Smart Production of Lipids as Bio-Fuel in Spirulina Platensis (= Arthrospira Fusiformis ), and Bio-Oxygen and Bio-Electricity in Media Cultured in Supernatant of Digested Poultry Waste

: An experiment was conducted to evaluate growth performances and production of bio-fuel of Spirulina platensis (Gomont), and bio-oxygen and bio-electricity of culture media in supernatant of three different amount of digested poultry waste (DPW), and Kosaric medium (KM) as control. Three different amounts (concentrations) such as 2.0, 4.0 and 6.0 g/L poultry waste were allowed to digest under aeration. After 17 days, 700 ml grayish coloured supernatant was taken with addition of 9.0 g/L NaHCO 3 and 0.50 ml/l micronutrient in 2.0 L conical flask with three replications and then autoclaved. Spirulina was inoculated to grow in these three treatments including KM (Control) after 72 hours of autoclave and then allowed to grow for a period of 14 days. This duration was estimated through repeated growth trials. The cell weight of spirulina was attained a maximum of 12.58 ± 1.25 mg/L (dry wt. basis) in KM followed by 11.46 ± 1.03, 9.16 ± 0.84 and 8.13 ± 0.73 mg/L in supernatant of 4.0, 2.0 and 6.0 g/L DPW, respectively on the 10th day of culture. Similar trend was also observed in the cases of optical density of the media contained spirulina, chlorophyll a content (mg/L), cell weight, total biomass (mg/l) and specific growth rates on the basis of cell weight and chlorophyll a . Cell weight of spirulina grown in these media had highly significant (P < 0.01) correlation with the chlorophyll a content (r = 0.993) of spirulina. Crude protein of spirulina grown in supernatant of DPW was lower than that of spirulina cultured in KM. Crude lipids as bio-fuel of spirulina cultured in supernatant of 4.0 g/L DPW was almost three times higher than that of spirulina grown in KM which may be due to high phospholipids bioaccumulation. Phosphate-P was decreased in media of DPW due to its use for high lipids biosynthesis as phospholipids. Bio-oxygen was produced higher ranged from 2.1 ± 11 mg/L on initial day to 10.20 ± 0.54 mg/L on 10th day grown in supernatant of 4.0 g/L DPW media. Bio-electricity as green electricity ranged from 135 ± 4 on initial day to 284 ± 7 mV on 10th day when spirulina cultured in supernatant of 4.0 g/L DPW which was higher than grown in other media. pH followed the similar trend like bio-oxygen and bio-electricity. It was found that the production of bio-oxygen, bio-fuel and bio-electricity had direct relation with pH in culture media of spirulina. Therefore, mass production of Spirulina platensis might be done in supernatant of 4.0 g/L digested poultry waste to get high total lipids as bio-fuel, bio-electricity and bio-oxygen.

In Bangladesh, a lot of waste materials and effluents of agro-industrial products are available which have nutritional values [28,36,37].Among these, waste effluents discharged from sugar mills, fertilizer factory, biscuit factory, sago factory, poultry industry etc. are important [35,36,37].Spirulina usually reduce chemical oxygen demand (COD), ammoniacal-N, and available-P upto 98.00, 99.90 and 99.40%, respectively from culture system [4].The above results show the way to use other wastes by spirulina to biosynthesize essential nutrients.Some other media were developed in the laboratory for domestic scale culture of spirulina in Bangladesh [38].It is proved that micro-algal fuel cells (MFCs) would provide a source of green electricity or bioelectricity to generate power and bio-fuel from lipids of spirulina by [39] using domestic and industrial wastes [40,41] due to photosynthesis.In photosynthesis, light energy splits water molecules into bio-oxygen, protons and electrons which usually known as bio-electricity [42].Among the wastes, poultry waste may help for biosynthesis of protein, lipids and carbohydrate.Poultry waste as an inexpensive medium is also one the major pollutants like other agro-industrial wastes.Huge amount of poultry waste approximately 1.8 million kg is discharging in the country every year from 90,000-1,00,000 poultry farms [43].This waste contains considerable amount of nitrogen, and high amount of calcium and phosphorus due to use of mollusk shell powder in feed of poultry [43].This phosphorus may help to produce high phospholipids in microalgae and ultimately increase the amount of total lipids [18,43].However, poultry waste is easily available nationwide and collected all the time.The information related to this work is scanty in the country.Therefore, the present study was conducted to culture spirulina in supernatant of this inexpensive waste material to record the growth performances of spirulina, to produce high lipids in spirulina, bio-oxygen and bio-electricity in media with the following specific objectives: a) To evaluate the growth performances of spirulina grown in supernatant of digested poutry waste; b) To analyze growth parameters, and c) To produce bio-fuel as lipids, bio-oxygen and bio-electricity in the culture system of spirulina.

