Foi desenvolvido um método para análise do ferro acumulado em células de fitoplâncton, usando ferrocianeto de potássio como agente complexante. Com esse objetivo montaram-se duas experiências: uma em que se mostra o acúmulo de ferro em relação à idade das células e uma segunda experiência onde a assimilação é plotada em função da concentração de EDTA no meio de cultura.A colorimetric method for the assessment of iron accumulated in the cells of phytoplankton utilizing potassium ferrocyanide was developed. Two experiences with Phaeodactylum tricornutum and Chlorella sp. were carried out: the first one, on uptake of the iron relatively to the age of the culture: the second one, on the iron uptake by the cells, relatively to different concentrations of the chelator EDTA

Paredes, Jorge Falcão

Schmidt, Gilda

Brazilian Journal of Oceanography

English

A colorimetric method for the assessment of iron accumulated in the cells of phytoplankton utilizing potassium ferrocyanide was developed. Two experiences with Phaeodactylum tricornutum and Chlorella sp. were carried out: the first one, on uptake of the iron relatively to the age of the culture: the second one, on the iron uptake by the cells, relatively to different concentrations of the chelator EDTA.Foi desenvolvido um método para análise do ferro acumulado em células de fitoplâncton, usando ferrocianeto de potássio como agente complexante. Com esse objetivo montaram-se duas experiências: uma em que se mostra o acúmulo de ferro em relação à idade das células e uma segunda experiência onde a assimilação é plotada em função da concentração de EDTA no meio de cultura

Jorge Falcão Paredes

Gilda Schmidt

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Analysis method of intracellular iron accumulated in phytoplankton

