Namrata Sharma 1*, Surendra Singh 2,Rekha Patel 2 ABSTRACT

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1 AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: , ISSN Online: , doi: /abjna , ScienceHuβ, Nitrate uptake and nitrite efflux in wild type and Li + -R and Na + -R strains of Spirulina platensis under different nitrogen concentration and lightdark condition. Namrata Sharma 1*, Surendra Singh 2,Rekha Patel 2 1 Mata Gujri Women s College, Jabalpur, M.P. India 2 Department of Biological Sciences, Rani Durgawati University, Jabalpur, MP, India ABSTRACT In the present study we have investigated that 4.0gL -1 NaNO 3 concentration is toxic to the growth of wild type Spirulina platensis and its Li + R and Na + R mutant strains. Highest level of extracellular nitrite release was shown by Na + R strain, when grown in 4.0gL -1 NaNO 3 concentration followed by Li + R and wild type strain of S. platensis whereas Li + R strain uptake nitrate more rapidly followed by Na + R and wild type strain of S. platensis, when estimated after 8d of incubation. Cultures assimilate nitrate more rapidly in light than in darkness which suggests that nitrate uptake process depend on photosynthetically generated light energy. The nitrate uptake pattern consisted of two distinct phase : initial rapid phase followed by the slower second phase. There is no lag in the initial uptake of nitrate in either of the culture suggesting that rapid phase is not dependent on reduction of nitrate whereas slower phase seemed to be dependent on metabolism. Among these three strains of S. platensis Li + R strain assimilate nitrate more rapidly and fix it into ammonia by sequential action of nitrate reductase and nitrite reductase. Keywords - Nitrate uptake, Nitrite efflux, Spirulina platensis, Li +- R and Na + R mutants. INTRODUCTION Spirulina also called Arthrospira is a microscopic, filamentous and non-nitrogen fixing cyanobacterium that has a long history of use as food (Abdulaqader et al., 2009; Belay et al., 1996). Spirulina is 50-70% protein by weight (Phang et al., 2000), and contain a rich source of vitamins, especially vitamin B 12, β- carotene, vitamin E. It also contain carbohydrates like rhamnose, fructose, ribose, mannose. Besides - linoleic acid (GLA), it contains a host of other photochemicals that have potential health benefits (Belay et al., 2002; Foster et al., 1999). Nitrate assimilation is a fundamental biological process of nitrogen acquisition in the cyanobacteria (Guererro et al., 1981). It is transported into the cells by an active transport system and reduced to ammonium by the sequential action of nitrate reductase (NR) and nitrite reductase (NiR) prior to fixation into amino acids through the glutamine synthetase (GS) pathway. The process of nitrite uptake and its regulation has been widely studied in higher plants and fungi (Raghuram et al., 2006; Lochab et al., 2007) algae (Berges, 1997), bacteria (Lin et al., 1998) and to a lesser extent in cyanobacteria (Herrero et al., 2001). Different responses to nitrate have been reported in literature between the non-nitrogen fixing (nitrate utilizing) and nitrogen fixing cyanobacteria. In nonnitrogen fixers, high levels of NR have been reported in media lacking combined nitrogen (Palod et al., 1990), suggesting constitutive expression. There is little information regarding the role of nitrate and nitrite in non-n 2 fixing cyanobacteria in general and Spirulina in particular. Therefore, we have examined the effect of nitrate concentration and its downstream metabolite nitrite on NR activity development and growth in Spirulina platensis and its Li + R and Na + R strains as a basis for further characterization of nitrate assimilation in this organism. MATERIALS AND METHODS Organism and Source: Wild type S.platensis used in present investigation was obtained from CFTRI, Mysore, India, and its Li + and Na + resistant mutant strains are isolates of Algal Biotechnology Laboratory, Department of Biological Science, R.D. University, Jabalpur M.P., India (Singh et al., 2000). Growth Conditions: Pure cultures of S. platensis and its Li + -R and Na + R strain were grown axenically in CFTRI medium at ph-11 with the following composition:- NaHCO 3 : 4.5g, K 2 HPO 4 : 0.5g,

