Evaluation of rotten orange as a source of inorganic nutrients for blue green algae, Spirulina platensis culture

: A study was conducted to examine the culture and growth performance of Spirulina platensis in three different concentrations (25, 50 and 75 percent) of digested rotten orange (DRO) and Kosaric medium (KM) as control for three months. Each experiment was done in triplicates under fluorescent light in light: dark (12 hour: 12 hour) condition for a period of 14 days. Cell weight, chlorophyll a content of Spirulina platensis and physico-chemical parameters of cultured media (temperature, pH, light intensity) were measured at every alternative day. During the culture period, temperature was more or less similar and ranged from 30.1 to 31.4˚C. The range of light intensities was recorded from 2660 to 2770 lux/ /s during the culture period. The maximum pH was recorded 9.9 in 25% digest rotten orange (DRO) on 8th day of the culture and minimum was recorded 8.2 in all the medias on the initial day of the culture. The growth rate of Spirulina platensis cultured in supernatant of DRO and KM were varied. The initial cell weight of Spirulina platensis was 0.0023 mg/L that attained maximum cell weight of 12.44 mg/L in Kosaric medium on the 10 th day of culture. Besides that, the maximum cell weight was 7.679, 12.366, and 9.455 mg/L when cultured in supernatant of 25, 50, and 75% in DRO on the 10th day of the culture. While, the initial chlorophyll a content of Spirulina platensis was 0.0015 mg/L which attained the highest content of 10.54 mg/L when grown in KM. The highest chlorophyll a content of Spirulina platensis was 6.926 mg/L when grown in 25% DRO, it was 10.476 mg/L when grown in 50% DRO, and it was 9.140 mg/L when grown in 75% DRO on the 10 th day of the culture. This study showed that, the growth performance of Spirulina platensis was higher in supernatant of 50% DRO than 25 and 75% DRO. This concentration of medium gave satisfactory results compared with standard KM. This variation probably occurred because of differences in nutrient concentrations and composition of the medium. Therefore, it can be concluded that, the concentration of 50% DRO is suitable and favorable for Spirulina platensis culture.


