Accepted Manuscript Control of CO2 input conditions during outdoor culture of Chlorella vulgaris in bubble column photobioreactors Zhi Guo, Wei Boon Alfred Phooi, Zi Jian Lim, Yen Wah Tong PII: DOI: Reference:

S0960-8524(15)00397-1 http://dx.doi.org/10.1016/j.biortech.2015.03.065 BITE 14755

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

22 December 2014 9 March 2015 12 March 2015

Please cite this article as: Guo, Z., Phooi, W.B.A., Lim, Z.J., Tong, Y.W., Control of CO2 input conditions during outdoor culture of Chlorella vulgaris in bubble column photobioreactors, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.03.065

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Control of CO2 input conditions during outdoor culture of Chlorella vulgaris in bubble column photobioreactors

Zhi Guo, Wei Boon Alfred Phooi, Zi Jian Lim, Yen Wah Tong * Department of Chemical and Biomolecular Engineering, National University of Singapore 4 Engineering Drive 4, Singapore 117576

*Corresponding Author: Yen Wah Tong Tel: (65) 65168467; Fax: (65) 67791936; E-mail Address: [email protected]

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ABSTRACT A study on the optimization of CO2 usage during outdoor microalgae cultivation in order to further maximize the CO2 to biomass conversion efficiency is presented. A constant supply of CO2 was found to be non-essential for culturing microalgae outdoors in 80 L (8 L×10 sets) bubble columns. Among the different CO2 input conditions that were studied, 2% CO2 with intermittent supply and 2% + 4% CO2 alternation did not affect the algal growth as compared to having a constant supply of 2% CO2. However, during both input conditions, the CO2 to biomass conversion efficiency was doubled while the amount of CO2 used was reduced by 50%. The algal biomass obtained was found to have a higher carbohydrate yield but a lower protein yield as compared to previously published studies. The findings from this study could be applied for large-scale microalgae production so as to minimize cultivation and energy costs.

Keywords: Chlorella vulgaris; CO2 input condition; outdoor microalgae cultivation; bubble column; photobioreactors.

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1. Introduction Energy security and global climate change are among the most important and pressing issues concerning the world today that has led to an ongoing research for more sustainable and environment-friendly energy sources as alternatives to fossil fuels (Chisti, 2008). In this regard, biofuels have emerged as one of the promising contenders having both renewable and carbon-neutral potential (Ragauskas et al., 2006). With diverse terrestrial crops being investigated as feedstock for biofuels production, the first-generation biofuels that used food crops (such as maize) as the feedstock have possibly aggravated food shortages, since increase of biofuel production capacity competes with food production. On the other hand, second-generation biofuels used non-food sources such as wood and food wastes as the feedstock and does not impact the food production. However, the second-generation biofuels are associated with prohibitive costs of feedstock pretreatment that limit their wide spread usage. Lately, microalgae have become the most promising source for third-generation biofuels that exhibit great potential to overcome the challenges encountered by the previous generation biofuels. Microalgae have a higher biomass yield per unit of light and area as well as exhibit a higher growth rate as compared to conventional crops (Dismukes et al., 2008). Besides, they do not compete with the food production industries for arable land. In addition, the nutrients required by the microalgae could be supplied by wastewater and the CO2 necessary for microalgal photosynthesis can be acquired from exhaust gases released from power plants (Parmar et al., 2011). In some species such as Botryococcus braunii, it was found that up to 75% of its dry weight consisted of lipids 3

(Banerjee et al., 2002), which can be converted to biodiesel via transesterification. Further, the carbohydrates in algal cells can be converted to bioethanol via fermentation (Choi et al., 2010) and the algal proteins can also be converted to biofuels using bacteria modified by metabolic engineering (Huo et al., 2011). Other than being a source of biofuels, numerous microalgal strains can be utilized for a wide range of applications. For instance, they can be used as microbioreactors to produce chemical products that are currently being used in food, cosmetics, nutrition and pharmaceutical industries (Gong et al., 2011). Chlorella vulgaris (C. vulgaris) is one such microalgal strain that has been studied extensively in lab-scale experiments (Ogawa and Aiba, 1981; Martínez and Orús, 1991; Liu et al., 2008). It exhibits high growth rate and its lipid content can be as high as 53% of its dry cell weight under certain stressed conditions (Mujtaba et al., 2012). Brányiková et al. (2011) reported 60% carbohydrate content in dry C. vulgaris biomass, which potentially makes it a viable feedstock for bioethanol production. In addition, C. vulgaris is able to tolerate temperatures of up to 40 °C, which is crucial in avoiding algal culture crashes in high outdoor temperature conditions (Ho et al., 2011). The growth of C. vulgaris has also been found to be enhanced when cultured with a symbiotic bacteria species (Guo el al. 2014). Finally, C. vulgaris is one of the few microalgae strains capable of developing suitable molecular mechanisms that allow it to adapt and efficiently utilize high concentrations of CO2 (Concas et al., 2012). Outdoor cultivation of microalgae is very advantageous since it makes the system more energy-efficient by directly utilizing sunlight as the light source, while CO2 from flue

