01 Rinanti.cdr Vol.10, No.3, September 2016, p 79-86 DOI: 10.5454/mi.10.3.1 Enhancing the Removal of Highly Concentrated CO Through Synergism 2 between Microalgae Consortium and Nutrient Ratio in Photobioreactor 1 2 ASTRI RINANTI * AND KANIA DEWI 1 Environmental Engineering Department, Universitas Trisakti, Jalan Kyai Tapa No. 1, Jakarta 11440, Indonesia; 2 Faculty of Civil and Environmental Engineering, Institut Teknologi Bandung, Jalan Ganesha No. 10, Bandung 40132, Indonesia This research was carried out by developing the carbon capture and storage (CCS) technology to determine the synergism between microalgae consortium and the optimum nutrient ratio as an effort to obtain higher CO 2 removal efficiency and CO utilization efficiency. The microalgae consortium consisting of Chlorella sp., 2 Scenedesmus sp., and Ankistrodesmus sp. have been selected previously as potential candidates for Microbial Carbon Capture and Storage (MCCS) agent and already cultured continuously in PHM (Provasoli Haematococcus Media) artificial medium, in vertical column photobioreactor. Pure CO gas at a high 2 concentration of 10% (v/v) flowed from the bottom of vertical column photobioreactor continuously with -1 optimum flow rate of 5 L.min . A growth medium (PHM) containing artificial nutrients was flowed continuously -1 at flow rate 7 L.day and detention time 3.8 days. Four fluorescent lamps were positioned outside the photo- bioreactor to obtain light intensity of 4000 lux, set for 16 hours light exposure and 8 hours dark, with operating temperature 30 °C maintained during the study. Three compositional variations of microalgae consortium were used. They are as follows; Ch : Sc : An = 1: 1: 1; Ch : Sc = 1: 1; and Ch : An = 1: 1, where Ch, Sc, and An were Chlorella sp., Scenedesmus obliquus, and Ankistrodesmus sp., respectively. The following variations of nutrient composition were used; C: N: P = 100: 10: 1, C: N: P = 100: 50: 1 and C: N: P = 100: 25: 1. The C, N, and P sources were CO (inorganic), KNO , and KH PO , respectively. This study proves that synergism between the types 2 3 2 4 making up the consortium also determined the ability to utilize inorganic carbon source. Without the presence of Ankistrodesmus sp., synergism between Scenedesmus obliquus and Chlorella sp. showed twice higher CO2 utilization efficiency in comparison to the synergism between Ankistrodesmus sp. and Chlorella sp. Increased nitrogen concentration in medium increased the growth of Chlorella sp. and Scenedesmus obliquus as a consortium, the CO removal efficiency, the CO utilization efficiency and the Carbon Uptake Rate. The nutrient 2 2 ratios C:N:P of 100:50:1 could increase CO utilization efficiency upto 50% higher than the C:N:P of 100:10:1.2 Key words: CO removal, CO utilization, constructed consortium, nutrient ratio, synergism2 2 Untuk memperoleh rasio nutrisi yang optimal sebagai upaya untuk mendapatkan efisiensi removal dan efisiensi pemanfaatan CO yang lebih tinggi, konsorisum miroba yang terdiri dari Chlorella sp., Scenedesmus 2 obliquus dan Ankistrodesmus sp. dipilih sebagai agen potensial untuk menerapkan metode MCCS (Microbial Carbon Capture and Storage) dan dikultur dengan sistem kontinyu di dalam fotobioreaktor kolom vertikal yang berisi media PHM (Provasoli Haematococcus Media). Gas CO 10% (v/v) dan media PHM dialirkan terus 2 -1 -1 menerus dengan laju alir masing-masing 5 L.menit dan 7L.hari dengan waktu detensi 3,8 hari. Empat lampu (4000 lux) ditempatkan di luar fotobioreaktor, paparan cahaya selama 16 jam terang dan 8 jam gelap, suhu dipertahankan 30 °C selama penelitian. Tiga variasi komposisi jenis mikroalga yang diuji Chlorella sp (Ch) : Scenedesmus obliquus (Ch) : Ankistrodesmus sp. (An) = 1: 1: 1; Ch: Sc = 1: 1; dan Ch: An = 1: 1. Variasi komposisi nutrien yang digunakan adalah Carbon (C) : Nitrogen (N): Phospor (P) = 100: 10: 1, C: N: P = 100: 50: 1, dan C: N: P = 100: 25: 1. Sumber C, N, dan P masing-masing adalah CO (anorganik), KNO , dan KH PO . 