Introduction
The number of species of micro algae is estimated at 22000 to 26000 out of which 50 have been studied in detail with regard to their biochemistry and eco physiology (Callegari, 1989).Spirulina is an important Cyanophyta known as Spirulina rich in high quality protein around (60-65%), essential lipids, vitamins, minerals and many biologically active substances (Becker, 1984).The ability of Spirulina to grow in hot and alkaline environments ensures its hygienic status, as no other organisms can survive to pollute the waters in which this alga thrives.Spirulina is one of the cleanest, most naturally sterile foods found in nature.It has been used as feed for fish, poultry, and farm animals (Tragut et al., 1995).Spirulina has reported to prevent oxidative damage and hence can indirectly reduce cancer formation in human body.In this respect, the increased consumption of foods characterized by free radical scavenging activity, leads up to a doubling of protection against many common types of cancer formation (Cooke et al., 2002;Romay et al., 2003;Anbarasan et al., 2011).Spirulina (Spirulina platensis) is a "super-food" among the most plants and even good quality animal food.It has a rich, vibrant history, and occupies an intriguing biological and ecological niche in the plant kingdom.Spirulina is a spiralshaped, blue-green microalgae that grows naturally in the wild in alkaline lakes, seawater, and saltwater.Its deep blue-green color is what gives the water its greenish hue.Spirulina is also cultivated and harvested in manmade reservoirs around the world.For centuries, civilizations the world over have cultivated and cherished Spirulina for its health-improving benefits (Habib, 1998).However, According to the researchers 1kg Spirulina is similar to 100 kg other vegetables in nutrition.These blue green algae contain 50-70% protein, 10-12% carbohydrate (in dry condition), 6% fat, 7% mineral, and a lot of vitamins (Shuvo, 2001).It was reported that Spirulina contains 6-11% of polyunsaturated fatty acids; the predominant are palmitic (16:0, 44.6-54.1%)gamalinolenic or GLA (18:3, 8.0-31.7%),linoleic (18:2, 10.8-30.7%)and oleic acids (18:1, 1-15.5%).Now a day, Spirulina platensis is gaining great interest for its cellular contents such as vitamins, minerals, polyunsaturated fatty acids, carotenoids and other pigments that have antioxidant activity (Cohen and Vonshak, 1991).The growth of Spirulina and the composition of the biomass produced depend on many factors, the most important of which are nutrient availability, temperature, and light (Cornet et al., 1992).In addition, Spirulina requires relatively high pH values between 9.5 and 9.8 (Belkin and Boussiba, 1971), which effectively inhibits contamination by most algae in the culture.Production of Spirulina with reduced costs is necessary when considering large-scale cultivation for industrial purposes.The cost of nutrients is considered the second major factors influence the cost of Spirulina biomass production after labor (Vonshak, 1997).Zarrouk's medium has successfully served as the standard medium (SM) for Spirulina culture for many years (Zarrouk, 1996).Consequently, many media have been developed using seawater (Faucher et al., 1979), sewage water (Saxena et al., 1982) and industrial effluents (Tanticharoen et al., 1993).Spirulina is most commonly found in natural lakes having high pH value i.e. 8 to 10 all over the earth.Spirulina has been consumed from a very long past time in many parts of the world as a food supplement for human as well as animals in various forms like health drink, tablets, etc. because of its alimentary value (Ruiz et al., 2003).Spirulina required light intensities during its growth phase.Currently, the commercial production of Spirulina is mainly oriented towards the health food market, utilizing a chemically defined medium (Belay et al., 1993).Mass culture of Spirulina platensis and used in various purpose in African country, some South East Asian countries, India etc.In Bangladesh the culture of S. platensis is first initiated by Dr. Flora Mazid (Chairman, Bangladesh Council of Scientific and Industrial Research, BCSIR).The ultimate goal of this experiment is to develop low cost media for large-scale production of Spirulina.Therefore, rotten orange can be considered as a media in producing Spirulina as rotten orange can be obtained cheaply from the market.In addition, rotten orange contains significant amount of inorganic nutrients, which can be used as the source of nutrient in Spirulina culture.This study was conducted to evaluate the growth performances of Spirulina in supernatant of digested rotten orange medium (DROM); and to analyze the proximate composition of Spirulina that grows on digested rotten orange medium (DROM).

Dissolved oxygen
It was determined using the digital oxygen meter (Model Nutron DO-5509).

Total suspended solids (TSS) and total dissolved solids (TDS)
This was analyzed by using the following method methods and procedure (HP Module, 1999).

Alkalinity
This was analyzed by using the following method -(APHA, 1976) and using alkalinity test kit (Thermo Fisher Scientific Company, 2008).

Phosphate-P (Available P)
This was analyzed by using the following method methods and procedure (HP Module, 1999).

Collection of Spirulina platensis
Microalgae, Spirulina platensis was collected from the imported stock of my respected teacher and research supervisor Dr. Md.Ahsan Bin Habib, Professor, Department of fish, Bangladesh Agricultural University, Mymensingh-2202.

Maintenance of pure stock culture of Spirulina platensis
Pure stock culture of Spirulina platensis was maintained in the laboratory in Kosaric medium (KM) (Zarrouk, 1996).Growth of Spirulina platensis were monitored at every alternative day and was checked under microscope to confirm it's purity following some keys given by Bold and Wynne (1978), Vymazal (1995) and Phang and Chu (1999).

Preparation of digested rotten orange medium (DROM) and Kosaric medium (KM)
Rotten orange medium (ROM) and Kosaric medium (KM) were prepared for culture of Spirulina platensis.Different concentration and composition of rotten orange medium and kosaric medium are shown in the table 2 and 3 respectively.For the preparation of supernatant of rotten orange collected samples were digested firstly by aerating it into a 5 liter volumetric flask under 4 liter distilled water.The concentration of rotten orange of 60g/L was maintained during digestion.After 47 days (27.02.18 -15.04.18), digestion of rotten orange was completed and its supernatant was taken from the flask by filtering it with plankton net.Then the digested rotten orange was diluted according to the above direction with three replications using distilled water.Then the medium was mixed well and sterilized at 115 o C for 15 minutes by high pressure bumping water autoclave.After autoclaving, the media were kept 3 days to be sure about any contamination free before culture of microalgae.For the preparation of Kosaric medium, the above mentioned amount (Table 3) of ingredients from no. 1 to 8 was weighted by the help of electric balance and took in a 1.0 L conical flask.Then 0.5ml micronutrient solution was pipetted in the flask and distilled water was added to make the volume 1.0 L. Mixing, autoclaving and cooling were carried out pursing the procedure used during the preparation of RSM.

