MACROALGAL NUTRIENT

RELATIONSHIPS.

Adrian B. Jones BSc.

1993

Department of Botany

University of Queensland

A literature review submitted to the Department of Botany,

University Queensland, in partial fulfillment of the

requirements of the Honours Degree of the

Bachelor of Science.

ii

CONTENTS

Title Page i

Contents ii

1. Introduction 1

2. Nutrients 4

2.1 Carbon : Nitrogen : Phosphorous Ratios 5

2.2 Nitrogen Versus Phosphorous Limitation 5

2.3 Nitrogen 8

2.3.1 Ammonium 9

2.3.2 Nitrate And Nitrite 9

2.3.3 Organic Nitrogen 10

2.3.4 Comparative Uptake Of Different Forms Of Nitrogen 11

2.4 Phosphorous 14

2.5 Plant Responses To Variations In Nutrient Concentrations 14

3. Nitrogen Storage In Algae 18

3.1 Nutrient Indicators 21

3.1.1 Amino Acids 22

3.1.2 Pigments

27

iii

3.1.3 Tissue Nutrients 29

4. Water Quality 32

4.1 Light 32

4.2 Temperature 34

4.3 Salinity 34

4.4 Water Movement 35

5. Use Of Bioindicators 36

6. Summary 37

7. References 39

1

1. Introduction

Macroalgae can be divided into three main groups, the Chlorophyta (green algae), the

Phaeophyta (brown algae), and the Rhodophyta (red algae). This literature review will

primarily deal with the Rhodophyta. This group typically inhabits rocky shores, or in the case

of some Gracilaria spp. may occur loose-lying or embedded on muddy shores (Norton and

Kain, 1990). Rhodophyta are widely dispersed in coastal marine waters and estuaries in both

tropical and temperate regions. This group is instrumental in the survival of many aquatic

organisms. They provide a food source for numerous herbivores and are essential for the

survival of coral reefs which depend on the cementing action of some species of red algae.

Further, the Rhodophyta has various commercial uses including the production of agar, food

colouring, pharmaceutical products, human bone implants and many others still relatively

undeveloped (Woelkerling, 1990).

A large proportion of the research outlined in this review concentrates on the nutrient

relationships of the species Gracilaria tikvahiae, a branching red algae. The red algae have

been observed to respond to fluxes in external ambient nutrient concentrations. This may be

due in part to their high surface area to volume ratio and the location of their phycobilisome

storage sites on the surface of the lipid bilayer of the thallus lamellae. In particular,

Gracilaria spp. are commonly used for nutrient studies because of their ability to tolerate

relatively large fluctuations in salinity, temperature and light, thereby making them ideal for

multiple site studies (Horrocks, 1993).

The effect of nutrients on macroalgae is governed by macroalgal N relationships, i.e.,

algal uptake, assimilation, storage and release (Hanisak, 1983). Photosynthetic production of

organic matter by algae is dependent on the assimilation of inorganic nutrients (Ryther and

Dunstan, 1971). Hanisak (1983) noted that in the 1970's the accepted view was that

macroalgal growth was not limited by its requirement for nutrients as moving water

surrounding the algae would supply sufficient nutrients. Whilst this holds for most of the

required nutrients, nitrogen (N) and phosphorous (P) are required by the algae in much higher

concentrations and therefore are often the limiting factors for productivity (Ryther and Dunstan,

1971).

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Strong seasonal variation in the N content of macroalgae has been observed in

numerous studies (e.g., Butler, 1936; Black and DeWar, 1949; Moss, 1950; Macpherson and

Young, 1952; Vinogradov, 1953; Chapman and Craigie, 1977; Hanisak, 1979b; Chapman and

Lindley, 1980; Lyngby, 1990). Their observations suggest that there is reduced N content

during summer when external supplies of N are usually at their annual minima. Due to the

number of observed exceptions, generalisations cannot be made about the seasonal trends in N

availability (Nixon and Pilson, 1983).

Nutrient concentrations are usually determined by sampling the water column for

dissolved nutrients. These nutrient concentrations can be used to predict the effects of specific

pollutants on the growth of algae and higher aquatic plants in lakes and streams (Lea et al.,

1954). The concentration of nutrients in the water column can fluctuate on short time scales,

rendering it difficult to assess potential nutrient limitation without intensive monitoring of the

water column (Wheeler and Björnsäter, 1992). With the continued increase in nutrient

concentrations of waterways, faster, easier and more accurate ways of measuring these

concentrations are sought.

The total N content of seaweeds varies with changes in the environment (Haas and Hill,

1933). Recent research by Vona et al. (1992) and Horrocks (1993) has indicated that an

accurate measure of the concentration of nutrients available to marine plants can be ascertained

by using algae as bioindicators. The advantage of such bioindicators is their ability to

integrate the nutrient regime over a period of time. This process results in a more reliable

indication of the water column nutrient status and the ability of plants in the system to utilise the

available nutrients. In addition, macroalgae absorb many of the heavy metal pollutants, and

hence can also be used to determine metal availability (Wheeler and Björnsäter, 1992).

This review will outline the nutrient relationships of a number of macroalgal species,

concentrating on the Rhodophyta and, in particular, the genus Gracilaria. It will deal with the

nutrients which are found to be limiting to its growth, and its responses to changes in the

concentrations of these nutrients. Particular emphasis will be placed on the changes in amino

acid composition, pigment concentrations and tissue nutrient concentrations as a result of

changes in the concentrations of the various inorganic and organic forms of N. It has been

shown by several researchers that changes in these internal parameters reflect changes in

external nutrient availability (Bird et al., 1982; Vona et al., 1992; Horrocks, 1993). Nitrogen

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has been identified by researchers as the main factor in limiting macroalgal growth (Ryther and

Dunstan, 1971). The effects of external factors such as light, temperature, salinity and water

motion on macroalgae are also reviewed as they can play a major part in affecting algal growth

and the levels of the various algal nutrient indicators.

The use of amino acid analysis on plants grown in situ is relatively unexplored, but

data on the relationship between amino acid concentrations and N availability from aquaria

studies indicates a definite correlation (Horrocks, 1993). The effectiveness of this method as

an indicator of water column nutrient availability in the field should be explored in future

research.

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2. Nutrients

Of the major nutrients required for algal growth, carbon (C) is needed in the highest

concentrations. Marine plants can obtain CO 2 from bicarbonate, which is rarely limiting in

full salinity seawater (Sand-Jensen and Gordon, 1984). Therefore, although large amounts of

C are needed, it is not usually limiting. However, P and N are usually only available at

concentrations which are limiting to algal growth. It is generally agreed that N is the limiting

nutrient to macroalgal growth in marine systems, while in freshwater systems it is usually P

(Ryther and Dunstan, 1971; Valiela, 1984). As this review concentrates primarily on marine

systems, the effects of N will be dealt with in greater detail.

Nitrogen is introduced to waterways by terrestrial weathering, plant decomposition,

oceanic mixing processes, regeneration from decomposing marine primary and secondary

producers (in the water column and sediments) and by air-sea exchange processes (including

dry deposition and rainfall) (UNESCO, 1990). The most common land-based sources are

domestic wastes (mainly sewage and detergents), agricultural run-off (either fertilisers or

increased erosion and weathering), animal wastes from intensive livestock units, industrial

effluents and atmospheric discharges from fuel combustion and agriculture (UNESCO, 1990).

Normal seasonal storm winds can cause localised increases in dissolved nutrient

concentrations and phytoplankton biomass in inshore water by resuspension of inshore

sediments (Furnas, 1988). As a result of phytoplankton's ability to absorb N at rates

equivalent to the standing crop within hours, human inputs of N may not produce an increase in

dissolved nutrient concentrations (Furnas, 1988). Any observed increases are likely to be due

to the ratios of nutrients entering the system being unbalanced for phytoplankton growth,

resulting in the depletion of one nutrient before all are consumed. This could lead to

accumulations of ’surplus’ nutrients (e.g., dissolved inorganic N, phosphate or silicate) in

particular situations (Furnas, 1988).

