Proceso

This section describes two methods for quantifying the functioning of phytoplankton cells in field populations, by measuring the phytoplankton response to light. The first method is the traditional measurement of 14_{C-labelled carbon fixation of a phytoplankton sample across a}

range of light conditions, the Photosynthesis vs. Irradiance (P vs. E) method. A detailed discussion of the physiological information gained by this method is described by MacIntyre et al. (2002) as well as Sakshaug et al. (1997) and Laws (2002).

A more recent technique is the use of active fluorescence here described in terms of the Fast Repetition Rate Fluorometer (FRRF). The measurement of the fluorescent response of phytoplankton to a sequence of excitation flashlets provides information on part of the

photosynthesis process (Falkowski and Raven 1997). Detailed discussion of this theory behind the technique is given by Kraus and Weiss (1991), Kolber and Falkowski (1993), Kolber et al. (1998) and reviewed by Falkowski and Raven (1997).

1.4.1 P vs. E Experiments

Measurement of photosynthetic rates for a phytoplankton sample over a range of light conditions provides a description of the inherent light-dependency of photosynthesis and photoacclimation state of the phytoplankton (Fig 1.3). This approach can be employed to measure the light-response of any variable, for example, 15_{N uptake, although focus here is on}

the measurement of photosynthetic rates by 14_{C-fixation or O}

2- evolution over the time course of

the incubation (Williams 1993). Photosynthetic rates are typically normalised to chl-a.

Figure 1.3. Schematic of Photosynthesis verses Irradiance (P vs. E) curve. Parameters indicated are described in text. α E_k P_m β P h ot osynt h esis Irradiance α E_k P_m β P h ot osynt h esis Irradiance

The shape of the P vs. E curve varies with the photo-acclimation state of phytoplankton and can vary both within and between species (Sakshaug et al. 1997). The shape of the P vs. E curve is described by the parameters indicated in Figure 1.3, the ecological relevance of these parameters is described below.

The intercept of the P vs. E curve is typically non-zero. In the case of oxygen evolution, the intercept is typically less than zero due to net community respiration and in the case of carbon fixation it is always positive resulting from light-independent processes and / or systematic experimental artefacts (Sakshaug et al. 1997).

Physiological Parameters

α (and ΦM) - The initial slope of the P vs. E curve is close to linear and represents the increase in the amount of photosynthesis per unit incident light. It is known as the maximum light utilization coefficient (Mauzerall and Herron 1970, Sakshaug et al. 1997) denoted α and has

units of (mg O2 evolved or CO2 fixed m-3 h-1) (μE m-2 s-1)-1. In this linear region of the curve the

rate-limiting process is the absorption of photons by the light harvesting antennae, and photosynthesis is light-limited.

The value of α is dependent on the spectral quality of the light in the incubator relative to the spectra of light absorbed by the phytoplankton. Although relative trends are informative, the absolute value has no ecological significance unless a correction is applied to account for the difference between the spectral light field in the P vs. E incubator compared to in situ. When the

photosynthetic rate is normalised to chl-a, α is denoted α* and has units (mol O2 evolved or CO2

fixed h-1_{) (mg chl-a)}-1 _{(μE m}-2 _s-1₎-1_.

The maximum quantum yield (Φm) is the amount of carbon fixed per unit light absorbed

(Mauzerall and Herron 1970, Sakshaug et al. 1997). It is related to α* by the ratio of incident light to light absorbed (Falkowski et al. 1985) and is calculated from Equation 1.6:

0231

.

0

*

1

*⋅

⋅

=

a

α

φ

(1.6)

where a* is the spectral light absorption by phytoplankton normalised to chl-a (m2 _{(mg chl-a)}-1₎

and hours to seconds. Thus Φm is independent of both chlorophyll-a and unit area and has units

of (mol O2 evolved or CO2 fixed (mol absorbed photons)-1). Since 8 photons are required to

derive one molecule of O2, it follows that Φm has a theoretical maximum of 0.125 (Kok 1948).

Variability in α* is theoretically largely genotypic as a result of changes in a* due to the pigment composition and pigment packaging (Geider et al. 1993a, Babin et al. 1996, MacIntyre et al. 2002). Significant decrease in the maximum quantum yield (Φm) may occur in surface

phytoplankton with increased proportions of photoprotecting pigments that absorb light but do not transfer energy to photochemistry (Marra et al. 2000). However, quantum efficiency is also seen to decline under nutrient stress (Welschmeyer and Lorenzen 1981, Geider et al. 1993b) but the role of light and nutrients in phenotypic variability of this parameter remains unclear

(MacIntyre et al. 2002).

Pm - The light-saturated photosynthetic rate, Pm, is the maximum rate of photosynthesis and has

units of (mg O2 evolved or CO2 fixed m-3 h-1). At this maximal photosynthetic rate a further

increase in irradiance will not cause and increase in photosynthesis. As such, it is the processes downstream of light capture (such as electron transfer or carbon fixation) that limit the rate of photosynthesis (Sakshaug et al. 1997). Pm is independent of the light capture processes,

including the absorption cross-section of the photosynthetic apparatus, and is therefore not spectrally dependant.

