4+ (a, e), NO3 (b, f),

NO2 (c, g) and urea (d, h) in light and dark respectively) for P. tetrastomatica. Curves are

fitted by using Michaelis Menten equation

53

Table 3.2: Values of the uptake kinetic constants (V maxN (g dry wt) -1 1.1-1 ), K (plA N) and

a for different species of alga during the incubations in constant light and dark.

Species Light Dark Nitrate Vmax Ka a Vmax Ks a S. marginatum 31.38 9.21 3.4 - - - S. tenerrimum 52.09 0.78 66.78 31.37 1.16 27.04 U. iactuca 84.20 8.27 10.18 85.05 11.61 7.32 D. dichotoma 105.47 7.77 13.57 102.65 24.00 4.27 P. tetrastomatica 32.67 6.77 4.82 34.71 4.39 7.91 Nitrite S. marginatum 47.44 10.64 4.45 - - - S. tenerrimum 59.33 10.71 5.54 50.22 3.01 16.67 U. lactuca 127.18 6.69 19.01 92.25 4.84 19.05 D. dichotoma 54.24 1.62 33.38 56.93 1.74 32.72 C. sertulariodes 163.65 1.29 126.76 - - - P. tetrastomatica 32.68 2.72 12.02 27.74 1.29 21.44 Ammonium S. marginatum 96.50 28.80 3.35 - - - S. tenerrimum 84.82 10.20 8.31 51.67 0.97 53.16 U. lactuca 38.58 0.59 65.16 105.78 5.64 18.73 D. dichotoma 161.95 29.56 5.47 54.15 10.75 5.03 P. tetrastomatica 64.29 17.80 3.61 56.36 2.15 26.22 Urea S. marginatum 46.89 21.68 2.16 - - - S. tenerrimum 21.32 1.12 19.08 16.07 4.83 3.32 U. lactuca 37.17 3.92 9.48 45.62 4.92 9.27 D. dichotoma 20.78 1.90 10.92 22.37 5.97 3.74

P. tetrastomatica 44.73 0.90 49.65 28.73 10.84 2.65

54

U)

(b)

4

2 101

0 20 40 60 80

(c)

3 2 1 0

0 20 40 60 80

(a)

0 1.0.5 kolio

0.0 0 50

(e)

1.0 love,

0.5 0.0

0 50

(C)

2.0

0 1.0

.0 0 50

(g)

2.0 1000841, 1.0

0.0 0 50

(d)

4.0 1.100,00 2.0

0.0 0 50

Concentration (RM)

Figure 3.7: Hanes- Woolf transformed plots of N uptake data for S. marginatum in light for

NH4+ (a), NO3 (b), NO2 (c) and urea (d). 95 % confidence intervals are shown for the linear

regressions.

(b)

(f)

2 ■

1 0

0

50

Concentration (p.M)

Figure 3.8: Hanes- Woolf transformed plots of N uptake data for S. tenenimum (NH4+ (a, e),

NO3 (b, f), NO2 (c, g) and urea (d, h) in light and dark respectively). 95 % confidence

intervals are shown for the linear regressions

55

(e)

0.4

0.2

0.0

0 20

(f)

1.0 ilekor

0.5

0.0

0 20 40 60

(c)

0.6

0 0.4

.2 reuvoneagoir n

0.0

0 50

(a)

1.0 11001

0.5

0.0

0 20

0 50

(b)

1 0.5

0

0 30 60

(c)

1.5 1000000 1.0

0.5 0.0

0 50

1

eis•-•°' 0.5

0

(f) (e)

01 10144° 0 50

2

(a)

0.6 0.4 0.2

0

0 50

Concentration (aM)

Figure 3.9: Hanes- Woolf transformed plots of N uptake data for U. lactuca (NH4+ (a, e),

NO3 (b, f), NO2 (c, g) and urea (d, h) in light and dark respectively). 95 % confidence

intervals are shown for the linear regressions.

Concentration (A)

Figure 3.10: Hanes- Woolf transformed plots of N uptake data for D. dichotoma (NH4+ (a, e),

NO3 (b, f), NO2 (c, g), and urea (d, h) in light and dark respectively). 95 % confidence

intervals are shown for the linear regressions.

56

3.0 2.0 1.0 0.0

0 50 100

(d)

2.0 ei:

1.0

0.0 • 0 50 100

1

0.5

0 20 40

(e)

Concentration (11M)

Figure 3.11: Hanes- Woolf transformed plots of N uptake data for C. sertulariodes for NO2-

(a) in light. 95 % confidence intervals are shown for the linear regressions.

Concentration (LiM)

Figure 3.12: Hanes- Woolf transformed plots of N uptake data for P. tetrastomatica (NI-14+

(a, e), NO3 (b, f), NO2 (c, g) and urea (d, h) in light and dark respectively). 95 % confidence

intervals are shown for the linear regressions

57

Table 3.3: Variance ratios computed for differences in uptake of different N compounds by

a given algal species in light (* p < 0.05; ** p < 0.01)

N species S. marginatum S. tenenimum U. lactuca D. dichotoma P. tetrastomatica

F ratio df F ratio df F ratio df F ratio df F ratio df

NO3" - NH4+ 54.78** 1,20 35.61** 1,16 1.42 1,18 11.45** 1,16 21.82** 1,20

NO3- - NO2- 4.73* 1,20 31.30** 1,16 2.0 1,20 22.87** 1,16 3.78 1,20

NO3- urea 10.04** 1,20 2.65 1,16 17.63** 1,20 32.41** 1,16 12.73** 1,20

NH4 - NO2- 26.19** 1,20 2.81 1,16 2.15 1,18 98.49" 1,16 39.02** 1,20

NH4+ - urea 25.66** 1,20 41.58** 1,16 1.51 1,18 - 1,16 54.96" 1,20

NO2- urea 0.27 1,20 40.03** 1,16 20.9** 1,20 5.42* 1,16 2.98 1,20

Table 3.4: Variance ratios computed for differences in uptake of different N compounds by

a given algal species in dark (* p < 0.05; ** p < 0.01)

N species S. tenerrimum U. lactuca D. dichotoma P. tetrastomatica

F ratio df F ratio df F ratio df F ratio df NO3" - NH4+ 1.07 1,16 35.28** 1,18 17.94** 1,16 0.49 1,20

NO3- NO2- 10.11** 1,16 1.38 1,20 56.65** 1,16 10.84** 1,20

NO3- urea 1.04 1,16 16.38** 1,20 69.45** 1,16 2.28 1,20

NH4+ - NO2- 5.96** 1,16 39.84** 1,18 12.35** 1,16 10.92** 1,20

N H4+ - urea 0.03 1,16 83.97** 1,18 18.79** 1,16 0.34 1,20

NO2- - urea 7.1** 1,16 6.18** 1,20 0.42 1,16 25.75** 1,20

Table 3.5: Variance ratios computed for differences in uptake of N compound in light and

dark by a given algal species (* p < 0.05; ** p < 0.01)

N species S. tenemmum U. lactuca D. dichotoma P. tetrastomatica

F ratio df F ratio df F ratio df F ratio df

NO3 0.25 1,16 0.3 1,20 0.44 1,16 0.38 1,20

NH4+ 33.06** 1,16 47.5** 1,16 49.5** 1,16 17.15** 1,20

NO2 9** 1,16 3.61 1,20 0.1 1,16 4.91* 1,20

Urea 5.62* 1,16 1.56 1,20 6.91** 1,16 21.3** 1,20

58

Table 3.6: Variance ratios computed for differences in uptake of a given N compound by

different algal species in light (* p < 0.05; ** p < 0.01) (S. marginatum- SM, S. tenerrimum - ST, U. lactuca- UL, Caulerpa sertulariodes- CS, D. dichotoma- DD, P. tetrastomatica- OT)

Algal species

NO3- NH4+ Urea NO2-

F ratio df F ratio df F ratio df F ratio df SM- ST 14.43** 1,16 1.15 1,16 71.58" 1,16 1.25 1,16 SM- UL 13.83" 1,16 14.8** 1,16 12.96** 1,16 1028** 1,16 SM- DD 17.24** 1,16 16.56** 1,16 58.84** 1,16 11.41** 1,16 SM- CS - 1,16 - 1,16 1,16 0.06 1,16 SM- PT 0.08 1,16 7.35** 1,16 50.4** 1,16 15.42** 1,16 ST- UL 29.82** 1,16 4.59* 1,16 14.48** 1,16 6.32" 1,16 ST- DD 30.68** 1,16 20.01** 1,16 1.43 1,16 17.07 1,16 ST- CS - 1,16 - 1,16 1,16 0.36 1,16 ST- PT 11.08 1,16 1.15 1,16 2.47 1,16 21.11** 1,16 UL- DD 0.83 1,16 23.73** 1,16 9.81** 1,16 22.62** 1,16 UL-CS - 1,16 - 1,16 1,16 7.43** 1,16 UL- PT 14.72** 1,16 5.07* 1,16 7.35" 1,16 24.46** 1,16 DD- CS - 1,16 - 1,16 1,16 5.94* 1,16 DD- PT 18.06** 1,16 37.53" 1,16 0.26 1,16 0.2 1,16 CS- PT - 1,16 - 1,16 1,16 7.34" 1,16

Table 3.7: Variance ratios computed for differences in uptake of a given N compound by

different algal species in dark (* p < 0.05; ** p < 0.01) (S. tenemmum - ST, U. lactuca- UL, Caulerpa sertulariodes- CS, D. dichotoma- DD, P. tetrastomatica- PT)

Algal species

NO3 NH4 urea NO2

F ratio df F ratio df F ratio df F ratio df ST- UL 42.21** 1,16 96.45** 1,16 11.33** 1,16 8.35** 1,16 ST- DD 89.4** 1,16 25.85** 1,16 2.76 1,16 0.71 1,16 ST- PT 9.61** 1,16 6.25" 1,16 17.29** 1,16 10.55** 1,16 UL- DD 1.98 1,16 55.11** 1,16 5.14* 1,16 11.84** 1,16 UL- PT 23.89** 1,16 71.89** 1,16 0.01 1,16 22.27" 1,16 DO-PT 54.79** 1,16 5.58* 1,16 7.11" 1,16 6.53** 1,16

59

Table 3.8: Calculated safety factors for NO3, NH4+, NO2 and urea for macroalgae in light

and dark (L- light; D- dark).