2 3 2 4 Studi ini membuktikan bahwa sinergisme ditentukan oleh kemampuan jenis mikroalga dalam memanfaatkan sumber karbon anorganik. Tanpa kehadiran Ankistrodesmus sp., sinergisme antara Scenedesmus obliquus dan Chlorella sp. menunjukkan efisiensi pemanfaatan CO dua kali lebih tinggi dibandingkan dengan sinergisme 2 antara Ankistrodesmus sp. dan Chlorella sp. Rasio nutrien C: N: P sebesar 100: 50: 1 dapat meningkatkan efisiensi pemanfaatan CO hingga 50% lebih tinggi dibandingkan rasio 100: 10: 1.2 Kata kunci: konsorsium terbangun, pemanfaatan CO , penyisihan CO , rasio nutrien, sinergisme2 2 MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone: +62-21-5663232 ext. 8765, Fax:+62-21-5602575; Email: astririnanti@trisakti.ac.id reduce excess amount of CO emitted into the 2 atmosphere. Due to the ability of microalgae to capture CO during the process of photosynthesis, the algae are 2 n o w b e i n g d e v e l o p e d i n t o b i o l o g i c a l o r biotechnological Carbon Capture and Storage (CCS) to reduce carbon emissions that would otherwise be The global warming issue, mainly caused by carbon dioxide (CO ) as the major contributor of 2 greenhouse effects, has triggered various efforts to released into the atmosphere. The carbon capture process works based on the carbon requirement for the process of photosynthesis. Microalgae consortium is a potential producer that requires CO continuously to 2 perform photosynthesis reaction. Compared to various terrestrial plants, microalgae are generally considered photosynthetically more efficient. Chojnacka and Marquez-Rocha (2004) described that there are various types of microalgae based on their metabolism, i.e. autotrophs, heterotrophs, mixotrophs, photohetero- trophs, and able to change its metabolism as a form of adaptation or response to changing environmental conditions. Autotroph microalgae in natural environment, that use inorganic CO from the air and 2 the sun as the main energy source, could change into mixotroph that utilize organic compounds and inorganic CO for growth in close system 2 photobioreactor. Beside that, autotroph microalgae able to change into heterotroph as well when only organic CO2 that naturally available to be transformed become another organic matters. It means the microalgae can change its metabolism as a response to changing environmental conditions. Microalgae can utilize inorganic CO from three different sources, i.e. 2 fixing CO directly from the atmosphere, taking up CO 2 2 from industrial waste gas, and taking up CO from 2 dissolved carbonate (Wang et al. 2008). Microalgae growth is strongly influenced by environmental conditions and the availability of nutrients. Carbon ©, nitrogen (N) and phosphorus (P) are macronutrients much needed by microalgae for making proteins. To achieve their optimum growth, source selection and concentration of nutrients must be adapted to the characteristics of strains of microalgae (Westerhoff et al. 2010). Many types of microalgae can utilize carbonate ions in the form of Na CO and 2 3 NaHCO as sources of C. Other research shows slow 3 algal growth in medium without N (Burkhard et al. 1999). Therefore we need extra N to increase the rate of cell proliferation. N sources commonly used for the cultivation of microalgae is in the form of nitrate, ammonia, urea, or a combination of them. Nitrogen can be obtained from KNO , NaNO , or NH Cl (Cuaresma 3 3 4 et al. 2010). Phosphorus is also the base material forming nucleic acids, enzymes, and vitamins that can be obtained from KH PO , NaH PO , or Ca PO , 2 4 2 4 3 4 whereas elemental sulfur can be obtained from NH SO , or CuSO . The metabolism of these elements; 4 4 4 C, N, and P, are related to one another. Phosphate (P) plays a central role in cellular energy transfer. Low concentration has an impact on the balance in C and N assimilation. Potassium (K) plays a role in the metabolism of carbohydrates and also a cofactor for several coenzymes. Potassium can be obtained from KCl, KNO , or KH PO . Iron (Fe) plays a role in the 3 2 4 formation of chlorophyll and is an essential component in oxidation. This element can be obtained from FeCl , 3 FeSO , or FeCaH O (Graham and Wilcox 2000). 