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2.1 Carbon:Nitrogen:Phosphorous Ratios

The Redfield molar ratio of C:N:P is 106:16:1 and is an average value for the

composition of aquatic particulate matter determined from phytoplankton studies (Redfield,

1934). These values were calculated from the chemical composition of plankton. As a

consequence, it was postulated that molar N and P uptake from seawater would also be 16:1.

The average N:P ratio in different species of macroalgae observed in Moreton Bay and the

Logan River, both in Queensland was 10.45 (Horrocks, 1993). The C:N ratio in macroalgae

increases during periods of N limitation, but the N:P ratio, as observed in a number studies

(e.g., Gordon et al., 1981; Björnsäter and Wheeler, 1990), does not alter since the phosphate

content declines due to enzymatic hydrolysis at times of nutrient limitation. This may indicate a

coupling of the metabolism of N and P (McGlathery, 1992).

The nutrients entering from the watersheds into freshwater bodies may have an N:P

ratio of 40:1 (Valiela, 1984). In addition to this, N-fixation rates are much greater in

freshwater systems, thereby further increasing the N:P ratio (Valiela, 1984). If N

concentrations are very high then P becomes the secondarily limiting nutrient, thereby

preventing growth at rates proportional to the concentration of N. In macroalgae the excess N

is stored as tissue N, pigments, or amino acids (Ryther et al., 1981). If both N and P occur at

high concentrations, then trace metals may limit the growth of macrophytes (Prince, 1974).

This is indicative of Liebig's law: there is always one limiting factor (Liebig, 1840).

2.2 Nitrogen Versus Phosphorous Limitation

The three main factors controlling whether N or P will be limiting to plant growth are:

(a) the ratio of N to P in external nutrient inputs (e.g., high N:P from sewage outfall results in

lakes being P limited); (b) the preferential loss from the photic zone of N or P due to

biogeochemical processes such as denitrification, preferential sedimentation of N in

zooplankton fecal pellets, or adsorption of P; and (c) the extent to which any relative deficit in

N availability is made up through N fixation (there is generally more N fixation in freshwater

systems which leads to P limitation) (Howarth, 1988). At least in the northern temperate zone

6

these three factors are more likely to give rise to estuaries and coastal marine waters which are

N limited. Howarth (1988) notes that some estuaries may be P limited, or may switch between

being P and N limited. Howarth (1988) also argues that the main reason for N limitation in

estuaries and not in lakes is planktonic N fixation, which makes up the deficit in lakes but not in

estuaries or marine coastal waters. The relative lack of N fixation in marine systems appears

to be due to a lower availability in seawater of molybdenum and iron, two trace metals

required for N fixation (Howarth et al., 1988). A combination of the above factors result in N

concentrations being much higher in freshwater than seawater (Valiela, 1984). Figure 1

presents a simplified version of the biogeochemical processes which control nutrient limitation

in aquatic ecosystems. It includes terrestrial input and input from nitrogen fixation, and

presents the interaction between nutrients and phytoplankton growth and decomposition, along

with sediment nutrient cycling.

Figure 1. Summary of biogeochemical processes controlling nutrient limitation in aquatic ecosystems. The ratio of nitrogen to phosphorous from terrestrial inputs varies greatly, generally being lower for ecosystems receiving more sewage inputs. Nitrogen and phosphorous are assimilated by phytoplankton in an approximate molar ratio of 16:1. The N:P ratio of nutrients released in the water column during zooplankton feeding and during decomposition frequently approximates 16:1 but can be as high as 30:1 in oligotrophic waters. Nutrients released from marine and estuarine sediments back to the water column tend to have low N:P ratios, but such releases in lake sediments often have N:P ratios near 16:1 or higher. The extent of nitrogen fixation varies but tends to be much greater in lakes than in marine systems (from Howarth, 1988).

7

Figure 2 shows how the N and P content of macroalgae and aquatic vascular plants is affected

by the varying proportions of N and P found in marine systems versus freshwater systems.

Figure 2. Nitrogen and phosphorous contents of marine and freshwater macroalgae and vascular plants. Compiled from many sources. The marine data from Vinogradov (1953) and articles in Limnology and Oceanography, Journal of Phycology, Aquatic Botany, Botanica Marina, Marine Biology of the last 10 years (from Valiela, 1984).

Further evidence of N (rather than P), being the critical limiting factor to algal growth and

eutrophication in coastal marine waters can be found in the distribution of inorganic N and P

and the results of bioassay experiments. Some of the results from these experiments found

seasonal variation between N and P limitation, with N being limiting during the annual peak in

primary production in summer (Lyngby, 1990). Although N appears to be the critical limiting

factor to algal growth, it has been found that N and P additions have a greater effect together,

which suggests a secondary limitation by P. It can be concluded therefore that excessive

amounts of N cannot act as a substitute for the P requirements of the plant (Howarth, 1988). In

a study by Boynton et al. (1982), 22 out of 27 surveyed sites had N:P ratios well below the

Redfield Ratio thereby indicating probable N limitation in the phytoplankton. Further, D'Elia

et al. (1986) found that in the Patuxent River (a tributary of Chesapeake Bay in North America)

the dissolved inorganic N (DIN) : dissolved inorganic P (DIP) ratio was 100:1 in late winter,

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and 1:1 in late summer. These ratios are consistent with bioassay results from studies such as

Ryther and Dunstan (1971) which indicate that N is limiting in summer and P is limiting in

winter.

2.3 Nitrogen

The N cycle in marine systems involves a number complex interactions. These have

been simplified and outlined in figure 3, which includes the major inputs of new N (N fixation,

terrestrial input, atmospheric nitrogen), nitrogen regeneration (death of organisms, input from

sediments), nitrogen outputs (transfer to atmosphere, fishing, guano) and the internal transfer

between different forms of nitrogen (denitrification, ammonification, decay, uptake by

organisms).

Figure 3. Simplified scheme of major transformations and transport of nitrogen in marine environments (from Valiela, 1984).

9

It has been demonstrated that at times marine macroalgal growth is definitely N limited

(Hanisak, 1983; Ryther and Dunstan, 1971). The limiting form of N is usually dissolved

inorganic N (DIN), rather than dissolved organic N (DON) (Hanisak, 1983). The principal

forms of inorganic N in the marine environment are dissolved molecular N (used by the

cyanobacteria), nitrate, nitrite and ammonium. Organic forms also occur in both particulate

and dissolved forms, for example as urea and amino acids (Hanisak, 1983).

The process of N uptake is as follows:

� Nitrogen diffuses across the boundary layer adjacent to the plant surface.

� It then passes across the cell membrane.

� Finally, the amino acids are assimilated directly, in the case of NH 4+, or after

reduction of NO 3- or NO 2

- to NH 3. (Hanisak, 1983).

2.3.1 Ammonium

Despite being the most easily assimilated form of N for most macroalgal species, NH

4+ is present in seawater at a much lower concentration than NO 3

- (Hanisak, 1983). The exact

mechanism effecting ammonium uptake is uncertain due to the coexistence in solution of NH 3

and NH 4+ (Raven, 1984). Although NH 4

+ constitutes over 90% of the total ammonia, NH 3 is

more highly permeable to lipid bilayers and NH 3 uptake is the main form when external pH is

high. Although NH 4+ is the most commonly assimilated form its uptake must be transport

mediated (Raven, 1984).

2.3.2 Nitrate And Nitrite

Nitrate (NO 3-) being the most oxidised form of N is taken up by algae under aerobic

conditions and is then reduced by assimilation processes to the amine form which can be used

10

in metabolic processes (Valiela, 1984). Nitrate is not as easily assimilated by algae as

ammonium (D'Elia and DeBoer, 1978). Nitrite (NO 2-) is present in much smaller

concentrations in seawater, and is therefore less likely to be assimilated by algae than

NO 3- (D'Elia and DeBoer, 1978). Nitrate is the most common form of DIN found in marine

systems, with values significantly higher than ammonium and nitrite (Norton and Kain, 1990).

2.3.3 Organic Nitrogen

Although less important to macroalgae, organic forms of N (e.g. urea) may play an

important part in systems where the inorganic forms are at limiting concentrations. It has been

estimated that the uptake of urea N by phytoplankton in the New York Bight could increase the

biological assimilation of N by about 40% (Eppley et al.,1977; Garside, 1981; McCarthy,

1972). This suggests that in some systems urea is a significant source of N for marine plants.