Algal species NO3- NH4+ NO2- urea Sf n Sf n Sf n Sf n

S. marginatum (L) 1.84 9 5.79 12 2227 11 9.68 13 S. tenerrimum (L) 3.06 12 2.70 12 22.47 16 1.44 8 S. tenenimum (D) 1.84 9 1.16 12 7.02 12 2.93 8 U. lactuca (L) 4.95 14 1.09 8 14.38 9 2.56 14 U. lactuca (D) 5.00 12 1.94 8 10.69 9 2.96 14 D. dichotoma (L) 620 12 5.93 10 4.25 14 1.76 10 D. dichotoma (D) 6.03 12 2.79 9 4.48 13 3.39 12 C. sertulariodes (L) - - _ - 3.58 18 - - P. tetrastomatica (L) 1.92 16 3.96 8 6.44 16 1.36 15 P. tetrastomatica (D) 2.04 17 1.35 6 3.58 12 6 .34 11

3.1.3.2. N uptake kinetics for macroalgae in an oligotrophic environment

3.1.3.2.1 Ambient N concentrations

The minimum and the maximum ambient nitrite, nitrate, ammonium and urea concentration

measured during the experimental period ranged from 0 to 0.23 (average 0.10 ± 0.01 1AM N,

n=8), 0.08 to 1.09 (average 0.61 ± 0.43 pM N, n=9), 0.79 to 1.44 (average1.09± 0.28 µM N,

n=9) and 0.34 to 3.3511M N (average1.70 ± 1.16 pM N, n=9) respectively.

3.1.3.2.2 Uptake kinetics

All the species of macroalgae namely, U/va lactuca, Dictyota dichotoma, Padina

tetrastomatica, Caulerpa sertulariodes and Soliera robusta under study exhibited saturable

kinetics within 40 pM of the substrate for all the four forms of N (Figs 3.13 to 3.17).

The maximum N uptake rates and the affinity for N differed from species to species. Among

the five species of algae, D. dichotoma exhibited highest Vmax values for ammonium and

urea; C. settula►iodes exhibited highest V„‘„ for nitrate and U. lactuca for nitrite. The

60

greatest affinity for ammonium and urea was shown by C. sertulariodes, for nitrate by U.

lactuca and for nitrite by D. dichotoma (Table 3.9). The N uptake rates in P. tetrastomatica

were lower compared to the uptake rates observed in other species, but it showed high

affinity for ammonium and nitrate. It was observed that D. dichotoma had highest Vmax

values for the reduced forms and high affinity for nitrite.

The preference (affinity) order of U. lactuca, D. dichotoma, P. tetrastomatica, S. robusta

and C. sertulariodes for N species were NO3 > NH4+ > NO2 > urea, NO2 > NO3 > urea >

NH4+, NO3- > NH4+ > urea > NO2, NH4+ > NO2- > NO3> urea and NH4+ > NO2- > NO3 > urea

respectively.

The nitrate was preferred by the different species in the following order in light: U. lactuca >

C. sertulariodes > P. tetrastomatica > D. dichotoma > S. robusta, whereas ammonium was

preferred by the following species: C. sertulariodes > S. robusta > U. lactuca > P.

tetrastomatica > D. dichotoma. The preference for urea was shown by C. sertulariodes > P.

tetrastomatica > D. dichotoma > U. lactuca > S. robusta and for nitrite it was shown in the

order of D. dichotoma > C. sertulariodes > S. robusta > U. lactuca. The above results

suggest that these species have dissimilar affinity for different N species. No fixed pattern

to take up a particular N species was observed.

Plots of the 95% confidence limits to the regression relating S/V with S (Figs 3.18 to 3.22)

demonstrate the statistical significance of the linear fits. Variance ratios computed for

differences in uptake of different N compounds by a given algal species are given in Table

3.10. Similarly, variance ratios computed for differences in uptake of the given N

compounds by different algal species are given in Table 3.11.

The safety factor calculated for all the algal species was within the range of 1 to 3 for

nitrate, for ammonium it was within the range of 1 to 3 except for high values for D.

dichotoma (30), for nitrite wide range in values (2 to 60) were observed and for urea it

ranged from 1 to 5 (Table 3.12).

61

Ulva lactuca

Substrate saturable kinetics for all the N species was observed in U. lactuca (Fig. 3.13).

Almost similar Vmax values were observed for ammonium (80 ILM N (g dry wt 1 h -1 ) and nitrite

(83 µM N (g dry wt)" lh -1). The lowest Vmax values were estimated for nitrate uptake (Table

3.8). It was also seen that the ammonium and nitrite uptake rates saturate at around 30 µM

with nitrate uptake saturating at about 10 p.M. Even though the Vmax (23.5 1AM N (g dry wt) -

1 1-11 ) for NO3 was the lowest compared to other N species, the affinity for NO3 (a = 51) was

more than twice of the other three N nutrients. The analysis of covariance showed that the

differences in the rate of uptake of ammonium and nitrite, nitrate and urea and nitrite and

urea were highly significant. Plots of the 95% confidence limits to the regression relating

SN with S (Fig. 3.18) demonstrate the statistical significance of the linear fits. The safety

factor calculated for nitrite was 61.53, which was the highest, compared to the other N

nutrients (Table 3.12).

Dictyota dichotoma

Substrate saturable kinetics has been observed for all the N species (Fig. 3.14) with the

highest Vmax values for ammonium (127 1.L.M N (9 dry wt) -'h -9 ) and lowest for nitrate (47 1.1.M

N (g dry wt)4h-1). Compared to other N species greatest affinity (a=129) was seen for

nitrite. The analysis of covariance showed that the differences in the uptake rates of

ammonium and nitrite was (F = 89.1, d. f. = 1, 7, p < 0.05) highly significant compared to

nitrate and urea (F = 31.75, d. f. = 1, 12, p < 0.05) and nitrite and urea (F = 23.75, d. f. = 1,

7, p < 0.05). Plots of the 95 % confidence limits to the regression relating SN with S (Fig.

3.19) demonstrate the statistical significance of the linear fits. The safety factor for

ammonium was 30.93 which was the highest compared to the other N nutrients (Table

3.12).

Padina tetrastomatica

Substrate saturable kinetics was observed for all the N species (Fig. 3.15). The V ma,, values

for all the N species are almost similar (Vmax values for nitrate, nitrite, ammonium and urea

are 18, 19, 22 and 10 1.1.M N (g dry wt) -1 1-1 respectively. The affinity for nitrate (a = 30) and

62

ammonium (a = 21) was ten times higher than that of nitrite and urea. Plots of the 95%

confidence limits to the regression relating SN with S (Fig. 3.20) demonstrate the statistical

significance of the linear fits. Similar to the safety factor calculated for ammonium in U.

lactuca even this species show the highest safety factor for nitrite (25.99) (Table 3.12);

Soliera robusta

Substrate saturable kinetics was observed for all the N species (Fig. 3.16). Although the

Vmax values for nitrate, ammonium and urea were similar, the greatest affinity (a = 47) was

shown for ammonium. Plots of the 95% confidence limits to the regression relating SN with

S (Fig. 3.21) demonstrate the statistical significance of the linear fits. Similar to U. lactuca

and P. tetrastomatica this species also showed high safety factor for the nitrite (11.35) but

the values were comparatively lower than the other two (Table 3.12).

Caulerpa sertulariodes

All the N species exhibited substrate saturable kinetics (Fig. 3.17). The highest and lowest

Vrn,, values were observed for nitrate (78 µM N (g dry wt) -'h -') and ammonium (36 OA N (g

dry wt)4 11-1 ) respectively. The analysis of covariance showed that the differences in the

uptake rates of most of the N species when compared were insignificant except for nitrate

and ammonium (F = 18.45, d. f. = 1, 8, p < 0.05) and ammonium and urea (F = 27.81, d. f.

= 1, 8, p < 0.05). Plots of the 95 % confidence limits to the regression relating SN with S

(Fig. 3.22) demonstrate the statistical significance of the linear fits. The safety factors for all

the N nutrients are almost similar except for NO2 (5.86) (Table 3.12).

63

0

■ 11P16-7;t.

