Arch Biol Sci. 2019;71(3):475-482 https://doi.org/10.2298/ABS190131030K Growth performance and biochemical profile of Azolla pinnata and Azolla caroliniana grown under greenhouse conditions Taylan Kösesakal1,* and Mustafa Yıldız2 1Department of Botany, Faculty of Science, Istanbul University, Istanbul, Turkey 2Department of Aquaculture, Faculty of Aquatic Sciences, Istanbul University, Istanbul, Turkey *Corresponding author: [email protected] Received: January 31, 2019; Revised: March 22, 2019; Accepted: April 25, 2019; Published online: May 10, 2019 Abstract: This study aimed to evaluate the growth performance, pigment content changes, essential amino acids (EAAs), fatty acids (FAs), and proximate composition of Azolla pinnata and Azolla caroliniana grown in a greenhouse. Plants were grown in nitrogen-free Hoagland’s solution at 28±2°C/21±2°C, day/night temperature and 60-70% humidity and examined on the 3rd, 5th, 10th and 15th days. The mean percentage of plant growth and relative growth rate for A. pinnata were 119% and 0.148 gg-1day-1, respectively, while for A. caroliniana these values were 94% and 0.120 gg-1day-1, respectively. Compared to day 3, the amount of total chlorophyll obtained on day 15 decreased significantly (p<0.05) for A. pinnata while the total phenolic and flavonoid contents increased significantly (p<0.05) from the 3rd to the 15th day. However, the total phenolic and flavonoid contents did not differ (p>0.0.5) in A. caroliniana. The crude protein, lipid, cellulose, ash values and the amounts of EAAs were higher in A. pinnata than A. caroliniana. Palmitic acid, oleic acid, and lignoceric acid were found to be predominant in A. pinnata and A. caroliniana. From the plant growth and pigment contents, we concluded that A. pinnata grew faster than A. caroliniana and its photosynthetic efficiency was more effective. Keywords: Azolla; chlorophyll; fatty acids; phenolics; essential amino acids INTRODUCTION growth promoter intermediaries. Therefore, with these nutritional values, Azolla species are a good source of Azolla is a floating fern that can grow in the absence feed for livestock [9]. of nitrogen in freshwater because of the symbiotic relationship between the heterocyst-forming, filamen- Industrial development and agricultural practices tous, nitrogen-fixing cyanobacteria Anabaena azollae, have negative climate and environmental impacts. To which lives in the dorsal lobe cavity of the leaves [1]. meet the requirements for sustainable agriculture there This symbiotic association has recently gained con- is a need for novel crops that require less or no nitro- siderable importance due to its potential for use as gen fertilizer, use nonarable land with high biomass an alternative to nitrogenous chemical fertilizers and yields, and provide for both the food and chemical animal feeding [2-4]. Azolla is very important for the industries [10]. The use of wastewater as a source of agricultural activities of developed and developing reclaimed water would significantly reduce the cost countries [5]. Azolla has great potential for biological and impact on the environment. Since the utiliza- N fixation (30-100 kg N ha−1) and thus Azolla species tion of wastewater is very limited for most terrestrial can be used effectively as a biofertilizer for paddy crops, attention has shifted towards the use of aquatic fields [6]. Furthermore, the use of Azolla species as plants [11]. Azolla species are one of the world’s most a biofertilizer in rice fields improves soil fertility by economically important macrophytes [12] because increasing the organic matter in the soil, thus improv- of their high growth rates, high biomass production, ing soil structure and environmental safety [7,8]. In bioremediation capacity, easy maintenance and easy addition, Azolla species are rich in proteins, essential harvest [13]. These plants can be used to improve water amino acids, minerals, vitamins, carotenoids and quality in view of their phytoremediation potentials. © 2019 by the Serbian Biological Society How to cite this article: Kösesakal T, Yıldız M. Growth performance and 475 biochemical profile of Azolla pinnata and Azolla caroliniana grown under greenhouse conditions. Arch Biol Sci. 2019;71(3):475-82. 476 Arch Biol Sci. 2019;71(3):475-482 In addition, Azolla species have been proposed as Determination of chlorophyll and carotenoid good candidates for phytoremediation of polluted contents freshwater areas [14-17]. Because of the multifaceted uses of Azolla species, especially in food, feed, biofuel To determine the chlorophyll and carotenoid contents, production, agriculture and phytoremediation, it would 200 mg of leaves were extracted in 80% acetone (Merck) be an ideal and environmentally-friendly factor in and the samples were centrifuged (Heraeus Labofuge sustainable agriculture [2]. 