Asian Journal of Fisheries and Aquatic Research 19(5): 29-38, 2022; Article no.AJFAR.92146 ISSN: 2582-3760 Proximate Composition Analysis of Spirulina platensis in Lab Scale Cultivation: Prospects of Digested Rotten Guava as a Culture Media Md. Hashibur Rahman a*, Mohammad Ashraful Alam b, Flura b, Md. Saiful Islam c, Md. Arifuzzaman d, Md. Moniruzzaman b, Asma Jaman a, Sharmin Sultana Mukti a, Anik Talukdar e and Md. Abu Kawser Didar b a Bangladesh Fisheries Research Institute, Headquarters, Mymensingh-2201, Bangladesh. Bangladesh Fisheries Research Institute, Riverine Station, Chandpur-3602, Bangladesh. c Department of Aquaculture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. d Bangladesh Fisheries Research Institute, Shrimp Research Station, Bagerhat-9300, Bangladesh. e Bangladesh Fisheries Research Institute, Freshwater Sub-Station, Jashore-7402, Bangladesh. b Authors’ contributions This work was carried out in collaboration among all authors. All authors read and approved the final manuscript. Article Information DOI: 10.9734/AJFAR/2022/v19i5475 Open Peer Review History: This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/92146 Original Research Article Received 09 July 2022 Accepted 19 September 2022 Published 23 September 2022 ABSTRACT An experiment was conducted to evaluate the proximate composition of Spirulina platensis and growth performance in supernatant of different concentrations (supernatant of 20, 40 and 60 rotten guavas) of digested rotten guava medium (DRGM). The growth rates in terms of optical density, dry cell weight and chlorophyll a of spirulina were varied from each other. The cell weight of Spirulina was attained a maximum of 0.818 ± 0.003 mg/L in 60% DRGM followed by 0.815 ± 0.0015 and 0.809± 0.0012 mg/L in supernatant of 20 and 40%, respectively on the 10th day of culture. Similar trend was also observed in the case of optical density of the media contained Spirulina, chlorophyll a content (mg/L), total biomass (mg/L), specific growth rates (on the basis of cell weight and chlorophyll a) and total biomass of Spirulina. Cell weight of Spirulina grown in these media had highly significant (P < 0.01) correlation with the chlorophyll a content (r = 0.746) and total biomass (r = 0.742) of Spirulina. The growth performance of Spirulina in supernatant of 60% DRGM was significantly (P < 0.01) higher than that of grown in 20% and 40% DRGM. The percentage of crude _____________________________________________________________________________________________________ *Corresponding author: Email: hasibkhan94bfri@gmail.com; Rahman et al.; AJFAR, 19(5): 29-38, 2022; Article no.AJFAR.92146 protein was found 53.35 ± 0.32% in supernatant of 40% DRGM. The crude lipid was attained of 10.15 ± 0.14% in supernatant of 60% DRGM which was significantly (P < 0.05) higher than that of grown in 20% and 40% of DGRM. Keywords: Spirulina platensis; proximate composition; growth performance; digested rotten guava medium (DRGM). temperature and light. The filamentous cyanobacteria such as spirulina are found to be most compatible microorganisms for the utilization of waste and wastewaters as they are able to produce large quantity of biomass and their harvesting is also relatively easy because of their structure. Also, these wastes reduce the cost of nutrient medium and act as a source of cheap nutrient medium for cultivation of spirulina” [8]. “The commercial production of spirulina can be made cost effective by reducing the input cost with cheap and readily available materials without sacrificing the production efficiency. They are very small a microscopic and 300-500 micrometer in length. Spirulina contains 50-70% protein, 10-12% carbohydrate, 6% fat, 7% minerals and a lot of vitamins” [8]. “However, according to the researchers, one kg of Spirulina spp is similar to 1000 kg of other vegetables” [9]. 