4 5 7 Elements Si and Ca are all ingredients of cell walls or shells. Micro nutrients are also needed to perform various functions in the growth of microalgae. Mn and Zn are necessary for photosynthesis. Mo, Bo, Co are required for the metabolism of nitrogen, while Mn, B, Cu are required to perform other metabolic functions (Westerhoff et al. 2010). The aim of this study was determining the composition of microalgae consortium and the optimum nutrient ratios to obtain higher CO removal 2 efficiency. MATERIALS AND METHODS Microalgae Consortium and Artificial Growth Medium. The consortium of green microalgae consisting of Chlorella sp., Scenedesmus sp., and Ankistrodesmus sp. was originally isolated from the Bojong Soang wastewater treatment plant, Bandung, Indonesia. The microalga was screened, and then a potential candidate was selected as Microbial Carbon Capture and Storage (MCCS) agent (Rinanti et al. 2013). The microalgal cells were cultured in PHM (Phovasoli Haematococcus Media) artificial medium, which was prepared as follows (per litre): 1.0 g KNO ,3 0.2 g KH PO , 0.2 g MgSO .7H O, 0.1 g Fe stock 2 4 4 2 consisting of (per litre) 189 g EDTA and 24.4 g FeCl .6H O. All nutrients were dissolved in distilled 3 2 water containing (per litre) 0.1 mL trace element made of (per 500 mL) 2.05 mg ZnCl , 30.5 mg H BO , 2.55 2 3 3 mg CaCl .6H O, 3.0 mg CuSO .5H O, 2.05 mg 2 2 4 2 MnCl .4H O and 19 mg (NH ) .Mo .O .4H O 2 2 4 6 7 2 4 2 (Provasoli and Pintner 1959). Cultivation of Microalgae in Vertical Column Photobioreactor. The vertical photobioreactor used in this experiment was made ​​of glass with a capacity of 10 L containing 8 L PHM growth medium and an initial 6 -1 cell density of 10 cell.mL . CO was injected from the 2 bottom of the column to allow gas mixing with the medium. Sparger was attached at the bottom of the photobioreactor to convert the gas into small bubbles. Air was bubbled at the bottom. Sparging with microbbuble allows thorough mixing, CO mass 2 transfer and also removes O produced during photo-2 80 RINANTI ET AL. Microbiol Indones synthesis. It was a strategy to provide good overall mixing, sufficient supply of CO , and efficient removal 2 of O . Pure CO gas at 10% (v/v) was flowed from the 2 2 bottom of vertical column photobioreactor -1 continuously with optimum flow rate 5 L.min . A growth medium containing artificial nutrients (PHM) -1 was flowed continuously at a flow rate 7L.day and the detention time of 3.8 days. Four fluorescent lamps were positioned outside the photo-bioreactor to obtain light intensity of 4000 lux, set for 16 hours light exposure and 8 hours dark with operating temperature of 30 °C maintained during the study (Rinanti et al. 2014). Three compositional variations of microalgae consortium were used. They are as follows; Ch : Sc : An = 1: 1: 1; Ch : Sc = 1: 1; and Ch : An = 1: 1, where Ch, Sc, and An were Chlorella sp., Scenedesmus obliquus, and Ankistrodesmus sp., respectively. The following variations of nutrient composition were used; C: N: P = 100: 10: 1, C: N: P = 100: 50: 1 and C: N: P = 100: 25: 1. The C, N, and P sources were CO 2 (inorganic), KNO , and KH PO , respectively.3 2 4 Measurement of Biomass Dryweight. To measure the biomass dryweight, the microalgal culture must be dried by evaporating the liquid in the culture. Prior to the evaporation, culture was centrifuged in 100mL tubes at 3,500 rpm for 10 minutes (Weldy and Huesemann 2007). Supernatant was then removed from the tube and the cell pellet was kept. The pellet was then placed in a petri dish that had previously been weighed (x). The sample-containing petri dishes were then kept in the oven set at 105 °C overnight until constant weight (y) was reached. Then, the sample was left in a desiccator for 30 minutes to evaporate any remaining liquid before re-weighed. Biomass (dry weight) was calculated according to the formula from Torzillo et al. (1991): dry weight (X; mg) = y (mg) - x (mg). -1 Specific growth rate (μ; d ) was calculated as follows: (1) Measurement of CO Concentration and 2 Determination of CO Removal Efficiency. The CO 2 2 concentration in the influent gas and effluent gas