Increased Lipids Production of Nannochloropsis Oculata And

MATEC Web of Conferences 154, 01009 (2018) https://doi.org/10.1051/matecconf/201815401009 ICET4SD 2017

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

Dianursanti*, Zenitha Lintang Agustin, and Dwini Normayulisa Putri

Chemical Engineering Department, Faculty of Engineering, University of Indonesia, Kampus Baru UI, Depok, West Java, 16424

Abstract. 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.

1 Introduction The selection of bicarbonate is considered to be an important point for increasing the lipid Transportation and electricity are the most crucial daily production of Nannochloropsis and Chlorella in the needs and must be met to support human welfare. On the upstream biodiesel synthesis study. According to - other hand, the increase population can causes an Devgoswami, the solubility of bicarbonate (HCO3 ) increasing of these sector demand and raising various is higher than carbon dioxide (CO2), so it can be problems in the energy field. To respond and address directly absorbed by the microalgae [4]. In addition, these issues, researchers are always looking for and bicarbonate acts as a carbon source that is closely developing renewable energy sources. The best candidate related to photosynthesis and the accumulation of of this potential source is microalgae because it can lipids in cells [5]. 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 2 Materials and Methods energy sources for biodiesel synthesis are Nannochloropsis and Chlorella [2]. Both microalgae can 2.1 Microalgae and Growth Medium produce a high lipid content in its cells, i.e 22.7-29.7% of Compositions the Nannochloropsis dry weight and 5-58% of the Chlorella dry weight [3]. One of the most common The microorganisms used during this study were problems is the acquisition of biomass and lipid Nannochloropsis oculata and Chlorella vulgaris microalgae that do not meet production targets for feed microalgae. The isolate is cultivated in the and biodiesel industries. Laboratory of Bioprocess Engineering, Department Based on the potential of these microalgae, University of Chemical Engineering, Faculty of Engineering, of Indonesia is actively researching and developing University of Indonesia, Depok, West Java. Isolates Nannochloropsis and Chlorella to contribute in the of both types microalgae are always maintained and production of biodiesel, especially in Depok, West Java. reproduced in the range of logarithmic-stationary One of effort to increase the lipid microalgae production growth phases. is re-optimize the regulation of growth medium using The growth medium of Nannochloropsis and bicarbonate based on previous studies, so that the Chlorella used in this study was the Walne medium University of Indonesia has valuable assets to play a role in seawater. Walne medium composition can be in biodiesel synthesis. seen in Table 1 [6].

* Corresponding author: [email protected]

© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). MATEC Web of Conferences 154, 01009 (2018) https://doi.org/10.1051/matecconf/201815401009 ICET4SD 2017

Table 1. Walne Medium Composition

Nutrient solution Trace element solution Vitamin solution Materials per litre Materials per litre Materials per litre Thiamine.HCl NaNO 100,0 g ZnCl 21,0 g 1,0 g 3 2 (vit. B1) Cyanocobalamin H BO 33,6 g CoCl .6H O 20,0 g 0,05 g 3 3 2 2 (vit. B12) Na2EDTA 45,0 g (NH4)6Mo7O24.4H2O 9,0 g NaH2PO4.H2O 20,0 g CuSO4.5H2O 20,0 g FeCl3.6H2O 1,3 g MnCl2.4H2O 0,36 g trace element solution 1 ml vitamin solution 100 µl

