Growth of mastigocladus (Cyanobacteria) HS-46 isolated from maribaya hot spring, indonesia in medium NPK as a potential biofuel feedstock

. One of cyanobacteria genera which isolated from Maribaya hot spring located in Indonesia is Mastigocladus HS-46 can be used as raw material for biofuel production. Mastigocladus is cultured in commercial NPK fertilizer. Commercial NPK fertilizer is one of the low cost medium that commonly used for microalgae growth. The research aims to know the best concentration of commercial NPK fertilizer for Mastigocladus HS-46 growth. In this research, Mastigocladus HS-46 was grown in commercial NPK fertilizer with concentration 80 ppm, 240 ppm, NPK 80 ppm+BBM, and BBM (Bold Basal’s Medium) as a control. This strain was incubated on 35 o C. Observation were made approximately 14 days with 2 sampling for each medium. The average of wet weight Mastigocladus HS-46 after 14 day observation was obtained at NPK 80 ppm: 0,019 g/L, NPK 240 ppm: 0.009 g/L, NPK+BBM: 0,014 g/L, and BBM: 0,015 g/L. The results showed that the best NPK medium concentration was 80 ppm for growth Mastigocladus HS-46. Commercial NPK fertilizer can be used for growth Mastigocladus HS-46 which has potential as biofuel feedstock .


Introduction
One of the genera of cyanobacteria that can live hot spring, namely Mastigocladus sp. Mastigocladus HS-46 was isolated from the Maribaya hot spring in Indonesia. Mastigocladus HS-46 lives at 42 ºC and pH 6 [1]. Mastigocladus is potentially as a raw material for the production of biofuels, because biomass Mastigocladus contain lipid which can be used as a raw material for the production of biofuels [2]. The benefits of using microalgae including cyanobacteria as a raw material for biofuels, that is in the process of harvesting can be done continuously, the cost of harvesting and transportation relatively low, and also does not require extensive land in breeding [3]. to be able to produce biofuel, it takes high amounts of biomass. Therefore, it needed a growth medium for growing cyanobacteria so it can produce a high amount of biomass that can be used as raw material for the production of biofuels.
Growth medium containing nutrient for the growth of cyanobacteria. The growth medium commonly used for the growth of microalgae including cyanobacteria is Bold Basal's Medium. The Bold Basal's medium has a complete macronutrient and micronutrient [4] to serve as a medium control for the study. Nevertheless, the use of medium BBM to growing cyanobacteria in large scale research that leads to the production of biofuels is not economically. Therefore, to produce a high biomass needed an economical alternative medium.
In addition to the Bold Basal's Medium, NPK fertilizers can be used as microalgae growth mediums [5]. NPK Fertilizer is an inorganic fertilizer with macronutrient composition consisting of N, P, and K which is essential and commonly used in plants to support its growth [6]. Other than as a plant fertilizer, NPK fertilizer can also be used to grow the cyanobacteria because it has enough content of nutrients for the growth of cyanobacteria.
This study is a basic study to find alternative medium, that is using NPK fertilizer to grow Mastigocladus HS-46 in order to produce high biomass. Research is expected to continue in large scale research leading to the biotechnology sector to produce biofuels.

Method
The first step of cultivation cyanobacteria is inoculated 30 mg biomass into growth medium. Amount 70 mL of growth medium were added into Erlenmeyer flask 100 mL. Mastigocladus HS-46 was incubated in an incubator at temperature 35 °C and light intensity ± 3300 lux, with initial pH 6-6.5 with two repetitions.

Measurement the weight of wet biomass Mastigocladus HS-46
Measurement of Mastigocladus HS-46 were done in 14 days with 10 times of observation. The sterile eppendorf tube 1.5 mL was measured at analytical measurement tool. Eppendorf tube with biomass were sentrifuged for 10 minute with 10.000 rpm. The supernatant were taken out and wet biomass weight were measured with analytical measurement tool.

Preparation and processing of data
This research is descriptive. Observation data include quantitative and qualitative data. Quantitative data in the form of weight biomass (g/L) and the qualitative data in the form of a color culture of Mastigocladus HS-46. Data weight biomass will be shown in the form of tables and curves while the color culture of Mastigocladus HS-46 will be shown in the photos of microscopic. Growth curve was made by comparing the value of biomass weight as ordinate axis Y by biomass weight counting time as abscissa X. The growth curves were made by Microsoft Excel.

Macroscopic observation Mastigocladus HS-46
Macroscopic observations of Mastigocladus HS-46 cultures by comparing the culture color on the Faber Castle standard color [7]. Based on the color chart, the color of the starter culture Mastigocladus HS-46 is sea green (Fig.1). The color appereance of Mastigocladus HS-46 in medium NPK 80 ppm dan 240 ppm was changed from sea green into apple green at day 14 (t14). while Mastigocladus HS-46 in BBM and NPK+BBM not changed and still colored sea green at day 14 (Fig.2)  The color change that occurs in the Mastigocladus HS-46 grown in medium NPK 240 ppm and 80 ppm, probably due to the chlorophyll degradations process. The process of chlorophyll degradation mechanism can occur due to the reaction of phaeophytin formation. Phaeophytin is a form of chlorophyll that loses the Mg 2+ ion. The process begins with the presence of Mg 2+ ions in the middle of the molecule. Furthermore, the Mg 2+ ion will be removed, and replaced by hydrogen ions. Consequently the color expressed becomes yellow [8].
Therefore Mastigocladus HS-46 grown in medium NPK 80 ppm and NPK 240 ppm change color to apple green.
The growth curve of this study still imperfect, because Mastigocladus HS-46 were still adapted with the condition of growth medium [5]. The growth curve can increase and decrease again so it has not reached stable. This is because the medium BBM, NPK 80 ppm, and NPK 240 ppm, has a high nitrogen content. Nitrogen contained in the medium is a major nutrient in the growth of microalgae including cyanobacteria. Nitrogen function in biochemical processes, such as the biosynthesis of nucleic acids (DNA and RNA) and amino acids (protein). Cyanobacteria use sources of nitrogen in the form of NH4 + , NO3 -, NO2 -, and NOand also organic molecules in the form of urea and amino acids [9]. The source of nitrogen in the medium BBM from NaNO3 while the nitrogen source in the NPK medium from amonium (NH4 + ); nitrat (NO3 -); and urea. High levels of nitrogen in the growth medium can lead to the deactivation of pigment production for photosynthesis [5]. Therefore, mediums that have high nitrogen levels take longer to reach a stable phase of growth. The nitrogen content in the BBM medium was 41.17 mg, while the nitrogen content of NPK medium 80 ppm and 240 ppm was 16 mg and 48 mg.

Conclusion
Based on the observation of the 14 day and the discuccion that has been done. The medium concentration of NPK 80 ppm is the best concentration of NPK for Mastigocladus HS-46 growth, because the average biomass weight Mastigocladus HS-46 in NPK 80 ppm on day 14 (t14): 0.019 g/L. The low average biomass weight in Mastigocladus HS-46 grown in NPK medium 240 ppm on day 14 (t14): 0.009 g/L.