Experimental design of Spirulina platensis culture
Two types of media viz., Rotten orange media (ROM) and Kosaric medium (KM) were used to culture Spirulina platensis.Inoculum Spirulina platensis was collected from the pure stock culture.Experimental design is shown in Table 4.  25, 50, and 75%) and one KM as control each with three replications were used to grow microalgae, S. platensis in 1.0 L volumetric flask.
Spirulina was inoculated into each culture flask to produce a culture containing 10% Spirulina suspension (Optical density at 620 nm = 0.20) (Habib, 1998).Twenty ml of Spirulina suspension needed for getting the required density.All the flasks were kept under fluorescent lights (TFC, FL-40 SD/38 day light, Taiwan) in light: dark (12h: 12h) conditions in live food culture laboratory.These culture flasks was continuously aerated using electric aquarium aerator (SB-348A).Eight sub-samplings (15ml vial) were carried out at every alternative day from each flask to record dry cell weight and chlorophyll a content of Spirulina, and properties of culture media.All the glassware used in the experiment was sterilized with dry heat at 70 o C overnight.

Estimation of Spirulina platensis cell weight (dry weight)
The cell weight of Spirulina was determined by the method of Clesceri et al. (1989).

Determination of Chlorophyll a of Spirulina
The chlorophyll a of Spirulina was determined by the method of Clesceri et al. (1989).

Total biomass of Spirulina (Spirulina platensis)
Total biomass was calculated using the following formula given by Vonshak and Richmond (1988), Total biomass = Chlorophyll a x 67.
2.6.Specific growth rate (SGR) on the basis of dry weight chlorophyll a content and total biomass of Spirulina 2.6.1.Specific growth rate (µ/day) of cultured Spirulina on the basis of dry weight SGR (µ/day) = In (X 1 -X 2 )/t 1 -t 2 Where, X 1 = Dry weight of biomass concentration of the end of selected time interval; X 2 = Dry weight biomass concentration at beginning of selected time interval; and t 1 -t 2 =Elapsed time between selected time in the day.

Specific growth rate (µ/day) of cultured Spirulina on the basis of chlorophyll a
Where, X 1 = Chlorophyll a at the end of selected time interval; X 2 = Chlorophyll a at the beginning of selected time interval; and t 1 -t 2 = Elapsed time between selected time in the day.

Specific growth rate (µ/day) of cultured Spirulina on the basis of total biomass
Where, X 1 = Total biomass at the end of selected time interval; X 2 = Total biomass at the beginning of selected time interval; and t 1 -t 2 = Elapsed time between selected time in the day.

Analysis of physicochemical parameters 2.7.1. Physical parameters
The physical parameters (temperature and light intensity) of the culture media were recorded as follows:

Temperature
Water temperature ( o C) of the culture media was measured during the time of sampling day by a Celsius thermometer.

Light intensity
Light intensity (lux/m 2 /s) was measured during sampling day by using a lux-meter [digital instrument, Lutron (LX-101)].

Chemical parameters
The chemical parameters such as pH of the culture media were recorded following the procedures given by Clesceri et al. (1989) in the laboratory.2.7.2.1.pH pH of the culture media was measured from each sub sample by an electric pH meter ( Conning pH meter 445).

Statistical analysis
Data of cell weight, chlorophyll a, total biomass, and specific growth rates in respect to dry cell weight, chlorophyll a total biomass and proximate composition of Spirulina in respect to four treatments were analyzed following one ways Analysis of Variance (ANOVA) and their significant differences using Turkey's test followed Duncan's New Multiple Range (DNMR) test at 5% level of probability (Zar, 1984).

Physico-chemical characteristics of rotten orange 3.1.1. Colour, Odour and Structure
The color of the rotten orange was yellowish with little bid bad smell (odour).The structure was almost semisolid (Table 5).