20 40

p (p

.M N

(g d

ry w

tyl F

il) (d)

40 -

20 -

0

(a)

200

1111

O 10 20 30 40

(b)

tto 20 ill,

0

.1141---11r—

0 20 40

Concentration (11M)

Figure 3.13: Uptake rate as a function of substrate concentration (NH4+ (a), NO3 (b), NO2 -

(c) and urea (d)) for U. lactuca in light. Curves are fitted by using Michaelis Menten

equation

(a)

(b)

(c)

(d)

100

50 sore_.-1—

0 O 20 40

100 10471_7

50

0

0 20 40

100

50

0

0 20 40

100 • 50 eir■

0 0 20 40

Concentration (JIM)

Figure 3.14: Uptake rate as a function of substrate concentration (NH 4+ (a), NO3 (b),

NO2- (c) and urea (d)) for D. dichotoma in light. Curves are fitted by using Michaelis Menten

equation

p (AM

N (g

dry

wti

l h-1

)

(b)

(c)

30 • 20 V 10 0

0 20 40

30 - 20 ■ 10 0

0 20 40

Concentration (µM)

Figure 3.15: Uptake rate as a function of substrate concentration (NH4 (a), NO3 (b),

NO2 (c) and urea (d)) for P. tetrastomatica in light. Curves are fitted by using Michaelis

Menten equation

64

(a)

60 40 20 0

0 20 40

p (g

M N

(g d

ry w

til (d)

30 20 10 0 0 20 40

Concentration (bM)

Figure 3.16: Uptake rate as a function of substrate concentration (NH 4+ (a), NO3 (b),

NO2- (c) and urea (d)) for S. robusta in light. Curves are fitted by using Michaelis Menten

equation

p N

(g d

ry w

til h

-.1)

(b)

(c)

100 ■

50

0 0 20 40

150 100 101,.,1

50• 0 0 20 40

Concentration (RM)

Figure 3.17: Uptake rate as a function of substrate concentration (NH4 (a), NO3 (b),

NO2- (c) and urea (d)) for C. sertulariodes in light. Curves are fitted by using Michaelis

Menten equation

65

Table 3.9: Values of the uptake kinetic constants N (g dry wt)"' hi), Ks (gM N) and

a for different species of alga during the incubations at constant light.

Macroalgat species Vmax Ks a Nitrate

U/va lactuca 23.51 0.46 51.10 Dictyota dichotoma 46.74 2.06 22.71 Padina tetrastomatica 18.03 0.58 30.76 Soliera robusta 28.47 3.06 9.30 Caulerpa sedulariodes 78.66 2.19 35.95 Nitrite

U/va lactuca 83.07 13.88 5.98 Dictyota dichotoma 51.04 0.39 129.87 Padina tetrastomatica 19.98 5.73 3.48 Soliera robusta 36.65 2.37 15.43 Caulerpa sertulariodes 68.92 1.12 61.65

Ammonium Utva lactuca 80.48 3.08 26.14 Dictyota dichotoma 127.77 43.11 2.96 Padina tetrastomatica 22.15 1.04 21.22 Soliera robusta 28.87 0.61 46.95 Caulerpa sertulariodes 36.67 0.57 64.33 Urea

Ulva lactuca 30.28 6.41 4.72 Dictyota dichotoma 87.43 14.36 6.09 Padina tetrastomatica 10.81 1.59 6.78 Soliera robusta 25.19 8.59 2.93

Caulerpa sertulariodes 59.28 4.79 12.35

66

1

0.5

0

0 20 40

(a) (b)

1 0.5

0

0 20 40

(b)

0.6

0.4

0.2

0

0 10 20

Concentration (LM)

Figure 3.18: Hanes- Woolf transformed plots of N uptake data for U. lactuca (NH4 (a), NO3-

(b), NO2 (c) and urea (d). 95 % confidence intervals are shown for the linear regressions

(c)

1

0.5

0

0 20 40

(d)

1

0.5

0

0 20 40

Concentration (I.LM)

Figure 3.19: Hanes- Woolf transformed plots of N uptake data for D. dichotoma (NH4+ (a),

NO3- (b), NO2- (c) and urea (d)). 95 % confidence intervals are shown for the linear

regressions.

Concentration (4M)

Figure 3.20: Hanes- Woolf transformed plots of N uptake data for P. tetrastomatica for NH4

(a), NO3 (b), NO2 (c) and urea (d). 95 % confidence intervals are shown for the linear

regressions.

67

Figure 3.21: Hanes- Woolf transformed plots of N uptake data for S. robusta for NH4+ (a),

NO3- (b), NO2- (c) and urea (d). 95 % confidence intervals are shown for the linear

regressions.

Concentration (AI)

Figure 3.22: Hanes- Woolf transformed plots of N uptake data for C. sertulariodes for NI-14+

(a), NO3 (b), NO2- (c) and urea (d). 95 % confidence intervals are given for the linear

regressions.

Table 3.10: Variance ratios computed for differences in uptake of different N compounds by

a given algal species (* p < 0.05; p < 0.01)

N U. lactuca D. dichotoma P. tettastomatica S. robusta C. settulatiodes

F ratio df F ratio df F ratio df F ratio df F ratio df

NO3- NH4+ 3.08 1,11 - 1.24 1,8 10.19" 1,12 18.45** 1,8

NO3- NO2- 10.32 1,2 3.74 1,7 9.59** 1,8 0.56 1,8 2.98 1,4

NO3-- urea 9.36** 1,7 31.75** 1,12 0.1 1,8 1.67 1,12 0.55 1,4

NH4+- NO2 12.05** 1,11 89.1** 1,7 10.01** 1,12 7.9** 1,8 4.99 1,8

NH4+- urea 0.03 1,16 2.01 1,12 122 1,12 23.19** 1,12 27.81** 1,8

NO2-urea 17.11*" 1,7 23.75** 1,7 15.98** 1,12 3.39 1,8 5.87 1,4

68

Table 3.11: Variance ratios computed for differences in uptake of a given N compound by

different algal species (* p < 0.05; ** p < 0.01) (U. lactuca- UL, D. dichotoma- DD, P.

tetrastomatica- PT, S. robusta — SR, Caulerpa sertulatiodes- CS)

Algal species NO3- NH4+ Urea NO2-

F ratio df F ratio df F ratio df F ratio df UL- DD 5.85* 1,7 51.95** 1,16 25.94** 1,12 0.79 1,2 UL - PT 0 1,3 5.63* 1,16 20.4** 1,12 29.18** 1,7 UL- SR 5.95* 1,7 6.17** 1,16 0.34 1,12 10.82* 1,3 UL- CS 8.87 1,3 5.68* 1,16 0.43 1,8 10.73* 1,3 DD- PT 7.07* 1,8 - - 59.04** 1,12 3.96 1,7 DD- SR 0.52 1,12 - - 31.72** 1,12 1.53 1,3 DD- CS 0.28 1,8 - - 10.26** 1,8 1.49 1,3 PT- SR 7.22* 1,8 0.3 1,12 26.64** 1,12 0.57 1,8 PT- CS 12.35** 1,8 0 1,12 35.83** 1,8 0.51 1,8 SR- CS 1.66 1,8 0.23 1,12 1.73 1,8 0 1,4

Table 3.12: Calculated safety factors for NO3, NH4 +, NO2 and urea in macroalgae.

Algal species NO3 NH4 NO2 urea

Sf n Sf n Sf n Sf n

U. lactuca 1.42 6 3.14 13 61.53 16 2.91 10 D. dichotoma 2.87 10 30.93 16 2.71 8 5.28 7 P. tetrastomatica 1.53 8 1.72 16 25.99 11 1.47 14 S. robusta 3.78 9 1.43 16 11.35 16 3.56 11 C. settulariodes 2.99 16 1.39 16 5.86 16 2.43 14

3.1.3.3 Comparison of the N uptake between three different algal species from two

different environmental conditions (eutrophic and oligotrophic)

When the kinetic constants for U. lactuca, D. dichotoma and P. tetrastomatica from

eutrophic (Goa) and oligotrophic (Lakshadweep) waters were compared it was observed

that the Vm,, and Ks for nitrate uptake were always higher in eutrophic waters but the affinity

was lower. The Vma, values for the nitrite uptake for U. lactuca and P. tetrastomatica from

oligotrophic waters was higher. D. dichotoma showed almost similar V,„,„ (50 RNA N (g dry

wt)-1 111 ) values from both the areas but affinity for nitrite in eutrophic (33 µM N (g dry wt)" 1 h-

1 ) was lower than the oligotrophic (129 1AM N (g dry wt1 1 h-1 ). The Vm,, values for the

ammonium uptake for D. dichotoma (161 1.1.M N (g dry wt) -'h -1 ) and P. tetrastomatica (64 RAA

69

N (g dry wt)"l h-1 ) were higher in eutrophic waters whereas for U. lactuca (80 RM (g dry wt)"'

h-1) the Va,,, values were higher in oligotrophic waters. The affinity for ammonium was

twice higher in D. dichotoma and U. lactuca and 7 times lower in P. tetrastomatica in

eutrophic system. Almost similar V max (30 RM N (g dry wtyl h-1 )) values for urea were

observed in U. lactuca from both the environments, but the affinity to take up urea was

twice higher in the species from eutrophic waters. The Vm,, values for D. dichotoma (87

(g dry wt)-' h-1 ) from oligotrophic waters was thrice higher than that of eutrophic waters. In

contrast to this the V m,, values for P. tetrastomatica (44 I.LM (g dry wtyl h-1) in eutrophic

waters were four times higher than the oligotrophic waters and the affinity for urea in

eutrophic water was also 8 times higher than the oligotrophic waters. It was observed that

the affinity for urea was high in all the three species of eutrophic waters. From the results it

was seen that affinity for nitrate in eutrophic species was always lower than the oligotrophic

species.