MATERIALS AND METGODS
Poultry waste was collected from a nearby poultry farm and then sun-dried for three days and packed in polythene bag for chemical analyses and used to prepare medium for spirulina culture.The proximate composition of poultry waste such as moisture, crude protein, crude lipids, ash and nitrogen free extract (NFE) were analyzed in triplicates following the standard methods [44] in the Fish Nutrition Laboratory, Department of Aquaculture, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh.Characteristics of poultry waste before and after digestion are shown in Tables 3 and 4, respectively.
a. Maintenance of pure stock culture of spirulina Pure stock culture of spirulina was maintained in the laboratory in Kosaric medium (KM) [45].Growth of spirulina was monitored at every alternate day and was checked under a microscope to confirm its purity following the keys of [46,47,48,49].

b. Preparation of supernatant of poultry waste
For perpetration of poultry waste medium, it was first collected from a local poultry farm, Mymensingh, Bangladesh and then screened through net (mesh size: 200 µm) to avoid solids, fibres and floating materials.Ten g/L dry poultry waste was allowed to decompose in 4.0 L of distilled water in 5.0 L glass bottle for 17 days under aerobic condition in the Live Food Culture laboratory (Table 1).Then a yellowish white coloured supernatant from bottle was filtered through Whatman filter paper of mesh size 0.45 µm to make free from suspended solids, and made three concentrations with distilled water at the rate of 2.0, 4.0 and 6.0 g/L decomposed poultry waste.Then 650 mL supernatant of three different concentrations were taken in 2.0 L flask with three replications.Sodium bicarbonate (9.0 g/L) and micronutrient solution (0.50 mL/L) were added in the flask to make the medium alkaline, mixed thoroughly and sterilized at 120°C for 15 minutes with moist heat by autoclave (Express Equipment, Dixon's Surgical Instrument Ltd.).After sterilization, the media were kept for 24 hrs to be sure whether any contamination before culture of microalgae.

c. Preparation of Kosaric medium
Kosaric medium (KM) was prepared for Spirulina platensis culture as a control.For the preparation of Kosaric medium, the mentioned amount (Table 2) of ingredients from no. 1 to 8 was weighed and taken in a 1.0 L conical flask.Then 0.5 ml 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 pursuing the procedure used during the preparation of poultry waste media.Culture laboratory.These culture flasks were continuously aerated using electric aerator (Aquarium Pump NS-8200).Twelve subsamplings of 20 mL each were taken at every alternative day from 12 flask to record dry cell weight and chlorophyll a content of spirulina and properties of culture media.All the microalgal samples were collected just before reaching the stationary phase.The stationary phase was recorded giving two growth trials of spirulina and then final experiment was conducted to collect spirulina before stationary phase.These samples were used to collect spirulina for the analyses of growth parameters and proximate composition of spirulina [44].All the analyses were done following the methods given by [2,44].Estimation of cell weight, chlorophyll a, specific growth rates (SGRs) on the basis of dry weight, chlorophyll a content and total biomass of spirulina were calculated using the methods given by [2].The physico-chemical properties of supernatant of digested poultry waste and leftover liquid residue were analyzed using different chemicals and equipments.These properties such as pH, total suspended solids, total dissolved solids, total alkalinity, nitarte-N (NO3-N) and phosphate-P (PO4-P) were analyzed in the laboratory of the Department of Aquaculture, BAU, Mymensingh following [2].
e. Estimation of bio-fuel, bio-oxygen and bio-electricity from culture media of spirulina Bio-oxygen (mg/L) and bio-electricity (mV) of culture media were recorded by digital meter (HEQEP CP 6014 G2) [50].Bio-fuel as lipids of spirulna was estimated using ether extraction method from the sample [44].
f. Statistical analysis Analysis of variance (ANOVA) of mean cell weight and chlorophyll a, and crude protein, crude lipids and ash of S. platensis cultured in different media (Treatments) were done and to find whether any significant difference among treatment means was done by Tukey test using statistical package following [51].