-3500 lux for Nannochloropsis and 2000 lux for 2.2 Stock Solution of NaHCO3 Chlorella. The both culture then harvested for lipid extraction after reaching the stationary phase. 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 2.4 Harvesting Biomass provide homogeneous conditions on each treatment and to obtain significant results in the work culture. After reaching the stationary phase, Nannochloropsis The NaHCO3 stock solution made for this study was oculata and Chlorella vulgaris cultures are harvested. 300 ppm which can be prepared by dissolving 300 mg The working culture is harvested by centrifugation at of NaHCO3 in 1000 ml of distilled water. Dilution for 4000 rpm for 15 minutes to obtain biomass [7]. The working culture using a mole ratio between NaHCO3 precipitated biomass then separated from the - and HCO3 . 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 2.3 Working Culture and Culture Condition equation: The working culture of Nannochloropsis and Chlorella (1) uses glass-reactor bottles with a capacity of 1500 ml. 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 (𝑔𝑔) − 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 (𝑔𝑔) - Variations of [HCO3 ] which added to Walne medium 𝑋𝑋 (𝑔𝑔/𝑙𝑙) = 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 (𝑙𝑙) were 25 ppm and 75 ppm, whereas treatment without - 2.5 Lipid Extraction addition of [HCO3 ] was used as a control. The composition of the working culture in this study can be The lipid extraction method used in this study is a seen in Table 2. modification of the Bligh & Dyer method [7]. Bligh & Dyer modification method can be done by means of Table 2. Working Culture Compositions wet biomass mixed with chloroform: methanol (2:4), Nannochloropsis then sonicated for 20 minutes. Then, the mixture was - Chlorella vulgaris [HCO3 ] oculata added with chloroform:aquadest (2:2) and re-sonicated treatment for 20 minutes. The mixture then centrifuged for 15 (ppm) 0 25 75 0 25 75 minutes to form three layers. The bottom layer is the extracted lipid. The extracted lipid then placed into a Inoculum 500 500 500 500 500 500 vial, which has been known the empty weight, for the (ml) solvent evaporation. After all the solvent has NaHCO3 evaporated, the vial is then weight. Microalgal lipid solution 0 125 375 0 125 375 (YL) can be determined by the following equation: addition (ml) (2) Walne 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 (𝑔𝑔) − 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑖𝑖𝑖𝑖𝑖𝑖𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 (𝑔𝑔) medium 1000 875 625 1000 875 625 𝑌𝑌𝐿𝐿 (%) = 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 (𝑔𝑔) 𝑥𝑥 100% (ml) 3 Results and Discussions With a working culture composition that looks like above, the Nannochloropsis and Chlorella culture are 3.1 Results 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- 3.1.1 Growth and Biomass Production

* Corresponding author: [email protected]

2 MATEC Web of Conferences 154, 01009 (2018) https://doi.org/10.1051/matecconf/201815401009 ICET4SD 2017

Biomass on [HCO -] treatments 3 The cultivation of Nannochloropsis oculata and 2 - Chlorella vulgaris with variations of [HCO3 ] addition Chlorella vulgaris 1.7233 in the Walne medium reached the stationary phase at Nannochloropsis oculata 288-336 hours. Growth of both microalgae can be seen 1.5 in Figure 1. Based on Figure 1, the control culture of Nannochloropsis oculata and Chlorella vulgaris - experienced the lowest growth, while the [HCO3 ] 75 0.9771 1 ppm experienced the highest growth. 0.8954 0.8838 0.9162 Growth of Chlorella vulgaris on [HCO -] treatments X (g/l) 3 0.743 6

8 9 Chlorella vulgaris [HCO -] 0 ppm Y = M0 + M1*x + ... M8*x + M9*x 3 0.5 M0 0.487 5 Chlorella vulgaris [HCO -] 25 ppm 3 M1 0.00715 Chlorella vulgaris [HCO -] 75 ppm ) 3 M2 4.96e-05

600 4 M3 -1.19e-07 0 R 0.997 0 ppm 25 ppm 75 ppm 3 [ HCO - ] Y = 3M0 + M1*x + ... M8*x8 + M9*x9 M0 0.385 2