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Bolm Inst. oceanogr., S Paulo, 26:1-10, 1977 ANALYSIS METHOD OF INTRACELLULAR IRON ACCUMULATED IN PHYTOPLANKTON JORGE FALCÃO PAREDES 1 & GILDA SCHMIDT Instituto Oceanográfico da Universidade de são Paulo SYNOPSIS A co1orimetric method for the assessment of iron accumu1ated in the ce11s of phytop1ankton uti1izing potassium ferrocyanide was de-ve1oped. Two experiences with Phaeodactylum tricornutum and Chlo-rella sp. were carried out: the first one, on uptake of the iron relatively to the age of the culture: the second one, on the iron uptake by the cells, re1ative1y to different concentrations of the chelator EDTA. INTRODUCTION Iron is one of the cations that plays an important role in the me-tabolism and behaviour of the phytop1ankton (Ryther & Kramer, 1961). It is constitutiona1 part of many enzimes and cytochromes and of certain porphy-rins. Thus, the growth of algae shou1d be closely re1ated with the iron up-take. Therefore, the concentration of this cation in the unicellular algae 1 Instituto Oceanogrãfico's visiting professor from Instituto de Investiga-ção Científica de Angola, financed by O.E.A. Present address: Centro de Pesquisas e Desenvolvimento. Salvador, BA-Brasil. PUBL. Nr 387 DO INST. OCEAN. DA USP. 2 Bo1m Inst. oceanogr., S Paulo, 26, 1977 should be much concerned to chelators concentration in the environment. ln-deed, it is known how the mineral euthrofication of aquatic environment fails out if it has not been accomplished by convenient concentration of chelators, which promotes its solubility (Doig & Martin, 1971). Some authors have pointed out that fluctuability of unicellular algae shows a great vari-ation during the culture growing and consequently with the environmental conditions. The ability of the phytoplankton buoyancy, according to Gross & Zeuthen (1948) and Paredes (1968a), would be related with the specific weight of cells. This will mean that the specific gravity variation may re-sult from ionic exchanges, ensured by permeability of the selective cellular membr ane, among its own chelators and environmental ones. From these points of view, the importance between the soluble iron concentration in water, and that one accumulated by cells, in their contents, can be quite relevant. Since the atomic absorption method is not often available, the method developed and here described seems to assume a simplified methodology con-cerning to the other known methods. METHODS 1. Species: Cultures of Phaeodactylum tricornutum and Chlorella sp. isolated from the Cananeia estuary (São Paulo State, Brazil) were utilized in the experiments. 2. Medium and culture conditions: Provasoli medium ASP 2 was used to culture those species which grew under fluorescent light, intensity of 5.10 3 lux, 24.0oC ± 1.0oC, and pH = 7.0, verified after autoclaving. 250 ml conical Pyrex flasks were used with 150 ml of medium. 3. Method developed 3.1. Reagents: Perchloric acid 70% Hidrochloric acid 1,5 N Potassium ferrocyanide 2% (made up just before using) PAREDES & SCHMIDT: Iron in phytoplankton 3 3.2. Procedure: 3~ Mi11ipore 25 mm white membrane with dry a1gae bu1k were digested in 3 m1 of perch10ric acid unti1 a1most total dryness in 50 m1 beakers by direct heating. After coo1ing, add 1 m1 of 1,5 N HCL and 3 m1 of potassium ferrocyanide solution. A b1ue co10r is, then, de-ve10ped. Its stabi1ity was reached in about 20-30 minutes. The opti-cal density was performed at 477 m~, as the most convenient wave 1ength achieved. The readings were obtained at a Beckman nu spectro-phometer using 1 em si1ica ce11s. 3.3. "B1ank" and standard solution: The "b1ank" was made up as the samp1es, with a membrane of the same package used for the samp1ing. The standard solution were prepared with 20 mg of FeC1 3 in 400 m1 of bidistilled water to give a so-1ution with 50 ~g/m1. Six alíquots of 1, 2, 5, 10, 25, and 50 ~g Fe were treated as the samp1es and these data were p10ted in the ca1i-bration curve showed in Figure 1. 4. Estimation of growth: Ce11s counting were performed 1n a counting slide and the density of the suspension was given by a Spectronic 20 Ab-sorptionmeter. RESULTS 1st E . xper1ment: Iron accumu1ation in Chlorella sp. regarding to age of cu1ture was test-ed. Three subcu1tures inocu1ated with 0.5 m1 of adense old stock of Chlo-rella sp. were harvested successive1y after two, four, and six days on 3~ Mi11ipore membranes of known weight. These membranes, dried under 700C for 60 minutes were, after coo1ing, weighted again. Treated by the method ear1ier described, it was revea1ed that Chlorella sp. uptakes much more iron during the ear1ier stages (lag phase) than during the exponentia1 phase 4 0,400 ~ E 0,300 " " 'It I-<:[ >-I-(f) ffi 0,200 a 0 , /00 Bolm Inst. oceanogr., S Paulo, 26, 1977 jJ 9 Fe Fig. 1 - Linear calibration of standard FeC1 3 solution, at 477 m]..1. (Fig. 2 and Tab. I). Iron rate re1ative1y to dry weight changed between 2.19 and 0.3%. The pH showed tend to increase during the growth. 