Biomass and nutrient composition of Spirulina platensis grown in goat manure media

The major costs of cultivating S. platensis are for media and labor. The aim of this study was to explore the use of goat manure as culture medium for S.platensis. A complete randomized block design was used in this study with 5 treatments of goat manure concentration, each of which was repeated 5 times. The results of the study showed that the growth of S.platensis at 25 g/L concentration of goat manure was significantly (P<0.05) lower than 50, 75, 100 g/L and Zarrouk media. S.plantesis productivity at a concentration of 25 g/L goat manure (0.128±0.011 g/L/day) was significant (P<0.05) lower than 50 (0.153±0.012 g/L/day), 75 (0.158±0.014 g/L/day), 100 g/L (0.153±0.009 g/L/day) and Zarrouk media (0.164±0.016 g/L/day). Protein content in S. platensis at 50 (59.03±4.31%), 75 (62.56±6.04%) and 100 g/L (61.22±4.54%) goat manure and Zarrouk (63.21±5.97%) media were significantly (P<0.05) higher than 25 g/L (47.35±5.16%). Lipid content in S. platensis at Zarrouk media (5.28±0.38%), 50 g/L (5.31±1.27%), 75 g/L (5.37±1.25%) and 100 g/L (5.42±1.35%) goat manure were significantly (P<0.05) lower than 25 g/L (7.86±1.16%). Carbohydrate content in S. platensis at Zarrouk (22.58±2.38%), 50 g/L (21.67±2.25%), 75 g/L (22.43±2.14%) and 100 g/L (22.67±2.38%) goat manure were significantly (P<0.05) lower than 25 g/L (27.69±1.78%). Chlorophyll content in S. platensis at Zarrouk and goat manure with 50, 75 and 100 g/L concentration were significantly (P<0.05) higher than 25 g/L (1.36±0.15%). We conclude that the concentration of goat manure 75 g/L in S.platensis cultivation media was the optimum concentration and has the potential to replace inorganic media.


Introduction
Spirulina platensis has the potential to be commercially produced and used as food and animal feed (Holman and Malau-Aduli, 2013;Muhling et al., 2005) because it is rich in protein, fat, carbohydrates and other important elements (Kerswell, 2006;Sherwood et al., 2010;Sánchez et al., 2003) such as chlorophyll (Moraes et al., 2011) and amino acids (Avila-Leon et al., 2012). High phytonutrient content in S. platensis biomass such as phycocyanin, carotene, xanthophyll, gamma linoleic acid, galactolipids, sulpholipids, chlorophyll, and minerals which have beneficial effects on human health such as antioxidation (Pleonsil et al., 2013), anti-cancer (Thangam et al., 2013), anti-inflammatory activity (Zhu et al., 2016), photo-induced cytotoxicity (Wang et al., 2012), stimulation of the immune system (Li et al., 2010), anti-malnutrition, anti-anemia, anti-oxidative, antiviral and anti-cancer properties (Estrada et al., 2001;Belay, 2002;Sánchez et al., 2003) attract market interest in the food industry. Nutrient content in S. platensis biomass has also attracted market interest in the animal feed industry, even Holman and Malau-Aduli (2013) reported that half of biomass production was used for livestock feed and fish. S. platensis has high adaptability and can be cultivated in various nutrient conditions. Generally, large-scale S. platensis cultivation used Zarrouk and Walne media as a source of nutrition. The both in-organic media are relatively complex, expensive and with limited availability. The main production costs for S. platensis cultivation are media and labor. The search for S. platensis cultivation media with lower cost and sufficient availability is conducted to minimize production costs and increase revenue. Some investigators have reported the replacement of several elements contained in the Walne and Zarrouk media. Organic matter (Andrade and Costa, 2009) and wastewater (Mezzomo et al., 2010) were reported to be used as a source of nutrition in S. platensis cultivation media. Livestock by-product is a potential source of nutrition for microalgae cultivation media and plant fertilizer. Goat manure is a livestock by-product that is quite abundant, especially in Indonesia but is not used conventionally as culture medium for S. platensis. Indonesian farmers generally use goat manure as growing media for plant. Goat manure contains 4.9% total nitrogen, 4.1% phosphorus, 1.9% potassium, 1.0% calcium, and 0.9% magnesium (Awodun et al., 2007) and has been studied to be used as a nutrient source for Chlorella microalgae cultivation media (Kinosraj et al., 2016;Agwa et al., 2012). Danmaigoro et al., (2019) reported that poultry manure had a significant effect on rice growth and yield. Awodun et al., (2007) reported that goat manure had a significant increase on pepper growth and yield parameters as number of leaves and branches, plant height, stem girth, number and weight of fruits. Livestock byproduct such as pig manure (Canizares et al., 1994;Olguin et al., 2001) and chickens manure (Iyoyo et al., 2010) has been reported as low-cost nitrogen sources for microalgae cultivation. However, information regarding the utilization of goat manure as a S. platensis cultivation medium is limited. The present study aims to explore the use of goat manure as a S. platensis cultivation medium.

