Poss ible so urces of error in the oxygen method

Quantitative estimation of dissolved oxygen content I

This is the basis of the oxygen technique, and if the conversion from titre of sodium thiosulphate to microlitres of oxygen were in error, there would be a corresponding error in estimates of photosynthetic (and

respiration) rates. Drew & Robertson (1974a, & Appendix 1) found, however,

that the method gave estimates of the oxygen content of seawater which ,

were in accordance with theoretical values (e.g. Richards & Corwin 1956).

Reactions of algal tissue with oxygen %

i By placing algal tissue samples in incubation bottles with seawater

and processing with the Winkler technique immediately, it was shown that :,';T 4

no,non-metabolie uptake of oxygen occurred (e.g. by oxidation of 4

exuded mucilages, etc) at least in the short-term.

Photosynthesis and/or respiration by microorganisms 7 J

^ %

^

3 ** i V

■

iÂ-

Photosynthetic rates of up to 26 yg C m h have been recorded X i

for phytoplankton in seawater in the temperate zone (Wallen & Geen 1971). ÿ

Such rates could account for up to ^10% of the oxygen produced in an 4

incubation bottle containing a macroalgal sample of low photosynthetic «

capacity, leading to a slight overestimate of photosynthesis for the tissue,

-à

However, on several occasions, when seawater controls were assessed for % oxygen content at the commencement of an experiment, and also after

incubation in the light and dark, changes in oxygen contents were negligible |

in comparison with the changes incurred by macroalgal samples. Sargent & ^

Lantrip (1952), however, estimated that oxygen uptake in dark bottles containing# discs of Macrocystis pyrifera was increased by bacterial oxidation of

exuded mucilage. They assumed that one half of the total oxygen uptake 'Ij

9

'3 % 6k

Effects of surfaces

Laevastu et al.(19^5) found that, in phytoplankton productivity experiments, oxygen uptake (in the dark) increased in direct proportion to the surface area of inert material (glass rods) present. In the present work, this could lead to higher respiration and lowered photo­ synthesis estimates in algae posessing high surface:volume ratios.

As Drew and Robertson (1974 a & Appendix l) found, the use of

plastic bottles in this technnique is inadvisable due to inward diffusion of oxygen.

J

c. Possible physiological sources of error in both methods | Photosynthetic quotient (PQ 4= AO^ ; -ACO^)

After analysing several phytoplankton species for carbon, oxygen and nitrogen content, Ryther (1956b) concluded that the PQ for long term growth must be greater than unity, and he suggested a value of 1.2 5.

Similarly, Westlake (I96 3) recommended the use of PQ = 1.2 for plants growing in unfavourable conditions and 1.25 when conditions were optimal. In the present study it is probable that, considering short-rterm photo­ synthesis of the red algae studied, most photosynthate is in the form of carbohydrate (see Majak et al.I96 6) and that a PQ of unity is thus a reasonable assumption. If the PQ was in reality around 1.25, and a PQ of 1 was used in conversion calculations (as here - Table 2.7) the carbon fixation rates calculated from oxygen method experiments would exceed the true values. This error does not occur in the direction

expected from the results in the present work, which implied that carbon |

fixation rates estimated from oxygen method experiments were, if anything, underestimates of the true values, since they were less than those of

14

65

Respiration in the light |

Dark or mitochondrial respiration, by definition occurring in the :l

:§

dark, was for long presumed to occur also in the light at a similar rate. $

1

This assumption, on which the basic light-dark bottle technique was based

(Gaarder & Gran 192?) led to computation of gross photosynthesis by adding •

dark oxygen uptake rate to rate of oxygen evolution in the light. This

f

method is now wholly invalidated (Sestak et al.1971, p.20) by the |

revelation that dark respiration does not invariably proceed unchanged in

the light, and that a separate form of respiration, photorespiration, occurs in the light exclusively. Mitochondrial respiration is considered to result from the complete oxidation of an organic compound (generally carbohydrate) # to 00g and H^O, with molecular 0^ as the ultimate electron acceptor (Gibbs

1962). In that the respiratory substrate consists of stored material, 4

oxidised by the Embden Myerhof Parnas and other pathways, this form of -.#

respiration is not immediately dependent upon photosynthesis.