This is supported by Asare (1980) and Hanisak (1981), who observed that growth of

Gracilaria tikvahiae was supported by forms of organic N from different types of organic

wastes. Other organic forms of N such as dissolved free amino acids (DFAA's), purines and

pyrimidines may be important sources of N for some macroalgae (Hanisak, 1983).

In considering organic sources of N, it should be noted that bacteria may be

assimilating forms such as urea, and in turn releasing inorganic N which is then taken up by the

algae (Hanisak, 1983). If experiments are performed under axenic conditions the effect of the

bacterial interference is removed. However, under in situ conditions the bacteria are an

integral part of the ecological unit of ‘algae and bacteria’. They are an integral part of the

process which facilitates algal utilisation of organic N, and need not be regarded as an

extraneous factor to be removed for correct analysis of algal N uptake (Hanisak, 1983).

2.3.4 Comparative Uptake Of Different Forms Of Nitrogen

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Ammonium (NH 4+) and nitrate (NO 3

-) are considered to be the most important sources

of N for macroalgae as they are generally the most easily assimilated forms available

(Hanisak, 1983). In a particular species one of these forms is usually preferred to the other.

However certain algal species have been found to prefer nitrite (NO 2-), which normally occurs

at much lower concentrations in seawater than NH 4+ and NO 3

-. Gracilaria tikvahiae has been

shown to prefer ammonium (DeBoer et al.,1978), while Codium fragile (Hanisak, 1979a),

Goniotrichum elegans and Nemalion multifidum (Fries, 1963) were found to respond much

better to NO 2- than NO 3

- or NH 4+. The acquisition rate of the different N forms is affected by

environmental conditions including light and temperature (Lapointe and Ryther, 1978; Valiela,

1984). The assimilation of the various forms of N by algae is dependent on the concentration

of each form in the surrounding water. For example, phytoplankton are able to utilise all forms

of N, although the less preferred forms are usually reserved for when the more easily

assimilated forms are no longer available. In highly productive marine ecosystems,

phytoplankton production is controlled by nitrate, although actual uptake is primarily

ammonium. In less productive systems ammonium may be a more important form, perhaps due

to the dependence of N regeneration within the system.

Studies have revealed that NH 4+ is taken up by macroalgae more rapidly than

NO 3- or NO 2

- and its presence usually inhibits the assimilation of NO 3- and NO 2

-(Hanisak

and Harlin, 1978; Harlin, 1978; Topinka, 1978; D'Elia and Deboer, 1978; Haines and

Wheeler, 1978; Ryther et al., 1981). Nitrate, the next most easily utilised form of N, may

inhibit the absorbance of NO 2-. The degree of inhibition is related to the concentration of the

inhibitor. However, some algae, especially the Phaeophyta, can assimilate NO 3- without

inhibition, even in the presence of NH 4+ (Haines and Wheeler, 1978). This characteristic is

more common to the brown algae, although some species from other groups of algae may also

be capable of preferentially utilising NO 3- over

NH 4+.

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Figure 4 illustrates the difference in assimilation of ammonium versus nitrate by Gracilaria

tikvahiae. It also demonstrates the difference in the quantity of ammonium which can be

assimilated by nitrogen-starved versus non-nitrogen starved individuals.

Figure 4. (A) Change in ammonium-nitrogen in tank containing no seaweed. (B) Change in ammonium-nitrogen in tank containing non-nitrogen-starved Gracilaria. (C) Removal of ammonium-nitrogen from water (solid circles) and calculated uptake (open circles) by nitrogen-starved Gracilaria. Triangles are measured increases in tissue nitrogen by the same plants. (D) Removal of nitrate-nitrogen from the water (solid circles) and calculated uptake (open circles) by nitrogen starved Gracilaria (from Ryther et al., 1981).

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Table 1 shows the preferred nitrogen form for a number of different macroalgal species. The

sources of data for many of the species are different resulting in some overlap, e.g., Gracilaria

tikvahiae being found in categories A and C.

A. Species that grow equally well on NO 3- or NH 4

+. Codium fragile Chondrus crispus Fucus spiralis Fucus vesiculosus Gracilaria tikvahiae Porphyra tenera B. Species that grow better on NO 3

- than NH 4+.

Gelidium amansii Gonotrichum elegans Nemalion multifidum Palmaria palmata C. Species that grow better on NH 4

+ than NO 3-.

Chordaria flagelliformis Gracilaria tikvahiae Neoagardhiella baileyi Pterocladia capillacea D. Species that grow equally well on urea or inorganic nitrogen. Codium fragile Enteromorpha linza E. Species that grow better on inorganic nitrogen than on urea, Asterocytis ramosa Chondrus crispus Gelidiella acerosa Gelidium amansii Neoagardhiella baileyi Rhodosorus marinus F. Species that grow better on urea than inorganic nitrogen. Pterocladia capillacea Ulva fasciata

Table 1. Reported nitrogen preferences for selected macroalgal species (adapted from Hanisak, 1983).

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Environments which are low in ammonium and relatively high in nitrate can cause an

acclimation process in the algae, whereby they adjust to absorbing nitrate. However it appears

that Gracilaria foliifera and Neoagardhiella baileyi will quickly return to absorbing NH 4+ if

a supply subsequently becomes available (D'Elia and DeBoer, 1978).

2.4 Phosphorous

Phosphorous in the marine system can occur in living organisms or as dissolved

inorganic P (DIP), dissolved organic P (DOP) and particulate P (part. P) (Valiela, 1984).

Phosphorous in seawater is mainly found incorporated in living particles, due to the rate at

which DIP is taken up by algae and bacteria (Valiela, 1984). Particulate Organic P (POP)

decays to DIP in the water column or on the sediment surface. As with N, P concentration in

surface waters is usually much lower than deeper waters, attributable mainly to uptake by algae

(Eppley et al., 1977; Valiela, 1984).

2.5 Plant Responses To Variations In Nutrient Concentrations

The growth of marine macroalgae is influenced by a number of environmental factors

including light, temperature, nutrient availability, water motion (this affects the uptake of N),

desiccation (for intertidal species), and salinity (Josselyn and West, 1985). In the aquatic

environment the principal limiting factors to primary productivity are light and nutrients. The

relative importance of each is affected by the type of organism being studied and the relative

availability of each factor. Species that are limited by light are frequently less affected by

nutrients, such as rhizophytic species which have access to nutrients directly from the

substratum (McGlathery, 1992). However when nutrients in the water column increase,

especially due to terrestrial runoff, opportunistic species (especially mat-forming macroalgae)

that were nutrient limited can grow rapidly and outcompete the rhizophytic species, as these

15

species do not exhibit as strong a response to an increase in water column nutrients. Over time

this will cause a change in the species composition (McGlathery, 1992).

Studies have indicated that the coastal ecosystems at various locations are N rather than

P or silicon limited (Furnas, 1988). The limitation is a biomass limitation rather than a kinetic

or growth limitation (Furnas, 1988; Howarth, 1988). There are three types of limitation in the

marine system; growth of current population limitation, net primary production limitation and

limitation of net ecosystem productivity (Howarth, 1988). Even if the growth rate of the

present species of phytoplankton is not limited, the potential net primary productivity may be

limited (Howarth, 1988). If the nutrient supply is increased a new species of phytoplankton

that is better adapted to the higher nutrient concentrations can outcompete the other species, and

have a higher production rate (Howarth, 1988).

Plants exposed to nutrient limiting conditions exhibit symptoms specific to the element

which is absent or growth-limiting (Vona et al., 1992). In particular, N-limitation in algae

results in depression of enzymes involved in N metabolism, such as nitrate reductase,

glutamine synthetase and glutamate synthase. Pigment concentrations are reduced, as are the

stores of free amino acids, and tissue N (Rigano et al., 1981). The plant uses stores of protein

to survive during periods of low N availability (Vona et al., 1992).