was measured by Portable Combination Gas Detector RIKEN Model RX-515. Efficiency of CO removal can 2 be calculated by the following formula: (2) Carbon Uptake Rate. A formula (CO H N P ) 0.48 1.83 0.11 0.01 suggested by Grobbelaar (2004) was used to make an expected estimate of the dry biomass yield and carbon uptake rate was determined by using the following equation: Carbon uptake rate = C x P (3) where C is the carbon content of the dried cell (g carbon.g -1 -1 -1 biomass ), P is the productivity (g biomass.L d ). Results of elemental analysis in our study showed that the carbon content in the mix culture was 67.56%. CO Utilization Efficiency. CO utilization 2 2 efficiency was determined by using the following equation (Ryu et al. 2009): (4) where C is the carbon content of the dried cell (g -1 - carbon.g biomass ), P is the productivity (g biomass.L 1 -1 .h ), 44 and 12 are the molecular weights of carbon dioxide and carbon, respectively, and V is the rate of CO supplied to the microalgal culture medium (g 2 -1 -1 CO .L .h ).2 RESULTS Species Variation of Microalgae Consortium. The three species of microalgae composing the consortium used in this study, Chlorella sp., Scenedesmus obliquus, and Ankistrodesmus sp., seemed to have different physiological characteristics. As mentioned by Chojnacka and Marquez-Rocha (2004), every species of microalgae carries specific physiological properties. Although these three species are capable of living synergistically by utilizing highly concentrated inorganic carbon source (10% v/v) together, synergism between Chlorella sp. and Scenedesmus obliquus showed better growth rate compared to the synergism between Chlorella sp. and Ankistrodesmus sp. (Fig 1, Fig 2, and Table 1). Determination of the Optimum Nutrient Ratios. Previous studies reported about algal cell vitality reduction due to the deficiency of various nutrients. This is related to the loss of the cell's ability to build functional structures associated with the limited amount of nutrients. Hence, the following attempt to improve the CO removal efficiency was done by adding 2 different concentrations of nitrogen to the growth medium (artificial PHM). The CO removal efficiency 2 increased in accordance to the increment of nitrogen addition. Nutrient ratio C : N : P of 100 : 50 : 1 was found to be the optimum ratio and was very sufficient for the Volume 10, 2016 Microbiol Indones 81 % CO utilization2 efficiency carbon content ´ P ´ MCO2 aeration rate of CO2 100%˟ = m = 1 X dX dt % CO removal2 efficiency Influent of CO - Effluent of CO2 2 Influent of CO2 100%˟ = 82 RINANTI ET AL. Microbiol Indones Fig 2 Profile of biomass dry weight (g/L) as a function of different species composition in microalgae consortium. (Ch = Chlorella sp.; Sc = Scenedesmus obliquus; An = Ankistrodesmus sp.). Fig 1 Profile of CO removal efficiency (%) as a function of different species composition in microalgae 2 consortium. (Ch = Chlorella sp., Sc = Scenedesmus obliquus, An = Ankistrodesmus sp.). Fig 3 Profile of biomass dry weight (g/L) as a function of different species composition in microalgae consortium. (Ch = Chlorella sp.; Sc = Scenedesmus obliquus; An = Ankistrodesmus sp.). Volume 10, 2016 Microbiol Indones 83 growth of Chlorella sp and Scenedesmus obliquus consortium, consequently the highest CO removal 2 efficiency of 87.63% was obtained when this ratio was applied (Fig 3 and Table 1). Increased dry weight of the microalgae biomass occured in all tested nutrient ratio variations. In general, the higher the concentration of nitrogen, the higher the dry weight of the biomass produced. Nutrient ratio C : N : P of 100 : 50 : 1 yielded an average -1 biomass dry weight of 8.86 (gL ) at steady state condition (Fig 4). All variations of nutrient ratio reached steady state starting from day 7 until the end of the study. DISCUSSION Species Variation of Microalgae Consortium. Synergism of Chlorella sp. and Scenedesmus obliquus gave the highest CO removal efficiency (84.33%) and 2 -1 biomass dry weight (8.1 g.L ) compared to the other synergism. Likewise, the synergism of these two green