Temperature (°C)
Temperature of rotten orange was little bid higher than normal ambient temperature.It was ranged from 28.10 to 28.30ºC (Table 5).

Total solid (TSS + TDS)
Total soild is the addition of total suspended solids and total dissolved soilds of semi liquid (rotten orange) which was ranged from 1523 to 1622 mg/L (Table 5).

pH
pH of digested rotten orange was ranged from 4.30 to 5.15 which was acidic in nature (Table 5).

Alkalinity
Alkalinity of digested rotten orange was quite high and ranged from 75 to 85 mg/L (Table 5).

Nitrate-N (NO 3 -N)
Nitrate-N (Available N) of digested rotten orange was ranged from 2.20 to 2.35 mg/L (Table 5).

Phosphate-P (PO 4 -P)
Phosphate-P (Available P) of the digested rotten orange was high and varied from 3.80 to 3.950 mg/L (Table 5).6).

pH
pH of supernatant of DRO used for Spirulina culture was found to range from 5.20 to 5.40 (Table 6).

Total solid (TSS + TDS)
Total solid (TSS + TDS) of supernatant of DRO used to culture Spirulina was reduced due to decomposition which was ranged from 115 to 133 mg/L (Table 6).

Alkalinity
Alkalinity of supernatant of DRO used to culture Spirulina was high which was ranged from 110 to 120 mg/L (Table 6).

Nitrate N (NO 3 -N)
Nitrate N (Available N) of supernatant of DRO used for Spirulina culture was also high in amount and varied from 2.55 to 2.80 mg/ (Table 6).

Phosphate P (PO 4 -P)
Phosphate P (Available P) of supernatant of DRO used to culture Spirulinawas very high and found to vary from 3.30 to 3.50 mg/L (Table 6).

Total N
Total N of supernatant of DROM used for Spirulina culture was found also high in amount and ranged from 2.30 to 2.45 mg/L (Table 6).Available P (PO 3 -P) (mg/L) 3.30-3.50

Proximate composition of rotten orange on dry basis 3.3.1. Moisture
It was measured from dry orange was 9.83% (Table 7).

Crude protein
Crude protein of rotten orange was 8.97% (Table 7) which quite high in percent.

Crude lipid
Crude lipid of rotten orange was 9.67% (Table 7) which quite high in percent.

Crude fiber
Crude fiber in rotten orange was not high in amount and it was 9.06% (Table 7).

Nitrogen free extracts (NFE)
The NFE of rotten orange was high in amount and it was 67.90% (Table 7).1).

Temperature
The temperature round the culture in T 1 was found 30.1 ± 0.24 (lowest) on the first day to 30.8 ± 0.26 at the end (14th day) of experiment with slight up on 2nd, 6th and 10th day of experiment.It was also follow the similar trend of fluctuation from first to last day in T 2 and T 3 .But, it was recorded 30.3 ± 0.32 °C on the first day of experiment to 31.2 ± 0.42 °C at the end of experiment in T 4 (Figure 2).

pH
During the 14 days experiment, it was increased from 8.2±0.18 on first day to 9.9±0.23 on 8th day in T 1 and then it was decreased to 8.5±0.20 on last day (14 th day) of experiment.last day (14 th day) of experiment.Similar trend of fluctuation of pH were observed in T 3 .Nevertheless, it was increased from first day (8.2 ± 0.33) of experiment up to 10 th day (9.9 ± 0.32) of experiment, and then decreased on the 12 th day and again decreased on the last day (14 th day) in T 4 (Figure 3).

Alkalinity
It was found 2260±102 mg/L on first day of experiment and then increased up to 2263±105 on the 2 nd day and then gradually decreased up to 1660±210 mg/L on the 8 th day, and then increased up to 1938±170 on the 14 th day (last day) in T 1 .Total alkalinity was recorded 1810±160 mg/L on first day of experiment and increased up to 10 th day (2045 ± 185 mg/L), and then decreased on 12 th day and again increased on 14 th day in T 2 .It was found 2110 ±170 mg/L on the first day of experiment, and then the concentrations of total alkalinity was followed a zig-jag trend up to 10 th day of experiment and then decreased up to 14 th day in T 3 .The concentrations of total alkalinity was followed a zig-jag trend from first day up to 6 th day of experiment and then decreased up to 14 th day in T 4 (Figure 4).

