Enzymic Determination of Nitrate by use of Frozen/Thawed Chlorella CellsAuthor(s): C. R. Hipkin and P. J. SyrettSource: New Phytologist, Vol. 72, No. 1 (Jan., 1973), pp. 47-49Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2430615 .

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New Phytol. (1973) 72, 47-49-

ENZYMIC DETERMINATION OF NITRATE BY USE OF FROZEN/THAWVED CHLORELLA CELLS

BY C. R. HIPKIN AND P. J. SYRETT

Department of Botany and Microbiology, University College, Swansea

(Received 5 july I972)

Frozen/thawed cells of Chlorella reduce nitrate quantitatively to nitrite which can be determined colorimetrically. I4-420 ng nitrate-N can be determined. The method is suitable for the deter- mination of nitrate in sea water.

INTRODUCTION

Various methods for the estimation of nitrate have been described which depend on the reduction of nitrate to nitrite, the nitrite then being measured colorimetrically by the sensitive Griess-Ilosvay reaction. Chemical methods for the reduction of nitrate (e.g. Chow and Johnstone, I962; Morris and Riley, I963; Wood, Armstrong and Richards, I967) frequently suffer from lack of reproducibility and specificity. Use of an enzymic method of reduction overcomes these problems and several such methods have been described (e.g. MacNamara et al., I971; Vennesland and Jetschmann, 197I; Relimpio et al., I972). The method we have used is an enzymic one but since it uses frozen/thawed whole cells of Chlorella, the 'enzyme' can be prepared very easily and no purification steps are involved. Cells can be stored frozen for at least 6 weeks and are still effective in the assay.

MATERIALS AND METHODS

Culture of the organism Chlorella fusca var. vacuolata (Cambridge Culture Collection strain 2 II/8p) was

grown autotrophically in a medium containing 2o mm KNO3, as described by Syrett (I973). The cells were harvested by centrifugation, washed three times with, and sus- pended in, o.i M phosphate buffer, pH 6.i to give a suspension containing about I.0 mg dry wt cells/ml. The suspension was then frozen in a deep freeze for at least i6 hours.

Estimation of nitrate The Chlorella cell suspension was thawed carefully, the temperature being kept below

30 C. Two ml cell suspension (2 mg dry wt cells) was pipetted into a microcentrifuge tube and the cells sedimented by centrifugation. The cells were resuspended in an assay volume of o.6 ml containing 20, mol tricine buffer, pH 8.5, 6o 0mol sodium hydrogen malate, pH 8.5, and the nitrate sample to be determined. The tube was incubated at 300 C for i hour; 0.5 ml Griess-Ilosvay reagent (Snell and Snell, I949) was then added. After centrifugation the optical density of the supernatant was determined at 530 nm.

D 47

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48 C. R. HIPKIN AND P. J. SYRETT

RESULTS

Table i shows that quantities of nitrate containing from I4 to 420 ng nitrate-N were re- covered quantitatively as nitrite. Such quantitative recovery was obtained with cell suspensions that had been stored overnight in a deep freeze and also with suspensions that had been stored for up to 6 weeks.

We have used the method in attempts to measure the nitrate present in hot water extracts of frozen/thawed cells of Ankistrodesmus braunii. We failed to detect any nitrate in extract from 5 mg dry wt Ankistrodesmus cells but 84 ng nitrate-N added to the extract was estimately quantitatively with ioo% accuracy by our method.

Table i. Quantitative reduction of known amounts of nitrate to nitrite ng NO'3-N ng NO'2-N

added recovered 0 0

I4 '4 28 28 42 42 70 69 84 83

I40 138 i68 i66 210 210 252 252 336 330 420 420

The quantities of nitrate shown were added to assay tubes set up as described in methods. The quantities of nitrite formed determined colorimetrically by reference to a standard curve constructed with known quantities of nitrite. The data given are the mean values derived from eight experiments. Recovery of nitrate as nitrite was never less than 96%4.

Table 2. Determination of nitrate in sea water and recovery of nitrate added to sea-water samples

Sea-water NO'3-content* NO'3-N added NO'3-N % recovery NO'3-content sample (ng NO'3-N/ml) to 0.2 ml determined of added NO'3 (ng NO'3-N/ml)

(ng) (ng) A 635 0 I26 630

I68 287 96 B 7I4 0 I5I 755

i68 3I5 98

Determinations by the frozen/thawed cell enzymic method were carried out on 0.2-ml volumes of sea water with or without the prior addition of I68 ng NO'3-N.

* As determined by U.V. absorption at 2I0 nm (Hoather and Rackham, I959).

We have also used the method to measure the nitrate in samples of sea water collected from Mumbles, Swansea. The nitrate in these samples had previously been estimated by measurement of U.V. absorption at 2IO nm in our laboratory. Table 2 shows that the Chlorella nitrate reductase method gave essentially the same values for the nitrate content of the samples.

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Enzymic determination of nitrate 49

DISCUSSION

One difficulty with methods of nitrate determination that depend on reduction of nitrate to nitrite is that reduction may proceed further; for example nitrite may be reduced to gaseous nitrogen or to ammonia. It is clear from Table i that as used by us, reduction of nitrate to nitrite is complete and proceeds no further since ioo% recovery of nitrate is obtained. The advantages of this method of enzymic assay of nitrate are its sensitivity, its simplicity and the fact that the source of enzyme can be stored for a relatively long period without affecting the sensitivity of the assay.

ACKNOWLEDGMENT

We thank Mrs J. Paulraj for providing the sea-water samples which she had analysed for nitrate by the U.V. absorption method. One of us (C.R.H.) acknowledges a research studentship from the Science Research Council.

REFERENCES

CHOW, T. J. & JOHNSTONE, M. S. (I962). The determination of nitrate in sea water. Analytica chim. Acta, 27, 441.

HOATHER, R. C. & RACKHAM, R. F. (1959). Oxidised nitrogen and sewage effluents observed by ultraviolet spectrophometry. Analyst, Lond., 85, 548.

MACNAMARA, A. L., MEEKER, G. B., SHAW, P. D. & HAGEMAN, R. H. (1971). Use of a dissimilatory nitrate reductase from Escherichia coli and formate as a reductive system for nitrate assays. 5'. agric. Fd Chem., 19, 229.

MomIs, A. W. & RILEY, J. P. (I963). The determination of nitrate in sea water. Analytica chim. Acta, 29, 272.

RELIMPIO, A. M., GUERRERO, M. G., PANEQUE, A. & LOSADA, M. (1972). Determination of nitrate with nitrate reductase from spinach leaves. Z. Pfl. Physiol., 66, 290.

SNELL, F. D. & SNELL, C. T. (Ig49). Colorimetric Methods of Analysis, Vol. II, 3rd edn, p. 785. Van Nostrand, New York.

SYRETT, P. J. (1973). Measurement of nitrate and nitrite reductase activities in whole cells of Chlorella. New Phytol., 72, 37.

VENNESLAND, B. & JETSCHMANN, C. (197I). The nitrate reductase of Chlorella pyrenoidosa. Biochim. biophys. Acta, 229, 554.

WOOD, E. D., ARMSTRONG, F. A. J. & RICHARDS, F. A. (I967). Determination of nitrate in sea water by cadmium-copper reduction to nitrite. J. mar. biol. Ass. U.K., 47, 23.

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