microalgae gave a higher CO utilization efficiency and 2 carbon uptake rate than the synergism of other microalgae species in this study. Extremely high CO concentration can cause 2 physiological and metabolic changes consisting of stomatal density reduction, low availability of Rubisco enzyme and chlorophyll, also low photorespiration -1 Fig 4 Profile of biomass dry weight (gL ) as a function of various nutrients composition. (C = carbon, N = nitrogen, P = phosphor). *) Ch = Chlorella sp.; Sc = Scenedesmus obliquus; An = Ankistrodesmus sp.) **) C = CO2 (inorganic), N = KNO3, P = KH2PO4 Table 1 The effects of species variation in microalgae consortium and nutrient ratios towards CO removal and utilization 2 efficiency, biomass dry weight and carbon uptake rate. 84 RINANTI ET AL. Microbiol Indones (Papazi et al. 2008). The microalgae species that is able to thrive in a highly concentrated CO environment may 2 still have the genetic ability to photosynthesize (Papazi et al. 2008). The species that can adopt transitional state in supporting the photosystem is able to grow well at high CO concentration (Miyachi et al. 2003). 2 Microalgae also perform molecular mechanism in response to high CO concentration by changing the 2 photosynthesis mode through Carbon Concentrating Mechanism. Saturated or half-saturated CO 2 concentration can change the characteristics of cellular photosynthesis, for example, the affinity of CO2 reached 0.5% in Chlorella kessleri (Papazi et al. 2008). Composition of microalgae species making up the consortium found in this study indicated that the highest CO utilization efficiency occurred in cultures 2 containing equal amount of Chlorella sp. and Scenedesmus obliquus, without Ankistrodesmus sp.. In the next stage, we tried to find the optimum nutrient ratio to obtain higher CO utilization efficiency by 2 culture containing equal amount of Chlorella sp and Scenedesmus obliquus. Determination of the Optimum Nutrient Ratios. A proper and sufficient nutrient ratio remarkably affects the CO removal efficiency. Nonetheless, it was 2 not significantly different from nutrient ratio with C : N : P = 100 : 25 : 1, which produced 86.03% CO removal 2 efficiency. Considering these results, adding higher nitrogen concentration were not expected to increase the CO removal efficiency, and hence was not studied.2 Microscopical observation showed that the size of Chlorella cells tends to be bigger in the culture with high concentration of CO gas (10% v/v). Therefore, 2 despite the relatively lower cell density in 10% (v/v) CO concentration, a higher biomass dry weight was 2 achieved (Fig 4). It is easily understandable because with adequate supply of carbon, microalgae have greater opportunities to thrive than with limited carbon supply. This result is similar to the studies carried out by Yoo et al. (2010) and Chiu et al. (2008), where pure 10% CO2 was also streamed into the reactor. Thus, an optimum CO concentration of 10% is needed to gain 2 high productivity of microalgae biomass. Zhu et al. (2010) explained that the right composition of nutrients is one of the main factors affecting microalgal biomass productivity, in addition to light intensity, temperature, and CO mass transfer 2 into the liquid (Ono and J. Cuello 2006). The efficiency of CO utilization improves in 2 accordance to the escalation of nitrogen source concentration in the growth medium. The highest CO 2 utilization efficiency of 5.22% occured in the culture with C : N : P ratio of 100 : 50 : 1. Chojnacka and Marquez-Rocha (2004) described that microalgae could perform various types of metabolism, i.e. a u t o t r o p h , h e t e r o t r o p h , m i x o t r o p h , a n d photoheterotroph, and able to perform metabolic alteration as an adaptation measure or response to shifting environmental conditions. During this study, it seemed that the microalgae consortium comprising of Chlorella sp and Scenedesmus obliquus was mixotroph in conducting photosynthesis as the main energy source, yet it could maximize the utilization of nitrogen, organic compounds, and inorganic CO for its 2 growth. Adequate