Nitrate N (NO 3 -N)
It was positively increased from 1.76±0.14mg/L (first day) to 3.37±0.26mg/L (6 th day) of experiment and then decreased to 2.60±0.20 (8 th day) of experiment and then again increased up to 2.80±0.20 (12 th day) of experiment and again decreased to 1.85±0.16(14 th day) of experiment in T 1 .The trend of nitrate-N was found to decrease from first day (2.26±0.15mg/L) to 8 th day (1.27±0.14mg/L) of culture and then increased up to 14 th day in T 2 .It was 3.34±0.20mg/L on the first day of the experiment.Lowest amount of nitrate-N (2.99±0.26mg/L) was recorded on the 2 nd day of the experiment and it was increased up to 6.05±0.47 on the 14 th day of the experiment which was highest amount of nitrate-N in T 3 .It was found lowest (2.04 ± 0.18 mg/L) on 4 th day of culture and it was found highest (3.63 ± 0.28 mg/L) 14 th day in T 4 (Figure 5).

Phosphate-P (PO 4 -P)
Phosphate-P (Available P) was high in amount in the media in first day (31.21 ± 3.03 mg/L) of experiment and gradually decreased in amount up to 10th day (10.55 ± 1.22 mg/L) in T 1 , but increased from 12th day up to 14 th day of culture.Similarly it was found to decrease from first day (46.40 ± 4.40 mg/L) of experiment to 10th day (15.55 ± 1.42 mg/L) but increased from 12 th to 14 th in T 2 .However, Phosphate-P (Available P) was found to decrease from first day (60.35 ± 5.40 mg/L) of experiment to 12 th day (18.20 ± 2.20 mg/L) and then again increased to 25.70 ± 2.40 mg/L on the 14 th in T 3 .Similar trend was sharply followed in T 4 (Figure 6).

Optical density of media contained Spirulina
Optical density (OD) of media contained Spirulina was found to increased up to 10 th day of culture of all the media of digested rotten orange (DRO) and Kosaric medium and then decreased up to 14 th day of experiment.However, highest OD of T 1 was 1.325 ± 0.11 mg/L, where highest OD of T 2 was found 2.285 ± 0.16 mg/L.The highest OD of T 3 was 1.577 ± 0.13.The highest optical density of T 4 was 2.63 ± 0.20 mg/L (Figure 7).

Cell weight of Spirulina
Cell weight (mg/L) of Spirulina cultured in all the media was found higher on 10 th day of culture than other days (Figure 8).Cell weight of Spirulina increased from initial day (first day) up to 10 th day (7.679 ± 0.23 mg/L) in T 1 and then decreased up to 14 th day (4.104 ± 0.18 mg/L ) of experiment.However, the highest cell weight was found to be 12.44 ± 0.21 mg/L in T 2 .Cell weight of Spirulina increased from initial day (first day) up to 10 th day (9.455 ± 0.40mg/L) T 3 and then decreased up to 14 th day 2.30 ± 0.13 mg/L of experiment.Highest cell weight of T 4 was 12.44 ± 0.21 mg/L on 10 th day and then decreased up to 14 th day of experiment.

Chlorophyll a of Spirulina
Chlorophyll a of Spirulina increased from first day up to 10th day (6.926 ± 0.15 mg/L) in T 1 and then decreased up to 14th day (3.137 ± 0.15 mg/L).However, chlorophyll a in T 2 was 10.476 ± 0.31 mg/L on 10th day and then decreased up to 14th day (last day) of culture.Chlorophyll a in T 3 was 9.140 ± 0.22 mg/L on 10th day and then decreased up to 14th day (last day) of experiment, where the highest chlorophyll a in T 4 was 10.54 ± 0.14 mg/L on 10th day and decreased up to 14th day (last day) of experiment (Figure 9).