2 NaNO 3 :1.5 g, K 2 SO 4 : 1.0 g, NaCl : 1.0 g, MgSO 4 : 0.2g, CaCl 2 :0.04, FeSO 4 : 0.01g in 1000ml of distilled water. The cultures were grown at 25±1 0 C under white light illumination (photon flux density - 47 Em -2 s -1 ) with a 16:8 light dark rhythm. Measurement of growth: Growth was measured by estimating changes in chlorophyll a content of the cultures at 24h interval upto 16 days by the method of Mackinney (1941). Determination of nitrate uptake: Uptake of nitrate was assayed by measuring the depletion of nitrate from the medium at 24 h intervals by the method of Nicholas and Nason (1957). Estimation of nitrite: Extracellular excretion of nitrite was estimated by the method of Snell and Snell (1949). Experimental Set: Impact of different concentrations of nitrate: Three sets of five sterilized Erlenmeyer flasks with 100ml CFTRI growth medium containing five different concentration of NaNO 3 : 1.0gL -1, 1.5gL -1, 2.0gL -1, 3.0gL -1, 4.0gL -1 were taken and equal amount of exponentially growing (6 d old) wild type S.Platensis and its Li + -R and Na + -R strains were inoculated to these flasks. One flask in each set containing 1.5gL -1 NaNO3 was taken as control. Flasks were incubated under photoautotrophic conditions. Cultures were allowed to grow for 16 days and samples were removed in duplicate everyday to determine growth, nitrate uptake and extracellular excretion of nitrite. Impact of light and dark conditions: Wild type S.Platensis and its Li + -R and Na + -R strains were preincubated for 24h in nitrate free medium for complete depletion of intracellular pool of nitrate nitrogen. Cultures were harvested (3000g, 10 min), washed in nitrate free medium thrice and inoculated in nitrate free CFTRT medium for 30 min under growth conditions to stabilize cultures. After stabilization, 100 mol KNO 3 was added to these flasks. One set of flask was incubated in light (photon flux density 47 E m -2 s -1 ) and another set under dark condition. Samples were removed every 10 min interval upto 100 min to estimate nitrate uptake and extracellular nitrite release. RESULTS AND DISCUSSION Impact of graded concentration of NaNO 3 on the growth of wild type S.Platensis and its Li + -R and Na + - R strains was monitored for 16 days. With increasing concentration of NaNO 3 wild type S.Platensis and its Li + -R and Na + -R strains exhibited increasing growth upto 3.0gL -1 concentration of NaNO 3. Concentration beyond 3.0gL -1 was growth inhibitory for both parent and mutant strains. There was an increase of 47% and 17% in chlorophyll a content of Li + -R and Na + -R strain over the parent strain, respectively, on 16 d of growth in 3.0gL -1 NaNO 3 (Fig : 1, 2,3). 78

3 Nitrate reductase activity (in terms of extracellular nitrite release) of wild type S.Platensis and its Li + - Rand Na + -R strains was also monitored along the growth cycle. Kinetics of extracellular nitrite release progressed similarly along the changes in the chlorophyll content of the cells. Highest level of extracellular nitrite was released by Na + -R strain when grown in 4.0gL -1 of NaNO 3 over the wild type (32%) and Li + -R (5%) of S.Platensis (Fig: 4, 5, 6). Enhanced levels of NR activity in presence of graded concentration of NaNO 3, suggesting inducing expression of NR in both parent and mutant strains of S. platensis. Jha et al., also reported the nitrate induction of NR activity in S. platensis (2007). 79

4 Utilization of nitrate by the cultures was also observed by measuring the depletion of nitrate from the medium. Li + -R strain utilized nitrate more rapidly followed by Na + - R strain and wild type of S.Platensis, when estimated after 8d of incubation. Increase in nitrate uptake and nitrite release with increasing concentration of NaNO 3 suggesting the role of nitrate concentration in regulating the development of nitrate reductase activity and nitrite release (Table-1). Table-1 Percent reduction in nitrate in medium by parent and mutant strains of S. platensis estimated after 8 th d of incubation. S.platensis strains Graded concentration of NaNO 3 ( gl -1 ) Wild type Li + R Na + R Time course of nitrate uptake and extracellular release was also examined in cultures preincubated for 24h in nitrogen free medium. The nitrate uptake pattern consisted of two distinct phases : initial rapid phase lasting upto 20 min in wild type strain whereas in Li + -R and Na + -R strains it lasted upto 10 min. followed by the slower second phase. Wild type S.Platensis exhibited 2.0 fold, Li + -R strain 2.5 fold and Na + -R 2.0 fold increase in nitrate uptake over the dark condition (fig: 7, 8, 9). No lag in the initial uptake of nitrate in either of the culture suggest that rapid phase is not dependent on reduction of nitrate whereas slower phase seemed to be dependent on metabolism. Results as shown in Fig: 7, 8, 9 clearly suggest that extracellular efflux of nitrite started after a lag of min as compared to first rapid phase of nitrate uptake which takes about min to complete. Evidently long lag period in the efflux of nitrite suggest that this could be due to time consumed in build up of critical level of intracellular pool of nitrate and nitrite leading to the activation of nitrite efflux system. The substantial difference in amount of nitrate uptake (6 mol) and release of nitrite (0.3 mol) suggest the involvement of intracellular pool of 80