While N plays a major role in macroalgal growth the concentration of N required for

growth in situ, is unknown. The importance of N is evidenced by the dramatic increase in

growth that occurs in regions close to wastewater outfalls (Tewari, 1972). This has been

confirmed by experiments which have shown that the addition of N to natural populations

results in a marked stimulation in growth (Chapman and Craigie, 1977). This indicates that, for

at least part of the year algal growth at these sites is limited by N (Tseng et al., 1955; Chapman

and Craigie, 1977; Harlin and Thorne-Miller, 1981).

Macroalgae have the ability to grow rapidly. If conditions are optimal and all nutrients

are in sufficient supply the biomass doubling time of most macroalgae can be as little as 12

days in summer (Masini et al., 1990). The optimum concentration of N for maximum growth of

algae, as determined by aquaria experiments, far exceed those found in situ (Boalch, 1961;

Fries, 1963; Hanisak, 1979b; Iwasaki, 1967; Mohsen et al., 1974; Steffensen, 1976).

Therefore, rather than measuring the optimum concentration, it may be more useful to measure

the growth kinetics at concentrations relevant to ambient N concentrations (Hanisak, 1983).

16

Research on phytoplankton has shown that there is a hyperbolic relationship between growth

and the external N concentration (Chapman et al., 1978; Deboer et al., 1978; Hanisak, 1979b;

Probyn, 1981). The uptake kinetics of NO 3- and NH 4

+ by N deficient Gracilaria tikvahiae

and Neoagardhiella baileyi, did not conform to the Michaelis-Menten model of saturable

uptake kinetics (D'Elia and DeBoer, 1978; Fujita, 1985). The rates of uptake were

independent of, and far in excess of uptake associated with growth. This is due to their ability

to store large amounts of N (D'Elia and DeBoer, 1978). Figure 5 is provides two examples of

Michaelis-Menten uptake curves for NH 4+ and NO 3

- uptake by two species of phytoplankton.

Figure 5. Michaelis-Menten curves (filled circles) and Lineweaver-Burk plots (open circles) fitted to data on

uptake ( µmole/hr) of ammonium and nitrate by two diatoms at different nutrient concentrations (µmole/litre).

S is the concentration of the nutrient being taken up, V is the uptake velocity. The χ-intercept provides the

estimate of K s (Adapted from Valiela, 1984).

The half-saturation constant (K s) of a number of macroalgal species has been

estimated, and for G. tikvahiae is 0.4 µM when grown on NO 3-, and 0.2 µM when grown on

NH 4- (DeBoer et al., 1978). This is a measure of a species' relative ability to take up nutrients

at low concentrations. These values are likely to vary with location and the corresponding

change in ambient N concentration. K s can be contrasted with Vmax , which estimates the

maximal uptake rate that occurs at significantly higher nutrient concentrations (Hanisak, 1983).

Vmax data for macroalgal species has confirmed that ammonium is more easily assimilated than

NO 3- or NO 2

- even in plants preconditioned to NO 3- (D'Elia and Deboer, 1978). Variations in

Vmax estimates can occur due to N preconditioning, environmental influence, and the portion of

17

the thallus which is used for analysis. It has generally been assumed that K s values are more

ecologically significant than Vmax , but D'Elia and DeBoer (1978) suggest that if short pulses of

N occur then Vmax becomes relevant. Further, they noted that G. tikvahiae and N. baileyi have

a dual phasic system involved in NH 4+ uptake. Below 10 µM, a high-affinity (low K s) system

was operative, while at the higher concentrations, there appeared to be a low-affinity

(high K s) system, or a strong diffusive element. MacFarlane and Smith (1982) have provided

confirmation for the existence of a dual-phasic system, in Ulva rigida. These dual phasic

systems of uptake don't adhere to Michaelis-Menten kinetics.

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3. Nitrogen Storage In Algae

Macroalgae are important in the cycling and transformation of N in the marine

ecosystem, due to their ability to absorb and store large amounts of N (Hanisak, 1979b; Ryther

et al., 1981). Nitrogen starved Gracilaria spp. assimilated ammonium-N rapidly doubling total

tissue N content in 8h or less, however, nitrate-N uptake was less rapid (Ryther et al., 1981).

G. tikvahiae has been demonstrated to take up sufficient N in 6 h to allow it to grow for up to 2

weeks at non-nitrogen-limited concentrations (Ryther et al., 1981). The brown algae,

Laminaria longicruris, has the ability to boost its N concentrations to 28,000 times that of

ambient external concentrations, which amounts to 2.1% of the dry weight. This supply was

sufficient to provide unlimited growth for 2 months after external concentrations had declined

(Chapman and Craigie, 1977). Rapid assimilation of N by starved algae may depend on

carbohydrate reserves and cease when these reserves are depleted (Ryther et al., 1981)

The storage of N in the inorganic form, or biomechanical metabolites, is known as

luxury consumption, and is an ecological adaption to N - limitation (Gerloff and Krombholz,

1966). In the case of L. longicruris, the main site of storage is most likely the vacuole.

However in species with smaller inorganic pools such as Chondrus crispus, Laycock and

Craigie (1977) found that half their organic N can be in the form of the dipeptide

citrullinylarginine. This suggests that amino acids and proteins are the major storage units

(Hanisak, 1983). The free amino acid composition and concentration of marine macroalgae

has been observed to be influenced by the type of externally available N (Nasr et al., 1968).

For example Nasr et al. (1968) found that urea produced the greatest increase in amino acid

concentrations for both Ulva lactata and Pterocladia capillacea, while for Dichtyota

dichotoma, ammonium produced the greatest increase. The most notable increases with urea

were 10 to 20 fold increases in asparagine,

α-aminobutyric and β-alanine. It appears that assimilation of urea by some macroalgae may

not involve urease. Hattori (1958) suggested that urea may combine directly with ornithine to

form arginine, rather than first being broken down into ammonium. While the type of available

nitrogen is important in determining amino acid composition, the species being analysed is also

important. For example, G. tikvahiae has been shown to store N as the dipeptide

citrullinylarginine (Bird et al., 1982), whereas U. lactata and

19

P. capillacea showed no sign of citrulline. Horrocks (1993) observed that the addition of N to

Gracilaria verrucosa increased the percentage of N as free amino acids from 10.5% to 17.6%.

Citrulline was found to be the most responsive to an increase in N, changing from 8.8% to

13.9% of tissue N. Citrulline was found to be 70 - 90% of the total free amino acids.

There are three types of N pools - structural, physiological, and storage. The structural

pool consists only of those molecules which make up the cell walls, and in

G. tikvahiae, it makes up approximately 0.8% of the dry weight (Hanisak, 1983). The

physiological pool contains N in forms such as enzymes and photosynthetic pigments, (those

which are important for physiological or metabolic processes). The storage pool contains the

critical internal N concentration, and any excess absorbed above this is directed to the storage

pool (Hanisak, 1983). Increased growth rates caused by external factors including an increase

in light availability may alter the balance between physiological and storage pools. Stored N

can be used to sustain life when the externally available N falls below the critical

concentration required. Algae that do not have these N reserves have to fragment or produce

propagules to survive the nutrient stress (Hanisak, 1983). Figure 6 shows the relationships

between the three nitrogen pools and their response to changes in internal nitrogen

concentration

Figure 6. Major nitrogen pools and their relationships to internal nitrogen concentration (e.g., percent of dry weight) and growth rate. The size of the "structural pool" is conservative and independent of growth rate; the size of the "physiological pool" increases as both internal nitrogen and growth rate increase; the "storage pool" contains nitrogen that is assimilated in excess of structural and physiological requirements (from Hanisak, 1983).

20

Research by Fujita (1985) indicates that G. tikvahiae has a higher storage capacity than

other species, but does not respond with increased growth when the supply of N is increased.

Species grown together with G. tikvahiae do not survive well when nutrients are supplied in

pulses every fourteen days. This is because they don't have the same storage capabilities as G.

tikvahiae, and therefore can't remain healthy for two weeks without nutrients (Ryther et al.,

1981).

Bird et al. (1982) demonstrated that the majority of the increase in thallus N, as a

result of NH 4+ supply to N deficient G. tikvahiae, is in the form of protein. The predominant

form was amino acids, whereas DNA and inorganic N appear to be minimal as stores for N.