Total biomass of Spirulina
Total biomass of Spirulina was increased from initial day (first day) (0.101 ± 0.003 mg/L) up to 10th day (464.04± 8.05 mg/L) in T 1 and then decreased up to 14th day (210.18± 4.45 mg/L) of experiment.However, the highest total biomass in T 2 was recorded 701.89 ± 4.33 mg/L on 10th day of culture and then decreased up to 14th day (209.84± 2.12 mg/L).Again, total biomass in T 3 was increased from first day up to 10th day (612.38 ± 4.30 mg/L) and then decreased up to 14th day (127.97± 2.17 mg/L).The highest total biomass in T 4 was found to be 706.18± 4.50 mg/L on 10th day and then decreased up to 14th day (278.05± 2.12 mg/L) (Figure 10).

. Optical density of media contained Spirulina
Optical density of T 2 and T 4 was significantly (P < 0.01) higher than that of two other media (25%) and (75%) (Table 8).There was no significant (P > 0.05) difference among optical density of T 1 and T 3 during the study.

Cell weight of Spirulina
Highest cell weight (mg/L) of Spirulina grown in T 4 was recorded (Table 8).Cell weight of Spirulina grown in T 4 and T 2 was varied significantly (P < 0.01) from that cultured in T 1 and T 3 (Table 8).However, there was no significant (P > 0.01) difference of cell weight of Spirulina grown in T 1 and T 3 .

Chlorophyll a of Spirulina
Chlorophyll a (mg/L) of Spirulina grown in T 4 and T 2 was significantly (P < 0.01) higher than that of Spirulina cultured in T 1 and T 3 (Table 8).There was no significant difference among the Chlorophyll a of Spirulina grown in T 1 and T 3 .

Total biomass of Spirulina (Spirulina platensis)
Total biomass (mg/L) of Spirulina cultured in T 4 and T 2 was significantly (P < 0.01) higher than that of Spirulina grown in T 1 and T 3 (Table 8).There was no significant difference found among the total biomass of Spirulina cultured in T 1 and T 3 .*Total biomass = Chlorophyll a x 67 (Vonshak and Richmond, 1988).Figures in common letters do not differ significantly at 5% level of probability.

Correlation among the growth parameters of Spirulina
Cell weight of Spirulina (Spirulina platensis) had highly significant (P < 0.01) direct correlation with chlorophyll a (r = 0.806) of Spirulina grown in the supernatant of different digested rotten media and Kosaric medium during the study (Figure 11).Similarly, total biomass of S. platensis was highly (P < 0.01) and directly correlated with chlorophyll a (r = 0.845) of Spirulina cultured in the supernatant of various digested rotten orange and Kosaric medium (Figure 12).Again, total biomass of Spirulina was found to be highly (P < 0.01) and directly correlated with the cell weight (r = 0.833) of Spirulina grown in the supernatant of different digested rotten orange and Kosaric medium (Figure 13).3.8.Specific growth Rates (SGR) of Spirulina (Spirulina platensis) 3.8.1.SGR in respect to cell weight of Spirulina Specific growth rate (SGR) in respect to cell weight of Spirulina grown in T 4 and T 2 was significantly (P <0.01) higher than that of Spirulina cultured in T 1 and T 3 (Table 9).There was no significant (P > 0.01) difference among the SGR of cell weight of Spirulina grown in T 4 and T 2 , and among the same of Spirulina cultured in T 1 and T 3 .

SGR in respect to Chlorophyll a of Spirulina (Spirulina platensis)
The SGR in respect to Chlorophyll a of Spirulina cultured in T 4 and T 2 was significantly (P < 0.01) varied from that of Spirulina grown in T 1 and T 3 (Table 9).It had no significant difference when Spirulina grown in T 4 and T 2 , and similar thing happened when Spirulina cultured in T 1 and T 3 .The SGR in respect to total biomass of Spirulina cultured in T 4 and T 2 was significantly (P < 0.01) varied from that of Spirulina grown in T 1 and T 3 (Table 9).There was no significant (P < 0.01) difference recorded among the SGRs on the basis of total biomass of S. platensis grown in T 2 and T 4 .Similarly, it had no significant variation among the SGR on the basis of total biomass of Spirulina when cultured in T 1 and T 3 .10).The moisture content (%) was suitable for the preservation of the samples for future analysis.

