Increased lipids production of Nannochloropsis oculata and Chlorella vulgaris for biodiesel synthesis through the optimization of growth medium composition arrangement by using bicarbonate addition

Chlorella vulgaris and Nannochloropsis oculata are a highly potential microalgae to be used in pilot-scale of biodiesel synthesis. The essential content from these microalgae is the fatty acid of lipid which is the main target for the feed and biodiesel industries. One of the key factor in improving lipid microalgae are the arrangemment of nutrients in the growth medium. Research on the regulation of nutrients using bicarbonate (HCO3-) as an additional inorganic carbon source has been done by many studies, but the yield of lipids obtained has not been much. The aim of the study was to improve the lipid yield of Chlorella vulgaris and Nannochloropsis oculata. Variation of [HCO3-] which added to Walne medium were 25 ppm and 75 ppm, while the Walne medium without the addition of bicarbonate acts as control. The results showed that [HCO3] 75 ppm could increase Chlorella vulgaris biomass by 0.9162 g/l with 17.0% wt, while Nannochloropsis oculata produced the greatest lipid content in [HCO3-] 25 ppm of 20.3% wt and the largest biomass on [HCO3] 75 ppm of 1.7233 g/l.


Introduction
Transportation and electricity are the most crucial daily needs and must be met to support human welfare. On the other hand, the increase population can causes an increasing of these sector demand and raising various problems in the energy field. To respond and address these issues, researchers are always looking for and developing renewable energy sources. The best candidate of this potential source is microalgae because it can produce a large number of lipid in a small area everyday, so it better than crop plants [1].
The most commonly used microalgae as a renewable energy sources for biodiesel synthesis are Nannochloropsis and Chlorella [2]. Both microalgae can produce a high lipid content in its cells, i.e 22.7-29.7% of the Nannochloropsis dry weight and 5-58% of the Chlorella dry weight [3]. One of the most common problems is the acquisition of biomass and lipid microalgae that do not meet production targets for feed and biodiesel industries.
Based on the potential of these microalgae, University of Indonesia is actively researching and developing Nannochloropsis and Chlorella to contribute in the production of biodiesel, especially in Depok, West Java. One of effort to increase the lipid microalgae production is re-optimize the regulation of growth medium using bicarbonate based on previous studies, so that the University of Indonesia has valuable assets to play a role in biodiesel synthesis.
The selection of bicarbonate is considered to be an important point for increasing the lipid production of Nannochloropsis and Chlorella in the upstream biodiesel synthesis study. According to Devgoswami, the solubility of bicarbonate (HCO3 -) is higher than carbon dioxide (CO2), so it can be directly absorbed by the microalgae [4]. In addition, bicarbonate acts as a carbon source that is closely related to photosynthesis and the accumulation of lipids in cells [5].

Microalgae and Growth Medium Compositions
The microorganisms used during this study were Nannochloropsis oculata and Chlorella vulgaris microalgae. The isolate is cultivated in the Laboratory of Bioprocess Engineering, Department of Chemical Engineering, Faculty of Engineering, University of Indonesia, Depok, West Java. Isolates of both types microalgae are always maintained and reproduced in the range of logarithmic-stationary growth phases. The growth medium of Nannochloropsis and Chlorella used in this study was the Walne medium in seawater. Walne medium composition can be seen in Table 1 [6].

Stock Solution of NaHCO3
The NaHCO3 solution is used as an additional carbon source solution in the Walne medium. The purpose of making NaHCO3 stock solution in this study was to provide homogeneous conditions on each treatment and to obtain significant results in the work culture. The NaHCO3 stock solution made for this study was 300 ppm which can be prepared by dissolving 300 mg of NaHCO3 in 1000 ml of distilled water. Dilution for working culture using a mole ratio between NaHCO3 and HCO3 -.

Working Culture and Culture Condition
The working culture of Nannochloropsis and Chlorella uses glass-reactor bottles with a capacity of 1500 ml. Variations of [HCO3 -] which added to Walne medium were 25 ppm and 75 ppm, whereas treatment without addition of [HCO3 -] was used as a control. The composition of the working culture in this study can be seen in Table 2. With a working culture composition that looks like above, the Nannochloropsis and Chlorella culture are set with initial optical density (OD) of ±0.4. The set of reactor then placed near the white lamp with light intensity that has been set for each microalga, i.e 3000--3500 lux for Nannochloropsis and 2000 lux for Chlorella. The both culture then harvested for lipid extraction after reaching the stationary phase.

Harvesting Biomass
After reaching the stationary phase, Nannochloropsis oculata and Chlorella vulgaris cultures are harvested. The working culture is harvested by centrifugation at 4000 rpm for 15 minutes to obtain biomass [7]. The precipitated biomass then separated from the supernatant and transferred to a container. Container with wet biomass then dried naturally. The dry weight of biomass (X) can be determined by the following equation:

Lipid Extraction
The lipid extraction method used in this study is a modification of the Bligh & Dyer method [7]. Bligh & Dyer modification method can be done by means of wet biomass mixed with chloroform: methanol (2:4), then sonicated for 20 minutes. Then, the mixture was added with chloroform:aquadest (2:2) and re-sonicated for 20 minutes. The mixture then centrifuged for 15 minutes to form three layers. The bottom layer is the extracted lipid. The extracted lipid then placed into a vial, which has been known the empty weight, for the solvent evaporation. After all the solvent has evaporated, the vial is then weight. Microalgal lipid (YL) can be determined by the following equation:

Growth and Biomass Production
The cultivation of Nannochloropsis oculata and Chlorella vulgaris with variations of [HCO3 -] addition in the Walne medium reached the stationary phase at 288-336 hours. Growth of both microalgae can be seen in Figure 1. Based on Figure 1, the control culture of Nannochloropsis oculata and Chlorella vulgaris experienced the lowest growth, while the [HCO3 -] 75 ppm experienced the highest growth.

Fig. 1. Growth of Nannochloropsis oculata and Chlorella vulgarisin [HCO3 -] treatments
Dry biomass of Nannochloropsis oculata and Chlorella vulgaris can be seen in Figure 2. According to Figure 2, the control treatment from Nannochloropsis oculata and Chlorella vulgaris also had the lowest biomass of 0.8954 g/l and 0.743 g/l, whereas the highest biomass was owned by [HCO3 -] 75 ppm treatment of 1.7233 g/l and 0.9162 g/l. Increased growth and biomass on the addition of [HCO3 -] into the culture medium has also been demonstrated in Devgoswami et al. [4], Ibrahim [8], Lin et al. [9], White et al. [10], and Agustin [11].

Lipid Production
Yield lipid of Nannochloropsis oculata and Chlorella vulgaris can be seen in Figure 3. According to Figure  3, the lowest lipid is controlled by the control treatment of both cultures, i.e. 12.1% dry weight of Nannochloropsis oculata and 11.8% dry weight of Chlorella vulgaris. The highest lipid was obtained from cultures of Nannochloropsis oculata with [HCO3 -] 25 ppm of 20.3% dry weight and Chlorella vulgaris with [HCO3 -] 75 ppm of 17% dry weight. Increased lipid in cultures that given of [HCO3 -] also been demonstrated in Devgoswami et al. [4] and Agustin [11].

Discussions
The lowest growth in control culture was caused by no addition of carbon source that can enhance the process of photosynthesis, so cultures rely only on air derived from aeration. This is in accordance with Gardner et