At the onset of N deficiency, amino acid concentrations declined rapidly, indicating these are

the first source of N to be utilised when ambient concentrations fall (Bird et al., 1982).

Inorganic forms of N are also rapidly utilised. Protein was utilised at a lesser rate than amino

acids or inorganic forms. After three weeks the total concentration of protein in G. tikvahiae

was still significant, being half the initial concentration (Bird et al., 1982). Nitrate raised

plants did not show the same decrease in the amino acid pool, as NH 4+ plants, probably due to

their N content being initially lower.

Healthy, well nourished G. tikvahiae plants are capable of growth for up to two weeks

in water which contains low to undetectable concentrations of inorganic N (Ryther et al.,

1981). G. tikvahiae appears to have a steady state pool of NH 4+ , which may exist from high

external NH 4+ supply, or NO 3

- which has been reduced to NH 4+. When their internal N

reserves are depleted, the algae start to lose their dark reddish-brown pigment, become a pale

straw-yellow colour, and cease growing (Ryther et al., 1981). These changes were shown to

be a result of a N deficiency, evidenced by a change in the plant tissue's total N concentration

from 3-4% to 1-2% of the total dry weight (Ryther et al., 1981). It was also noted that the loss

of colour was accompanied by an increase in the C:N ratio from 6:1 to nearly 30:1 (Lapointe

and Ryther, 1979; Ryther and Hanisak, 1981).

From the data presented in this section it is apparent that the ability of macroalgae to

store nutrients can provide a faster, easier and more accurate way to monitor the concentrations

of nutrients in the water column.

21

3.1 Nutrient Indicators

The nutrient concentrations of waterways can be determined directly from periodic

water column analysis, or by analysing various characteristics related to the nutrient storage of

inhabitant macroalgae (Wheeler and Björnsäter, 1992). The use of algae allows integration of

the nutrient regime over a period of time, rather than providing merely an instantaneous value

(Wheeler and Björnsäter, 1992). The characteristics of algal tissue used for analysis are

referred to as algal nutrient indicators. These indicators are affected by, and therefore have

been shown to reflect the concentrations of externally available nutrients. Other environmental

factors, including the type of the available nutrients and the availability of light, are also

capable of influencing the quantity or composition of the various indicators (Hanisak, 1983).

Consequently the results obtained from algal bioindicators may not be consistent across all

environments, and most certainly not between different species. To use plants as bioindicators

it is necessary to determine their critical concentration for each nutrient, i.e., the minimum

tissue nutrient content in each species which is necessary for maximum growth (Hanisak,

1979b). Concentrations below this are limiting to growth, while concentrations above are

referred to as luxury consumption (Gerloff and Krombholz, 1966).

The effectiveness of macroalgae as bioindicators is optimal under conditions where

other environmental parameters such as temperature, salinity, and light, are in no way limiting

to growth. It is therefore important to select species which are tolerant to changes in salinity or

temperature, and conditions of variable light (Masini et al., 1990; Hanisak, 1979a). Of the

macroalgae, Rhodophyta, appear to be the most effective bioindicators of water quality. A

primary reason for this is due to their ability to store nutrients and other trace elements, and

heavy metals in their accessory photosynthetic pigment structures. These phycobilisome

storage sites are located on the surface of the lipid bilayer of the thallus lamellae, and as such

are highly responsive to external conditions (Kursar and Alberte, 1983; Gantt, 1990).

As discussed in this section, internal concentrations of stored N reflect the

concentrations of externally available nutrients. The main three methods of determining water

column nutrient concentrations using plants as bioindicators involves the analyses of their

22

amino acid composition, pigment concentrations, or tissue nutrients (Bird et al., 1982;

Horrocks, 1993).

3.1.1 Amino Acids

The production of amino acids from N involves firstly the reduction of nitrogen.

Nitrate and nitrite are reduced to ammonium, while urea is hydrolysed to ammonium. In algae

this reduced N is assimilated into amino acids by either the glutamate dehydrogenase (GDH)

pathway or the glutamine synthetase (GS) / glutamate synthase (GOGAT) pathway (Wheeler,

1983; Turpin, 1991). The reductive amination of

α-ketoglutarate, catalysed by GDH, was previously believed to be the major pathway for

ammonium assimilation. The discovery of GOGAT has since shown that NH 4+ incorporation

by GS is possible (Falkowski, 1983). Ammonium is incorporated into glutamate by GS to

form glutamine. Finally, glutamine is reduced with α-ketoglutamate to form two molecules of

glutamate by GOGAT (Falkowski, 1983). The GS/GOGAT pathway is the major mechanism

for uptake of nitrogen by macroalgae, except for some green algae which employ the GDH

pathway when external NH 4+ levels are high (Wheeler, 1983).

Amino acid biosynthesis involves the production of a few key intermediates which are

either metabolised to waste products or used further for biosynthetic purposes (Arnstein,

1975). Glutamic acid and glutamine are the primary initial products of ammonium

incorporation (Falkowski, 1983). The glutamate family of amino acids (glutamate, glutamine,

arginine and proline) are perhaps the most important, as the N atoms of all other amino acids

are obtained via glutamate or glutamine (Arnstein, 1975). The synthesis of glutamate is the

means by which N is assimilated from ammonium, and as such is stimulated by the presence of

the ammonium (Arnstein, 1975; Kanazawa et al., 1970).

23

Figure 7 shows the biosynthesis of the glutamate family of amino acids including citrulline and

arginine which appear to act as major stores of nitrogen in the red algae.

Figure 7. Biosynthesis of the glutamate family of amino acids. The reactions shown for the biosynthesis of glutamate, glutamine, proline and arginine are assumed to occur in all organisms. Also included in the figure are the reactions leading from α-oxoglutarate to lysine which are found in certain fungi (from Arnstein, 1975).

24

Figure 8 illustrates the pathway of nitrogen assimilation in algae using the GS/GOGAT

pathway.

Figure 8. Simplified schematic diagram showing the proposed pathway for the assimilation of nitrate, nitrite, and ammonium in algae. The inorganic nitrogen species is first transported into the cell via some active transport system(s) involving reversible ATP hydrolysis. Nitrate is reduced by nitrate reductase (NR) to nitrite in the cytoplasm. Nitrite is reduced to ammonium by nitrite reductase (NiR) in the chloroplast. Ammonium is incorporated into glutamate by glutamine synthetase (GS) to form glutamine. Finally, glutamine is reduced with α-ketoglutamate to form two molecules of glutamate by glutamate synthase (GOGAT). GS and GOGAT appear to be located in both the chloroplast and cytoplasm (from Falkowski, 1983). In addition to the protein amino acids, plants contain large numbers of non-protein

amino acids (Fowden, 1990). A significant proportion of the N assimilated via plant roots is

incorporated into amino acids and proteins (Fowden, 1990). In particular, Steward and

Pollard (1962) found that asparagine, glutamine and arginine are N-rich storage or mobile

substances. Additionally, alanine, aspartic acid, and glutamic acid were found to be main

starting points for N metabolism (Steward and Pollard, 1962). Research by Vona et al. (1992)

has shown that in the case of Cyanidium caldarium, resupplying N-limited plants with

ammonium caused cell concentrations of glutamine, citrulline, arginine, alanine, serine, δ-

aminole-vulinic acid (δ-ALA) and putrescine to increase. Citrulline showed the most

noticeable increase upon addition of ammonium. In Anabaena cylindrica the synthesis of

citrulline is an important means of ammonium assimilation through carbamyl phosphate

formation (Chen et al., 1987). Addition of phosphate, however, yielded different responses in

the concentrations of the various amino acids. The type of nitrogen being assimilated as well

as the availability of light also affects the algal free amino acid pool (Bird et al., 1982).

25

In figure 9 the changes in total amino acid concentration are graphed against the nitrogen type

and whether the plants were grown in the light or dark.