1. INTRODUCTION “With the aim of increased aquaculture production through applying adequate feed, there are a large numbers of feed industries are developed in Bangladesh. Due to increased aquaculture practice, demand of good quality feed is increasing day by day. Prime quality feed is essential for fish growth. Maintain feed conversion ratio (FCR) close to 1 is highly depends upon good feed. Feed should have adequate protein content which facilitates high growth. Net protein utilization should be around 27%” [1]. But fish meal and bone meal are not available in Bangladesh. So, we can find to alternative sources. We can use to alternative fish meal to spirulina. Spirulina is a “superfood” which is the most nutritious, rich in protein and concentrated whole food known to humankind [2]. “It has a vibrant history occupied an intriguing biological and ecological niche in the plant kingdom. Spirulina is a spiral-shaped, blue-green microalgae that grows naturally in the wild in freshwater alkaline lakes, natural springs, and saltwater. Its deep blue green color is what gives the water greenish hue” [3]. “Spirulina is also cultivated and harvested in man-made reservoirs around the world. For centuries, civilizations the world over have cultivated and cherished spirulina for its health-improving benefits. The Aztecs harvested the spirulina from Lake Texcoco in Mexico. It grows well in supernatant of different digested agro-industrial wastes available in Bangladesh” [4]. Spirulina has been studied for single cell protein (SPC), vitamins, minerals, proteins and polyunsaturated fatty acids (γ-linolenic acid), therapeutic properties, antioxidant activity. Several cultivation methods like open ponds, tubular photo bioreactors, inclined glass panels have been tried. Cost and composition of cultivation media along with growth rate of the algae we challenging factors for commercially viable production. The most convincing trials are of course those conducted among populations which traditionally eat spirulina. Culture and growth performance of Spirulina platensis in supernatant of digested rotten guava to evaluate the proximate composition of spirulina; to analyze growth parameters 30 in supernatant digested rotten guava; and to find out the suitable concentration of the medium for maximum growth of Spirulina platensis. Spirulina may be grown in agro-industrial wastes [5], rotten fruits [6], chicken wastes [7]. Among fruits huge quantity of guava spoils (rotten) in different markets in the country. Therefore, market is allowed to digest (aerobic & anaerobic) and supernatant may be used for the growth of spirulina. This inexpensive low-cost medium may be used to produce Spirulina platensis culture which can contribute significantly for the development of fisheries and fish production. It takes inorganic nutrients for the supernatant and grows. “Many factors are important for the production of spirulina at large scale, of which most important factors are nutrient availability, 2. MATERIALS AND METHODS 2.1 Study Area The study carried out in Live Food Aquaculture Laboratory, Department Aquaculture, Faculty of Fisheries, Bangladesh Agricultural University (BAU), Mymensingh-2202, and Bangladesh. 30 Rahman et al.; AJFAR, 19(5): 29-38, 2022; Article no.AJFAR.92146 the reading for the samples and blank were recorded. The readings were calculated the following formula: 2.2 Collection of Rotten Guava The rotten guava was selected as medium for Spirulina platensis culture. It was collected from Kamal Ronjit market (K.R.) of Bangladesh Agricultural University, Mymensingh-2202, and Bangladesh. It was thought that the proximate composition of this media might be suitable for the growth of culture species. Percentage of nitrogen For animal, % Protein = % Nitrogen x 6.25; and For plant, % Protein = % Nitrogen x 5.85 2.3 Analysis of Proximate Composition of Rotten Guava (RG) 2.6 Crude Lipid Before media preparation, the proximate composition of rotten guava was analyzed to know its nutritional status. The analysis was performed in Fish Nutrition Laboratory, Department Aquaculture, Faculty of Fisheries, Bangladesh Agricultural University (BAU), Mymensingh-2202, and Bangladesh, following standard methods [10]. Lipid content was estimated by solvent extraction of lipid using Soxlet apparatus. 