steps (&ssham and Kirk, 1964; Grant and Turner, 1969; Thacker and Syrett, 1972; .... the dark-grown cells :lIsa showed a slight increase in the amounl of ...
Simultaneous utilization of nitrate- and ammonium-nitrogen by Scenedesmus obJiquus (Turpin) Kutzing A. K. Rai and u. N. Rai O"partm.nt of 80t.,y. Sa...r.. Hindu
Uni,,",r~ity.
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Abstract Nitrogen starved Scenedesmus obIlquus celts, whettlllr grown on n;trate-(lltrogeo or ammonium-. nitrogen, always showed an intracellular nitrite pool, a product of oxidative convert.ion of organic compounds. 80m ammonium- and nitrate·nitrogen wh,m present together were simultaneously available for the algal growth; the nitrogenous substrate wim greater conce"trat;ons had II preferential utilizatio" over the other with lower conca"tratiom. Lack of any remarkable increase in nitritl! lev&l at me light ,nte"sities ra"lling from 0.5 to 1.0 klux hl!s been interpreted in terms of charlge or remodulalio" of nitrate IransPOl"tfassimilation at those specific light irnensitias.
Introduction Recent nutritional studies, particularly those concerned with nitrogen.nutrition being the major limi ting factor having a direct impact on phytoplankton productivity and on species distribution pat terns, have attracted much interest. Most of the phytoplankton except some of the nitrogen-fixing blue-green algae, utilize many commonly available inorganic nitrogenous compounds, such as nitrates, nitrites, ammonia and other organic compounds, for their growth. It is commonly agreed that ammonium-nitrogen being the most reduced state of inorganic nitrogen is taken up preferably over the other forms such as nitrite- and nitrllte-nitrogen (Morris, 1974). Prior to assimilation, nitrate is reduced to nitrite, catalysed by the enzyme nitrate reductallC (a molybdo-protein), and nitrite to ammonia in the presence of the enzyme nitrite reductase (an iron protein). Thus, nitrate reduction is the first step in nitrogen assimilation. Despite the controversy regarding the role oflight in nitrate assimilation, whether direct or indirect, it is evident that nitrate assimilation depends on light and saturates at high light intensities (Owens and Esaias, 1976). This dependency is supposed to be for: (I) the reducing co-factor produced by photosynthesis, (2) ATP derived from cyclic or noncydic phosphorylation for uptake process, and (3) the supply of carbon skeletons for amination steps (&ssham and Kirk, 1964; Grant and Turner, 1969; Thacker and Syrett, 1972; Owens and Esaias, 1976; Solomonson and Spehar, 1977). In the present investigation, nitrogen utilization by the unicellular green alga, Sccncdcsmm obliquus. at different light intensities, with simultaneous utilization of inorganic nitrogenous forms and the possible assimilation preference, have been studied.
Materials and methods The unicellular alga Sccnedesmus obliquus (Turpin) Kutzing (Chloropl.yceae), used in the present investigation was grown in modified Chu 10 nutrient medium (Saffennan and Morris, 1964). Cultures low in nitrogen contained 0.06 mg per ml of potassium
13
Microbios Letters 5 13-18
nitrate, i.e. 1/10 of the nonnal amount of nitrogen. Cultures were illuminated continuously from below with nuorescent tubes giving an intensity of2ldux. Algal celli harvested during exponential growth were washed thoroughly with sterile distilled water and pre-incubaled in the basal growth medium without any combined nitrogen souru for 12 h. Cells, thus obtained, staNed of nitrogen, were rewashed prior to incubation for further experimentation. All these steps were carried out aseptically. Nitrate utilization was estimated by measuring the amount of nitrite fonned (expressed as ~ nitrite produced per mg of protein) after 8 h of algal incubation, a stage when nitrite production reaches its maximum (Figure I) by the azo-coupling method (Snell and Snell, 1949). Totll protein was estimated according to the method of Lowry er al. (1951). However, the present experiments can only account for the available nitrite at a particular time and not its incorporation in the metabolic pool since nitrate and nitrite reduction are going on slmuJtaneously in the organism and no experiments were done to exclude one from the other.
Results Nitrogen starved cells always showed a basa1level of intracellular nitrite (0.5 pg NO, -N/mg protein). When nitrogen starved cells were incubated in nitrate medium (added at the level of 0.6 mg per m1 as KNO l ) nitrate production increased rapidly and reached its maxima within 8 h followed by a sharp fall, and finally to a steady state somewhere after 48 h (Figwe 1). Table I shows the highest level of nitrite in the cells incubated with 0.6 mg/ml nitrate followed by ammonium nitrate (0.15:0.45 mg/rol), nitrate (0.3 mg/ml), and ammonium nitrate (0.3:0.3 mg/ml and 0.45:0.15 mg/ml). Nitrite was also detected (although in lesser quantity) even in the cells grown on ammoniacal nitrogen. Furthermore, the results obtained clearly showed the simultaneous utilization of ammonium-
Table 1 level of intracellular nitrite (initial level was 0.007 pg NO~·N per ml algal culture) after B h of algal incubation with different concentrations of ammonium--
and nitrate·nitrogen added in the nutrient solution Solution number
Nitrogltnous source lmglml):
NO,·N l.IIg/mlalglll culture)
NO,
(0.61
0.202
2
NO,
10.31
0.144
3
NO, + NH. 10.45+ 0.151
0.162
4
NO, + NH. 10.3 + 0.31
0.126
5
NO.+NH.IO.15+0.45J
0.58
6
NH.
0.02
14
Mkrobios Letters
10.31
A. K. Rai and U. N. Rai
0 I
1.0
400
0.6
240
I
;
E
~
•
3
,•
;
'!•
3•
0