Figure 9. Changes in the percentage (dry wt. basis) of amino acids of Gracilaria tikvahiae after immersion for 24 h in either NH 4

+ or NO 3- enriched seawater in darkness or in light. Controls were held in ambient

daylight. Day 1 represents the data for thalli immediately after 24 h immersion in the N supplements, at which time the N supplement was drained and the thalli were returned to unenriched ambient seawater at one turnover per day (adapted from Bird et al., 1982). In the green algae Chlorella spp., ammonium stimulates the synthesis of glutamine and

other amino acids associated with the TCA cycle (Hattori, 1958; Hiller, 1970; Kanazawa et

al., 1970; Miyachi and Miyachi, 1980). Further, it stimulates dark respiration, dark CO2

fixation (Miyachi et al., 1977) and starch breakdown (Miyachi and Miyachi, 1980). In an

experiment conducted by Ohmori et al. (1984), Chlorella spp. grown on nitrate (50 mM) for a

week contained asparagine, glutamate and arginine as the major free amino acids. Asparagine

constituted approximately 30% of the total free amino acids. These cells were subsequently

subjected to N starvation. After two days the total cellular amino acid concentrations had

fallen to approximately 20% of the original concentrations. Asparagine and arginine fell

markedly, indicating that they may be important N sources for the cells during starvation

26

(Ohmori et al., 1984). In these N-starved cells, glutamate and alanine became the major free

amino acids, while glutamate could not be detected. Ohmori et al. (1984) also found that the

addition of NH 4+ to Chlorella vulgaris before N starvation caused no significant changes in

amino acid composition or the level of dark respiration. In the N starved cells however, the

addition of NH 4+ caused dark respiration to increase 3 to 4 fold, and the amino acid

composition to change markedly, especially those related to the TCA cycle. Increases were

observed in alanine, while the concentrations of glutamine and glutamate varied with time after

addition of the NH 4Cl. Glutamine synthesis via glutamine synthetase is thought to be the

primary step in ammonium assimilation by algae (Ohmori et al., 1984). Hattori (1958)

observed that on addition of ammonium or urea to Chlorella ellipsoidea II , the most marked

increase was in arginine. However the increase with urea was not as rapid as with ammonium.

The absence of glutamine in the assimilation of urea may allow easy detection of urea

assimilation by macroalgae. The process of N assimilation for the two forms of N is as

follows:

� Ammonia → amide (glutamine) → arginine → cell substance

� Urea → arginine → cell substance

The prevalence of dissolved free amino acids (DFAA) in the water may be linked to

the concentrations found in algae in the area (Jørgensen, 1982). These concentrations were

found to be partially reflected in the water column chlorophyll a concentrations (Jørgensen,

1982). However it was noted that the occurrence of nitrate and ammonium in the region was

independent of DFAA concentrations, and the major source of nitrate and ammonium was the

river. The major amino acids present were serine, glutamic acid, glycine, ornithine and

alanine. Estuarine heterotrophic assimilation of DFAA's can account for approximately 10%

of primary production in the summer, whereas in offshore waters it has a range of 1 to 10%

(Jørgensen, 1982). The natural range of concentrations of DFAA's are therefore higher in

coastal and estuarine waters and are positively correlated with the level of primary production.

Assimilation of free amino acids was found to be quantitatively important in the cycling of

carbon when compared with primary production (Jørgensen, 1982). This may affect the C:N

27

ratio in the marine environment, as low C:N values in algae are representative of periods of

rapid increase in biomass (Niell, 1976).

3.1.2 Pigments

In the red algae, such as Gracilaria spp. there are a number of accessory pigments

which capture light and transfer it to chlorophyll a (Raven et al., 1987). These pigments are

referred to as phycobiliproteins, and include phycoerythrin, phycocyanin and allophycocyanin.

They exist together in a macromolecular complex called a phycobilisome which is attached to

the photosynthetic lamellae (Bird et al., 1982; Gantt, 1990; Lapointe, 1981). These

phycobilisome structures enable efficient transfer of energy from phycoerythrin to phycocyanin

(Kursar and Alberte, 1983).

Figure 10 illustrates the structure of the phycobilisome with the photosystems. It should be

noted that presence of phycoerythrin at the outside of the complex makes the algae very

sensitive to external changes in nutrient concentrations.

Figure 10. Schematic arrangement of phycobiliprotein organisation in a phycobilisome of Porphyridium purpureum and the presumed arrangement of the phycobilisome with the photosystems. The terminal pigment may mediate excitation energy transfer between phycobilisome and the photosystem II - antennae chlorophyll (from Gantt, 1990).

28

Pigment concentrations in algal tissue provide a strong representation of the

concentration of N available from the water column. Vergara and Niell (1993) demonstrated

that in Corallina elongata the proportion of pigmented and nonpigmented proteins varies with

N concentrations and light availability. This appears to function as a mechanism for storage of

N for subsequent periods, when nutrients are limiting. While plant pigments are primarily light

absorbing substances, during times of nutrient limitation they are often used as a source of

protein (Lapointe, 1981; Bird et al., 1982).

Phycoerythrin is assimilated at a slower rate than the total protein pool (Bird et al.,

1982). However, as its production also occurs in the dark, it is synthesized at a faster rate than

chlorophyll. This indicates that it is an important storage pool for N, as well as a light

absorbing protein (Bird et al., 1982; LaPointe, 1981; and Gantt, 1981). Wyman et al. (1985)

noted that phycoerythrin in the marine cyanobacterium Synechococcus strain DC2 is used as a

N reserve and a collector of quanta for photosynthesis. Chlorophyll a and phycoerythrin

concentrations Gracilaria foliifera and Gracilaria conferta have been shown to be inversely

correlated to the light level and growth rate (Lapointe, 1981; Friedlander et al., 1991).

However, Algarra and Niell (1990) showed that in Corallina elongata phycoerythrin was

inversely correlated to light irradiance, while chlorophyll was positively correlated. Lapointe

(1981) observed that the phycoerythrin and chlorophyll a content correlated positively with

%N content. Growth of Gracilaria foliifera was shown to correlate positively with the carbon

: phycoerythrin and carbon : chlorophyll values, but negatively with %N and phycoerythrin and

chlorophyll a concentrations. When grown close to its maximum rate at high light intensities,

G. foliifera exhibits a significantly lower phycoerythrin : chlorophyll a ratio than when

growing more slowly under lower light (Lapointe, 1981). Further examination of these studies

suggests that plant responses to changes in light are often variable between species.

Pigment production (like growth), is stimulated differentially with the various nutrient

types. For example, Gracilaria tikvahiae, when exposed to NH 4+, produces greater

concentrations of phycoerythrin than when exposed to NO 3- (Bird et al., 1982). This study

also demonstrated that ammonium fed G. tikvahiae contained less chlorophyll and

phycoerythrin in dark treatments than in the light. However, nitrate fed plants in the dark

29

showed an increase in phycoerythrin relative to the light treatment. Experiments by Bird et al.

(1982) involving N deprivation and resupply, also indicated that within the protein pool of G.

tikvahiae, phycobiliproteins are an important N reserve. At low irradiance levels

phycobiliprotein synthesis (both r-phycoerythrin and r-phycocyanin) occurred when N supply

was sufficient. Saturating irradiances resulted in pigment degradation under N - limiting

conditions, however, some phycoerythrin production was observed under N - sufficient

conditions. N - limitation stimulates the flow of internal N metabolites towards synthesis of

nonpigmented proteins.

As pigment production is enhanced by low light and sufficient nutrients, it would be

expected that algae growing in nutrient enriched rivers will have high pigment concentrations.

Lapointe (1981) found that G. foliifera grown under low light had almost double the

concentrations of phycoerythrin and chlorophyll a than when grown under high light. Further,

with increasing growth the concentrations of phycoerythrin and chlorophyll a are reduced.

Percent N values correlated with phycoerythrin and chlorophyll a, but were inversely

correlated with the C:N ratio. For example, Gracilaria spp. changed from its natural light

green-brown colour to a deep red with increases in N supply (Lapointe et al., 1976; Horrocks,

1993). Harlin and Thorne-Miller (1981) reported that

G. tikvahiae didn't always increase its growth rate with added ammonium or nitrate, but a

reddening of the tissue was observed. This suggests that Gracilaria spp. stores as much N in

reserves as possible before using the other available N for growth. Measuring the storage of N

in pigments provides a more sensitive measure of increases in N supply than simply observing

increases in growth.