2.0 g dried samples were taken into extraction thimbles to place into the extraction unit along with the weighed extraction cups having 50 ml of solvent as acetone. Extraction of first 15 minutes was in the boiling position and the cups were released and dried in the oven for 30 minutes. The percentage of crude lipid was determined using the following equation: 2.4 Moisture The sample was weighed in a previously preweighed small crucible in triplicates. The samples contained in the crucible were dried to o moisture from it 105 C for 24 hrs. After drying, the samples with crucible were cooled with the help in a desiccator. Then, cooling at room temperature and weighed in a sensitive balance. The percentage of moisture of the sample was calculated using the following equation: % moisture  % Lipid  Weight of cup with lipid  initialweight of cup 100 weight of sample (g) Ash: The pre-weighed crucible containing dried sample from the moisture determination was preashed. The samples were kept into a muffle furnace at 550°C for 6.0 hrs. The crucible containing ash was cooled in a desiccator. The percentage of ash was determined by using the following: XY 100 X % Ash  Where, Weight of ash with preweighed crucible  weight of crucible 100 weight of dried sample X = Weight of sample before drying; and Y= Weight of sample after drying. NFE: NFE were calculated the following formula: Nitrogen Free Extract = 100 - (Moisture + Crude protein + Crude lipids + Ash). 2.5 Crude Protein 2.7 Estimation of Cell Weight (Dry Weight) of Spirulina (Clesceri et al., 1989) Kjeldhal Auto 1030 Analyzer was used for determination of crude protein content of samples. A sample of 0.5g and a blank were digested in the digestion tube. 10 ml of concentrated sulphuric acid (H2SO4), 2.0 ml of H2O2 and one Kjeldhal tablet were added in the tubes and mixed gently by electric mixer. Then, the digestion tubes were set in digestion chamber fixing at 420°C for 45 minutes. The digestion the tubes were allowed to cool and 75 ml of distilled water was added in each tube. 50 ml of 40% NaOH was added before titration. After titration with 1% boric acid and 0.2 N HCl Sample containing 15 ml spirulina suspension was filtered through a Sartorius filter paper of mesh size 0.45 µm and diameter 47 mm. The filter papers were dried in an oven for 24 hrs. overnight at 70°C and weighed prior to filtration. The filtered samples were washed three times to remove insoluble salts. After that the filter papers were put in a glass petri dish and kept in the oven at 70°C overnight. For cooling, petri dish 31 Rahman et al.; AJFAR, 19(5): 29-38, 2022; Article no.AJFAR.92146 SGR (µ/day) = In (X1-X2)/t1-t2 was put into desiccator for 20 minutes and then filter papers were weighed. The dry weight of algae on the filter paper was measured using the following equation: Where, X1 = Dry weight of biomass concentration of the end of selected time interval; X2 = Dry weight biomass concentration at beginning of selected time interval; And t1-t2 =Elapsed time between selected time in the day. Dry weight (mg/L), W FFW  IFW 100 Amount of sample taken for filtration (ml) Where, Specific growth rate (µ/day) of cultured Spirulina on the basis of chlorophyll a W = Cell dry weight in mg/L; FFW = Final filter paper weight in g; and IFW = Initial filter paper weight in g. 2.8 Estimation of Chlorophyll a Spirulina (Clesceri et al. 1989) SGR (µ/day) = In (X1-X2)/t1-t2 of Where, X1 = Chlorophyll a at the end of selected time interval; X2 = Chlorophyll a at the beginning of selected time interval; And t1-t2 = Elapsed time between selected time in the day. The samples of Spirulina platensis were collected in different times and chlorophyll a content of S. platensis was estimated. Ten ml of S. platensis sample were filtered with an electric filtration unit using filter papers (Sartorius filter paper of 0.45 µm mesh size and 47 mm). These filtered samples together with filter paper was taken into test tubes, ground with glass rod and finally mixed with 10 ml of 100% redistilled acetone. Each of the test tubes was wrapped with foil papers to inhibit the contact of light. The wrapped test tubes were kept into a refrigerator (LMS Laboratory Refrigerator) over night. Then the refrigerated samples were homogenized for 2 minutes followed by centrifugation at 4000 rpm for 10 minutes. After centrifugation, the supernatant was isolated and taken for chlorophyll a determination. Optical densities of the samples were determined at 664 nm, 647 nm and 630 nm by using UV spectrophotometer (Milton Roy, Spectronic 1001 plus) [Clesceri et al. 1989]. A blank with 100% acetone was run simultaneously. Specific growth rate (µ/day) of cultured Spirulina