2nd . Exper~ment: Four subcu1tures with four different concentrations of EDTA in ASP 2 medium were inoculated with few cel1s of Phaeodactylum tricornutum and Chlo-rella sp. (Fig. 3 and Tab. 11). When these subcultures have reached the final stage of exponentia1 phase, 15 ml samp1es were harvested on 3]..1 Mi11ipore membranes of known weight. PAREDES & SCHMIDT: Iron in phytoplankton o 05 ::lo F o ~ 0 .04 .... lO o 6 0 .03 QI .... lO ~ 0.02 ~ o <!) 001 Age of culture 'i OI 2-. +-~ 2 .0 .~ QI ~ ~ :; ~ ao - 1. 5 o ; +- ~ OI ,., C ~ c: " 1. o ~ 2 u c -' o " u .. OI ~ +-lO ! 0.5 ~ c " ~ ~ ~ 5 4 EJ - Chlorel/a ~ - Phaeodaefy/um frieornufum 3 2 o lO 30 60 100 E . D.T. A . cone (jJ9/ml) Fig. 2 - Experiment of growth rate and iron a·ssimilation of ChZoreZZa sp. in relation to the age of culture. 0---0 growth rate: _____ iron as-simi lation. Fig. 3 - Iron assimilation by ChZoreZZa sp. and PhaeodaatyZum triaornutum as a function of the concentration of EDTA. TABLE I - Data showing the iron uptake' variation in relation to the age of ChZoreZZa sp. culture AGE IN DAYS 2 4 6 OPTICAL DENSITY 0.015 0.025 0.045 AT 600 IDll CELLS NUMBER/m1 447 x 10 3 745 x 10 3 1341 x 10 3 MEAN OF CELL 7.0 x 10-5 4.7 X 10-5 8.5 X 10-5 DRY WEIGHT (llg) VARIATION OF INITIAL +0.1 +0.2 +0.2 pH = 7.0 IRON RATE CONCERNING 2.19 1.54 0.30 TO DRY WEIGHT (%) IRON CONC. CELL (ll~g) 1.5 x 10- 3 0.7 X 10- 3 O .26 X 10- 3 6 Bolm Inst. oceanogr., S Paulo, 26, 1977 TABLE I I - Data concerning to the same aged cells suspension given by optic-al density and to the dry weight given by algal bulk harvested from 15 ml ChZoreUa sp. Ph. trwo!'nutum EDTA CONC. llg/m1 O.D. 600 111ll DRY WEIGHT in llg O.D. 600 111ll DRY WEIGHT in llg (from 15 m1) (from 15 rnl) 10 0.033 1.000 0.048 800 30 0.045 800 0.048 1.300 60 0.042 1.450 0.048 1.400 100 0.040 500 0.048 1.200 The particulate iron uptaken by both species, in terms of iron rates re-garding to dry weight, decreases in the opposite way relatively to EDTA concentration. DISCUSSION After Davies (1970) the iron can be present in cultures in four differ-ent forms: "that adhering to the culture vessel, that in free suspension in the medium, that adhering to the externaI surfaces of the cells and that within the cells (as an integral part of the iron porphyrins, cytochromes, and certain enzymes associated with subcellular particles". The iron 1n free suspension can still be 1n ionic or complexed formo Adequated concentrations for the growth of Chlorella sp. have been situated between 1.8 x 10-7 to 2.6 X 10- 8 M. The high cellular metabolic iron content at the first stage of the growth, i.e. at lag phase and at its sharp decreasing during the exponential phasefound out in the conditions described for the first experiment, is PAREDES & SCHMIDT: Iron in phytoplankton 7 rather a lot in agreement with results obtained by this author relatively to the growth kinetics of Dunalliela tertiolecta against the ,iron available in the culture medium. It is well known that media deficient in iron, above alI in darkness, drives to a minor photosynthesis rate reducing consequently the relative growth and becoming the cells chlorotic (Hayward, 1968). The iron deficience seems closely related with a reduction 1n the synthesis of proteins within the choroplasts, as well as with the falling back in the incorporation of magnesium into the molecule of protoporphyrin. So long we know, the protein synthesis is high during the first stages of the growth decreasing during the logarithmic growth and further on (Paredes, 1968b). However, Davies (1970) showed that Dunaliella tertiolecta under iron deficient conditions is able to adapt gradually to such an environment, being the growth rate much concerned to the metabolic iron content of the cells. Nielsen & Wium-Andersen (1970) have pointed down that copper could be an inhibitor for phytoplankton growth in upweling water, where may lie as about 10 ~g Cu/I. The copper, if it is complexed in sea water by organic matter, is not poisonous to algae, but if it is in such concentration at ionic form as shown by these authors, the photosynthesis and the phyto-plankton growth is rather inhibited. Goldberg (1952) showed that phytoplankton are not able to uptake the iron which is chelated. On the other hand, unchelated iron may adhere to the glass of culture vessels (Davies, 1970). The maximum concentration of soluble iron, i.e., in cationic form at the normal pH of sea water is 5 x 10-7 ~g/l. Therefore, iron is mainly present as colloidal ferric hidroxide, ferric phosphate and silicate or still as complexes with organic substances (Riley, 1965). In order to avoid an over-estimate of flocculated iron, 3~ Millipore membranes were utilized. However, the absorbed iron on the cells of both species, will come in the assessment together with the metabolized iron, masking the results. 