3.1.3 Tissue Nutrients

Plant tissue analysis can be used to determine a plant's internal N or P content. This is

considered to be the most accurate method for determining the plant's nutrient status, i.e.,

limited, sufficient or excessive (Hanisak, 1983). This method has traditionally been used for

analysis in terrestrial and aquatic vascular plants (Gerloff and Krombholz, 1966). Tissue

analysis allows determination of a species' critical nutrient concentration, i.e., the

30

concentration which just limits its maximal growth (Hanisak, 1983). For example, the critical

N concentration of Gracilaria tikvahiae is approximately 2% dry weight (Hanisak, 1981,

1983). The minimum N content for growth was found to be 0.8% dry weight. Therefore,

between 0.8% and 2%, growth will be proportional to internal N content. At concentrations

above 2%, there is the opportunity for luxury storage of nutrients. Figure 11 shows how

Codium fragile continues to absorb and store nitrogen at rates above what it can use for growth

i.e. the critical nitrogen concentration.

Figure 11. The relationship of the growth of Codium fragile as the increase in dry weight over a 21-day period in culture and its internal nitrogen concentration, as percent of dry weight (from Hanisak, 1979 b).

Nitrogen and P are the most frequently measured nutrients in macroalgae, as these are

generally the limiting nutrients (Lyngby, 1990). The composition of plant tissue varies between

the algal groups on both temporal and spatial scales, with internal N content reflecting the

external ambient concentrations. The species used for analysis is very important as some

provide a better representation of the water column nutrient status than others. In general, the

red algae provide the most accurate correlations, for example Lyngby (1990) used Ceramium

rubrum which provided an R-squared value of 0.60. However, studies using the green

macroalgae Cladophora spp. resulted in an R-squared value of only 0.14, indicating no

correlation (Lohman and Priscu, 1992). The concentrations of nutrients also vary between

different morphological forms of algae (Littler and Littler, 1980). Accurate determination of

the nutrient status of macroalgae can be hindered by a number of problems. For example, algae

can be subject to toxicity from high concentrations of ammonium or organic N, if used in place

of nitrate (Lapointe and Ryther, 1979). It was found for example, that while NH 4+ is usually a

31

better source of N than NO 3-, at times it may be toxic (Lapointe and Ryther, 1979), resulting in

uncoupling of photophosphorylation (Raven, 1980).

Studies conducted in the laboratory may give deceptive results regarding the algae's

ability to use in situ concentrations of different N sources. Iwasaki (1967) noted that the

preference of one N source over another was dependent on the concentration used in the

medium. Algae may utilise different sources of N as a result of other environmental factors, of

which light appears to be the most important (Bird et al., 1979). Under low-light laboratory

conditions, G. tikvahiae grew more successfully on NH 4+ than NO 3

- (DeBoer et al., 1978).

However in high-light outdoor tanks, the species grew equally as well on either nutrient source

(Lapointe and Ryther, 1978).

Macroalgal C:N ratios are reported to be between 5 and 40 (Niell, 1976 and Hanisak,

1979b). These ratios have been used in an attempt to assess the N status of macroalgae.

Whilst this is an appropriate method for interpreting the physiological state of algae, it must be

noted that changes in the ratio may be due to changes in carbon as well as N metabolism

(Hanisak, 1979b). Studies on the critical C to N ratio for macroalgae have revealed ratios

between 10 and 15. Higher values are indicative of N limitation, while lower ratios represent

N storage (Hanisak, 1983). Above the critical C:N value of between 10 and 15, NH 4+ uptake

increased as the C:N ratio increased, however, below the critical concentration there appeared

to be no change in uptake rates. Unfortunately C:N ratios may be influenced as much by light

intensity as by N availability (Lapointe, 1981; Lapointe and Tenore, 1981). Protein :

carbohydrate ratios have been used to detect N limitation in Gracilaria verrucosa, and it

appears that, in general, there is an inverse relationship between the concentrations of N and

the carbohydrate content of the thallus (Bird et al., 1982). Hanisak (1983) agrees that internal

N concentration, carbon : nitrogen ratios and protein : carbohydrate ratios are effective

methods for determining the N status of macroalgae. Enzymatic methods may also be used to

determine a plant's N status. Such methods rely on the process in which the algae's internal N

concentration regulates enzyme activities and hence its metabolic capacity (Küppers and

Weidner, 1980).

Algal nutrient indicators, as outlined in this section, appear ideal as a means to

integrate the nutrient regime of a waterway. As well as nutrients there are a number of other

32

factors which can affect the levels of these indicators. These factors can be classified as

parameters of water quality.

33

4. Water Quality

Water quality includes a wide range of parameters, including nutrient concentrations,

light availability, temperature, salinity and water movement. Waters that are high in nutrients

as a result of human inputs (e.g., sewage and fertiliser plant waste) are commonly highly turbid

due to increases in terrestrial sediment runoff from land clearing, and more direct impacts such

as dredging (Moss, 1987). These impacts result in large decreases in the levels of light being

made available to all submerged aquatic vegetation. The middle reaches of a river generally

exhibit the highest turbidity. This is due in part to the usual concentration of dredging efforts in

this region, and a low level of fresh water input into the river (Moss, 1987). The natural

effects of the saltwater - freshwater boundary cause all estuaries to be more turbid in this

region, due to the various internal cycling processes (Postma, 1967). The resulting low light

availability prevents the usual increases in algal growth associated with increased nutrients

(Moss, 1987).

The vertical profile of oceanic waters usually shows a marked decrease in nutrients

near the surface. This is due to the uptake of nutrients by phytoplankton in this photic zone

(Valiela, 1984). The level of nutrient flux in the coastal environment is frequently high due to

river surges (Valiela, 1984). Hence using water column analysis can yield significantly

different values depending on the time of sampling.

Human inputs into the river can be categorised into point and non-point (diffuse)

discharges. In the Brisbane River, Queensland one of the main sources of terrestrial input is

secondarily treated sewage. Other inputs include waste from agricultural practices and

industry. Industrial input includes heated water (causing thermal pollution), high nutrient output

and toxic inputs from heavy industry (Moss, 1987). These inputs affect the concentrations of

nutrients accessible to the macroalgae and the availability of light.

4.1 Light

Light is a major factor limiting the growth of macroalgae in the marine environment

(Hanisak, 1983). The quantity of light which is received by benthic attached algae is

34

determined by water clarity and depth (Valiela, 1984). The intensity, and the quality of the

light changes as it passes through the water column. Species that are subjected to low light

have developed compensatory mechanisms to survive successfully in these conditions. Such

mechanisms include high pigment content, high accessory pigment : chlorophyll a ratios

(Reiskind et al., 1989), mechanisms or morphological differences to cope with the varying

wavelengths that are received in deeper waters, and low photosynthetic saturation levels

(Hanisak, 1979b).

Light differentially affects the uptake of the different forms of inorganic N. The

intensity, as well as the duration of low light periods affects the N uptake by the algae. For

example, the uptake of ammonium by Gracilaria tikvahiae in the dark is initially the same as in

the light, but decreases with time (Ryther et al., 1981). Light exposed plants can continue to

take up ammonium for a longer period of time (Ryther et al., 1981). This will increase the

stores of N available for times when ambient concentrations are limiting. The production of

amino acids in macroalgae with a high NH 4+ nutrient supply is enhanced in the dark (Bird et

al., 1982). Carbohydrate concentrations in the dark were lower, suggesting that the available

carbon was being used to fuel increased amino acid and protein synthesis. This has been found

to be a common mechanism in other plants (Kanazawa et al., 1970; Platt et al., 1977). Despite

their dependence on light, the saturation light levels for N uptake in macroalgae are fairly low,

e.g., 7-28 µ mol quanta m-2 s-1 for Codium fragile (Hanisak and Harlin, 1978).