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gases can be used as the carbon source to further reduce production costs (Doucha et al., 2005). Also, outdoor cultivation is more suitable for scaling up to pilot-scale or to commercial large-scale algal biomass production. In this study, C. vulgaris was cultivated in pilot-scale 80 L (8 L×10 sets) bubble column photobioreactors (PBRs) in a tropical outdoor environment without temperature and pH control. The feasibility of C. vulgaris outdoor cultivation was investigated using different CO2 input conditions in order to study the effects of CO2 on C. vulgaris growth and algal metabolites accumulation, with the eventual aim of optimizing CO2 usage and maximizing the CO2 to biomass conversion efficiency.

2. Methods 2.1. Microalgae strain and medium C. vulgaris ATCC® 13482TM used in this study were purchased from the American Type Culture Collection (ATCC, Rockville, MD). The stock cultures were maintained in both agar plates and liquid medium of 3N-BBM+V (modified Bold Basal Medium with 3-fold Nitrogen and Vitamins). The 3N-BBM+V medium consisted of macro-nutrients: 0.75 g NaNO3, 0.025 g CaCl2•2H2O, 0.075 g MgSO4•7H2O, 0.075 g K2HPO4•3H2O, 0.175g KH2PO4, 0.025 g NaCl and micro-nutrients: 4.5 mg Na2EDTA, 0.582 mg FeCl3•6H2O, 0.246 mg MnCl2•4H2O, 0.03 mg ZnCl2, 0.012 mg CoCl2•6H2O, 0.024 mg Na2MoO4•2H2O, 1.2 mg Thiamine hydrochloride as well as 0.01 mg Cyanocobalamin, per liter of DI water. All chemicals were purchased from Sigma-Aldrich (Singapore).

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2.2. Cultivation of C. vulgaris in the lab The inoculum of C. vulgaris for outdoor cultivation was cultured indoors in a 4 L glass conical flask containing 3 L autoclaved 3N-BBM+V medium. The contents of the culture flask was magnetically stirred to provide good mixing under room temperature and the algal culture was fed with sterile air at a gas flow rate of 0.4 vvm (volume gas per volume medium per min). The illumination was provided by four cool fluorescent lamps using a 12 h light/ 12 h dark cycle. The pH of the medium was adjusted to be 6.5 using 1 M NaOH solution. When a cell concentration of 1 g/L was obtained, the algal culture was used as inoculum for the subsequent outdoor cultivation.

2.3. Outdoor cultivation of C. vulgaris in bubble columns Culture medium 3N-BBM+V (7.5 L) was inoculated with the above prepared C. vulgaris culture in pilot-scale bubble columns (0.1 m diameter and 1.014 m height). The initial density of C. vulgaris was adjusted to be at 0.25 g/L. Pure CO2 was mixed with sterile air and pumped into the bottom of the bubble column at a flow rate of 0.4 vvm regulated by a gas flow-meter (Cole-Parmer, Illinois, USA). CO2 concentrations (v/v) of 2%, 4% and 8% were also controlled by the gas flow-meters. Different CO2 input conditions but with the same total amount of CO2 (112 L) was adjusted manually as shown in Fig. 1. The following CO2 input conditions were used: Condition A of 2% CO2 provided intermittently (1 h 2% CO2 enriched air/1 h air); Condition B of 4% CO2 provided intermittently (40 min 4% CO2 enriched air thrice per day while sparging only

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air during the rest of the cultivation time); Condition C of 2% & 4% CO2 alternatively supplied (30 min 4% CO2 enriched air twice and 1 h 2% CO2 enriched air twice per day while sparging only air during the rest of the cultivation time) and Condition D of 2% CO2 provided corresponding to the increase of algal biomass concentration. CO2 was provided only during daytime. All the experimental facilities were located on the roof of a building for sunlight & weather exposure and the cultivation period was 7 days.

2.4. Analytical methods 2.4.1. Outdoor cultivation parameters measurement Light intensity on the column surface was measured with a photometer (STI&BAL, China) and temperature of the algal culture in the bubble column was measured using a thermometer (Brannan, England). The pH of the algal culture was determined using a pH meter (Thermo Scientific, USA) and the dissolved oxygen (DO) concentration was measured using a DO meter (Eutech Instrument, USA). The official weather data including sunlight hours and maximum/minimum environmental temperature was obtained

from

the

National

Environment

Agency

(NEA)

of

Singapore

(www.nea.gov.sg).