The comparative chart showing the variance ratios for differences in uptake of the given N

compound by different algal species from both the study sites are given in Table 3.13. The

analysis of covariance showed that the differences in the uptake rate for nitrate in all the 3

species in both the waters were significant (for U. lactuca F = 9.49, d. f. = 1, 9, p < 0.05, D.

dichotoma F = 11.45, d. f. = 1, 14, p < 0.05 and P. tetrastomatica F = 7.7, d. f. = 1, 10, p <

0.05), but for nitrite it was insignificant (Table 3.13). The analysis of covariance comparing

the differences in the uptake rates for ammonium from eutrophic and oligotrophic species

revealed significant differences only for P. tetrastomatica (F = 36.53, d. f. = 1, 14, p < 0.05)

whereas for urea significant differences were observed only for D. dichotoma (F = 82.14, d.

f. = 1, 14, p < 0.05).

Table 3.13: Comparative chart showing the variance ratios computed for differences in

uptake of a given N compound by U. lactuca, D. dichotoma and P. tetrastomatica from the

oligotrophic (0) and eutrophic (E) waters (* p< 0.05, ** p< 0.01).

Species NO3 F df

NI-14+ F df

Urea F df F

NO2 df

U. lactuca (0-E) 9.49** 1,9 0.5 1,16 1.43 1,14 0 1,9

D. dichotoma (0-E) 11.45** 1,14 2.49 1,14 82.14** 1,14 1.55 1,9

P. tetrastomatica (0-E) 7.7** 1,10 36.53** 1,14 2.25 1,14 0.35 1,14

70

3.1.4 Discussion

Nitrogen, an element for growth can potentially be taken up as dissolved inorganic N (DIN)

and dissolved organic N (DON). From the present study, it is clear that the uptake rates of

N differ in different species (species specific). The macroalgae growing in oligotrophic and

eutrophic habitats have different capacities to take up different N nutrients. The uptake

rates are also dependent on external N concentrations. Several authors have also shown

that uptake kinetics for a given algal species are plastic and can vary under different

environmental conditions (Wallentinus, 1984; Thomas at at., 1987; Lavery and McComb,

1991; Fong et al., 1994). Some of them have shown dependency on the external N

concentrations (Haines and Wheeler, 1978; Harlin, 1978; Harlin and Craigie, 1978).

In this study the relationship between nutrient uptake and external nutrient concentration

were hyperbolic and could be fitted to Michaelis Menten equation. This substrate saturable

kinetics (N uptake) indicated active transport in both light and dark for the algae collected

from Goa, concurring the hypothesis that saturable N uptake is the characteristic of the

macroalgae growing under high N conditions. The N uptake as well showed substrate

saturable kinetics for the algae collected from Lakshadweep (from where only light uptake

was studied), thus partially obeying the hypothesis that nonsaturable uptake is a

characteristic of macroalgae growing under low N conditions. So the hypothesis that was

earlier put forth is partially valid.

There are few other studies where macroalgae directly comparable to the ones which are

used in this study have been used. Furthermore, the enhancement in the uptake rates as

observed in figs 3.1 to 3.6 and 3.13 to 3.17 is real and not simply the result of our arbitrarily

starting the uptake curves at the origin. In this case the origin must be considered a valid

datum point because at time zero there is no accumulation of 15 N.

Moreover, Wallentinus (1984) had earlier reported that the higher nutrient uptake of the

macroalgae have implications for the competitive outcome in nutrient enriched

environments. Rosenberg and Ramus (1982) concluded that high nitrogen uptake rates are

necessary for the strategy of the opportunistic macroalgae. The high uptake rates should be

looked upon as the potential, through luxury uptake, to utilize nutrients which are suddenly

71

made available from sources such as animal excretion and leaching of fertilizers from land

or from sewage treatment plants. The fact that the macroalgae can use temporary

increases in nutrient concentrations for growth later on has been shown in aquacultures of

macroalgae (e.g. Morgan and Simpson, 1981; Ryther et at, 1981; Tseng, 1981).

The half saturation constants (K s) - the concentration at which half the maximum uptake

rate is achieved. It is generally believed that low values of K s express a competitive

advantage at low nutrient concentrations t ,Table 3.2 and 3.9 , showed that the different

species of algae were adapted for using N nutrients from the ambient as well as from

pulsed addition of N. For example, some alga which were adapted for using a particular

water column N nutrients had its K, values similar to the concentration found in the water

column (K, values for P. tetrastomatica for nitrate in oligotrophic and eutrophic waters are

0.6 and 6µM respectively and the K s values for U. lactuca for nitrate in oligotrophic and

eutrophic waters are 0.5 and 8µM respectively), whereas in some species K s values are

order of magnitude greater, which may correspond to the pulsed concentration of N added

by the different process in the system or by organisms in and around the algae.

However, in the natural ecosystem, the half saturation constant for the algae, could be an

integrated unit of above and below ground biomass influenced by high ammonium

concentrations of sediment pore waters. For example, in the study carried out by Williams

and Fisher (1985), the half saturation constants obtained for C. cupressoides growing in

Tague Bay, with two primary sources of ammonium; one was the ammonium in sediment

pore waters (14 - 388 1.1M) and the second was ammonium in the water column (< 1 11M)

available to the blades, ranged from 19 -1111.1,M ammonium which were among the highest

reported for seaweeds (Probyn and Chapman, 1982; Wallentinus, 1984). This could be

more applicable to the alga adapted for deriving N from the sediment pore waters at a much

higher rate than from water column.

It was expected that the K G values for the macroalgae would be higher in eutrophic and

lower in oligotrophic waters. However, the results of this study showed contrasting

behaviour in most of the algae when compared with the K s values from two different waters.

The K, values for different N species ranged from 0.3 to 43 µM N in species collected from

Lakshadweep and 0.5 to 28 µM N in species collected from Goa. This deviation in K s

72

values from what was expected is evidence for adaptation of the alga to the N source of

nutrient available in any form over wide concentration ranges.

Although the results of the present study have shown that the N uptake was the function of

substrate concentration and there was no negative effect, various studies have however

suggested that N addition can have different effects on macroalgal N uptake. It was

reported by Waite and Mitchell (1972) that 60 JAM ammonium was the critical concentration

for U/va sp. Other species highly intolerant to high N were also reported by Prince (1974),

Byerrum and Benson (1975) and Thomas et. a/. (1980), whereas Harlin (1978) found no

negative effect of even 100 !AM ammonium with Laminatia longicniris. Some algal species

show an ability to grow in full strength sewage effluent, such as the one reported by Chan

et. a/. (1982) with E. lima with the uptake of NH 4+ concentrations > 500 ja.M

The results of the calculation of Vmax and Ks values of the present study when compared

with the other studies shows that the differences are large within the same species of

macroalgae (Tarutani et al. 2004; Pedersen and Borum, 1997; Naldi and Viaroli, 2002).

This can be explained partly, by the differences in conditions such as temperature, light,

etc. at which the experiments were conducted. The V max and K5 values obtained using the

enhanced N nutrient concentrations could be higher than those found in the natural

environment. This data could be used to predict a species response to high nutrient pulses

and could be used to develop ecological models to predict the competitive advantage of

one species over another when the nutrients are limiting (Fong et al., 1994; Smith et al.,

1999).

Nitrite uptake

Nitrite supply to the coastal ecosystems is caused due to the processes such as summer

upwelling events (Valiela, 1995), phytoplankton excretion (Collos, 1998), bacterial oxidation

of ammonium to nitrite or primary nitrification (McCarthy et. al., 1984) and sewage inputs to

coastal areas (Brinkhuis et. al., 1989). Not much importance was given to this N species in

the uptake process of autotrophs.

The references available on the nitrite uptake by phytoplankton are also few (Wafar et al.,

2004; Collos, 1998; Conway, 1977). Nitrite uptake exceeded nitrate uptake in several

73

marine phytoplankton species in culture and natural populations (Collos, 1998). However

Wafar et al. (2004) in his studies on nitrogen uptake by size-fractionated plankton in

permanently well-mixed temperate coastal waters found that nitrite uptake did not exceed

the other three N forms in any of the three phytoplankton fractions (net-, nano- and

picoplankton).

Similar to the nitrite uptake studies on phytoplankton, the nitrite uptake kinetics has also

been largely ignored in macroalgae and restricted to few studies (Hanisak and Harlin, 1978;

Topinka, 1978; Harlin and Craige, 1978; Martinez and Rico, 2004) with different uptake

patterns. For example, Hanisak and Harlin (1978) reported that the maximum uptake rate

for nitrite was similar to nitrate uptake rates but was lower than ammonium uptake rates in

Codium fragile. In yet another instance the nitrite uptake for Pa!merle palmate was linear at

concentrations as high as 76 times greater than the maximum mean concentration in the

area studied (0.52 1.1.M) (Martinez and Rico, 2004).