on the basis of total biomass SGR (µ/day) = In (X1-X2)/t1-t2 Where, X1 = Total biomass at the end of selected time interval; X2 = Total biomass at the beginning of selected time interval; and t1-t2 = Elapsed time between selected time in the day 2.10 Culture and Collection of Spirulina platensis Spirulina platensis was collected from the stock in the live food culture laboratory, Department of aquaculture, Faculty of Fisheries, Bangladesh Agricultural University (BAU), Mymensingh-2202, and Bangladesh. Nine conical flasks (2L capacity) were used for the culture of spirulina. Chlorophyll a content was calculated by the following formula: Chlorophyll a (mg/L) = 11.85 (OD 664) – 1.54 (OD 647) – 0.08 (OD 630) 2.9 Total Biomass platensis) of Spirulina (S. 2.11 Maintenance of Pure Stock Culture of Spirulina platensis Total biomass was calculated using the following formula given by Vonshak and Richmond [11]: Total biomass = Chlorophyll a x 67. Pure stock culture of Spirulina platensis was maintained in the laboratory [12]. “Growth of Spirulina platensis were observed at every alternative day and was checked under microscope to confirm its purity following some keys given” by Bold and Wynne [13], Vymazal [14] and Phang and Chu [15]. Specific growth rate (SGR) on the basis of dry and total weight, chlorophyll a content biomass of spirulina (Clesceri et al. 1989): Specific growth rate (µ/day) of cultured spirulina on the basis of dry weight. 32 Rahman et al.; AJFAR, 19(5): 29-38, 2022; Article no.AJFAR.92146 2.12 Preparation of Digested Guava Media (DRGM) in different media (treatments) were done and to find whether any significant among treatment mean was done by Ducan’s Multiple Range Test (DMRT) at 5% level of probability” [16]. Rotten The compositions of Rotten Guava Medium (RGM) were prepared for culture of Spirulina platensis. To decompose in 5.0 L glass bottle 50 g/L rotten guava was allowed for 34 days under aerobic condition in the Live Food Culture laboratory, Department of Aquaculture, BAU, Mymensingh. Then a Light reddish white colored supernatant from bottle was diluted and made three concentrations at the rate of 20%, 40% and 60% digested rotten guava. Then the supernatant of three different concentrations were taken in 1.0 L flask with three replications. For the preparation of rotten guava medium, digested and continuous aeration 5 litter volumetric flask was filtered with plankton net after (10.10.18 to 14.11.2018) 34 days left. Then, using distilled water in three replications, the filtered rotting guava was diluted and added 0.8 g (0.2 g/L) urea in accordance with the aforementioned instructions. The medium was then thoroughly mixed before being sterilised in a high pressure, bumping water autoclave for 15 minutes at 115°C. Before growing microalgae, the media were left in the autoclave for three days to ensure that there were no contaminants. The method used during the creation of digested rotten guava media was mixed, autoclaved, and cooled. 3. RESULTS 3.1 Proximate Composition (%) Spirulina (Spirulina platensis) of There was no significant variation among the crude protein of Spirulina grown in the supernatant of three different DRG (Table 2). The percentage of crude protein of Spirulina was 53.25 ± 0.32, 53.35 ± 0.34 and 53.28 ± 0.32% when grown in the supernatant of 20, 40 and 60% DRG media, respectively. There was no significant (P > 0.05) variation among the crude protein percent of spirulina cultured in three different media of DRG. There was no significant (P > 0.05) difference of crude lipids of spirulina when cultured in the supernatant of 20, 40 and 60% digested rotten guava. Ash (%) of Spirulina grown in supernatant of 20% (10.22 ± 0.13%) 40% (10.16 ± 0.17%) and 60% (10.33 ± 0.21%) digested rotten guava. There was no significant (p> 0.05) difference among the ash of spirulina grown in supernatant of 20, 40 and 60% DRG. There was no significant variation among the NFEs of spirulina grown in supernatant of 20, 40 and 60% DRG as the values found for 20% (16.28 ± 0.32%), 40% (16.10 ± 0.18%) and 60% (15.99 ± 0.22%), respectively. 