nutrient availability for microalgae is a precondition for optimum photosynthesis. Nutrient deficiency will cause interferences with metabolism and production discrepancy in intermediary phase of photosynthesis. However, the correct ratio of carbon, nitrogen, and phosporus as the main nutrients also immensely affects the metabolism, for instance affecting Carbon Uptake Rate. In this study, carbon uptake rate was defined as the absorption rate of CO 2 dissolved in the PHM growth medium by microalgae consortium. When the photosynthesis took place, pure CO gas flow of 10% at 5 L/min flow rate was the 2 primary source of inorganic carbon consumed by microalgae to generate energy. The rate of CO uptake 2 by microalgae cells was allegedly stimulated by an increase of CO content in the media, so that the culture 2 without CO addition absorbed less CO , escalating the 2 2 culture pH, yet had never exceeded value of 8. Stepan et al. (2002) stated that media pH ranging from 7.0–8.0 is good enough for laboratory microalgae culture. Accordingly, the media pH was maintained at 7 during the study. Microalgae commonly use nitrate as the primary nitrogen source. Shi et al. (2007), Bich et al. (1999), and Qing-xue et al. (2010) explained that nitrogen compounds are strongly influenced by dissolved oxygen concentration in the water. Nitrogen turns into ammonia (NH ) at low-level dissolved 3 oxygen and into nitrate (NO ) at high-level dissolved 3 oxygen. Nonetheless, when the environmental conditions are not favorable, then ammonia or urea can serve as nitrogen source (Sassano 2007; Soletto 2005). The lack of nitrogen content will result in the limited production of proteins needed for new cells (Westerhoff et al. 2010). This study proved that in addition to the specific characteristics of microalgal cells' physiology, synergy between the types making up the consortium also Volume 10, 2016 Microbiol Indones 85 determine the ability to utilize inorganic carbon source. Chlorella sp have the highest synergy to interact together well with the Scenedesmus obliquus or Ankistrodesmus sp. Without the presence of Ankistrodesmus sp, synergism between Scenedesmus obliquus and Chlorella sp. provides twice higher CO2 utilization efficiency than the synergism between Ankistrodesmus sp and Chlorella sp without the presence of Scenedesmus obliquus. Increased nitrogen proved to increase the growth of Chlorella sp and Scenedesmus obliquus as a consortium in line with the increase of CO2 removal efficiency, CO2 utilization efficiency and carbon uptake rate. The availability of macro nutrients, inorganic carbon, nitrogen, and phosphorus, in the appropriate ratio (C: N: P of 100: 50: 1) may increase CO utilization efficiency 50% higher than when the C: 2 N: P ratio was 100: 10 : 1. However, the efficiency was not significantly different from when the C: N: P was 100: 25: 1. ACKNOWLEDGMENT We thank DIKTI's (General Directorate of Higher Education Indonesia) Decentralization Program 2015 for funding some parts of this research. REFERENCES Anjos M, Bruno D. Fernandes, António A. Vicente, José A. Teixeira, Giuliano Dragone. 2013 Optimization of CO 2 bio-mitigation by Chlorella vulgaris. Bioresour Technol. 139: 149–154. http://dx.doi.org/10.1016/ j.biortech.2013.04.032. Burkhard S, I. Zondervan, U. Riebesell. 1999. Effect of CO 2 concentration on C : N : P ratio in marine phytoplankton : A species comparisons. Limnol Ocean. 44 (3): 683 - 690. Chiu SY, Kao CY, Tsai MT, Ong SC, Chen CH, Lin CS. 2009. Lipid accumulation and CO utilization of 2 Nannochloropsis oculata in response to CO aeration. 2 Bioresour Technol. 100: 833-838. doi: 10.1016/ j.biortech.2008.06.061. Chojnacka K, Marquez-Rocha FJ. 2004. Kinetic stoichiometric relationships of the energy and carbon m e t a b o l i s m i n t h e c u l t u r e o f m i c r o a l g a e . Biotechnology 3(1): 21–34. doi: 10.3923/biotech. 2004.21.34. Cuaresma M, Casal C, Forjan E, Vilchez C. 2010. Productivity and selective accumulation of carotenoids of the novel extremophile microalga Chlamydomonas acidophila grown with different carbon sources in batch systems. J Ind Microbiol Biotechnol. 