In the present study the nitrite uptake showed substrate saturable kinetics in both light and

dark for the algae collected from Goa and Lakshadweep (where only light uptake was

studied). Substrate saturation for different species of algae collected from both the sites

occurred at the concentrations between 10 and 40 µM nitrite. The uptake rates for nitrite

was almost within the range of the rates measured for the other N species for all the algae

studied.

Compared to the lower Vmax obtained for U. lactuca and P. tetrastomatica of Lakshadweep,

the species collected from Goa has higher Vmax . But the Vmax for D. dichotoma from both the

sites showed similar nitrite uptake rates. However when the Vmax values were compared for

the same site (Goa) in light and dark it was similar. This suggest that even though they

show differences in the uptake rates from two dissimilar sites, there was no effect (similar)

on the Vmax when incubated in light and dark for the same site. This implies that it is

substrate dependent rather than light. In other words they can harvest nitrite at the same

rates irrespective of the availability of light. Analogous to these results were the nitrite

uptake rates for U. lactuca and S. tenemmum (Table 3.2).

The algal species collected from Goa show different maximum uptake rates in light and

dark. The variance ratios calculated for the differences in the uptake of nitrite (Table 3.5) in

74

light and dark were not significant in U. lactuca and D. dichotoma but were significant in S.

tenemmum and P. tetrastomatica (p < 0.01 and p < 0.05 respectively). Topinka (1978)

however reported that the light stimulate nitrite uptake rates in Fucus spiralis. He reported

that the for nitrite was lower in dark (0.084 J.LM cm -2 h -) compared to the values

obtained in light (0.275 Ai cm 2 h -1 ).

a has been used as a measure of uptake capacities at low concentrations (Healey, 1980).

C. sertulariodes (a = 126) in light and D. dichotoma = 32) in dark collected from Goa and

in D. dichotoma (a = 129) collected from Lakshadweep showed highest affinity for nitrite

when compared with the other N species. However the preference order (affinity) to take up

nitrite amongst the other N species was the maximum in S. marginatum and D. dichotoma

in light and U. lactuca and D. dichotoma in dark collected from eutrophic waters and D.

dichotoma collected from oligotrophic waters. These preferences for NO2 uptake suggest a

good ability to use it at lower concentrations. The wide range of a values among the

different algal species suggest that the affinity for nitrite vary from species to species.

The variance ratios calculated for the differences in nitrite uptake rates for different algal

species (Table 3.6) in light collected from eutrophic waters were significant except for the

uptake rates between S. marginatum and S. tenerrimum, S. marginatum and C.

sertulariodes, S. tenerrimum and C. sertulariodes and D. dichotoma and P. tetrastomatica.

However the variance ratios calculated for the differences in nitrite uptake rates for different

algal species (Table 3.11) in light from the oligotrophic waters were not significant except

for the uptake rates between U. lactuca and P. tetrastomatica, U. lactuca and S. robusta

and U. lactuca and C. sertulariodes. This verifies that the nitrite uptake rates are species

specific. Similarly when the variance ratios were computed for the differences in nitrite

uptake rates for the three common algal species (Table 3.13) collected from eutrophic and

oligotrophic waters were not significant suggesting that the uptake rates were almost

similar. The analysis of covariance showed that the differences in the nitrite uptake rates in

dark when compared for different algae collected from Goa (Table 3.7) were significant

except for S. tenemmum and D. dichotoma.

75

Nitrate uptake

The sources of nitrate supply to the coastal ecosystems is associated with the

oceanographic processes such as upwelling (Valiela, 1995), advection along with the land

runoff, bacterial oxidation of ammonium to nitrate or primary nitrification (McCarthy et. al.,

1984. These are the important nitrate sources for the autotrophs, however the negative

charge on nitrate means that nitrate uptake would always be energy-dependent (Ritchie,

1988).

Extensive studies carried out on the nitrate uptake in macroalgae have shown that they

follow saturable, linear and biphasic uptake patterns. Generally the nitrate uptake exhibits

saturation kinetics for Laminaria longicruris (Harlin and Craigie, 1978), Fucus spiralis

(Topinka, 1978), F. distichus (Thomas et al., 1985), Ceramium tenuicome (Wallentinus,

1984), Stictosiphonia arbuscula, Apophlaea Iyaffii, Sctohamnus austrails and Xiphophora

gladiata (Phillips and Hurd, 2004), Himantothallus grandifolius and Laminaria solidungula

(Korb and Gerard, 2000), Macrocystis pyrifera and Hypnea musciformis (Haines and

Wheeler, 1978) indicating active transport but linear uptake with the increase in nitrate

concentration for Laminaria groenlandica (Harrison et al., 1986), Chaetomorpha linum

(Lavery and McComb, 1991), Gracilaria pacifica (Thomas et al., 1987), Chondrus crispus

(Amat and Braud, 1990) has also been reported. Biphasic nitrate uptake kinetics is also well

known in marcroalgae (Conolly and Drew, 1985; Lavery and McComb, 1991; Collos et al.,

1992).

In this study the nitrate uptake was substrate saturable in all the species collected from both

the sites and the saturation occurred at the concentrations between 10 to 40 1AM nitrate.

The maximum uptake rates measured for U. lactuca collected from Goa showed that the

values of maximum uptake rates (84 and 85 11M N (g dry wt) -' h".1 in light and dark

respectively) are comparable with the values reported by Tarutani et al. (2004) for U.

pertusa (59-85 1.1.M N (g dry wt) -1 h-1 ) in light. But the maximum uptake rates for U. lactuca

collected from Lakshadweep and incubated in light showed comparatively low values (23

JAM N (g dry wt) -1

The Vmax and Ks for U. lactuca studied in Denmark and reported by Pedersen and Borum

(1997) are 20 WIN (g dry wt) -1 h-1 and 5µM respectively. This Vm,, value is equivalent with

76

the one reported in this study from Lakshadweep but the half saturation constant for nitrate

(Ks = 0.46 Al) is less than that reported by Pedersen and Borum (1997). Similarly Vmax

reported for another species Ulva rigida (72 11M N (g dry wt)"' h -1) from West Australia by

Lavery and McComb (1991) are closer to the values reported here for U. lactuca (85 RIVI N

(g dry wt) -' h -1 ) collected from Goa. Naldi and Viaroli (2002) studied the nitrate uptake in U.

ngida from an highly eutrophic coastal lagoon (Sacca di Goro, Italy) and reported the Vmax

(68.61 }AM N (g dry wt) -' h -1 ) values quite lower than those reported in this study for U.

lactuca in the eutrophic waters but the K s (38 gM) value reported from the same study by

them were thrice higher than the values reported for U. lactuca in this study.

The half saturation constant for U. lactuca collected from Goa was almost similar in both

light and dark (8 and 11 pM respectively) but for the same species from Lakshadweep very

low value (0.4611M) were observed. All these values reported here in this study are

comparatively less than those reported (18 - 34 gM) by Tarutani et al. (2004).

The Vmax value for nitrate in D. dichotoma collected from Goa (Table 3.2) is more than

double the value for nitrate collected from Lakshadweep (Table 3.9). The K s value for

nitrate (Table 3.2) for D. dichotoma collected from Goa are thrice higher in dark than in light

showing that it has higher affinity for nitrate during light incubations. This result suggests

that the nitrate uptake is a light dependent process. Also the affinity for nitrate is more for D.

dichotoma collected from Lakshadweep which highlights its adaptation to N procurement in

an environment where N supply may be limited. Also the low uptake efficiency for nitrate in

dark for S. tenerrimum and U. lactuca collected from Goa suggest the light dependent

process.

The variance ratios calculated between the differences in nitrate uptake rates in light and

dark for different algal species (Table 3.5) collected from eutrophic waters were not

significant in any species. However when the variance ratios were calculated for the

differences in the uptake rates between two different algal species (in light) showed

significance only in few species (Tables 3.6 and 3.11), but the differences in the uptake

rates between two different species in dark (Table 3.7) showed significance in the uptake

rates in all the species except for the combination of U. lactuca and D. dichotoma. This

indicates that similar to nitrite, the nitrate uptake rates are also species specific. The

77

variance ratios for the differences in the nitrate uptake rates between the common species

of the eutrophic and oligotrophic waters were significant (Table 3.13) suggesting that the

nitrate uptake rates were dissimilar.

Ammonium uptake

The important ammonium source for autotrophs (macroalgae) is the ammonium excreted

as the waste product by heterotrophs (Kautsky and Wallentinus, 1980; Probyn and

Chapman, 1983; Taylor and Rees, 1998; Hurd et al., 1994; Williamson and Rees, 1994;

Bracken, 2004; Bracken and Nielsen, 2004) and land runoff and ground water seepage

(Naim, 1993).

The uptake rate for ammonium in macroalgae generally exceeds that for nitrate (Topinka,

1978; Haines and Wheeler, 1978; Wallentinus, 1984; Pedersen and Borum, 1997; Phillips,

2001; Naldi and Wheeler, 2002; Phillips and Hurd, 2004).

