Impact Of Nitrogen and Phosphorus on The Synthesis of Carotenoids in Cladophora Glomerata (Linnaeus) Kützing, A Green Macroalga

Algae are diverse group of single to multicellular eukaryotic organisms that generally inhabits water or humid environments including damp soils, and are capable of photosynthesis as they possess photosynthetic pigment chlorophyll, a class of green pigment that transform sunlight into chemical energy and food along with water, therefore considered as an autotrophic group, capable of producing their own food (Arora and Sahoo, 2015). Algae are distinguishable from seagrass and angiosperms as they lack vascular system and exhibits thalloid structure (Pereira et al., 2021). Some algae grow to a length of several meters, due to which they are called macroalgae (Ross et al., 2018). Few of these species have efficient nitrogen absorption capabilities ranging to 19 to 96.6%, increased CO2 utilization and high growth rates from 7.1 to 11.7% per day (Eustance et al., 2016). Also, biomass generated through cultivation or natural harvesting may be converted into biofuels or used as food, fertilizer and high value products which ultimately increase economical value of macroalgae (Ross et al., 2018). The genus Cladophora Kützing is ubiquitous in occurrence and distributed worldwide in fresh, brackish, and marine waters (Ennabili et al., 2025). The green alga Cladophora is considered as one of the most suitable candidate for research in biotechnological applications, including bioremediation (Rojas-Villalta et al., 2024). In some studies, it was also concluded that genus Cladophora which is highly branched, herbivory resistant, produces noticeable growths near shore, along rocky shorelines of pristine to eutrophic, marine, estuarine, brackish and freshwater environments (Dodds and Gudder, 1992). The genus Cladophora has been described as an ecological engineer because of its intricate structure which efficiently increases its functional and taxonomic diversity of benthic microflora (Zulkifly et al., 2013). The Cladophora glomerata (Linnaeus) Kützing is reported to have proteins and carbohydrates in good amount which is 9-17.3% protein and 62.8-74.5% carbohydrates respectively. The genus Cladophora is added to regular bread to increase its nutritional value because it contains vital amino acids and minerals. The green alga Cladophora also has more necessary amino-acids comparatively to Porphyra and Spirulina (Munir et al., 2019). The present communication deals with the evaluation of the effect of different concentrations of di-potassium hydrogen orthophosphate as a sole source of phosphate and sodium nitrate as a sole source of nitrogen in the culture medium for the synthesis of photoprotective pigment carotenoid in the filamentous, a truly branched green macroalga C. glomerata. The present work is focused on evaluating the effect of different concentration of nitrogen and phosphorus in the form of sodium nitrate (NaNO3) and di-potassium hydrogen orthophosphate (K2HPO4) respectively on the synthesis of total carotenoids content of C. glomerata (Linnaeus) Kützing.

MATERIALS AND METHODS

Sampling site and collection of algal samples:

Algal growth containing samples were collected in polybags from the cemented water tank located at Department of Botany, Chaudhary Charan Singh University, Meerut, Uttar Pradesh, Bharat.

Isolation, purification and culturing of experimental organism:

Collected algal samples were inoculated in liquid BG-11 medium (Stanier et al., 1971) and their enrichment cultures were raised. From enrichment culture petridishes 1-2 filaments were picked up carefully with the help of inoculation needle, and were transferred and streaked out into semisolid petridishes containing BG-11 medium. For getting pure culture of the experimental organisms, culturing and sub culturing method followed by Kant et al. (2005) and sub culturing process were followed repeatedly until the pure cultures of experimental organism were obtained.

Morphological observations and Identification of the experimental organism:

One filament of unialgal cultures of the green alga was taken and their temporary slides were prepared, and observed with the help of Research Microscope (Trinocular, Olympus-CH20i) and their morphological observations were recorded with help of digital camera (Magnus, Magcam DC 10) fitted with Research Microscope and finally the alga was identified as Cladophora glomerata (Linnaeus) Kützing. Isolated culture of C. glomerata is deposited in the Algal Biotech Laboratory, Department of Botany, Chaudhary Charan Singh University, Meerut, Uttar Pradesh, Bharat.