Crude protein
There was no significant variation among the crude protein of Spirulina grown in the supernatant of three different digested rotten oranges (Table 10).But, crude protein of T 4 (58.55 ± 0.40%) was (P < 0.01) higher than that of Spirulina grown in the supernatant of three other digested rotten orange media.The percentage of crude protein of Spirulina was 53.74 ± 0.38%, 57.25 ± 0.42% and 56.35 ± 0.52% when grown in T 1 , T 2 and T 3 .

Crude fiber of Spirulina
Very small amount of crude fiber (%) was found in Spirulina grown in the supernatant of three different digested rotten orange media (DROM), and Kosaric medium (Table 10).However, it was varied from 0.71 ± 0.04% when Spirulina grown in T 1 and T 3 to 0.72 ± 0.03% when cultured in T 2 .

Discussion
One of the most important microalgae Spirulina platensis was cultured in supernatant of three concentrations of digested rotten orange media (DROM), such as 25, 50, and 75% and Kosaric medium (KM) as control.This experiment was conducted in order to evaluate culture and growth performance of S. platensis in the laboratory.The cell weight of S. platensis in three concentrations of DROM and KM were ranged from 0.0022 ± 0 mg/L to 7.679 ± 0.23, 0.0025 ± 0 mg/L to 12.366 ± 0.50 mg/L, 0.0022 ± 0 mg/L to 9.455 ± 0.40 mg/L, and 0.0023 ± 0 mg/L to 12.44 ± 0.21 mg/L when grown in 25% DROM, 50% DROM, 75% DROM and KM.Nevertheless, the cell growth was found to be varied in different media.Most likely, this variation in cell weight occurred because of composition of varied media and the differences in nutrient concentration.S. platensis growth rate was comparatively higher in KM than digested rotten orange media (DROM) in various concentrations.Besides that, higher growth rate was observed in concentration of 50% DROM than other concentrations of DROM (concentration 25% and 75%).This was probably happened due to suitable and favorable amounts of nutrients in 50% DROM than other concentrations of DROM during the culture of S. platensis.The concentration of 25 and 75 % DROM saw lower growth of Spirulina platensis due to lack of nutrients in 25 % DROM and more nutrients contain in 75% DROM than 50% DROM.The exponential phase was found up to 10 th day the beginning and then the cell weight declined i.e. stationary phase started.The physico-chemical properties such as light intensity, aeration, temperature played a crucial role to the entire culture system.During the culture system, the climatic condition was more or less suitable and favorable for the growth of S. platensis.Similar type of work was carried out by Mario et al. (1986) where the annual yield of biomass of Spirulina maxima strain 4MX grown in fertilized sea water in out door system was 7.359 mg i.e. 0.39 g which was higher than the present study.At present study the cell weight of S. platensis in DROM and KM were lower than the findings of Mario et al. (1986).The variation in result probably happened because of different nutrient component of media used in culture, different culture technique and different species cultured.An experiment conducted by Becker (1984) on algal culture in a series of different horizontal ponds and recorded that yield of Spirulinasp.Was 8 to 12 g/ /d.The yield found from the experiment was also much higher than the present findings.From the findings of Li and Qi (1997) it was reported that the biomass output rate in Chinese production plant was 7.0g/ /d which was much higher than the results of present study.Similarly, Tanticharoen et al. (1990) reported that the addition of NaHCO 3 and nirogen fertilizer in waste water from the stabilization pond of topics orange factory raise the productivity up to 7-10 g which was much higher than the findings of the present study.The variation in results occurred due to nutrient composition of different media and physico-chemical factors involved in the culture.On the other hand, Satter (2017) studied on culture and production of housefly larvae and Spirulina using poultry waste, and their use as food for catfish postlarvae.He produced Spirulina and used as important feed ingredient to replace fish meal up to 100% but got very good growth of catfish post-larvae fed diet contained 25% fish meal, 50% Spirulina meal and 25% maggot meal.He also got good results when post-larvae fed diets contained 25% fish meal and 75% Spirulina meal, and another diet contained 100% Spirulina meal.During culture of Spirulina platensis in 25, 50 and 75% of digested of poultry waste (DPW) which was better growth in 25% DPW at the same result found in the present study.From above all the discussion it may be concluded that the chemical parameters, the physical properties and technical facilities used in the present study where more or less similar to those used by other researchers.The findings of the present work differed with other findings mentioned above due to the nutritional variation of the culture media.