However Lapointe and Tenore (1981) found that light levels and growth rates of Ulva

fasciata were inversely proportional to NO 3- uptake at medium and high concentrations of N

loading. The uptake of nitrate and nitrite in algae is more strongly dependent on light than

ammonium (Hanisak, 1984; Falkowski, 1983). This suggests that in regions of low light algae

will be more efficient at utilising ammonium than nitrate or nitrite. Although ammonium uptake

is strongly reduced under low light conditions, it has been found that in C. fragile, for example,

it is still greater than the light uptake of nitrate and nitrite (Hanisak and Harlin, 1978). For data

collected under experimental conditions, it is important to note the effect of continual supply by

artificial lights versus natural light on a day/night cycle. Ryther et al. (1981) demonstrated that

under natural light on a day\night cycle, the uptake of ammonium by N-starved G. tikvahiae

was significantly higher in the light than in the dark. However the same experiment conducted

35

with the light treatment exposed to artificial lights for the entire period resulted in greater

assimilation of ammonium in the dark treatment.

4.2 Temperature

Temperature is a significant factor controlling N uptake in macroalgae. It has a marked

effect on respiratory and dark reaction photosynthetic enzyme activity (Kain and Norton, 1990).

Cells at lower temperatures have higher concentrations of photosynthetic pigments, enzymes

and carbon (Valiela, 1984). Uptake of nutrients and the maximum photosynthetic rate of algae

at a particular light level, increased with an increase in temperature (Valiela, 1984).

Temperature thresholds vary between species. Hanisak and Harlin (1978) have shown

that for the green algae Codium fragile, intermediate temperatures of approximately 12°- 24°C

provide the maximal conditions for the uptake of all forms of inorganic N. Higher temperatures

of approximately 30°C resulted in slightly lower uptake, and temperatures of approximately

6°C produced the lowest uptake. Whilst most other species exhibit similar thresholds to C.

fragile, G. tikvahiae has the ability to withstand much wider temperature ranges (Bird et al.,

1979). G. tikvahiae also demonstrated an ability to respond rapidly to changes in temperature

with a 20-fold increase in growth occurring between 10°C and 20°C (Bird et al., 1979).

4.3 Salinity

The tidal cycle is the main factor in controlling variation in salinity in estuarine

environments. Changes in freshwater runoff, fluctuations in the currents, storms, winds and the

solar cycle, also affect salinity (Yarish and Edwards, 1982). Species in estuarine

environments must withstand relatively high changes in salinity over a short time period.

These rapid changes can have marked effects on macroalgal growth. This is a harsh

environment in which only a few well adapted species can flourish (Josselyn and West, 1985).

In particular, G. tikvahiae has one of the highest tolerances, being capable of relatively high

growth rates at salinities of between 17 and 40‰ (Bird et al., 1979). As well as interspecific

36

differences, G. verrucosa displays intraspecific physiological adaptations to the local salinity

conditions (Yarish and Edwards, 1982). The number of species of macroalgae usually

decreases with declining salinity, which indicates that macroalgae are typically marine

organisms (Lavery and McComb, 1991).

4.4 Water Movement

Water movement is important for algal growth as it changes the algae's surrounding

water, thereby replenishing its nutrient supply, removing waste products and preventing the

settling of silt (Kain and Norton, 1990). Water movement also results in downstream

deflection of the thallus. This places the algae closer to the surface and perpendicular to the

sun and therefore increases growth (Jones, 1959).

The boundary layer, being the layer of water closest to the plant surface, experiences a

reduced flow rate due to adhesion. Diffusion of nutrients across the boundary layer must

therefore be facilitated by water movement. Gerard (1982) demonstrated that with Macrocystis

pyrifera the rate of movement of 2.5 cm s -1 for maximum nutrient uptake is always exceeded in

situ. However, Wheeler (1980) using water tunnel experiments, calculated that for M.

pyrifera photosynthetic output could be increased 300% by increasing the water speeds over

the algal blade from 0 to 4 cm sec -1. Above 4 cm sec -1 the assimilation of carbon was

responsible for limiting algal growth.

37

5. Use Of Bioindicators

Nutrient enrichment of the world's waterways is increasing with increasing levels of

agricultural and industrial wastes being deposited into rivers and oceans. The concentration of

the dissolved nutrients found in waterways are dynamic, changing on tidal and diel timescales,

with seasonal and longer term changes over many years. Standard water column analysis

techniques are inadequate as they only detect the instantaneous nutrient concentrations at the

time of sampling.

Macroalgae, together with seagrasses, and mangroves are capable of integrating the

concentrations of these nutrients over an extended period of time. Changes in macroalgal

pigment content, amino acid composition, and tissue nutrient concentrations may be used to

determine the nutrient status of the water body in question.

Water quality can include the nutrient status of a water body, but also includes or can

affect a number of other factors which limit the growth of aquatic plants. Some of these factors

include nutrients, temperature, salinity, light, and water movement. It is therefore important to

use a species which is tolerant to significant changes in the above parameters so that an

accurate representation of the nutrient status of the water is achieved. The red algae are

considered ideal as bioindicators due to their tolerance to the abovementioned parameters, the

nature of their pigments, and their ability to store large quantities of nutrients during times of

high nutrient availability.

Most studies involving the use of macroalgae, and indeed all plant forms have

concentrated on analysing the tissue nutrient concentrations. However it has recently been

shown the analyses of the pigment content of macroalgae can provide an accurate

representation of the nutrient status of water body in question. The use of amino acid analysis

is at present only new, and often limited only to in vitro studies. However data collected so

far (e.g. Horrocks, 1993) suggests that it may prove to be a more accurate and simple method

than pigment or tissue nutrient analysis.

38

6. Summary

Nitrogen has been identified as the major limiting nutrient to macroalgal growth in

coastal and estuarine ecosystems. Ordinarily, dissolved inorganic nitrogen (DIN) is the

limiting form. For the majority of algal species, ammonium is the most easily assimilated type

of DIN, although some species may prefer nitrate, nitrite or an organic form such as urea. As

opposed to marine systems, freshwater systems are typically phosphorous limited. There are

three types of nutrient limitation in the marine system, growth of current population limitation,

net primary production limitation and net ecosystem productivity limitation. Plants which are

exposed to nutrient limiting conditions exhibit symptoms specific to the element which is

missing or growth limiting.

Uptake kinetics show that macroalgae can assimilate nitrogen at rates independent and

far in excess of uptake associated with growth. The algae stores the extra N for periods of

nutrient limitation. In species with large inorganic pools nitrogen is stored in the vacuole,

whereas species with smaller inorganic pools store nitrogen in the form of proteins or amino

acids. In addition to storage pools, algae also contain N in structural (cell walls etc.) and

physiological (enzymes and photosynthetic pigments) pools. Amino acid, pigment and tissue

nutrient stores are used as a protein source. Amino acids are the first to be utilised at the onset

of N deficiency.

The composition and concentrations of tissue N, pigments and amino acids in algae can

be used as bioindicators of water column nutrient availability, as they respond to changes in the

concentration of externally available nutrients. The type of nutrient, e.g., nitrate, nitrite,

ammonium or urea also modifies the algal indicators. The amino acid composition is

potentially the most responsive indicator to the type of nutrient supplied. Further research is

required to qualify the exact relationships between nutrient type and the composition and

quantities of the various free amino acids present in the algae.

Additional water quality parameters including light, temperature, salinity and water

movement influence the composition and concentration of the various algal bioindicators.

Temperature, salinity and water movement mainly affect the speed at which the algae grow.

Light influences the type of nutrient which is assimilated, the chlorophyll : phycoerythrin ratio

and the C:N ratio of the tissue nutrients. These parameters can be significantly influenced by

39

human inputs. Agricultural runoff and disposal of waste from sewage treatment plants and

industry contribute to the eutrophication of waterways. The effect of these nutrient inputs is

regulated to some degree by the hydrodynamic regime of the region.

Nutrient enrichment of the world's waterways is an ever increasing problem which

cannot be fully understood with standard water column analysis techniques. The use of

macroalgal bioindicators to integrate the nutrient regime over time is an ideal solution. The

red algae, and in particular the genus Gracilaria because of its ability to survive in a wide

range of habitats and the nature of its nitrogen storage pools, has proved to be the most

effective bioindicator. Future research may employ the Gracilaria spp. in the analysis of free

amino acids to yield an effective and accurate representation of the quantity and type of

nutrients capable of being utilised by the aquatic flora.

40

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