Absorbance (OD Absorbance Fig. 2. Dry biomass of NannochloropsisM1 oculata0.0135 and - Chlorella vulgarisin [HCO3 ] treatmentsM2 -6.28e-07 8 9 R 0.995 Y = M0 + M1*x + ... M8*x + M9*x 1 M0 -11.6 Y = M0 + M1*x + ... M8*x8 + M9*x9 M1 0.095 3.1.2 Lipid ProductionM0 0.401 M2 -0.000138 0 M1 0.00707 Growth of Nannochloropsis oculata on [HCO -] treatments R 1 0 50 100 150 200 250 3 300 350 400 Yield lipid of NannochloropsisY = M0 + M1*xM2 + ... M8*x 8oculata + M9*x5.59e-059 and Chlorella 6 t (hour) M3 -1.39e-07 vulgaris can be seen inM0 Figure 3. According32.7 to Figure M1 R -0.3530.997 3, the lowest lipid is controlled by the control treatmentY = M0 + M1*x + ... M8*x8 + M9*x9 M2 0.00134 8 9 5 8 9 Y = M0 + M1*x + ... M8*x + M9*x Y = M0 + M1*x + ... M8*x + M9*x M0 0.43 of both cultures, M3i.e. 12.1%-1.63e-06 dry weightM0 of 0.398 M0 0.428 M1 0.0152 ) Nannochloropsis oculataR and 11.8%0.999 dry weightM1 of 0.0144 M1 0.0165 M2 -0.000263 540 4 M2 -4.13e-05 Chlorella vulgaris. TheM2 highest -0.000317lipid was obtainedM3 3.18e-06 M3 - 8.4e-08 from cultures of NannochloropsisM3 oculata3.45e-06 with [HCO3R 0.998 R 0.987 3 R 0.999 ] 25 ppm of 20.3% dry weight and Chlorella vulgarisY = M0 + M1*x + ... M8*x8 + M9*x9 - with [HCO3 ] 75 ppm of 17% dry8 weight9 . IncreasedM0 -0.0851 Y = M0 + M1*x + ... M8*x + M9*x - 2 lipid in cultures that given of [HCO3 ] also beenM1 0.0184 Absorbance (OD Absorbance M0 -0.209 Nannochloropsis oculata [HCO -] 0 ppm M2 1.69e-05 3 demonstrated in DevgoswamiM1 et al.0.0204 [4] and Agustin M3 -6.8e-08 Nannochloropsis oculata [HCO -] 25 ppm 1 3 M2 -4.49e-06 [11]. - R 0.994 - YieldM3 lipid on [HCO ] -3.58e-08treatments Nannochloropsis oculata [HCO ] 75 ppm 3 3 R 0.99 30 0 0 50 100 150 200 250 300 350 400 Chlorella vulgaris t (hour) 25 Nannochloropsis oculata Fig. 1. Growth of Nannochloropsis oculata and Chlorella 20.3 - vulgarisin [HCO3 ] treatments 20 17.8 17

Dry biomass of Nannochloropsis oculata and 14.4 15 13.3 Chlorella vulgaris can be seen in Figure 2. According 12.1 (% dry weight)

to Figure 2, the control treatment from L

Nannochloropsis oculata and Chlorella vulgaris also Y 10 had the lowest biomass of 0.8954 g/l and 0.743 g/l, - whereas the highest biomass was owned by [HCO3 ] 75 5 ppm treatment of 1.7233 g/l and 0.9162 g/l. Increased - growth and biomass on the addition of [HCO3 ] into the 0 culture medium has also been demonstrated in 0 ppm 25 ppm 75 ppm Devgoswami et al. [4], Ibrahim [8], Lin et al. [9], White [ HCO - ] 3 et al. [10], and Agustin [11]. Fig. 3. Yield lipid of Nannochloropsis oculata and Chlorella - vulgarisin [HCO3 ] treatments