2.13 Experimental Design of Spirulina platensis Culture Three types media viz., Rotten guava (RG) were used to culture Spirulina platensis. Inoculum of Spirulina platensis was collected from the pure stock culture. Experimental design is shown in (Table 1). Very small amount of crude fibre (%) was found in spirulina grown in the supernatant of three different digested rotten guava (DRG). However, it was varied from 0.69 ± 0.0% when Spirulina grown in the supernatant of 40% DRG to 0.71 ± 0.04% and 0.71 ± 0.03% when cultured in the same of 60% DRG, respectively (Table 2). 2.14 Analysis of Proximate Composition Spirulina Best optical density of spirulina (S. platensis) was found on 10th day of culture. In that day, spirulina was filtered, collected in petridish and kept in an oven at 40°C for overnight for drying. Then the proximate composition of cultured S. platensis was analyzed in the Fish nutrition Laboratory, Faculty of Fisheries, BAU, Mymensingh by standard methods [10]. All the procedures for determination of proximate composition, the procedures as described in the Chapter 3.2.2 were followed. 3.2 Optical Density of Media Contained Spirulina Optical density (OD) of media contained spirulina was found to increased up to 10th day of culture in all the media of digested rotten guava (DRG), and Kosaric medium and then decreased up to 14th day of experiment (Fig. 1). However, highest OD of 20% DRG culture contained spirulina was 0.631 ± 0.0023, where highest OD of 40% DRG culture contained spirulina was found 0.704 ± 0.0015. The OD of supernatant of 60% DRG contained spirulina was 0.725 ± 0.0012. 2.15 Statistical Analysis “Analysis of variance (ANOVA) of mean cell weight and chlorophyll a of S. platensis cultured 33 Rahman et al.; AJFAR, 19(5): 29-38, 2022; Article no.AJFAR.92146 (0.725 ± 0.0012). There was no significant (P > 0.05) difference among optical densities of 20, 40 and 60% DRG during the study. There was no significant (P > 0.05) difference of cell weight of spirulina grown in 20, 40 and 60% DRG. Chlorophyll a of spirulina grown in 60% DRG (0.862 ± 0.0012 mg/L) was significantly (p < 0.05) higher than that of spirulina cultured in 40% (0.768 ± 0.0012 mg/L) and 20% DRG (0.770 ± 0.14 mg/L). There was no significant difference among the Chlorophyll a of spirulina grown in supernatant of 20, 40 and 60% DRG during the study (Table 3). 3.3 Chlorophyll a of Spirulina Chlorophyll a of spirulina was found also higher on 10th day of culture than other days of culture in supernatant of all the media (Fig. 2). Chlorophyll a of spirulina increased from first day (0.00158 ± 0 g/L) up to 10th day (0.770 ± 0.0012 g/L) of culture in 20% digested rotten guava media (DRTM) and then decreased up to 14th day (0.710 ± 0.0012 g/L) of experiment. However, chlorophyll a of spirulina cultured in supernatant of 40% DRG was 0.768 ± 0.0012 g/L th on 10 day and then decreased up to 14th day (last day) of culture. Chlorophyll a of spirulina grown in supernatant of 60% DRG was 7.365 ± 0.20 g/L on 10th day from first day (0.0016 ± 0) and then decreased up to 14th day (last day) of experiment (Fig. 2). 3.6 Growth Cell weight of spirulina (Spirulina platensis) had highly significant (p< 0.05) direct correlation with chlorophyll a (r = 0.746) of spirulina grown in the supernatant of different digested rotten guava. Similarly, total biomass of S. platensis was highly (p< 0.05)and directly correlated with chlorophyll a (r = 0.795) of spirulina cultured in the supernatant of various digested rotten guava. Again, total biomass of spirulina was found to be highly (p< 0.05) and directly correlated with the cell weight (r = 0.742) of spirulina grown in the supernatant of different digested rotten guava. 3.4 Total Biomass of Spirulina Total biomass of spirulina was increased from first day (0.106 ± 0.003) mg/L up to 10th day (67.77 ± 0.44 mg/L) in the culture of 20% digested rotten guava media (DRGM) and then decreased up to 14th day (47.57 ± 0.42 mg/L) of experiment. The highest total biomass of spirulina grown in the culture of 40% DRGM was recorded 51.46 ± 0.28 mg/L on 10th day of culture and then decreased up to 14th day (42.35 ± 0.19 mg/L) during the experiment. Total biomass of spirulina cultured in the culture of 60% DRGM was increased from first day (0.107 ± 0.04 mg/L) up to 10th day (57.75 ± 0.20 g/L) and then decreased up to 14th day (41.88 ± 0.14 mg/L) of experiment (Fig. 3). 