RESULTS AND DISCUSSION
Poultry waste was dense liquid (semi-liquid) contained high amount of total solids, chemical oxygen demand (COD), pH, phosphate-P and alkalinity (Table 3).The amount of total solids and COD were decreased drastically after digestion (Table 4).It contained high percentage of moisture, ash, % nitrogen and denatured nitrogen free extract (Table 5).Most of the animal based wastes specially agro-industries contain high amount of pH, available N, available P, total solids, COD and total N which agrees with the present findings of [9,6,28,36,52].Spirulina platensis, very nutritious blue-green microalgae has grown very well in supernatant of aerobically digested 4.0 g/l poultry waste.During the study, pH showed almost an increasing trend in culture media except in supernant of 2.0 g/l DPW (Fig. 1).The decomposed agro-industrial waste help to grow microalgae as chlorella and spirulina, and mosquito larvae due to presence of high nutrients, pH values and chemical oxygen demand which has the similarity with the present findings [4,8,10,11,13,53].Bio-oxygen (Dissolved oxygen) was found to increase gradually upto 10th day of culture and then decreased in most of the cases (Fig. 2).It has been observed that available P (PO4-P) was gradually decreased up to highest peak of growth (Fig. 3) where available N (Nitrate-N) did not follow a definite trend (Fig. 4).Phosphate-P (Available P) was gradually decreased in amount up to 8th day in the culture of supernatant of 2.0 g/L digested poultry waste (DPW) contained spirulina and again increased from 10th day up to 14th day of culture.But it was found to decrease from first day (46.80 ± 4.70 g/l) of experiment up to 10th day (15.40 ± 1.60 g/L) but increased from 12th to 14th day of experiment in the culture of 4.0 and 6.0 g/l DPW, and KM contained spirulina (Fig. 3).Both available N and available P were utilized by spirulina during its growth and ultimately these were decreased as spirulina increased up to the exponential phase of growth [37,54,55].Optical density of media contained spirulina and cell weight and chlorophyll a of spirulina grown in supernatant of 4.0 g/L digested poultry waste significantly higher (P < 0.05) than those of spirulina cultured in other media except KM (Table 6, Figs. 5, 6 and 7).The specific growth rates on the basis of cell weight and chlorophyll a of spirulina cultured in supernatant of 4.0 g/L poultry waste had significant (P < 0.05) differences from those of spirulina grown in other media except KM (Table 6).The growth parameters of S. platensis grown in the supernatant of digested 4.0 g/L poultry waste were higher than other cultures in the supernatant of 2.0 and 6.0 g/L digested poultry waste (Figs.other environmental parameters that agree with the findings of [6,9,16,56,57].Cell weight was directly and highly significant (P < 0.01) and correlated (r = 0.993) with chlorophyll a of spirulina grown in supernatant of different digested poultry waste and Kosaric medium during the study (Fig. 9).[58] Found that S. platensis grown in digested mustard oil cake media in the concentrations of 3.0, 4.0, 0.5 mg/l, and KM, the maximum total biomass were 451.0, 614.33, 403.5 and 719.0 mg/l, respectively.[4,37] recorded almost similar cell weight and chlorophyll a of S. platensis when cultured in digested sago waste water which has the similarity with the present findings.Researchers [13,59] recorded good growth of spirulina when cultured in supernatant of digested rotten potato and apple which almost agree with the present results.
A very important result was found that the crude lipids of spirulina were sharply increased when cultured in digested poultry waste (DPW) (Table 7).Spirulina contained higher amount (almost three times) of lipids when grown in supernatant of 4.0 g/L DPW than 2.0 and 6.0 g/L DPW, and Kosaric medium (Table 7).But, the crude protein of spirulina grown in PWM was lower than that cultured in Kosaric medium.Bioaccumulation of lipids in spirulina grown in poultry waste is a very promising result so that a high quality and quantity of lipids might be produced in plenty in spirulina when grown in the supernatant of digested poultry waste which may be due to phospholipids biosynthesized in spirulina.[4,8] also recorded high lipids in spirulina cultured in digested sago starch factory waste water and fermented waste water of noodle factory, respectively due to presence of high carbon.
Poultry waste contains high amount of phosphorus and calcium due to use of mollusk shell powder as source of minerals in poultry feed.This phosphorus came out from intestine with poultry waste (droppings) which ultimately helped to biosynthesis of phospholipids in spirulina.Almost three times more lipids may be biosynthesized in spirulina if it is cultured in the supernatant of 4.0 g/L digested poultry waste.But, [60] used in tapioca wastewater, [4] used digested sago starch factory waste water and [8] used waste water of Thai rice noodles factories to grow spirulina and recorded higher protein and lipids than cultured in Kosaric medium has similarity with the present findings in case of lipids.Nutrient manipulation in media helps to bioaccumulate lipids as bio-fuel in high amount [43] in microalgae.Nutrients manipulated with sodium acetate which may increase lipids biosynthesized in microalgae, Ankistrodesmus convolutes [61] and Chlorella vulgaris [62].
It was recorded that pH, bio-oxygen and bio-electricity were found to increase gradually upto 10th day of culture and then decreased in most of the cases (Figs. 1, 2 and 8) which has direct relation with gradual increment of optical density, cell weight and chlorophyll a of spirulina.Phosphate-P was decreased up to 10th day of culture which might be used due to bioaccumulation of lipids (Phospholipids) (Fig. 3).The production of bio-oxygen, bio-electricity, bio-fuel (lipids) were directly related with each other which were not similar when compared with other culture media [4,40,41,42,50].The most important thing is the production of bio-fuel, bio-oxygen and bio-electricity using agroindustrial wastes for the production of spirulina [4,42,50].