Culturing media formulation

The present study was conducted in triplicates using 150ml conical flasks (Borosil) containing 100ml phosphorus and nitrogen free nutrient medium (Table-1 & 2).

Table-1: Chemical constituents of nitrogen and phosphorusfree nutrient medium

Table-2: Chemical constituents of Trace Metal Mix

Exponential growth of the experimental organism

Axenic culture of C. glomerate was inoculated at BG-11 media (Stainer et al., 1971) under 4Klux light for 14:10 h light: dark regime at 28±2°C for 10 days on multi-position magnetic stirrer for even and exponential growth of the culture organism.

Experimental design

Exponentially growing culture of C. glomerata was batch cultured in triplicates under 4K Lux light for 14:10 h light: dark regime at 28±2°C using three different concentrations (0.04g/l, 0.06g/l, 0.08g/l) of di-potassium hydrogen orthophosphate (K₂HPO₄) as a sole source of phosphate and three different concentrations (1.5g/l, 2.25g/l, 3.375g/l) of sodium nitrate (NaNO₃) as a sole nitrogen source and harvested every 10^thday till 30 days to evaluate the effect of di-potassium hydrogen orthophosphate and sodium nitrate on the synthesis of photo-protective pigment total carotenoids. Culture medium containing 0.4g/l of di-potassium hydrogen orthophosphate as a sole phosphate source and 1.5g/l sodium nitrate as nitrogen source was used as control. Detailed concentration of different variants of di-potassium hydrogen orthophosphate (K₂HPO₄) and sodium nitrate (NaNO₃) used in the experiment for evaluation of carotenoid pigment is given in Table-3 and Table-4.

Estimation of Carotenoids

Estimation of total carotenoids pigment was determined by Jensen (1987) and the absorbance was measured at 450nm against 90% acetone blank. The whole experiment was conducted in very low light condition to avoid photoreaction and loss of pigment.

Analysis of Variance

The data obtained was subjected to statistical analysis of variance (ANOVA) carried out in MS office 2019. Each mean value of the data was calculated in triplicate. Standard deviation and Standard error were calculated against the obtained value (Mondal and Mondal, 2016).

RESULTS AND DISCUSSION

Evaluation of photoprotective pigment under different conc. of di-potassium hydrogen orthophosphate and sodium nitrate was done and it was found that the higher amount of di-potassium hydrogen orthophosphate in the culture media decreases the carotenoid content in the cells while high concentration of sodium nitrate as nitrogen source boosts carotenoid content. Maximum total carotenoid content was observed 0.003972µg/ml under the influence of culture media containing treatment flask C3 after 20days of batch culturing. Minimum concentration of carotenoid was observed 0.00314µg/ml under the influence of treatment P3 after 30 days of batch culturing. Results obtained indicated that high nitrogen content in the culture media could proliferate carotenoid content in the C. glomerata cells. A detailed result of carotenoid content in C. glomerata cells under different concentrations of di-potassium hydrogen orthophosphate is given in Fig.-1 while Fig.-2 illustrates carotenoid content under different concentrations of sodium nitrate. Carotenoid content under treatment flasks C1 and C2 under the influence of sodium nitrate increased by 1.70% and 7.04% respectively in compared to the control flasks on 20^th day while under the effect of treatment flasks P2 and P3 containing different concentrations of di-potassium hydrogen orthophosphate, carotenoid content decreased by 0.98% and 3.47% respectively in compared to the control flasks after 20 days of batch culturing.

Figure-1: Carotenoid content in C. glomerata cells under different concentrations of di-potassium hydrogen orthophosphate (K₂HPO₄) as a sole source of phosphate

Figure-2: Carotenoid content in C. glomerata cells under different concentrations of sodium nitrate (NaNO₃) as nitrogen source