Material and Methods
This study has been carried out at the Laboratory of Biology, Study Program of Biology, Faculty of Mathematics and Natural Sciences, University of PGRI Adi Buana Surabaya. A completely randomized block design with 4 concentrations of goat manure in S. platensis medium follows A (25 g/L), B (50 g/L), C (75 g/L) and D (100 g/L) and Zarrouk media was use as a control. All treatments in this study were repeated 5 times.

Preparation of goat manure
Goat manure was obtained from local farmers in Sidoarjo, Indonesia. A total of 2500 g of goat manure was dried in the sun for 2 days and milled using mechanical grinder to obtain goat manure powder.

Cultivation of S. platensis
A total of 500 g of goat manure powder is divided into 4 parts, 50, 100, 150 and 200 g, respectively. Each part was dissolved in 20 L of tap water, stirred and filtered by Whatman paper No. 42 to obtain the concentration of goat manure 25, 50, 75 and 100 g/L. To the filtrates 0.2% NaCl was added and pH was adjusted to 10 with NaHCO 3 and the solution was stirred until homogeneous. In the present study, we have used the media of Zarrouk (Zarrouk, 1966) as a control cultivation media with 5 replications. All media were sterilized at 121 o C for 15 minutes and after being cooled in the room until the temperature reached 24 o C, 0.25 L of S. platensis brood stock was inoculated into each 1 L of media. Cultivation of S. platensis was carried out for 14 days in an Erlenmeyer flask (1500 mL) at 25-35 o C, salinity 30-70 ppt, pH 9.0-9.5, light intensity 5000 lux (Benetech GM 1020 illuminance meter) and agitation at 60 rpm in the shaker (Gesellschaft fur Labortechnik, Germany).

Assays of chemical composition of goat manure media
The assays for chemical composition of the goat manure media included total organic carbon, nitrate and phosphorus was carried out before S. platensis inoculation. The content of total organic carbon (TOC) in the S. platensis biomass was analyzed using the wet oxidation method guided by Walkey and Black (1965). The level of nitrate (NO 3 -N) analysis was done using the method of spectrophotometer at 410 nm. The spectrophotometer method has been carried out to determine the phosphorus content in S. platensis biomass.

Biomass S. platensis
The method of Yap et al. (2018) used a spectrophotometer (Thermo Fisher Scientific, Genesis 20, USA) at a wavelength of 680 nm and was repeated 3 times to be used to observe the development of daily S. platensis biomass.One milliliterof Spirulina culture was took out every day for the OD 680 measurement. The biomass daily Spirulina was analyzed using regression analysis as the Spirulina biomass is 0.8274 optical density value plus 0.0393. The standard curve has been made by diluting a series of 1.6 g/L of Spirulina dry biomass and was subsequently used to calculate the biomass of individual samples. At the 14 days cultivation Spirulina was harvested by filtration through synthetic fiber cloth with a mesh size of 50 microns to separate the biomass of Spirulina from the liquid culture, washed by fresh water followed by filtration using a mesh size of 200 microns to remove any salt from the culture medium. The filters with the Spirulina cells were dried at 80°C in an oven overnight. Analysis of the composition biomass of S. platensis including protein, lipid, carbohydrate and chlorophyll contents were carried out.

Assays the biomass of S. platensis nutrient composition
Nutrient composition in S. platensis biomass was analyzed for proximate composition in accordance with Association of Official Analytical Chemists (AOAC, 1990) recommendations consists of crude protein, crude fat and carbohydrates. Chlorophyll-a content in the biomass of S. platensis was determined by the method of Jeffrey and Humphrey (1975).