Conclusions
After observing the results of the present study, it can be concluded that the growth performance of Spirulina platensis was higher in supernatant of 50% digested rotten orange (DRO) than 25% and 75% DRO.This concentration of media gave satisfactory results compared with standard Kosaric media.This variation probably occurred because of differences in nutrient concentrations and composition of the media.Therefore, the concentration of 50% DRO is suitable and favorable for Spirulina platensis culture.

Conflict of interest
None to declare.

Figure 1 .
Figure 1.Mean values of light intensity (Lux/m 2 /s) during culture of Spirulina platensis.

Figure 2 .
Figure 2. Mean values of temperature (°C) during culture of Spirulina platensis.

Figure 3 .
Figure 3. Mean values of pH of culture during Spirulina platensis.

Figure 4 .
Figure 4. Mean values of alkalinity (mg/L) during culture of Spirulina platensis.

Figure 5 .
Figure 5. Mean values of Nitrate-N (mg/L) during culture of Spirulina platensis.

Figure 6 .
Figure 6.Mean values of Phosphate-P (mg/L) during culture of Spirulina platensis.

Figure 7 .
Figure 7. Mean values of optical density of media contained Spirulina platensis.

Figure 8 .
Figure 8. Mean values of cell weight (mg/L) of Spirulina platensis.

Figure 9 .
Figure 9. Mean values of chlorophyll a (mg/L) of Spirulina platensis.

Figure 11 .
Figure 11.Correlation coefficient (r) of cell weight (mg/L) of Spirulina platensis with chlorophyll a (mg/L) of Spirulina grown in supernatant of three digested rotten orange, and Kosaric medium.

Figure 12 .
Figure 12.Correlation coefficient (r) of total biomass (mg/L) of Spirulina platensis with chlorophyll a (mg/L) of Spirulina grown in supernatant of three digested rotten orange, and Kosaric medium.

Figure 13 .
Figure 13.Correlation coefficient (r) of total biomass (mg/L) of Spirulina platensis with cell weight (mg/L) of Spirulina grown in supernatant of three digested rotten orange, and Kosaric medium.

Culture of Spirulina platensis in supernatant of digested rotten orange medium DROM and Kosaric medium
KMFour treatments, three from supernatant of DROM for three different concentrations (

Physico-chemical properties of supernatant of digested rotten orange 3.2.1. Temperature
Temperature of supernatant of digested rotten orange (DRO) used to culture Spirulina was varied from 28.40 to 29.70°C (Table

Physico-chemical properties of different media contained Spirulina (Spirulina platensis) culture 3.4.1. Light intensity
It was varied slightly in different days in all the four culture media.However, light intensity (lux/m 2 /s) was varied from 2754 ± 24 on first day to 2770 ± 27 lux/m 2 /s on the last day with slight variation in other days in T 1 .It was varied from 2740 ± 26 on first day to 2766 ± 25 lux /m 2 /s on the last day in T 2 .Similarly, it was observed 2725 ± 30 on the first day and 2727 ± 30 on the last day (14th day) in T 3 .Light intensity was found to be 2725 ± 26 lux /m 2 /s on first day in T 4 and 2660 ± 15 lux /m 2 /s on the last day (14th day) of experiment (Figure

Table 9 . Specific growth rates (SGRs) on the basis of cell weight, chlorophyll a and total biomass of Spirulina platensis grown in supernatant of three different concentrations of digested rotten orange (DRO), and Kosaric medium
Spirulina grown in the supernatant of three different digested rotten orange and Kosaric medium was varied from 8.22 ± 0.06 to 8.27 ± 0.07 % (Table

Table 10 . Proximate composition (% in dry matter basis) of Spirulina platensis cultured in supernatant of three different concentrations of digested rotten orange (DRO), and control as Kosaric medium.
*NFE (Nitrogen Free Extract) = 100 -(Moisture + Crude protein + Crude lipids + Ash).Figures in common letters in the same row do not differ significantly at 1% level of probability.