The nutrient past history of the algae plays an important role in the N uptake rates. For

example the uptake rates of U. lactuca grown at the high ammonium flux was much slower

than uptake by field collected algae at all the concentrations upto 60 µM ammonium

following the Michaelis Menten kinetics. However this species could take up ammonium

rapidly at all concentrations upto 60 1.IM immediately after collection from the field following

the linear uptake (Fujita, 1985). Runcie et al. (2003) reported that Ulva lactuca and

Catenella nipae exhibited saturable kinetics and no evidence were found for ammonia

toxicity over the range of concentrations upto 1200 I.LM ammonium. Conversely there are

reports of ammonium toxicity for example in Ulva lactuca ammonium was found to be toxic

at high concentrations (> 30 - 50 g,M) (Waite and Mitchell, 1972).

Saturable kinetics was reported by Probyn and Chapman (1982), Korb and Gerard (2000),

,Rosenberg et al. (1984), Fujita (1985), Pedersen (1994), Pedersen and Borum (1997),

Schaffelke and Klumpp (1998), Campbell (1999), etc. Codium fragile has shown ammonium

saturation kinetics, suggesting active transport (Hanisak and Harlin, 1978). Campbell

(1999) determined the kinetics of ammonium for macroalgae from nutrient enriched waters

of Port Phillip Bay and reported that at the highest concentration examined (28.6 µM N),

78

Hincksia sordida had a higher rate of uptake (435 1AM N (g dry wt) -' h' 1 ) than Ulva sp. (108

p.M N (g dry wt)"' h -1 ) or Polysiphonia decipiens (53 µM N (g dry wt) -' h -1 ).

Sargassum baccularia from Great Barrier Reef subjected to land run off (Schaffelke and

Klumpp, 1998) have the substrate saturation at the concentrations above 30 RM ammonium

with Vmax of 111 p.M N (g dry wt) -' h -1 and Ks of 15.6 ji.M. However, from the present study it

was observed that the Vmax and VC values of S. tenerrimum collected from Goa were much

lower (Vmax 84 and 51 1.M N (g dry wt) -' h -1 in light and dark respectively) than the values

reported by Schaffelke and Klumpp (1998) for Sargassum baccularia.

Some nonsaturating kinetics with linear increase in the uptake rates has also been reported

for many macroalgae. The linear uptake rates of ammonium upto the concentrations of 60

11M for Fucus distichus (Thomas et al., 1985), 50 tiM for Neoagardhiella baileyi and 50 pLM

for Gracilaria foliifera (D'Elia and DeBoer, 1978), 60 1.1M for Enteromorpha sp. and U.

lactuca (Fujita, 1985), between 20 - 30 1.1M for Macrocystis pyrifera (Haines and Wheeler,

1978) and 20 - 2000 1.1,M for G. tikvahiae (Friedlander and Dawes, 1985) were reported. In

addition nonsaturable ammonium uptake was also observed in Laminaria groenlandica

(Harrison et al., 1986), Gracilaria pacifica (Thomas at al., 1987), Chondrus 'crispus (Amat

and Braud, 1990), U. rigida and Chaetomorpha sp. (Lavery and McComb, 1991).

One important example that can be cited is that of the experiments conducted by

Friedlander and Dawes (1985) which showed that the ammonium uptake rates of G.

tikvahiae were linear over a wide concentration range (20 — 2000 ILM). This agreed with the

diffusion component proposed by D' Elia and DeBoer (1978) in the study where G. foliifera

(= G. tikvahiae) were exposed to a considerably lower concentration range of ammonium

(20-60 IIM). However, in this study all the species from both the environments showed

saturable uptake (saturation occurred between 10 to 50 p.M ammonium) which could be

due to the rapid filling of the internal N pools and hence feedback inhibition or due to the

inhibition of glutamine synthetase (the primary N assimilatory enzyme) by the elevated

levels of glutamate accumulated in the cells.

The ammonium uptake rates of Chordaria ilagelliformis (Probyn and Chapman (1982)

measured in batch mode and continuous mode experiments generally showed a hyperbolic

79

relationship, although the batch mode uptake curve showed the biphasic tendency,

indicating the possible existence of more than one carrier system or a single multiphasic

carrier. This type of a dual system however was not observed in any of the species in the

present study.

Maximum uptake rate and half saturation constant for ammonium in U. lactuca from the

present study are within the range of uptake rates reported for U. pertusa (19 - 240 t.LM N (g

dry wt) -' h -1 and 5 - 27 p.M respectively) by Tarutani et aL (2004). Also the maximum uptake

rates for U/va sp. reported by Campbell (1999) can be compared with the U. lactuca (Table

3.2) from the present study collected from the eutrophic waters and incubated in dark.

The ammonium uptake rates differ depending upon the ammonium levels upto which it is

incubated and at what concentration it saturates. For example the mean ammonium uptake

rates of U. lactuca, collected from the Waquoit Bay (Massachusetts) (Rivers and Peckol,

1995) undergoing eutrophication were 51 µM N (g dry wt) -' 11-1 and 219 11M N (g dry wt) -'

in subsaturating (15 RM) and saturating (75 AD ammonium levels respectively. Hence to

avoid such differences in the uptake rates due to ammonium levels, in this study, we have

carried out the experiments well above the saturation level of each species.

Uptake rates are often higher in light than in dark (Hanisak and Harlin, 1978; Topinka,

1978). For example, the uptake of ammonium by Codium fragile in dark was less than that

in light, but was still greater than the light uptake of nitrate and nitrite (Hanisak and Harlin,

1978). The length of dark period and also the source of light could be important in

determining the increase or decrease of N uptake in light. For example, the dark uptake of

ammonium by nitrogen starved G. tikvahiae was initially the same as that in light, but it

decreased with time (these experiments were carried out in natural daylight for 25 h),

however in another experiment it was observed that during the entire 25 h period (these

experiments were carried out in light provided by fluorescent lamp) the dark uptake rates

were higher compared to the light uptake rates but during the first hour the light and dark

uptake rates were similar (Ryther et al., 1981). In this study the algae namely S.

tenerrimum, 0. dichotoma and P. tefrastomatica collected from the eutrophic waters

exhibited the dark uptake rates lower than the light uptake rates except for U. lactuca which

showed the dark uptake rates almost thrice higher than light uptake rates. Such results are

not reported so far for any of the species.

80

The affinity for ammonium in U. lactuca collected from Goa is thrice higher in light than in

dark suggesting that light plays an important role in the ammonium uptake rates of this

species. However the affinity (a) for ammonium in U. lactuca collected from Lakshadweep

and incubated in light is almost similar to the affinity shown by the same species collected

from Goa and incubated in dark. Such behaviour of algae is yet to be analysed.

Even though the Vmax for P. tetrastomatica collected from Lakshadweep was less than that

of those collected from Goa, it was interesting to note that the affinity for ammonium is

higher suggesting its adaptability in the N limited waters. The preference order for

ammonium in different algae varies in light and dark and also varies depending upon the

region in which it was growing.

The variance ratios calculated for the differences in ammonium uptake rates in light and

dark for different algal species (Table 3.5) collected from eutrophic waters were significant

for all the species. However when the differences between the uptake rates of two different

algal species (in light) were tested by analysis of covariance showed that it was significant

for only few (Tables 3.6 and 3.11), but in dark all the species (Table 3.7) showed

significance in their uptake rates. Thus similar to nitrite and nitrate, the ammonium uptake

rates are also species specific. The variance ratios for the differences in the ammonium

uptake rates between the common species of the eutrophic and oligotrophic waters showed

that the uptake rates were significant (Table 3.13) only in P. tetrastomatica. Thus these

contrasting differences in the uptake rates between different species of algae indicate that,

the ecological relevance of the uptake rates may be dependent on the species of algae that

may compete successfully for ammonium.

The capacity of macroalgae, to take up ammonium at high rates observed in this study,

could be important in ammonium limited microhabitats, such as intertidal pools, where

invertebrate (Bracken, 2004; Uthicke and Klumpp, 1998; Wafar et al., 1990) and also the

vertebrate (Meyer et al., 1983) excreted ammonium are likely to be the important local scale

ammonia- N contributors to the microhabitat contributing indirectly to the macroalgal

productivity.

81

Urea uptake

Urea is supplied to the intertidal seaweeds through excretion by animals such as mollusks

(Berman and Bronk, 2003), agricultural runoff (Berman and Bronk, 2003), bacterial

degradation of purines and pyrimidines (Antia et at, 1991, Berman et al., 1999) etc.

Although the research on the urea uptake in macroalgae is limited, it is apparent that

macroalgae are capable of using urea (Probyn and Chapman, 1982; Phillips and Hurd,

2003). Phillips and Hurd (2004), determined the uptake rates by measuring the depletion of

urea from the flasks where the macroalgae were incubated. Tarutani et al. (2004) had

measured urea uptake by 15N incorporation in U. pertusa. Similarly in this study I have

detected the incorporation of ' 5 N into algal tissue at all the urea concentrations used.

Urea is a small-uncharged molecule that can diffuse in and out of algal cells (Rees and

Syrett, 1979) by passive diffusion. Evidence shows that urea is also taken up by active

process. Active uptake mechanisms for urea have been demonstrated in microalgae (Kirk

and Kirk, 1978; Rees and Syrett, 1979). The studies on urea uptake by McCarthy (1972) in

several marine phytoplankton have reported high affinities for urea. Hattori (1957)

demonstrated that N- deficient cells of Chlorella ellipsoidea Gemeck readily took up urea.