Recently, however, certain effects of quantity and quality of light upon dark respiration have been demonstrated. Hoch et al.(I96 3) found that

oxygen uptake by Anacystis nidulans (Cyanophyta) was less at low irradiances ■.# "j than in the dark, the phenomenon being termed the Kok effect (see Heath I9 6 9), Î

'

'1

since discovered in Chlanydomonas (Chlorophyta) by Healey & Myers (1971) at |

irradiances less than 0.3 m ¥ cm Kowallik (I96 7) found that low-level |

f irradiance of blue wavelengths resulted in reduced Og uptake in Chlorella

(Chlorophyta). These are both factors which could be of importance to 4

algae growing in the low-level blue-green irradiance at the lower extreme of the photic zone. At higher irradiance levels, rate of mitochondrial respiration is generally held to be the same as (Raven 1972) or less than

(Brown & Tregunna I9 6 7) in the dark. However, Mangat etlal,(l974) found mitochondrial respiration in the light to be four times the rate in the

dark,in Phaseolus leaves. 7

66

Because it involves stored carbohydrates, dark respiration is held to result in no great loss of fixed during short-term experiments (^4h duration). However, Steeman-Nielsen

&

Aabye-Jensen (1957) suggested that dark respiration was so low compared with photosynthesis (in photoplankton)

that loss of would be negligible in any case. Steemann-Nielsen (1955)

found that phytoplankton lost 0.6-3% of previously fixed through

respiration in the dark, and concluded from this that the C method t X

measured something between gross and net photosynthesis. Jupp (1972), after

1 li j

incubating Laminaria hyperborea in C for two hours in the light, found that .7

0 . 2 % was lost after a third hour of incubation in the dark rising e x p o n e n t i a l l y

14 #

to 6.6% after 4h in the dark. In the light 75^ of this lost 0 was apparently re-fixed, leaving ^^2% total loss after 4h. Thus it appears that in an

experiment on only 1h duration, this method measures effectively gross -f

carbon fixation. A t an incubation time of 4h, something between net and 4

gross fixation will be measured, tending towards net measurement at incubation C 14

times in excess of this, as C becomes increasingly incorporated into the |

pool of dark respiratory substrate. 7

Clearly, since the oxygen method integrates the evolution of oxygen V

by photosynthesis and the uptake of oxygen by respiratory processes, there #

can be no dispute that this method measures net oxygen evolution, and hence, #

net photosynthesis. g

Considering, briefly, photorespiration - defined by Tolbert (1974) #

as "light dependent oxygen uptake and carbon dioxide release occurring in |i photosynthetic tissues". Photorespiration involves the oxidation of

recently-formed photosynthate, generally glycolate, and so would appear

14 ,

to concern the C method more than does mitochondrial respiration in short-

term experiments. However, because it is linked directly to photosynthesis #

it cannot theoretically outpace photosynthesis, and a light compensation |

6T

concurrently with photosynthesis, and does not involve stored substrates, photorespiration incurs no net loss of carbon from the plant but is merely

l4

a wasteful part of the photosynthetic process. Thus, in the C method, it is probable that has quite a high turnover rate by being fixed by photosynthesis, "lost" as by photorespiration, and re-fixed by photosynthesis. Assuming that oxygen production by photosynthesis and uptake by .photorespiration are both directly related to irradiance,

photorespiration should not affect measurement of photosynthesis by the

oxygen method, which will still measure a net rate of oxygen evolution. Thus, both and oxygen methods will be equally unaffected by photorespiration. Photorespiration has been found to occur in macroalgae by Brown & Tregunna (1967) and discussed with regard to the algae in general, by Jackson & Volk (1970), Tolbert (1974) and Chollet & Ogren

(1975).