3.5 Correlation among the Parameters of Spirulina 3.7 Specific Growth Rate (SGR) in Respect to Total Biomass of Spirulina The SGR in respect to total biomass of spirulina was significantly (P < 0.05) varied from that of spirulina grown in the supernatant of 20, 40 and 60% DRG. There was no significant (P < 0.05) difference recorded among the SGRs on the basis of total biomass of S. platensis grown in the supernatant of 20, 40 and 60% DRG (Table 4). Comparison of Growth Parameters of Spirulina Optical density of 20% DRG (0.631 ± 0.002) ,40% DRG (0.704 ± 0.0015) and 60% DRG Table 1. Experimental design for Spirulina platensis culture using supernatant of three different concentrations of digested rotten guava (DRG) Types of medium Treatments Replications Supernatant of DRGM 1 2 3 3 (101, 102 and 103) 3 (201, 202 and 203) 3 (301, 302 and 303) 34 Amounts rotten guava (%) 20 40 60 Duration of culture (days) 14 Rahman et al.; AJFAR, 19(5): 29-38, 2022; Article no.AJFAR.92146 Table 2. Proximate composition (% in dry matter basis) of Spirulina platensis Treatments Moisture Crude Protein Crude Lipids Ash NFE* Crude Fiber T1 (20% DRG) 9.44 ± 0.05 b 53.25 ± 0.32 a 10.10 ± 0.16 b 10.22 ± 0.13 a 16.28 ± 0.32 0.70 ± 0.04 T2 (40% DRG) 9.55 ± 0.04 b 53.35 ± 0.34 a 10.14 ± 0.17 b 10.16 ± 0.17 a 16.10 ± 0.18 0.69 ± 0.03 T3 (60% DRG) 9.53 ± 0.05 b 53.28 ± 0.32 a 10.15 ± 0.14 b 10.33 ± 0.21 a 15.99 ± 0.22 0.71 ± 0.04 *NFE (Nitrogen Free Extract) = 100 - (Moisture + Crude protein + Crude lipids + Ash). Figures in common letters in the same row do not differ significantly at 5% level of probability . Fig. 1. Mean values of optical density of media contained Spirulina platensis Fig. 2. Mean values of chlorophyll a (mg/L) of Spirulina platensis 40% DRGM, 0.0023 to 0.818 mg/L in 60% DRGM. The growth performance of Spirulina platensis in supernatant of 60% DRGM was found better than 20% and 40% DRGM. This variation might be due to the differences in nutrient concentrations and composition of varied 4. DISCUSSION “The cell weight of Spirulina platensis in supernatant of digested rotten guava were found 0.0023 to 0.815 mg/L in 20% digested rotten guava media (DRGM), 0.0024 to 0.809 mg/L in 35 Rahman et al.; AJFAR, 19(5): 29-38, 2022; Article no.AJFAR.92146 Fig. 3. Mean values of total biomass (mg/L) of Spirulina platensis where is distribution total biomass (0 to 100) Table 3. Comparison of cell weight, chlorophyll a and total biomass of Spirulina platensis Parameters Optical density Cell weight (mg/L) Chlorophyll a (mg/L) Total biomass(mg/L) * T1 (20% DRG) b 0.631 ± 0.002 b 0.815 ± 0.0015 b 0.770 ± 0.14 b 67.77 ± 0.43 T2 (40% DRG) b 0.704 ± 0.0015 b 0.809 ± 0.0012 b 0.768 ± 0.0012 c 51.46 ± 0.28 T3 (60% DRG) b 0.725 ± 0.0012 b 0.818 ± 0.0013 b 0.862 ± 0.0012 bc 57.75 ± 0.20 *Total biomass = Chlorophyll a x 67 [11]. Figures in common letters do not differ significantly at 5% level of probability Table 4. Specific growth rates (SGRs) on the basis of cell weight, chlorophyll a and total biomass of Spirulina platensis Parameters SGR of cell weight SGR of Chlorophyll a SGR of total biomass T1 (20% DRG) b 0.21 ± 0.010 b 0.22 ± 0.004 b 0.54 ± 0.012 T2 (40% DRG) b 0.22 ± 0.009 b 0.21 ± 0.003 b 0.52 ± 0.012 T3 (60% DRG) b 0.21 ± 0.011 b 0.23 ± 0.004 b 0.53 ± 0.013 Figures in common letters in the same row do not differ significantly at 5% level of probability Similarly, Sharker [21] conducted “an experiment on the culture of Spirulina platensis in various concentrations viz., 0.3, 0.4 and 0.5 g/L of papaya skin powder medium (PSPM) and Kosaric medium (KM) in the laboratory for three months carried out for a period of 12 days”. In the present study, the chlorophyll a content of inoculated Spirulina platensis was 0.0015 mg/L which attained a high content of 0.862 mg/L in 60% DRGM at the 10th day of culture. These findings are not more or less similar with the findings of Phang et al. [22], Habib et al. [23] and Satter [4]. This might be due to lower nitrogen and phosphate concentration of the nutrients in the media. Dineshkumar et al. [19] studied that Spirulina platensis grew well in natural medium such as Conway medium, Zoarrouic medium and kosaric medium. media” [17]. “On the other hand 40% DRGM showed lower growth performance of Spirulina platensis in relation to 20 and 60% DRGM. This might be due to lower nitrogen and phosphate concentration of the nutrients in the media” [18]. During the present study, digested organic medium like rotten guava which has the similarity with the findings of Dineshkumar et al. [19] and Sukumaran et al. [20]. During culture of Spirulina platensis, the exponential phase was found up to 10th day from the beginning and then the cell weight declined i.e., stationary phase started. Satter [4] recorded “the cell weight and chlorophyll a content of S. platensis was significant (P<0.05) higher in 4.0 g/L digested poultry waste than other media where light intensity, aeration and temperature played significant role to the culture system”. 