Pm is typically normalised to chl-a, and is denoted P*m with units (mg O2 evolved or

CO2 fixed (mg chl-a)-1 h-1). In order to obtain a measure of phytoplankton growth rates, Pm

would require normalisation to carbon biomass, however, due to the difficulties in measuring carbon biomass (or chl-a:C) in the field photosynthesis is typically normalised to chl-a.

In contrast to the light-limited quantum efficiency, variability in light-saturated growth is typically observed to be largely governed by irradiance rather than between species (Geider et al. 1993a). However, a variety of phenotypic responses in P*m is also evident in nutrient stressed

cultures, as well as some variability observed between species (Cullen et al. 1992, Geider et al. 1993b).

Ek - The light saturation parameter, Ek, is a theoretical point on the P vs. E curve representing

the incident light at which the rate limiting processes of light capture and photochemical energy conversion and reductant utilization are balanced (Talling 1957, Escoubas et al. 1995, Sakshaug et al. 1997). Ek has units of light (μE m-2 _s-1_{) and is calculated by Equation 1.7.}

* *

α

P

Ek = (1.7)

Ek is independent of the variable used to normalise photosynthesis (typically chl-a). Like α*, Ek is spectrally dependant and as such its absolute value has no ecological relevance unless spectral differences between the light source used in the incubator and in situ irradiance are accounted

for. Resulting from processes controlling P*m and α*, Ek occurs at an irradiance just below that

of maximal photosynthesis and represents the balance between maximising growth rates and limiting the potential of photo-damage under very high irradiances (Geider et al. 1998, Laws et al. 2002).

β - Increasing irradiance beyond the level of saturation, eventually leads to a decline in photosynthetic rate. Quantified by parameter β* this is known as photoinhibition.

Photoinhibition reflects photo-damage in the phytoplankton antennae and is dependant on both the level of irradiance and the duration of exposure (Falkowski and Raven 1997).

1.4.2 Fast Repetition Rate Fluormeter

The Fast Repetition Rate Fluorometer (FRRF) is used to investigate photosynthetic rates via measurement of the fluorescence response to a prescribed series of stimulating flashes (Falkowski and Raven, 1997). In contrast to 14_{C-uptake or O}

2-evolution photosynthetic rate

measurements described above, the FRRF measures processes associated with PSII only rather than the entire photosynthesis process. The following is a brief overview of the relevant photosynthetic processes described more fully by Falkowski and Raven (1997).

As mentioned above, on capturing a photon of light at PSII (and subsequent excitation of an electron at the associated reaction centre) the first electron acceptor (Qa) of the electron transport chain between PSII and PSI becomes reduced, but only if the reaction centre is initially ‘open’ (i.e. Qa is initially oxidized). When the reaction centre is ‘closed’ (Qa is already reduced) there is a high probability that the absorbed light energy will be re-released as fluorescence.

At room temperature algae fluoresce at about 685 nm (Falkowski and Raven 1997). The stimulating flashes given by the FRRF induce a change in the ratio of fluorescence emitted to light absorbed at PSII, a parameter known as the fluorescence yield (F) (Kolber and Falkowski 1993). Figure 1.4 illustrates the change in fluorescence yield of phytoplankton given a sequence of flashlets by the FRRF over time.

Figure 1.4. Schematic of the fluorescence yield resulting from a sequence of saturation and relaxation flashlets given by the FRRF. A full description of this process (and identification of the parameters indicated) is described in text.

Under low illumination algae fluoresce at a constant low level, denoted F0. With increased light,

as reaction centres become closed, fluorescence increases until it reaches a maximum level, Fm.

The difference between the maximum and minimum levels of fluorescence (Fm – F0) is called

the variable fluorescence Fv. It follows that fluorescence is closely related to the oxidation state

of Qa as this affects the rate at which electrons are transferred from PSII. The excitation flashes from the FRRF are of equal intensity but are delivered in close repetition designed to

progressively close reaction centres and raise fluorescence from F0 to Fm.

At the plateau of the fluorescence yield at Fm and the achievement of the closure of all

RCIIs, PSII is light-saturated. Under conditions of PSII light-saturation the rate of light absorption of PSII is equal to or exceeds that of steady-state electron transport away from Qa, i.e. the rate limiting process for the turnover of electrons through PSII when PSIIs are light saturated is this oxidation rate of Qa. In order to measure the fluorescence yield as reaction centres become open a series of sub-saturating ‘relaxation flashes’ are given. The decrease in fluorescence yield illustrates the re-opening of reaction centres as electrons are passed from Qa to PQ, the timescales of which relates to the rate of electron transfer (1/τ).

Physiological Parameters

Fv/Fm - The variable fluorescence yield, Fv, is commonly normalized by Fm to give an

expression, Fv/Fm, known as the quantum efficiency of photochemistry. Fv/Fm represents the

amount of energy absorbed by PSII to that which is emitted (as fluorescence), and is therefore a σPSII F_m F luo re s c e n c e Yi el d

Saturation Flash Number

F0