Results and Discussion
In this study, some minerals content in goat manure consisted of 531.42 g/kg of total carbon, 3.78 g/kg of total nitrogen, 1.76 g/kg of phosphorus and 0.52 g/kg of potassium. Some previous investigators have reported the different chemical composition of goat manure (Mnkeni and Austin;Tennakoon andBandara, 2003: Omisore et al., 2018). Variations in the chemical composition of goat manure can be due to diet, age, weight sex (Powell et al., 1994), type of livestock, growth and feeding practices, weather, and storage time and conditions (Smith and Frost, 2000) . Some of the mineral content of goat manure in this study is presented in table 1. The concentration of goat manure in cultivation media can affect mineral content such as total carbon, total nitrogen, phosphorus and potassium S. platensis. The mineral content (total carbon, total nitrogen, phosphorus and potassium) in S. platensis cultivation media with goat manure concentrations 25, 50, 75 and 100 g/L was significantly (P<0.05) different from each other. The values and standard deviation (±) with different superscripts ( a , b , c and d ) in same column are different significant (P<0.05).

Biomass of S. platensis
The response of S. platensis daily biomass to Zarrouk and goat manure media in this study is presented in Figure 1. As shown in Figure 1, the rate of increase in S. platensis biomass in all media during the first 7 days was almost similar. But on day 8 to day 14 cultivation, the rate of increase in S. platensis biomass from medium 25 g/L goat manure was lower than Zarrouk, 50, 75 and 100 g/L goat manure media. The decreasing rate of S. platensis biomass is thought to be due to the depletion of nutrient availability and the increase of S.
platensis cell mass, especially in the medium of 25 g/L goat manure. The slowing down of the rate of increase in S. platensis biomass for all media in this study occurred on the 12 th day of cultivation. As shown in table 2, the productivity of S. platensis during the first week of cultivation did not different significantly (P>0.05) in all experimental media. But in the second week of cultivation, the productivity of S. platensis in the medium 25 g/L goat manure was significantly (P<0.05) lower than Zarrouk, 50, 75 and 100 g/L.