400 R) at 3000 x g (4°C) for 15 min. The pigment contents (chlorophyll a and b, total chlorophyll, and The main purpose of this study was to elucidate carotenoid) were measured using a Shimadzu 1601 growth performance, pigment content changes, proxi- UV-Visible Spectrophotometer) and expressed in µg/g mate composition, fatty acids and essential amino acids fresh weight [20]. of Azolla pinnata and Azolla caroliniana grown under greenhouse conditions. Also, the potential use of A. Analysis of total phenolics and flavonoids pinnata and A. caroliniana for further experimental studies has also been evaluated. Total phenolic and flavonoid contents of Azolla were measured as described [21]. The leaves were extracted MATERIALS AND METHODS with 1% HCl-methanol (5 mL), the extract was filtered and the filtrate was diluted with 1% HCl-methanol Azolla pinnata and Azolla caroliniana plants were used, to 10 mL. Absorbance of the solution was measured which were grown in modified Hoagland’s nutrient at 280 nm for total phenolic and at 325 nm for the -1 solution containing (in mg L ): KCl, 74.55; KH2PO4, flavonoid contents. The total phenolic content was 136.08; CaCl2•2H2O, 147.02; MgSO4•7H2O, 246.08; calculated from a standard curve made with gallic acid ZnSO4•7H2O, 0.22; H3BO3, 2.86; Na2MoO4•2H2O, 0.09; as a standard and expressed in GAeq. The flavonoid CuSO4•5H2O, 0.09; MnCl2•4H2O, 1.82; FeCl3•6H2O, content was expressed as the absorbance at 325 nm/g 4.84 and Na2EDTA, 15. The pH value of the nutrient fresh weight of Azolla. solution was adjusted to 6.0. Plants were grown at 28±2°C/21±2°C, day/night temperature and 60-70% Crude protein, lipid and cellulose analysis humidity under greenhouse conditions. To ensure the transfer of only healthy and young plantlets, the The crude protein, lipid, cellulose and ash contents were procedure described in [18] was applied. The percent- determined according to the standard methodology age of plant growth, relative growth rate (RGR), total [22]. Crude protein was determined as total nitrogen chlorophyll, carotenoid, total phenolic and flavonoid (N) using a semi-automatic Kjeldahl (Gerhardt VA- content changes were examined on the 3rd, 5th, 10th PODEST, 45s) technique (N×6.25). The lipid content and 15th days. was determined by ether extraction using a Soxtherm Multistat/SX PC (Gerhardt, Germany). The ash content Plant growth was obtained from the weight loss after incineration of dried samples in a muffle furnace. Cellulose was Prior to application, the plants were weighed. The determined using sulfuric acid, then sodium hydroxide growth rate was measured by comparing the weight (12.5%, w/w), and the final residue was washed with of the plants before and after the experimental times. 5% HCl and water, then filtered, dried, and weighed. Also relative growth rate (RGR) (g g−1 d−1) of the plants All samples were analyzed in triplicate. was calculated using the formula: Analysis of amino acids RGR = (ln W2−ln W1)/t, where W1 and W2 are the initial and final fresh weights, Amino acids were determined as described [23]. One respectively, and t is the experimental time [19]. hundred mg of freeze-dried plant samples (Alpha 1-2 LD plus, Christ, Germany) were digested in sealed glass tubes under nitrogen with 6 N HCl for 24 h at 110°C. Arch Biol Sci. 2019;71(3):475-482 477 The samples were then filtered and the excess acid from the hydrolysate was removed by flash evapora- tion under reduced pressure and resuspended in 0.02 N HCl. Amino acid analysis was performed using HPLC (Agilent 1100) [24]. Fatty acid (FA) analysis Fatty acid methyl esters were transmethylated with 2 M potassium hydroxide (KOH) (Merck, Germany) in methanol and n-hexane (Sigma-Aldrich, Germany) [25], with minor modification. Ten mg of extracted oil were dissolved in 2 mL of hexane followed by the Fig. 1. Plant growth percentages and RGRs of A. pinnata (A) and addition of 4 mL of 2 M methanolic KOH. By vor- A. caroliniana (B) on days 3, 5, 10 and 15. The bars represent the texing the tube for 2 min at room temperature and standard deviation. Significant differences determined by Tukey’s a centrifugation at 4000 x g for 10 min, the resulting multiple comparison test (p<0.05) are indicated by different letters. hexane layer was taken for GC analyses. By means of a gas chromatograph (Auto System XL Perkin Elmer, FID days were 44%, 107%, 129% and 194%, respectively. detector), using a 30 m x 0.25 mm x 0.25 µm capillary The RGR’s of the A. pinnata plants at the experimental column (CP-2380 Supelco, USA), the FA composition times were determined as 0.137, 0.180, 0.139 and 0.135 was analyzed. The conditions of the method were as gg-1day-1, respectively. The average percentage growth follows: carrier gas, helium; flame ionization detection and RGR for A. pinnata were 119% and 0.148 gg-1day-1, temperature, 260°C; split rate was 1/0, oven tempera- respectively.