5 nitrate and its further incorporation in the cell skeleton. Cultures assimilate nitrate more rapidly in light than in darkness. So it is evident from the result that reductants or energy produced by photosynthesis are involved either by photophosphorylation or ATP hydrolysis in nitrate uptake. Analogous results on light dependent nitrate uptake have also been reported in Anabaena doliolum and Anacystis nidulans (Singh, 1992) and Nostoc MAC (Singh et al., 1996). The development of the uptake process was completed well before the start of development of the nitrite process, indicating a possible role of a functional nitrate uptake system in regulating the development of nitrite efflux system. The time lag obtained for nitrite efflux was consistent for nitrate to be reduced to nitrite. Conclusion: It can be concluded from the ongoing discussion that Li + R mutant strain of S. platensis uptake nitrate more rapidly and incorporate it further into cell skeleton followed by Na + R mutant and wild type strain of S. platensis. Inducing expression of NR activity was shown by both the parent and wild type strains of S.platensis. Further characterization of these preliminary findings may be helpful to study the role of nitrate in expression of NR and development of nitrite efflux system in parent and mutant strains of S. platensis and may also reveal the basis of high protein content in these strains. REFERENCES: Abdulaqader G, Barsanti L, Tredici, M, (2009), Harvest of Arthrospira Platensis from lake kossorom (Chad) and its household usage among the Kanembu-J. Appl. Phycol, 12: Belay A, Kato T, Ota Y, (1996), Spirulina (Arthrospira) : Potential Application as an animal feed supplement J. Appl. Phycol, 18: Belay A, (2002), The potential application of Spirulina (Arthrospira) as a nutritional and therapeutic supplement in health management. J.Am Neutraceutical Assoc 5: Berges, JA (1997), Algal nirate reductases, Eur. J. Phycol, 32:3-8. Guerrero, MG, Vega, JM and Losada, M. (1981), The assimilatory nitrate reducing system and its regulation. Annu. Rev. Plant Physiol 32: Foster S., Tyler, VE (1999), Tyler s honest herbal: A sensible guide to the use of hergs and related remedies. 4 th ed. New York, Haworth, Herbal Press. Herrero, A. Muro-Pastor, AM. Flores, E (2001), Nitrogen control in cyanobacteria. 8. Jha, P. Ali, A. and Raghuram, N.(2007), Nitrate induction of nitrate reductase and its inhibition by nitrite and ammonium ions in S. platensis, Physiol Mol. Biol. Plant13(2): Lin, J.T. and Stewar, V. (1998), Nitrate assimilation by bacteria, Adv. Microb Physiol 39:1-30. Lochab, S. Pathak, RR and Reghulan, N. (2007), Molecular approaches for enhancement of nitrogen use efficiency in plants. In Agriculture use and its environmental implications. (Eds. Abrol, YP, Raghulam N, and Sachdev, Ms). I K International, Delhi. Mackinney, G (1941). Absorption of light of chlorophyll solutions, J-Biol Chem 140: Nicholas, DJ and Nason, A, (1957), in Methods in enzymolgy, Vol-III ed. By colowick, SP, Kalpal, No. Academic Press, New York, 982. Palod, A. Chauhan, VS and Bagchi, SN, (1990), Regulation of nitrate reduction in a cyanobacterium Phormidium uncinatum : Distinctive modes of ammonium repression of nitrate and nitrite reductases. FEMS Microbial Lett. 68: Phang, SM, Miah, MS, Chu, WL, and Hashim, M (2000), Spirulina Culture in digested sago starch factory waste water, J.Appl. Phycol 12: Raghuram, N, Pathak, RR and Sharma, P (2006), Signalling and the molecular aspects of N-use efficiency in higher plants. In Biotechnological approaches to improve nitrogen use efficiency in plants. (eds. Singh, R.P. and Jaiswal, P K), Studium Press LLC., Houston, Texas, USA, Singh, B B, Pandey, P K, Singh and Bisen P S, (1996),Regulation of nitrate uptake and nitrite efflux in the cyanbacterium Nostoc MAC. Curr-Microbiol 31:1-5. Singh, S, Patel R. Dutta, P and Choubey, A. (2000). Isolation and characterization of high salinity tolerant (Li + -R) strain of Spirulina platensis for biotechnological exploitation. Microbiotech AMI, 41: Singh, S, (1992), Regulation of nitrate uptake in cyanobacteria : effect of nitrogen starvation. Biomed Lett. 47: Snell, FD, Shell, CT (1949). Colorimetric methods of analysis. Vol. 3, New York: Von Nostrand. 81