Zarrouk and goat manure media
Referring to the results of analysis of some mineral content in goat manure media (table 1), the low concentrations of nitrogen especially nitrate and phosphorus may be the cause of lower biomass and S. platensis productivity from goat manure 25 g/L than other cultivations media in this study. As shown in table 1, the content of nitrogen, nitrate and phosphorus in the medium 25 g/L goat manure were 0.87±0.13 g/L, 5.22±2.04 mg/L and 0.34±0.09 g/L, respectively. Several investigators reported that the growth and productivity of S. platensis was determined by phosphorus concentration in cultivation media (Xin et al., 2010;Dammak et al., 2017). Markou et al. (2012) reported that the optimum productivity of spirulina biomass was obtained at a 250 mg/L phosphate concentration in the form of K 2 HPO 4 . Meanwhile, nitrogen plays a role in the formation of macromolecules and cell biomass (Perez-Garcia et al., 2011). Nitrogen concentration of 2.5 g/L was the optimum concentration recommended for cultivation of S. platensis (Celekli and Yavuzatmaca, 2009).  which ranges from 60 to 70% dry biomass weight. Nevertheless, differences in protein content in S. platensis biomass cultivated under different nutritional conditions have been reported by Marrez et al. (2014). Lipid content in S. platensis biomass from 5 media at both the early stages and the end of cultivation are presented in figure 3. At the early stages of cultivation, there is no significant difference between lipid content in S. platensis biomass from all concentration of goat manure and Zarrouk media. At the end stages, lipid content S. platensis biomass from Zarrouk media (5.28±0.38%), 50 g/L (5.31±1.27%), 75 g/L (5.37±1.25%) and 100 g/L (5.42±1.35%) goat manure were significantly (P<0.05) lower than 25 g/L (7.86±1.16%). From the medium of 25 g/L of goat manure, the lipid content in S. platensis biomass increased about 2.55% from 5.31% at the early stage to 7.86% at the end stage. Referring to the results of the analysis of some mineral content in goat manure media (table 1), the low concentration of nitrogen especially nitrate and phosphorus may be the cause of higher lipid content in S. platensis biomass from goat manure 25 g/L than other cultivation media in this study. According to some investigators, the concentration of nitrogen, phosphate and iron in cultivation media has an effect on the lipid content in the S. platensis biomass (Nyabuto et al. 2015;Liu et al., 2008). The low nitrogen concentration in cultivation media can increase lipid content in some microalgae biomass (Illman et al., 2000). Uslu et al. (2011) reported that 50 and 100% nitrogen deficiency in Spirulina increased total lipid ratio by 13.66 and 17.05% and reduced in protein of 53.5 and 5.6%, respectively. The lipid content in Spirulina biomass around 5-10% of its dry weight varies with cultivation conditions and analytical methods (Gutiérrez-Salmeán et al., 2015). Several investigators reported lipid content in Spirulina biomass ranged from 6 to 15% (Xue et al., 2002;Colla et al., 2007;Promya et al., 2008;Madkour et al., 2012). Optimal concentration of lipid content in Spirulina biomass for food or feed has not been documented. Polyunsaturated fatty acids are considered as important fractions of Spirulina lipid biomass.  (table  1), low nitrogen especially nitrate and phosphorus may be the cause of higher carbohydrate content in S. platensis biomass from goat manure 25 g/L than other cultivation media in this study. The carbohydrate content in S. platensis biomass is influenced by nitrogen concentration (Menegol et al., 2017). At low nitrogen concentrations in cultivation media, lipid and carbohydrate biosynthesis increases (Fan et al., 2012;Ho et al., 2012;Wang et al., 2014;Moraes et al., 2018) while protein synthesis decreases (Zhu et al., 2014). Markou et al. (2012) reported that the phosphorus concentration of 10 mg/L in the form of K 2 HPO 4 in cultivation media could increase the carbohydrate content in S. platensis biomass. The carbohydrate content of S. platensis biomass is generally around 2-20% (Vonshak, 2002). However, some researchers have reported an increase in carbohydrate content in S. platensis biomass. Increased carbohydrate content in S. platensis biomass can be caused by phosphorus deficiency in cultivation media (Dean et al., 2008;Cade-Menun and Paytan, 2010) or algal cell stress due to nitrogen deficiency cultivation media (Sassano et al., 2010). Chlorophyll in S. platensis biomass from 5 media at both early stages and the end of cultivation are presented in figure 5. At the early stages of cultivation, there is no significant difference between chlorophyll content in S. platensis biomass from all concentration of goat manure and Zarrouk media. At the end stages, there was no significant (P>0.05) difference between chlorophyll content in S. platensis biomass from Zarrouk (1.65±0.07%), 50 g/L (1.62±0.09%), 75 g/L (1.65±0.08%) and 100 g/L (1.61±0.12%), but chlorophyll content in S. platensis biomass from Zarrouk and goat manure with 50, 75 and 100 g/L concentration were significantly (P<0.05) higher than 25 g/L (1.36±0.15%). In the 25 g/L goat manure medium, the chlorophyll content in S. platensis biomass decrease about from 0.3% from 1.66% at the early stage to 1.36% at the end stage. According to the results of analysis of some mineral content in goat manure media (table 1), the low nitrogen content, especially nitrate, phosphorus and phosphate may be the cause of lower chlorophyll content in S. platensis biomass from goat manure 25 g/L than the other cultivation media in this study. The chlorophyll content of microalgae is determined by the concentration of minerals and nitrogen in cultivation media (Daughtry et al., 2000). Furthermore, Esen and Ürek (2014) reported that nitrate concentrations of 10 Asian J Agric & Biol. 2020;8(2):158-167 mM in the form of sodium nitrate were insufficient for pigment production algae. Some investigators have reported that low concentrations of nitrogen and phosphorus in cultivation media can inhibit photosynthesis, decrease protein and chlorophyll content and increase carbohydrate and lipid content in biomass S. platensis (George et al., 2014;Wykoff et al., 1998).

Conclusion
The present study demonstrated that goat manure can be used as a S. platensis cultivation medium. Goat manure can be a cheap alternative cultivation medium for Spirulina production. The optimal concentration of goat manure for S. platensis cultivation in this study was 75 g/L. The concentration of goat manure of less than 50 g/L in cultivation media can cause a decrease in productivity and protein and chlorophyll content in Spirulina biomass because the concentration of nutrients, especially nitrogen and phosphorus is not enough to support the maximum growth of Spirulina.