3.2 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

3 MATEC Web of Conferences 154, 01009 (2018) https://doi.org/10.1051/matecconf/201815401009 ICET4SD 2017

al., that culture with the availability of low carbon References sources will lead to low cell growth because - photosynthesis is inhibited [12]. When [HCO3 ] is 1. A. Demirbas, M.F. Demirbas. Algae energy: added to the cultivation medium, the bicarbonate Algae as a new source of biodiesel. Springer- - (HCO3 ) will induce the process of photosynthesis. The Verlag (2010) induction occurs in photosystems II (PS II) of light 2. Y. Chisti. Biotechnol. Adv., 25, 3 (2007) reactions and resulting in a large number of NADPH 3. T.M. Mata, A.A. Martins, N.S. Caetano. Renew. and ATP [5]. - Sust. Energ. Rev., 14, 1 (2010) In addition, bicarbonate (HCO3 ) also serves as a 4. Ch.R. Devgoswami, M.C. Kalita, J. Telukdar, R. carbon to replace source of CO2 in the medium and in the dark reaction of photosynthesis because the Bora, P. Sharma. Afr. J. Biotechnol., 10, 61 (2011) - solubility of bicarbonate (HCO3 ) in water is greater 5. W.G. Hopkins, N.P.A. Huner. Introduction to - than that of CO2. When [HCO3 ] is added to the plant physiology, 4th ed. John Wiley & Sons cultivation medium, the carbon source will increase to (2009) bind to the Rubisco enzyme (Ribulose-1,5- 6. R.A. Andersen. Alga Culturing Techniques. biphosphate), resulting in G3P synthesis Elsevier Academic Press (2005) (glyceraldehyde-3-phosphate or triose-3-phospate) at 7. D.N. Putri. Sintesis Biodiesel dari Mikroalga The Calvin cycle will increase. Increased of G3P will Nannochloropsis sp. melalui Reaksi increase the formation of glucose and lipids, because Transesterifikasi menggunakan Katalis Heterogen 28 G3P molecules will produce 14 molecules of CuO/Zeolit. Undergraduate Thesis (2016) glucose and 1 molecule TAG C18 [13]. According to Agustin, glucose is used as a source of energy for cell 8. A.I. Ibrahim. Pertumbuhan dan produksi lemak growth, so that biomass increases [11]. selular Scenedesmus sp. Undergraduate Thesis - Furthermore, the remaining bicarbonate (HCO3 ) in (2008) the medium still serves to induce lipid synthesis to 9. Q. Lin, N. Gu, G. Li, J. Lin, L. Huang, L. Tan. form energy reserves because the nutrient conditions Bioresour. Technol., 111 (2012) reduced when culture entered the stationary phase. 10. D.A. White, A. Pagarette, P. Rooks, S.T. Ali. J. - Bicarbonate (HCO3 ) will induce the formation of Appl. Phycol., 25 (2013) malonil-CoA which acts as a substrate in lipid 11. Z.L. Agustin. Pengaruh penambahan variasi synthesis. Carbohydrates will be broken down into konsentrasi HCO - terhadap produksi lemak acetyl-CoA which is a precursor of respiration and 3 Nannochloropsis. Undergraduate Thesis (2014) lipid synthesis. Acetyl-CoA then interacts with - bicarbonate (HCO3 ) to form malonil-CoA. According 12. R.D. Gardner, E. Lohman, R. Gerlach, K.E to Foster, increased production of malonil-CoA will Cooksey, B.M. Peyton. Biotechnol. Bioeng., 10 cause acetyl-CoA for the Krebs cycle then diverted to (2013) lipid synthesis, since the enzyme activity in the Krebs 13. L. de Jaeger, R.E.M. Verbeek, R.B. Draaisma, cycle is inhibited [14]. D.E. Martens, J. Springer, G. Eggink, R.H. Wijffels. Biotechnol. Biofuels, 7, 1 (2014) 4. Conclusion 14. D.W. Foster. J. Clin. Invest., 122, 6 (2012)

- The results showed that [HCO3 ] 75 ppm treatment could increase Chlorella vulgaris biomass by 0.9162 g/l with 17.0% wt, while Nannochloropsis oculata - produced the largest biomass on [HCO3 ] 75 ppm treatment of 1.7233 g/l and the greatest yield lipid - content in [HCO3 ] 25 ppm treatment of 20.3% wt. The addition of bicarbonate into the Walne medium with the composition are set in such way on this study has been able to induce the production of biomass and lipids of Nannochloropsis oculata and Chlorella vulgaris significantly compared with control treatments and prior studies.

The authors would like to thank the financial support provided by Universitas Indonesia through the PITTA 2016- 2017 funding scheme under Grant managed by the Directorate for Research and Public Services (DRPM) Universitas Indonesia, West Java, Indonesia.