WWW.WA S .ORG • WORLD AQUACULTURE • JUNE 2022 59 Wet phytoplankton slurry was also pooled and divided into two equal portions. One portion was stored in the refrigerator and the other portion was mineralized by acid digestion using a Digesdahl Digestion Apparatus (HACH©, Loveland, CO). Briefly, 10-g samples of wet microalgae were digested with 6 mL of concentrated sulfuric acid at 440 C for six minutes. Then, calcium hydroxide was added to neutralize the solution (pH range 6.8–7.2) and the volume brought up to 100 mL with distilled water. This mineralized algae solution contained all the minerals in the algae but no organic biostimulants. This process was repeated weekly to obtain fresh samples. Two pot experiments to assess the effects of algae on plant growth were performed. Experiment with Radish Radish Raphinus sativus was chosen because it allows for assessment of growth in a short period of time. Pots were filled with air-dried soil and peat moss at a volumetric ratio of 1:1. Ten radish seeds were sown around the periphery of each pot, four cm from the edge, and irrigated with distilled water. Seedlings were thinned out after the emergence keeping only three plants per pot. The experiment consisted of five treatments with nine replicate pots per treatment, distributed in a greenhouse in a completely randomized design. The treatment designations were: • DW (distilled water). Pots were watered with 0.2 L of distilled water each irrigation event (control). • T1 (green aquaculture water). Pots were watered with 0.2 L of green aquaculture freshwater for ten irrigations and then watering was continued with distilled water. • T2 (supernatant). Pots were watered with 0.2 L of supernatant aqueous extract for ten irrigations and then watering was continued with distilled water. • T3 (wet microalgae). Pots received 20 g of wet microalgae poured onto the soil surface once and then watered with 0.2 L distilled water for ten irrigations. • T4 (mineralized microalgae). Pots were watered with 0.2 L of mineralized microalgae once and then watered with 0.2 L distilled water for ten irrigations. Treatments were designed such that for every liter of green aquaculture water (T1) supplied to the pots, 1 L of supernatant (T2), 10 g of wet microalgae (T3) and 100 mL of mineralized microalgae solution (T4) were supplied. Succeeding irrigations were continued with distilled water. Irrigation schedule and volume were calculated based on the water holding capacity of the soil to avoid deep percolation. Plants were harvested seven weeks after seeds were planted. Radish plant growth parameters (shoot weight, root weight and plant biomass). Plants were then dried at 60 C in a forced oven for 72 h (until constant weight) and dry weight recorded. All treatments significantly increased root weight and shoot weight of radish plants compared to the DW control. No differences were observed among the other treatments (Table 1). Similar results were observed among dry weights of the plants, where plants in the DW treatment were significantly smaller than in all other treatments but there were no differences among the other treatments. acid-containing products” (du Jardin 2015). It is well documented that microalgae enhance crop growth as biofertilizers but their properties as plant biostimulants are not well studied. Nevertheless, microalgae as plant biostimulant has been suggested by some researchers (Adam 1999, Kulik 1995, Rodríguez et al. 2006, van der Voort et al. 2015. Kulik 1995). Growth promotion of rice in response to the application of microalgae Nostoc muscorum may be attributed to nitrogenase and nitrate reductase activities of microalgae associated with plants (Adam 1999). Additionally, amino acids and peptides produced in the microalgae filtrate and/or other compounds may stimulate plant growth. Rodríguez et al. (2006) reported that S. hofmanni may counteract hormone disturbance of rice seedlings under saline conditions by synthesizing and liberating growth regulators which act like gibberellin. AgroValley Inc. (USA), Agroplasma S.A. and AlgaEnergy S.A. (Spain) and Soley Biotech (India) market microalgae as biostimulants. These companies produce several microalgal products such as Spirulina, Scenedesmus, Chlorella sp. and Nannochloropsis sp. for use in crop production as organic fertilizers and biostimulants. AlgaEnergy S.A. state that farmers all over the world already rely on their biostimulants (AgriAlgae®) to take care of crops and maximize yields. Agroplasma S.A. market their product (Ferticell) by stating that it “stimulates the plant systems, resulting in more root mass, increased leaf area, increased number of flowers and fruit and early maturation.” However, there are no empirical data supporting the plant biostimulant properties of freshwater microalgae from aquaculture effluent. A study was conducted to investigate whether freshwater microalgae in aquaculture effluent stimulates crop growth and flowering and whether the response of crops to microalgae is a result of biostimulation or biofertilization. This investigation was conducted at the aquaculture laboratory and plant research facility of the American University of Beirut (AUB). Two pot experiments were designed to study the effect of microalgae from freshwater tilapia aquaculture on crop growth and flowering. Various treatments were assayed to determine whether microalgae act as biofertilizers, biostimulants or both. Soil was obtained from a local supplier and used in both pot experiments. The soil was sandy loam, non-saline, slightly alkaline and with low amounts of nutrients. In general, the soil was adequate for crop growth. Preparation of Microalgae Six 1-m3, cylindrical fiberglass tanks (Fig. 1) were filled with fresh water and each stocked with 30 tilapia (individual weight ~ 100 g). Water flowed through the bottom drain of each tank into a common settling tank fromwhere it was pumped into a biofilter and returned to individual tanks. Solids from the settling tank were flushed out twice weekly and lost water was replaced with clean well water. Fish were offered a commercial tilapia diet at 4 percent body weight daily divided into two feedings a day. When water became green, 100 mL from three tanks was collected and centrifuged at 5000 rpm for six minutes to estimate phytoplankton concentration in the water. When algal biomass reached 10 g/L, water was collected from tanks and used in the experiments. Part of the water was stored at 5 C and another part in 500-mL aliquots was centrifuged at 5000 rpm for six minutes. Supernatant from each aliquot was pooled and stored in a refrigerator. ( C O N T I N U E D O N P A G E 6 0 )
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