8 Bolm Inst. oceanogr., S Paulo, 26, 1977 Paredes (1968c) refers that Exuviella baltica was brought in culture only when an over chelated medium based in aged sea water was used. Also, only under this condition he was able to appreciate the ascension and ac-cumulation of the cells at the surface of culture vessels, which was due to their high buoyancy ability. Another relevant role of the EDTA is to convert, into soluble, the iron that had became particulated by autoclaving. Concentrations between 10-100 ~g Fe/l have been recorded in sea water, although concentrations up to 3 mg Fe/l have been cited (Riley, 1965). After Wiessner (1962) "when EDTA is present in the nutrient solution, larger quantities of iron must be supplied", otherwise no iron will be available for the cells, since it is in complex form. This seems to be confirmed with some evidence by the second experi-ment. Indeed this shows a sharp decrease in the particulated iron concen-trations in opposite way to EDTA contents in the culture medium (Fig. 3 and 'rabo II). The dry weights were somewhat contradictory, particularly concerning to c.hlorella sp. which will limit, at least, the meaning of the figures on the iron rates. Although, Chlorella sp. has shown a preferencial growth in 30 ~g/ml of EDTA given in terms of cellular density at 600 m~. On the other hand Ph. tricornutum has replied with the same growth rate in alI EDTA concen-trations. Davies (1970) reports that there is a quantitative relationship between the cellular iron and chlorophyll contents which can be governed by iron chelators. Unfortunately no chlorophy11 contents were assessed. In the 100 ~g/m1 of EDTA subcu1ture no iron response was obtained from the ce1lular bu1k harvested. It is, therefore, inferred that the iron avai1able and that one uptaken, shou1d be in so 10w concentration that wou1d be under the accu-racy of the described method. However, we did not pay attention to the iron "luxury consumption" which, in the 1acking of uncomp1exed iron, cou1d be enough to support the growth of this subcu1ture. PAREDES & SCHMIDT: Iron in phytoplankton 9 RESUMO Foi desenvolvido um metodo para análise do ferro acumulado em ce1u1as de fitop1âncton, usando ferrocianeto de potássio como agente comp1exante. Com esse objetivo montaram-se duas experiências: uma em que se mostra o acúmulo de ferro em relação ã idade das ce1u1as e uma segunda experiência onde a as-similação e p10tada em função da concentração de EDTA no meio de cultura. REFERENCES DAVIES, A. G. 1970. Iron, che1ation and the growth of marine phyto-p1ankton. J. mar. bio1. Ass. U. K., 50:65-86. DOIG 111, M. T. & MARTIN, D. F. 1971. Effect of humic acids on ~ron ana1ysis in natural waters. Wat. Res., 5:689-692. GOLDBERG, E. D. 1952. Iron assimi1ation by mar~ne diatoms. Bio1. BulI. mar. bio1. Lab., Woods Role, 102:243-248. GROSS, F. & ZEUTREN, E. 1948. The buoyancy of p1ankton diatoms: a prob1em of ce11 physio1ogy. Proc. R. Soe., sêr. B, 135:382-389. HAYWARD, J. 1968. cornutum. J. mar. Studies on the growth bio1. Ass. U. K., 48:295-302. of Phaeodactylum tri-NIELSEN, E. S. & WIUM-ANDERSEN, S. 1970. Copper ions as poison in the sea and fresh water. Mar. Bio1., 6(2):93-97. PAREDES, J. F. 1968a. Studies on cu1tures of marine phytop1ankton. I. Diatom Ditylum brightwellii West .. Mems Inst. Invest. ciente Moçamb., s~r. A., 9:156-183. 1968b. Studies on cu1tures of 111. Ch1orophycean Micromonas squamamata. Mems Moçamb., ser. A., 9:249-292. marine phytop1ankton. Inst. Invest. ciente 1968c. Studies on cu1tures of marine phytop1ankton. 11. Exuviella baltica bIoom with reference to a red tide occurred in the coast of Angola. Mems Inst. Invest. ciente Moçamb., ser. A., 9:184-247. RILEY, J. P. 1965. P. & Skirrow, G., vo1. 2, p. 378. Ana1ytica1 chemistry of sea water. In: RiIey, J. ed. - Chemica1 oceanography. London, Academic Press, 10 Bolm Inst. oceanogr., S Paulo, 26, 1977 RYTHER, J. H. & KRAMER, D. D. 1961. Re1ative iron requirements of some coas tal and offshore p1ankton a1gae. Eco1ogy, 43:444-446. WIESSNER, W. 1962. ed. - Physio1ogy and 929 p. Inorganic micronutrients. In: Lewin, R. A., biochemistry of a1gae. New York, Academic Press, (Recebido em 27/maio/197S) 

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Analysis method of intracellular iron accumulated in phytoplankton

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