Excretion of photosynthate

Excretion of recently-formed photosynthate would lead to under­ estimation of photosynthesis, measured by the method. Sieburth (I9 6 9) found that exudation of organic carbon from macroalgae could reach maxima of 5 .4 yg mg ^h ^ in Ascophylluoi nodosum (Phaeophyta) and 2.0 yg mg ^ h ^ in Ulva lactuca (Chlorophyta) but was much lower in the rhodophytes

Chondrus crispus (0.4 yg mg ^ h ^) and Polysiphonia lanosa (0.04 yg mg ^ h ^). However, Khailov^(I9 6 9) found no correlation between exudation rate and < taxonomic position, Hhodymenia palmata (Rhodophyta) exuding 9.8 yg mg ^ h whilst Laminaria saccharina (Phaeophyta) exuded only 1.7 yg mg ^ h ^. The value for Hhodymenia given would account for 47% of the photosynthesis measured in this species in the present work, implying that the measured

rate should in fact be 1.5 times the value found. Since Hhodymenia had ||

a very high carbon fixation rate as determined in the present study i

I

68

-4

Drew (pers, comm. ) studying the brown alga Laminaria hyperborea with the technique as used, in the present study, found, no radio­ activity in organic constituents in the bathing water from

experimental incubations of 6h duration. In a study of the direct effects of exudation on the measurement of photosynthesis in

2k

species of phytoplankton, using the method, Nalewajko (1966) found that only %5% of fixed was excreted as organic carbon during a 1h duration period. Ryther & Menzel (I9 6 5) found that in phytoplankton^photosynthesis measured by the method was the same as that computed gravimetrically

from growth measurements, concluding that if excretion did occur, it must be of immediate products of photosynthesis.

Thus, although the findings of Sieburth (I9 6 9) and Khailov (I9 6 9) do indicate that substantial exudation or excretion of organic carbon can occur in macroalgae, it appears that this may not incur a significant

l4

loss of C-labelled compounds in short-term experiments. If it were to. it would mean that rates measured by the present technique were under- estimates. The oxygen method should be unaffected by exudation.

Photooxidation processes

Franck & French (I94l) found that oxygen uptake by Hydrangea was higher in thellight than the dark, and, since this occurred in boiled as well as live tissue, they concluded that it was due to the photooxidation of intermediate products of photosynthesis, sensetised by chlorophyll. Griffiths et al. (1955) found that carotenoid-less mutants of photosynthetic bacteria underwent photooxidation of their bacteriochlorophyll when

irradiated under aerobic conditions, and proposed a protective function for caroteniods, by this means, in all plants. Cholnoky et al.(1956) described how caroteniods in higher plants underwent reversible oxidation as part of the oxygen transport system of green plant organs. It is

69

1

possible, then, that oxygen uptake by such processes may occur in the

algae studied (without production of COg and loss of and would >|

reduce the estimated rate of photosynthesis by the oxygen method, without effect upon the method. Such effects would be most pronounced at high irradiances. McAllister (I9 6I) found that in the coccolithophore

Syracosphaera, photosynthesis measured by the oxygen method was similar -ï

to that measured by the method at low irradiances but dropped

dramatically at irradiances a b o v e m ¥ cm ^ PAR; using the I^C technique “2

photosynthesis remained at a steady rate above 10 m¥ cm . He suggested - 4

that this was due to uptake of oxygen by photooxidation processes occurring at the higher irradiances. Drew (pers. comm.) However, found that photo-

• 1 Î4 • f i

synthetic rates of L.hyperborea measured by the C method were consistently # ; higher, by a factor of ^\2, than rates determined using the oxygen method, at J

ot

both-low and high irradiances, indicating the photooxidation was not the X #

cause for the discrepancy between the methods.

Thus, of all the above-cited possible methodological and physiological causes for the observed discrepancy between and oxygen estimates of

photosynthetic rate, none emerges as a single explanation. In the present

work the I^C method has been taken to measure near to gross photosynthesis, :i|

and the oxygen method to measure net photosynthesis. These have been

interconverted using a PQ of unity, although, for long term growth, the PQ value is probably closer to 1.2.

Due to the uncertainties concerning respiration in the light, in no case have rates derived from the method been "converted" to net rates by subtraction of dark respiration rates, nor have rates derived from the oxygen method been converted to gross fixation by addition of a dark respiration rate.

TO

Qualitatively, the two methods were in very close agreement, as will he shown, and the quantitative disparity must he taken as a reminder of the difficulties associated with indirect measurements of physiological processes. It may he that the variability of the discrepancy between the

two techniques could be of use in investigating its nature. I

I

T1

CHAPTER 3

Factors affecting the supply of oxygen and

inorganic carbon

CONTENTS

1. Introduction... 72

2. Note on m e t h o d s ... 74

In document Light, photosynthesis and growth of sublittoral macroalgae in Britain and the Mediterranean (Page 86-94)