Effect of Indole Acetic Acid on Growth and Physiology of a Cyanobacterium Spirulina Platensis

Spirulina platensis has been prominently catalogued as a rich source of proteins and micronutrients in the pyramid of the marketed nutraceuticals (Belay, 1997; Bhatia et al. 2016). Its original use as a dietary supplement is credited to natives of Central Africa around Lake Chad, and in East Africa along the Great Rift Valley. After the discovery of its immense nutritive potential, widespread commercial production had already jump started in different parts of the world. Nutraceutical importance of Spirulina has rapidly increased over last two decades with emerging role in healthcare (Belay 1997; Bhatia et al., 2014). Vast amount of research is directed to optimize Spirulina growth and production for its emerging demand worldwide (Vonshak and Tomaselli, 2000; Danesi et al., 2001; Ogbonda 2007; Celekli et al., 2009; Sena et al., 2011; Bhatia et al 2017). Availability of improved production techniques and better understanding of underlying biochemical properties are indeed vital for its commercial use. Growth and developmental processes of cyanobacteria can be influenced by a variety of environmental factors. Indole-3-acetic acid acts as growth promoter of various cyanobacteria has a wide range effect on growth and morphogenesis. Enhanced effect of IAA (1-6μg/ml) on antioxidant phenolic compounds has been indicated (Mohammed, 2011). Indole-3-acetic acid neither increased the nitrogenase activity nor enhanced the heterocyst frequency in cyanobacteria (Leganes et al., 1987). However, little is known about the possible effect of Indole-3-acetic acid on growth and physiological performance Spirulina. Various studies on algal systems (Czerpak et al., 1994; Czerpak et al., 1999; Ahmad and Winter (1970) reported increase in cell count and dry mass of Chlorella sp. due to exogenous application of auxins, auxin precursors and analogs. Single study on effect of IAA on carotenoids of Spirulina reported increased production of carotenoid content with exogenous IAA supplementation (Mohammed and Mohammed, 2011). In this context, role of phytohormones has recently attracted attention in optimizing growth of Spirulina. The present study, therefore, aimed to assess the efficacy of IAA in stimulating Spirulina growth.

The experimental cultures were propagated in Zarrouk medium (Zarrouk 1966) at 35 ± 2°C, pH of 9 – 9.5. The cultures were illuminated for 14 h light at 1800 lux on the surface of culture vessels. Wide range of Indole-3-acetic acid concentration was initially tested to identify the lowest and highest endpoints of concentrations showing no or inhibitory

on biomass, protein content, chlorophyll content, carotenoids and phycocyanin content. Estimation of Growth: The growth was enumerated on 1st day and then every 4th day till 20 days. Growth was quantified as an increase in spectrophotometric absorbance. The absorbance was recorded at 550 nm. The experiments were run in triplicate Estimation of Biomass: The Homogenous cultures were centrifuged and washed in double distilled water and dried at 70℃. The growth was expressed in terms of dry weight (µg ml-1). Estimation of Total Protein Content: The total protein content of the cultures was estimated by the method of Herbert et al. (1971),

Estimation of Caroteins Carotenoids were extracted in 90% acetone by cold extraction method. The concentration of carotenoids was determined using specific absorbance coefficient (α) of 12 (Weber and Wetton 1981). Estimation of Phycocyanin: The Phycocyanin was estimated by the method given by Tandeau de Marsac (1977) and was measured by the equations developed by Bennet and Bogorad (1973)

Cell morphology was observed for microscopically detectable morphological alterations, in Spirulina culture. The cultures were viewed under light microscope at 400X till the development of hormogonia in control cultures. The structural modifications at finer scale were studied using scanning electron microscopy. The specimen was fixed with 2.5% gluteraldehyde. Dehydration was done in increasing grades of ethanol. Critical point dried specimens were loaded on metallic specimen stubs and sputtering was done in JOEL JFC -1100 (Bozzola and Russell 1992). The specimens were examined under JOEL-JSM 6100.

All values were expressed as mean ± standard error mean (SEM) of the three replicates. Serial measurements of growth from baseline to day 20 were stratified by various ascorbic acid groups. They were further analyzed and compared by summary measures of area under concentration time curve (AUC0- t max),

growth at 25 g ml–1. There was significant increase in growth after 1st day of culture indicating effective rapid response. The growth of Spirulina showed 26% increase after 24 hours, however, 21% increase was observed at the end of 20 days. The growth levels measured serially from day 0-20 showed overall statistically significant differences when stratified by different doses of IAA (F-statistics 6.38; p value < 0.01). On further posthoc pair-wise analysis, IAA showed statistically significant difference from control. Comparison of serial measurement of growth between 0 hour and 1 hour across different groups was highly significant (p value < 0.01) indicating that IAA induced immediate increase in the growth of Spirulina. There was significant increase in growth of Spirulina supplemented with IAA at 25-50 g ml–1 by 20th day (p value < 0.01) thus indicating positive correlation between IAA supplementation and growth of Spirulina (F-statistics 351, p value < 0.01). At relatively higher concentrations, IAA showed inhibitory effect on Spirulina (Fig. 1)

Effect on dry weight: Dry weight increased by 7% after 1 hour of IAA supplementation indicating its enhancing effect however these values were statistically not significant implying that IAA showed early and faster effect on growth of Spirulina however the increase in dry weight was recorded on 4th day of experimentation. There was about 22% increase at 25 and 50 g ml–1 on 20th day of growth indicating its overall beneficial effect on the growth of this cyanobacterium. Spirulina showed significant increase in dry weight (F-statistics 451; p value < 0.01). Maximum cell productivity of about 0.57 g ml–1/day was observed with IAA supplemented group in comparison with control at 45 g ml–1/day (Fig.2).