In this study urea uptake by all the species exhibited saturable kinetics indicating active

transport. Substrate saturation occurred at the concentrations between 10 and 40 }AM urea.

Saturated urea uptake kinetics has been reported for the first time by Probyn and Chapman

(1982) for the macroalgae Chordaria flagelliformis in batch mode and continuous mode

experiments. In batch mode experiment (perturbation experiment) the maximum uptake

rate and half saturation constant of C. flageffiformis were 932 ig N (g dry M) -' Ill and 22.5

1.ig at 1:1 respectively upto the concentration of 90 1.1.g at O. Phillips and Hurd (2004) have

reported saturated urea uptake kinetics for Stictosiphonia arbuscula, Apophlaea lyallii,

Scytothamnus australis and Xiphophora gladiata during winter and summer whereas

Stictosiphonia arbuscula showed linear uptake during winter.

Even though the Vmax values for D. dichotoma (Goa) are similar in light and dark,

comparatively low values of a in dark suggest that the affinity to take up urea in light was

thrice higher than that in dark. Similarly in S. tenerrimum also the affinity for urea in light

82

was higher than that of dark. But in case of U. lactuca the affinity for urea uptake was not

affected by light or dark. The Vmax for S. tenerrimum and D. dichotoma are similar in light

and dark which suggest that the capacity to take up urea are almost similar but the Vm

values for all the algal species collected from lakshadweep are markedly different from

each other.

Another important result was that, the affinity of C. sertulariodes (collected from

Lakshadweep), growing on the sediments was highest compared to the other algae

attached to the coral rubbles, without well-developed rhizoides. This could be because of

the adaptability of this alga to harvest the nutrients faster with the rhizoids, which helps in

sequestering the nutrient from the sediments and the thallus, which helps nutrient uptake

from seawater during its growth in the field. Thus the uptake rates for such algae are the

integrated unit of above and below ground biomass, influenced by high N concentrations of

sediment pore waters which indicate to be an adaptation to sequester urea at low

concentration available. Such above and below ground biomass, influenced by high N

concentrations of sediment pore waters are reported by Williams and Fisher (1985).

The variance ratios calculated for the differences in urea uptake rates in light and dark for

different algal species (Table 3.5) collected from eutrophic waters were significant for all the

species except for U. lactuca suggesting different uptake rates. However when the

differences between the uptake rates of two different algal species (in light and dark) were

tested it showed significance for only few (Tables 3.6, 3.7 and 3.11). Thus, like nitrite,

nitrate and ammonium, urea uptake rates also exhibited species specific behaviour.

The variance ratios for the differences in the urea uptake rates between the common

species of the eutrophic and oligotrophic waters were significant (Table 3.13) only for D.

dichotoma suggesting that the urea uptake rates were dissimilar. Thus these contrasting

differences in the uptake rates between different species of algae indicate that the

ecological relevance of the uptake rates may be dependent on the species of algae that

may compete successfully for urea.

83

Safety factor

The major sources of N available for macroalgae are nitrate, ammonium, nitrite and urea

but how much is their capacity to take up these nutrients from nature was not known until

Diamond (1998, 2002) provided the safety factor, which is the measure of the amount of

surplus capacity relative to the maximum concentration of the nutrient that an alga is likely

to encounter in nature. As discussed earlier safety factor is defined as the ratio of the

maximum nutrient uptake rate to maximum ambient concentration of nutrient and provides

a simple estimate of the amount of surplus capacity of a nutrient uptake system.

The safety factors calculated for the different species of algae from Lakshadweep (Table

3.12) and Goa (Table 3.8) showed relatively higher values for nitrite except for ammonium

in D. dichotoma from Lakshadweep. Comparing the high Ks and safety factor values for

nitrite in U. lactuca (Ks = 13.87 IAA; safety value = 61.53) and ammonium in D. dichotoma

= 43.11 ILM; safety value = 30.93) collected from Lakshadweep to the other values of

different algal species, suggest that these algae have high affinity and also high surplus

capacity for particular N nutrient.

Ecologically in contrast to other N nutrients high safety factors for nitrite uptake suggest that

the concentration of nitrite in the vicinity of seaweed is unpredictable. The unpredictable

increase in nitrite concentration may occur due to phytoplankton excretion. Similarly high

surplus capacity for ammonium was observed by Rees (2003) in seaweeds which suggest

that the concentration of ammonium in the vicinity of a seaweed is unpredictable which is

likely to occur due to excretion by animals associated with seaweeds (Rees, 2003).

The concentration of N species may vary both temporally and spatially and any patch of

elevated N would cover a small proportion of the macroalgal thallus surface. Thus the

chances that these attached algae would not be able to harvest the available nutrient within

a short time is more. The efficient way of overcoming this problem is to ensure that the

uptake rate is doubled and for this to happen the algae should possess a high safety fator.

Generally the macroalgae with high safety factors have a greater potential to take

advantage of the increased nutrient concentrations than macroalgae with low safety factors

(Rees, 2003).

84

Conclusion

As a result of different efficiencies of uptake for different algal species, N can be

incorporated in the form of any of the four N sources (nitrate, nitrite, ammonium and urea).

The uptake of N by all the species from the two sites was a function of external

concentration in which it was incubated. It was also species specific showing wide

variations in the uptake rates. All the macroalgal species from both the study sites

exihibited substrate saturable kinetics. Thus the experiments carried out in this study

partially support the hypothesis (See 3.1.1) laid out in the beginning.

85

3.2 SURGE AMMONIUM UPTAKE RATES OF MACROALGAE IN LIGHT AND DARK

FROM AN OLIGOTROPHIC ENVIRONMENT

3.2.1 Introduction

Maintenance of high levels of biological productivity (up to several gC m-2 d"1 ) (Crossland at

al., 1991) by coral atolls in oligotrophic oceanic waters with low ambient concentrations of N

nutrients (typically of the order of few hundreds of nmols of inorganic N L -1) has been

explained variously in the last 2-3 decades. As far as corals themselves are concerned, the

high production could be maintained by a tight recycling of N between the coral and its

zooxanthellae (Wafar et al., 1985). Most other autrotrophs would have to depend on

sources such as upwelling near the reef (Andrews and Gentien, 1982), endogenous

upwelling within the reef frame (Rougerie and Wauthy, 1993), ground water seepage

(Naim, 1993) and regeneration in sediments (Williams et al., 1985), besides nitrification in a

range of animal-bacterial associations (Wafar et at., 1990; Corredor et al., 1988; Welsh and

Castadelli, 2004). Besides these 'steady' fluxes, sporadic supply is also important for N

economy of the reefs. For example, fish shoals migrating onto the reef could raise ambient

ammonium concentrations in the vicinity of corals up to 0.9 µM (Meyer et al., 1983) and

area-specific N regeneration by actively feeding holothurians (Uthicke and Klumpp, 1998)

could be in a range similar to that reported for nutrient fluxes in coral reefs.

The ability of autotrophs to capture the sporadic or pulse addition of N would, therefore, be

critical for maintaining the high productivity of the reefs. This is especially true with

macroalgal and seagrass assemblages on atolls that account often for as much as 50% of

the total production in a reef. N-starved algae are known, under experimental conditions, to

take up more N than their immediate requirements (Rosenberg et aL, 1984; Thomas and

Harrison, 1987; Costanzo et al., 2000; Dy and Yap, 2001). Such a transient or surge uptake

capability in the case of macroalgae in coral reefs would confer on them a competitive

advantage since they would be able to make complete use of nitrogen when provided with

in excess as would happen with excretion by large heterotrophs in the vicinity.

Notwithstanding this possibility, the potential for, and intensity of, surge uptake in

macroalgae of atolls has received little attention so far, with the exception of one study

(Schaffelke and Klumpp, 1998) where Sargassum sp. from a fringing reef has been shown

to respond with time-averaged increase in photosynthesis and growth following pulse

86

addition of N and P. However, this does not demonstrate how rapidly the algae could

remove N from the medium in short term.

In this work, rates of surge uptake of ammonium measured in 3 common species of

macroalgae namely U/va lactuca, Soliera robusta and Dictyota dichotoma from the

Kavaratti atoll in Lakshadweep archipelago are reported. Unlike in earlier studies (Dy and

Yap, 2001) where surge uptake was deduced from loss of N in the medium, here

accumulation of 15N in the algal cells was measured, thus minimizing interference from

bacterial removal of ammonium from the medium.

3.2.2 Methodology

The algae (U. lactuca, S. robusta and D. dichotoma) from subtidal lagoon waters were

removed with their holdfasts intact and brought to a shore laboratory where they were

cleaned of epiphytes and sediments. They were then held in filtered seawater with aeration

for few hrs before experimentation.

Uptake measurements were carried out with approximately 0.5 g of algal tissue placed in

the beakers with 250 cc of seawater to which ammonium was added to give final

concentrations of 2.5, 5, 10 and 2011M. The spike in each case was prepared by mixing 9

parts of unlabelled ammonium with 1 part of 15N—labelled ammonium (99 atom % excess).