2.4.2. Biomass concentration measurement The algal biomass was harvested and centrifuged at 5600×g for 10 min. The supernatant was discarded and the cell pellets were washed thrice with DI water. The pellets were dried in a vacuum oven and the weight of dry algal biomass was determined by

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gravimetric analysis. The algal growth curve was expressed in terms of dry biomass weight (g/L). The biomass productivity (g/L•d) was determined from equation (Eq. (1)): PBiomass =

Ct − C0 (1) t

where Ct was the biomass concentration after 7 days, C0 was the initial biomass concentration and t was the cultivation time. The dry algal biomass was pulverized to a fine powder using a mortar and pestle for subsequent analyses.

2.4.3. CO2 fixation rate and CO2 to biomass conversion efficiency The CO2 content in the inlet and the effluent gas mixture was determined by Gas Chromatography (GC Agilent 6890N, USA). Physically dissolved CO2 in the medium or algal culture was measured by a CO2 analyzer (OxyGuard, Denmark). The CO2 fixation rate (g CO2/L•d) was calculated using equation (Eq. (2)):

 M CO2  PCO2 = Ccarbon Pbiomass   (2)  MC  where MC was the molecular weight of carbon, MCO2 was the molecular weight of CO2 and Ccarbon was the carbon content in the algal biomass.

CO2 to biomass conversion efficiency (%) was calculated from equation (Eq. (3)):

ECO2 =

PCO2Vcolumnt

ρCO VCO 2

(3)

2

where Vcolumn was the working volume of the bubble column, VCO2 was the volume of total CO2 consumed during cultivation and ρCO2 was the density of CO2.

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2.4.4. Fatty acids quantification and analysis In situ transesterification was applied for fatty acids (FAs) quantification and composition analysis. The dry algal biomass was homogenized in methanol and disrupted through sonication using an ultrasonic processor (Sonics® Vibra-CellTM, VCX130, 130 watts, 20 kHz) for 15 min. After sonication, hexane was added and transesterification was performed at an acid concentration of 2% (v/v) using concentrated sulfuric acid (98%). The mixture was stirred at room temperature for 15.5 h. After that, DI water was added to the mixture and vortexed for 10 min before being centrifuged at 1200×g for 5 min. The hexane layer containing fatty acids methyl esters (FAMEs) was extracted and analyzed by GC-MS (Agilent 7890A Gas Chromatography coupled with an Agilent 5975C Mass Spectrometry). The GC-MS was equipped with a HP-5ms capillary column (5% phenyl methyl silox, 30 m × 0.25 mm inner diameter (ID) and 0.25 µm film thickness). Helium was injected as the carrier gas into the column. The oven temperature was programmed at an initial temperature of 50 °C and it was raised linearly at a rate of 15°C min-1 to 180°C, then to 230 °C at 2 °C min-1 and finally to 300 °C at 45°C min-1. By comparing the retention times of the corresponding peaks and their fragmentation pattern with known standards (AccuStandard, USA) and with the NIST library, the FAMEs were identified. Internal standard (heptadecanoic acid methyl ester) and calibration curve was used to quantify the amount of FAMEs.

2.4.5. Carbohydrate extraction and quantification

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The carbohydrates in the dry algal biomass were extracted using 2 mL of 80% sulfuric acid.

The mixture was stirred for 20 h at 4°C (Myklestad and Haug, 1972). The

mixture was then filtered and the filtrate was collected. DI water was used to wash the cell debris thrice and harvested to mix with the previous solution. The total amount of carbohydrates in the solution was determined using the phenol–sulfuric acid method (DuBois et al., 1956) using glucose as the standard.

2.4.6 Protein extraction and quantification The proteins in the dry algal samples were extracted based on the method from Barbarino and Lourenco (2005), which was modified accordingly in this work. Firstly, 1 mL of DI water was mixed with the dry algal biomass powder and stirred for 24 h at room temperature. The mixture was then centrifuged and the supernatant was collected. Subsequently, 1 mL 0.1 N NaOH was used to resuspend the residual cell pellet and this suspension was stirred for 1 hour. The supernatant was collected after centrifugation and mixed with the previously obtained supernatant. Finally, the algal cell pellet was washed three times with DI water and the supernatant after every wash was collected after centrifugation. The proteins in the supernatant solutions were quantified by using the Micro- BCATM Protein Assay Kit (Thermo Scientific, USA) using bovine serum albumin as the standard.

2.4.7. Elemental analysis

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Dry pulverized algal biomass powder was sent to the Elemental Analysis Laboratory, Department of Chemistry, Faculty of Science, National University of Singapore for analysis of carbon, hydrogen and nitrogen elemental contents. The instrument used was Elementar Vario Micro Cube (ELEMENTAR Analysensysteme GmbH, Germany).