36 Rahman et al.; AJFAR, 19(5): 29-38, 2022; Article no.AJFAR.92146 In the present study, supernatant of digested rotten guava was used as a media of three concentrations for the culture of Spirulina platensis. The supernatant of 60% digested rotten guava showed maximum optical density on the 10th day of culture comparing with 20% and 40 % supernatant of DRGM which has the similarity with the findings of Habib et al. [23,24], Satter [4]. From the above discussion, the growth performance of Spirulina platensis in supernatant of 60% DRGM was found better than 20% and 40% DRGM. It can be said that the physical properties, chemical parameters and technical facilities used in the present study were more or less similar to those used by other researchers. This study finds out a suitable concentration of digested rotten guava medium (DRGM) as an organic nutrient medium for culture and growth of Spirulina platensis. the NFEs of Spirulina grown in supernatant of 20, 40 and 60% DRG as the values found for 20% (16.28 ± 0.32%), 40% (16.10 ± 0.18%) and 60% (15.99 ± 0.22%), respectively. Specific growth rate (SGR) in respect to cell weight of spirulina grown in 60% digested rotten guava (DRG) medium was significantly (P <0.05) higher than that of spirulina cultured in the supernatant of 20, 40% DRGM. This medium may be used commercially as the collection and preparation of these organic media require little cost, less labour and is available throughout Bangladesh. However, it might be suggested that more research and cost-benefit analysis have to be performed to evaluate the grow-out potential of spirulina in lab-based cultivation. The culture techniques may dwindle the cost of production and might be considered as a media for Spirulina platensis cultivation. The experiment revealed the proximate composition, growth performance of Spirulina platensis where the initial cell weight was 0.0023 mg/L which attained a maximum cell weight of 0.818 mg /L in 60% DRGM, 0.809mg/L in 40% DRGM and 0.815 mg/L in 20% DRGM on the 10th day of the culture period. Similarly, the chlorophyll a content of inoculated S. platensis was 0.0015 mg/L which attained the highest content of 0.862 mg/L in 60% DRGM, 0.768 mg/L in 40% DRGM, 0.770 mg/L in 20% DRGM on the 10th day of culture period. COMPETING INTERESTS Authors have interests exis. declared that no competing REFERENCES 1. 5. CONCLUSION 2. The percentage of crude lipids (10.15 ± 0.14%) of spirulina cultured in supernatant of 60% DRGM was significantly (P < 0.05) and almost two times higher than that of spirulina grown in 20% and 40%, respectively. The percentage of crude protein of Spirulina was 53.25 ± 0.32, 53.35 ± 0.34 and 53.28 ± 0.32% when grown in the supernatant of 20, 40 and 60% DRG media, respectively. There was no significant (P > 0.05) variation among the crude protein percent of spirulina cultured in three different media of DRG. There was no significant (P > 0.05) difference of crude lipids of spirulina when cultured in the supernatant of 20, 40 and 60% digested rotten guava. Ash (%) of Spirulina grown in supernatant of 20% (10.22 ± 0.13%), 40% (10.16 ± 0.17%) and 60% (10.33 ± 0.21%) digested rotten guava. There was no significant (p>0.05) difference among the ash of spirulina grown in supernatant of 20, 40 and 60% DRGM. There was no significant variation among 3. 4. 5. 37 Talukder M. Culture and growth performance of S. platensis in different concentrations of saline and kosaric medium. An Ms. Thesis Submitted to the Department of Aquaculture, Bangladesh Agricultural University, Mymensingh. 2003;66. Torxillo G, Pushparaj, Florenzano. A new procedure for obtaining pure cultures of S. maxima and S. platensis. Ann. Microbiol. 