Chlorophyll a content also showed similar trend as an increase of 19% was observed in chlorophyll a content after 1 day of growth (Fig.3). On 20th day, there was approximately 30% increase in chlorophyll a content at 25 and 50 g ml–1 of IAA application. The mechanism of IAA action on chlorophyll a has not been elucidated so far but some protective role of IAA on chlorophyll a content of Spirulina seems plausible. The chlorophyll a level measured serially from 0-20th day showed statistically significant difference when stratified with different IAA concentrations (F-statistics 313). Further within groups contrast analysis the chlorophyll shows significant difference between the groups (p value < 0.01).

Spirulina showed 17% increase in carotenoid content at the end of 20th day at 50μg/l IAA concentration (Fig. 4). The carotene content measured serially from 0-20th day showed significant difference when stratified with different IAA regimes (F-statistics 4.52; P value < 0.01). On further posthoc pairwise analysis, IAA supplementation showed statistically significant differences in carotenoid concentrations at 25 g ml–1 (p value < 0.05) and 50 g ml–1 (p value < 0.01) from the control. Carotenoid content showed significant increase on 4th day of growth with Spirulina supplementation indicating effective immediate response of IAA on carotenoid production of Spirulina.

significant increase on first day of growth, However, there was 14% increase in phycocyanin content after 20th day of growth at 50 g ml–1 concentration (Fig. 5). Analysis of phycocyanin content measured serially from initial day to 20th day when stratified by different IAA doses groups, overall differences showed significant differences (F-statistics 3.37; p value < 0.01) on further posthoc pairwise comparison 25 and 50 g ml–1 (p value < 0.01). IAA supplemented groups showed significant difference over control.

The protein content observed on 20th day with different concentrations of IAA was found to be more than the control (Fig. 6). Maximum increase was recorded with supplementation of 25 g ml–1 and closely followed by 50 g ml–1 and 10 g ml–1, concentrations above 75 g ml–1 resulted in protein content equal to or less than control. The protein content measured serially from 0-20 day showed significant difference when stratified with different IAA regimes (F-statistics 77.39; p value < 0.01). On further posthoc pairwise analysis 25 and 50 g ml–1 dose groups of IAA showed statistically significant difference from the control. IAA resulted in significant increase in protein content in general.

Morphological study: Optical microscopy showed that IAA supplementation resulted in rapid growth after 1st day of inoculum. The helical trichomes showing different degree of coiling and straight uncoiled filaments were observed after repeated subculturing (Fig. 7 a,b). The development of hormogonia was observed on 24th day in control as well as IAA supplemented group. Further scanning electron microscopy (SEM) illustrates some structural alterations and enlargement of trichomes when supplemented with IAA (Fig. 7 c,d).

One of the known auxins identified as a plant growth hormone, indole acetic acid (IAA) is involved in nearly every aspect of the plant development including cell division, expansion and differentiation (Tsavkelova et al., 2006; Zhao, 2010). The present study indicated that IAA supplementation resulted in immediate increase in growth after just 24 hours of inoculation, which may be due to the fact that IAA causes fairly rapid increase in cell wall extensibility. Importantly, IAA causes responsive cell to extrude protons actively into the cell wall resulting in decreased pH which is responsible for activation of wall loosening enzymes that promote the breakage of cell wall bonds thus resulting in increased wall extensibility (Kutschera and Schopfer, 1986; Hussain et al., 2015). Further, it could also increase osmotic solutes of the cells thus reducing the wall pressure thereby, promoting the permeability of the cells to water. Ahmad (1971) also reported similar results where they observed 77% increase in growth of Nostoc muscorum (cyanobacterium) in dry weight, which continued even after 24 days. Our results indicated that addition of appropriate quantities of IAA, resulted in significant positive effect on growth and pigments of Spirulina which could be due to widening of anion channels present in various cell membranes and may have central role in cell signaling, osmoregulation, plant nutrition and metabolism (Barbier-Brygoo et al., 2000). In present study, IAA supplemented Spirulina indicated rapid increase in its growth in short span extending even up to 20 days of IAA supplementation. Subsequently, growth of Spirulina showed decline possibly due to overutilization of resources which caused scarcity and disequilibrium in demand and supply of the required nutrient resulting in death of filaments after 20 days. Latter observation, however, was not in consonance with study by Ahmad (1971) where the growth continued even after 24 days of growth. The enhancement in pigments of Spirulina with IAA supplementation was in corroboration with Yin (1937) who reported that IAA distinctly affects the rate of production of photosynthetic pigments. This effect is indirect through changes in chlorophyll content and size of cells. In young cultures, the enhancement in directly affected by chlorophyll content while in older cultures it is affected by chlorophyll content and the cell surface. The present study observation mirrors the findings by Mohammaed and Mohammaed (2011) who studied the effect of IAA on carotenoid content of Spirulina. This may be attributed to the fact that maximum amount of nitrogen enters into the cells of Spirulina due to elevation of osmotic solutes uptake. Luxuriant uptake of nitrogen and its utilization in the synthesis of aromatic acids, result in the increase of pigments. The altered morphology of Spirulina may be due to the cell wall extensibility on IAA supplementation as well study demonstrated positive correlation of IAA supplementation and chlorophyll a, carotenoids and phycocyanin production. Based on results, it is concluded that IAA, in appropriate quantity, can be used as growth promoter to increase the quantity of Spirulina produced with higher amount of photosynthetic pigments like chlorophylls, carotenes and phycocyanin thus enhancing antioxidant potential of Spirulina.

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Assistant Professor, Department of Botany, Sri Guru Gobind Singh College Sector 26, Chandigarh, India kawalpreet38@gmail.com