CONCLUSION
Therefore, it is recommended that spirulina may be grown in supernatant of 4.0 g/l decomposed poultry waste (DPW) to increase percentage of lipids as bio-fuel in spirulina during mass or industrial production.With very little cost, these agroindustrial wastes may be prepared easily for media of culture which are available throughout the country.Thus poultry as well as other wastes may be used commercially and economically for the culture of S. platensis to produce high amount of lipids as bio-fuel, bio-oxygen and bio-electricity.The most important activity of spirulina is to use carbon and CO2 from the environment during photosynthesis for the production of very important things such as bio-oxygen, lipids as bio-fuel, protein and bio-electricity.It will also help to reduce at least partially the environmental pollution, carbon and CO2 emissioned from animal activities and industries, and ultimately minimize climate change which will create peaceful environment in the world.

Table 7 .
Proximate composition (% in dry matter basis) of Spirulina platensis cultured in supernatant of three different digested poultry waste and control as Kosaric medium (values are mean ± standard error, n = 4± 0.04 *NFE = 100 -(Moisture + Crude protein + Crude lipid + Ash).Figures in common letters in the same row do not differ significantly (P < 0.01).

Table 1 .
Experimental design for Spirulina platensis culture using supernatant of three different concentrations of digested poultry waste.

Table 3 .
Characteristics of raw poultry waste just after collection.

Table 4 .
Chemical properties of supernatant of poultry waste after 17 days of digestion in aerobic condition (values are mean ±

Table 5 .
Chemical composition (%) of poultry waste on wet basis (values are mean ± standard error, n = 4)

Table 6 .
Specific growth rates (SGRs) on the basis of cell weight, chlorophyll a and total biomass of Spirulina platensis grown in supernatant of different digested poultry waste (DPW) and Kosaric medium (values are mean ± standard error, n = 4)