1985;135:165-173. Peerpompiral Y, Sunithong, Promkutkaew. Cultivation and protein content of S. platensis grow in sugar cane molasses distillery slops mixed with water hyacinth compost extract. Program and abstracts. th The 4 Asia-Pacific Conference on Algal Biotechnology, Hong Kong. 2000; 141. Satter A. Culture and production of housefly larva and spirulina using poultry waste, and their use as food for catfish post-larvae, Ph. D Thesis, Department of Aquaculture, Bangladesh Agricultural University, Mymensingh. 2017;143. Mario R, Papuzzo T, Tomaselli S. Outdoor mass culture of Spirulina maxima in sea water. Applied Microbiology and Biotechnology. 1986;24:47-50. Rahman et al.; AJFAR, 19(5): 29-38, 2022; Article no.AJFAR.92146 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Cohen Z, Vonshak A. Fatty acid composition of Spirulina and Spirulinalike cyanobacteria in relation to their chemotaxonomy. Phytochemistry. 1991; 30:205. Ruan JS, Guo BJ, Shu LH. Effect of spirulina polysaccharides on changes in white blood corpuscles induced by radiation in mice. Journal of Radiation Research Technology. 1990;8:210-213. Kebede E, Ahlgren G. Optimum growth conditions and light utilization efficiency of Spirulina platensis, (Arthrospira fusiformis) (Cyanophyta) from lake Chitu, Ephiopia. Hydrobiologia. 1996;32:99-109. Kato T. Chemistry of microalgae and their application to food. Food Chemistry. 1991; 8:30-35. Horwitz W. (Editor): Official Methods of Analysis of the Association of Official Analytical Chemists. 14th Edition. Association of Official Analytical Chemists, Washington DC. USA. 1984;1018. Vonshak A, Richmond A. Mass production of the blue green alga spirulina: An overview. Biomass. 1988;15:233-247. Zarrouk C. Contribution a l’etude d’une cyanobacterie: Influence de divers facteurs physiques et chimiques sur la croissance et la photosynthese de Spirulina maxima (Setchell et gardner) Geitler. Ph. D Thesis, University of Paris, France. 1996;412. Bold HC, Wynne MJ. Introduction to the algae. Structure and Reproduction. Englewood Cliffs. New Jersey. 1978;706. Vymazal J. Algae and Element Cycling in Wetlands. CRC Press, Inc., Boca Raton, Florida, USA. 1995;689. Phang SM, Chu WL. University of malaya algae culture collection (UMACC). Catalogue of strain. Institute of Postgraduate Studies and Research, University of Malaya, Kualalumpur, Malaysia. 1999;77. Zar JH. Biostatistics. Prentice-Hall Inc., Englewood Cliffs, New Jersey, USA. 1984;718. 17. 18. 19. 20. 21. 22. 23. 24. Richmond A. Spirulina. In: Borowitzka MA, Borowitzka L. (Eds.). Microalgal Biotechnology, Cambridge U.P., Cambridge, UK. 1988;85-121. Murugan T, Manikantavelu T, Saranraj P. Growth and bio-pigment production of three microalgal species in organic and inorganic media and determination of generation time-a comparative study. Original Research Article; 2012. Dineshkumar R, Narendran R, Sampathkumar P. Cultivation of Spirulina platensis in different selective media. Indian Journal of Marine Science. 2016;45 (12):1749-1754. Sukumaran P, Nulib R, Halimmon N, Simoh S, Omar H, Ismail A. Formulation of cost-effective medium using urea as a nitrogen source for Arthrospira platensis cultivation under real environment. Annual Research and Review in Biology. 2018;22(2):1-12. Sharker MGU. Study of the culture of Spirulina platensis in various concentrations using papaya skin powder medium. MS. Thesis Submitted to the Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh-2202. 2002;58. Phang SM, Miah MS, Chu WL, Hashim H. Spirulina culture in digested sago starch factory waste water. Journal of Applied Phycology. 2000;12: 395-400. Habib MAB, Yusoff FM, Phang SM, Mohamed S.: Growth and nutritional values of Moina micrura fed on Chlorella vulgaris grown in digested palm oil mill effluent. Asian Fisheries Science. 2003;16 (1-2):107-119. Habib MAB, Kohinoor AHNM. Culture and production of house fry larvae and spirulina using poultry waste and their use as food for catfish postlarvae. Report on Advanced Research, Ministry of Education, Govt. of People Republic of Bangladesh. 2018;2: 66-70. © 2022 Rahman et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Peer-review history: The peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/92146 38