For each set of experiments with a given concentration, 16 incubations with algal tissues

were prepared and spiked with ammonium. Incubations were carried out at a light intensity

of 800 ILE m -2 s-1 and at an ambient temperature of 27 - 28°C. Duplicate sets of algal tissues

were withdrawn at 0, 2, 4, 6, 8, 10, 20 and 30 min after the beginning of the incubation.

Dark incubations were carried out in the same manner, with algal samples withdrawn at the

same time intervals. The beakers were shaken periodically to prevent localized depletion of

ammonium in the incubation medium. Algal material removed from the incubation medium

was briefly rinsed with deionized water and dried to a constant weight at 70°C. Dried

samples were then processed for 14N: 15N isotopic ratios in a Jasco N-151 Heavy Nitrogen

analyzer as discussed in chapter II. The uptake rates were calculated with the equation

(Dugdale and Wilkerson, 1986) where PON concentration at the end of the incubation is

used. Control algal samples were used for the determination of natural abundance of 15 N.

87

3.2.3 Results

Addition of ammonium to levels greater than ambient (-0.2- 0.3 AM) led to a pronounced

uptake in light within 10 min in all the three species (Fig. 3.23). While the timing of the peak

surge uptake varied between 2 and 10 min in each experiment, the increase in uptake rate

as a function of the spike concentration was remarkable. In D. dichotoma, it was from 329

N (g dry wt)" l til at 2.5 p.M N to 704 Al N (g dry wt) -'h-1 at 20 RM N. In S. robusta, it was

from 90 to 577 11.M N (g dry wty l h-1 at corresponding levels of spike. With U. lactuca,

however, the increase was seen only up to 10 tiM spike, from 230 to 335 µM N (g dry wt) -

1 h-1 . The increase in surge uptake with increasing concentrations of spike suggested that

substrate-limitation could have occurred at lower concentrations. Calculation of residual

substrate concentrations with the measured uptake rates and the algal biomass used did

suggest a substrate-limitation beyond 10 min at 2.5 and 5 1AM N spikes but not at higher

spikes. Compared with the Vmax calculated for substrate-saturable ammonium uptake with

similar algal biomass and during 1 h incubation (65, 31 and 69 11M N (g dry wt) -1 11 -1

respectively for U. lactuca, S. robusta and D. dichotoma), the surge uptake rates were up to

an order of magnitude higher.

Surge uptake was also pronounced in dark within the first 10 min (Fig. 3.23), though the

absolute uptake rates at any given time, in comparison with light, were variable. Unlike with

the uptake rates in light, those in dark did not appear to increase with the increase in spike

concentration. While uptake in dark, on an average, was between 25 and 50% of that in

light, the pattern of changes in the light-to-dark uptake ratios was interesting (Table 3.14).

U. lactuca and S. robusta took up more ammonium in light at lower spikes, with a

progressive increase in dark uptake at higher spikes whereas with D. dichotoma, dark

uptake was more pronounced at lower spikes and decreased in importance at higher

spikes. The changes in the UD ratios as well as their averages (Table 3.14) show that the

ability to take up ammonium in dark was higher in U. lactuca than in S. robusta and D.

dichotoma.

88

p N

H4 (

p,M

N (g

dry

wt)-'

h-1)

Table 3.14: Ratios of light to dark uptake in the three algal species during 30 min incubation

Ammonium spike OM N) U. lactuca S. robusta D. dichotoma

2.5 3.55 6.59 2.1

5 2.37 2.98 0.54

10 1.39 0.93 5.65

20 1.0 1.84 6.81

Average 2.08 3.08 3.71

Time (min)

Figure 3.23: Ammonium uptake rates at spikes of 2.5 (closed circle), 5 (diamond), 10 (square) and 20 µM (triangle) in U. lactuca (a and d), S. robusta (b and e) and D. dichotoma (c and f) in light and dark respectively.

89

3.2.4 Discussion

Non-linear uptake of ammonium when provided in excess of immediate requirements has

been shown in phytoplankton from nutrient-poor natural waters and N-deficient culture

media (Conway et al., 1976; Conway and Harrison, 1977; Harrison et al., 1989). This ability

for rapid uptake of ammonium, in dark as well as in light (Packard, 1979), enables

phytoplankton to grow at rates close to their maximal potential in N-impoverished waters

(Goldman and Gibed, 1982). The extent of departure from linear uptake is therefore a

measure of the degree of N-limitation.

Macroalgae, because of their proximity to sites of ex-situ nutrient supply (land sources) or

regeneration (benthic sediments), could generally be regarded as less N-limited than

phytoplankton. Nonetheless, transient rapid uptake of ammonium has been observed in

algae grown in cultures or collected from natural habitats. Neoagardhieffa baileyi and

Gracilatia foliifera, grown under severe N-limitation, took up ammonium in the first 5

minutes at rates nearly twice higher than later (D'Elia and DeBoer, 1978). Ammonium

uptake in cultured brown seaweeds (Chordaria ffageffiformis and Fucus distichus) was non-

linear in the first 30 min at ammonium additions up to 80 1.1,M N, with highest initial rates of

depletion in thalli with lowest N content (Rosenberg et al., 1984). Measurements with algae

from field follow this pattern. A rapid ammonium uptake in four intertidal algae from British

Columbia (Thomas and Harrison, 1987) was seen in the first 15 min after exposure to 15

pM NH4 . Ulva lactuca from Roskilde Fjord showed highest ammonium uptake within 15 min

of incubations at substrate concentrations >10 pM N, with the initial rate of surge uptake

increasing as the function of nitrogen limitation (Pedersen, 1994). In Kappaphycus alvarezii

ammonium uptake tended to be higher with higher initial ammonium concentration (Dy and

Yap, 2001) and there were no significant differences between uptake in light and dark in at

least one concentration (30 pM N). More recently, it was shown that ammonium uptake

within the first 15 min was significantly higher than during the next 75 min in intertidal algae

Stictosiphonia arbuscula and Scytothamnus australis from New Zealand (Phillips and Hurd,

2003). In all these studies, cell quota of N and high C: N ratios were determinants for the

strength of surge uptake. For example, decrease in cell N quota from 4 to 2 % caused a 2-4

fold increase in surge uptake in Chordaria flagelliformis and Fucus distichus (Rosenberg et

al., 1984). Low ambient concentrations could also be important: field data, where available

90

(Dy and Yap, 2001; Phillips and Hurd, 2003), show that ambient ammonium concentrations

were generally < 2 I.LM N.

What is interesting in our results is not surge uptake per se but its strength. The initial

uptake-rates-are several times higher than those reported in most other algae (see above),

probably with the exception of U/va lactuca where the surge uptake rate measured (D'Elia

and DeBoer, 1978) is comparable to our data. Besides, ratios of uptake during surge to

uptake post-surge are quite high, up to 10. Such high surge uptake rates can only partially

be explained by low cell N quota (Fujita et a/., 1988; McGlathery at a/., 1996). Surge uptake

measured in Chordaria flagelliformis and Fucus distichus with a cell N quota of 1%

(Rosenberg at al., 1984) was still much less than in our study. It is likely; therefore, that

oligotrophy is an equally important cause of high surge uptake. Atoll waters are extremely

poor in dissolved inorganic N and the ammonium concentrations in Kavaratti atoll (and

elsewhere in the Lakshadweep atolls) are often below limits of detection. By contrast,

ambient ammonium concentrations, where available in similar studies (Dy and Yap, 2001;

Phillips and Hurd, 2003), were between 1 and 2 jiM N, typical of nearshore waters. The

intensity of surge uptake, therefore, could be a proportional response to the extent of (or

chronic) ambient N-privation before a fresh pulse supply. The rapid increases in surge

uptake rates in response to increasing concentrations of ammonium as well as the

significant amount of N taken up in dark with respect to light in all cases add further support

to this. In that case, ratios of surge to post-surge uptake rates could be useful indicators of

the extent of previous N-limitation.

Though surge uptake of ammonium is not frequently measured, it could be an important

mechanism of N acquisition for macroalgae in nutrient-poor reef waters given the diversity

of heterotrophs habiting on them and in the vicinity. Seaweed surfaces are inhabited by a

large number of small mobile and sessile organisms (Seed, 1986) and their excretion, as

was shown in the case of epifauna on Carpophyllum plumosum (Taylor and Rees, 1998), is

an important source of N for the alga. Other examples include fish shoals (Meyer et a/.,

1983), bryophyte colonies associated with Agarum fimbriatum and Macrocystis integrifolia

(Hurd et a/., 1994) amphipods and gastropods associated with Enteromorpha intestinalis

(Barr and Rees, 2003), echinoderms (Dy and Yap, 2001) and the sponge Haliclona

cymiformis symbiotic with Ceratodictyon spongiosum (Simon et al., 2002). While a

physically close association, as was the case in the symbiosis between C. spongiosum and

91

H. cymiformis (Simon et al., 2002), may provide all of or more than the quantity of N

required by the alga, removal by dilution (Taylor and Rees, 1998) could substantially reduce

the amount of N that actually becomes available to the alga from the heterotrophs. Efficient

acquisition of N from heterotrophic sources thus would depend not on the release rates but

rather on the rapidity of uptake. As seen from the highest surge uptake rates measured in

this study, the macroalga from the atoll reefs would seem to be better adapted than others

from elsewhere to achieve this.

92