38(1):167- 177. doi: 10.1007/s10295-010-0841-3. Graham LE, Graham JM, Wilcox LW. 2009. Algae, 2nd ed., San Francisco: Pearson Education, Inc. Grobbelaar JU. 1989. Do light/dark cycles of medium frequency enhance phytoplankton productivity. J Appl Phycol. 1(4): 333-340. doi: 10.1007/BF00003470. Kong QX, Li L, Martinez B, Chen P, Ruan R. 2010. Culture of microalgae Chlamydomonas reinhardtii in wastewater for biomass feedstock production. Appl Biochem Biotechnol Journal 160:9–18. doi: 10.1007/s12010-009-8670-4. Miyachi S, Iwasaki I, Shiraiwa Y. 2003. Historical perspective on microalgal and Cyanobacterial acclimation to low-and extremely high-CO conditions. 2 Photosynth. Res. 77: 139-153. doi: 10.1023/ A:1025817616865. Ono E, Cuello JL. 2009. Mini Review : Selection of optimal microalgae species for CO sequestration. Departemen 2 of Agricultural and Biosystem Engineering. USA Papazi A, Makridis P, Divanach P, Kotzabasis K. 2008. Bioenergetic changes in the microalgal photosynthetic apparatus by extremely high CO concentrations induce 2 an intense biomass production. Physiol. Plant. 132: 338-349. doi: 10.1111/j.1399-3054.2007.01015.x. Provasoli L, Pintner IJ. 1960. Artificial Media for Freshwater Algae: Problems and Suggestions. In: The Ecology of Algae. Tryon, C.A. and Hartman, R.T. (ed.). Special Publication No. 2. Pymatuning Laboratory of Field Biol., University Pittsburgh, USA. Rinanti A, Kardena E, Astuti DI, Dewi K. 2013. Screening of potential photosynthetic microalgae from wastewater treatment plant for carbon dioxide capture and storage (CCS) agent. Asian Transactions Sci Technol J. 03(1): 1-8. Rinanti A, Kardena E, Astuti DI, Dewi K. 2014. Improvement of carbon dioxide removal through artificial light intensity and temperature by constructed green microalgae consortium in a vertical bubble column photobioreactor. Malaysian J Microbiol. 10 (1): 29-37. http://dx.doi.org/10.21161/mjm.56713. Sassano CEN, Gioielli LA, Almeida KA, Sato S, Perego P, Converti A, and Carvalho JCM. 2007. Cultivation of Arthrospira (Spirulina) by continuous process using ammonium chloride as nitrogen source. Biomas B i o e n e r g . 3 1 : 5 9 3 - 5 9 8 . d o i : 1 0 . 1 0 1 6 / j.biombioe.2007.04.001. Shi J, Podola B, Melkonian M. 2007. Removal of nitrogen and phosphorus from wastewater using microalgae immobilized on twin layers: an experimental study. J Appl Phycol. 19(5): 417-423. doi: 10.1007/s10811- 006-9148-1. 86 RINANTI ET AL. Microbiol Indones Stepan DJ, Shockey RE, Moe TA, Dorn R. 2002. Subtask, 2.3 carbon dioxide sequestration using microalgae systems. Energy and Environmental Research Center. University of North Dakota. Torzillo G, Sacchi A, Materassi R. 1991. Temperature as an important factor affecting productivity and night biomass loss in Arthrospira (Spirulina) platensis grown outdoors in tubular phtobioreactors. Bioresour Technol. 38: 95-100. Wang B, Li Y, Wu N, Lan CQ. 2008. CO bio-mitigation 2 using microalgae. Appl Microbiol Biotechnol. 79(5): 707-718. doi: 10.1007/s00253-008-1518-y. Wang J, Wang J, Zhou W, Yang W, Ruan R. 2016. Application of nitrogen sufficiency conversion strategy for microalgae-based ammonium-rich wastewater treatment. Environment Technol J. (37) 20. http://dx.doi.org/10.1080/09593330.2016.1158744. Weldy CS, Huesemann M. 2007. Lipib production by Dunaliella salina in batch culture: effects of nitrogen limitation and light intensity. US Department of Energy. J Undergraduate Res. 7(1): 115–122. Westerhoff P, Hu Q, Esparza-Soto M, Vermaas W. 2010. Growth parameters of microalgae tolerant to high levels of carbon dioxide in batch and continuous-flow photobioreactors. Environment Technol J. 31: 523-532. doi: 10.1080/09593330903552078. Yoo C, Jun SY, Lee JY, Ahn CY, Oh HM. 2010. Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour Technol. 101(2010): 571- 574. doi: 10.1016/j.biortech.2009.03.030. Zhu XG, Long SP, Ort DR. 2010. Improving Photosynthetic Efficiency for Greater Yield. Annu. Rev. Plant Biol.61: 235-261. doi: 10.1146/annurev-arplant-042809-112206. Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8