2.5. Statistical analysis The data presented in Fig. 2c was normalized using 0 d dry biomass under sparging air only condition and expressed as normalized biomass ratio. The data shown in Fig. 5b as well as in Tables 1, 2 and 4 were normalized correspondingly using data of sparging air only each day. Results were expressed as mean ± SD of triplicate independent cultures. Differences between the groups were statistically analyzed by using ANOVA and statistical significance was denoted by * (p90% of total FAs) and contained a perceptible amount of C14:0 and C15:0. Higher content of C18:1 was

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obtained than C16:1. The FAMEs composition under different CO2 input conditions were similar with the above results, but some FAs, such as C16:17 and C16:2, were not detected in conditions C and D. In addition, the lipids of C. vulgaris did not contain C16:4 in condition D (Table 4). The composition of microalgal FAs determines the quality of the microalgae-based biodiesel. SFAs and MUFAs are desirable while PUFAs may affect the oxidative stability of the biodiesel (Stansell et al., 2012). The C. vulgaris obtained in this study with higher SFAs and lower PUFAs would be a suitable feedstock for biodiesel production.

3.5. Carbohydrate yield of C. vulgaris under outdoor conditions Carbohydrates are desirable biomass compounds for the production of several biofuels. In this study, the carbohydrate yield of C. vulgaris under different CO2 input conditions fluctuated as shown in Fig. 5. Carbohydrates tend to accumulate when the microalgae are subjected to stressed conditions (Markou et al., 2012). The starch content in C.

vulgaris can go up to 60% of the dry biomass weight induced by sulfur limitation and cycloheximide treatment (Brányiková et al., 2011). The highest carbohydrate yield obtained in this study was 521 mg/g dry biomass under outdoor conditions which was similar to the results published by Brányiková et al. Since the outdoor cultivation was operated without temperature control, high temperatures (up to 40 °C) inside the bubble columns could have stressed the algal cells and may have altered the carbohydrate yield of C. vulgaris. A significant amount of carbohydrate-rich algal biomass residual was

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often disposed after lipid extraction. However, the residual algal biomass can also be used to produce bioethanol as reported previously (Lee et al., 2013). Anaerobic digestion for methane production could also be a solution to recover more energy from microalgal residues and further enhance the economic feasibility of a microalgal biorefinery (Sialve et al., 2009).

3.6. Protein yield of C. vulgaris under outdoor conditions Microalgae have been used as microbioreactors to produce recombinant proteins and more than 20 therapeutically important proteins have been successfully expressed in microalgae (Specht et al., 2010). Previously, C. vulgaris has been investigated as feedstock for protein production and the protein content can reach as high as 55% of the dry biomass weight (Li et al., 2013). In this study, variation in the protein yield values of C. vulgaris was observed under different CO2 input conditions (Fig. 5) and the values were much lower as compared to prior studies (Li et al., 2013). The residual algal biomass obtained after lipid extraction was still found to contain proteins. Anaerobic digestion of protein-rich microalgal residues may release ammonia, which could be toxic to the digestion process. In this study, the residual biomass of C. vulgaris obtained after lipid extraction having a lower protein yield would be highly suitable for follow-up methane production.

4. Conclusion In this study, CO2 usage was successfully optimized and CO2 to biomass conversion efficiency was greatly improved in C. vulgaris outdoor cultivation. The amount of CO2

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used was reduced by 50% and CO2 to biomass conversion efficiency was doubled when 2% CO2 was intermittently provided, while the growth of C. vulgaris was not affected as compared to constant sparging of 2% CO2. CO2 enriched aeration was found to promote FAMEs yield and modify the FAMEs composition of C. vulgaris under outdoor conditions. The yield of FAMEs, carbohydrates and proteins under different CO2 input conditions were not significantly different.

Acknowledgments We acknowledge the funding support under the grant number R302000011112 and the research scholarship for Mr Zhi Guo from the National University of Singapore. This research programme is funded by the National Research Foundation (NRF), Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) programme.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at (website link).

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Figure Captions Fig. 1. Different CO2 input conditions: Condition A: 2% CO2 provided intermittently (1 h 2% CO2 enriched air/1 h air) (a); Condition B: 4% CO2 provided intermittently (40 min 4% CO2 enriched air thrice per day) (b); Condition C: 2% & 4% CO2 alternatively supplied (30 min 4% CO2 enriched air twice and 1 h 2% CO2 enriched air twice) (c); Condition D: 2% CO2 provided corresponding to the increase of algal biomass concentration (d). Fig. 2. Biomass concentration (g/L) of C. vulgaris ATCC® 13482TM cultivated in bubble columns under outdoor conditions: Effect of gas flow rates (a), Effect of different CO2 concentrations (b), Effect of different CO2 input conditions (c).

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Fig. 3. The change in sunlight intensity, dissolved oxygen concentration and pH during

C. vulgaris culture on a typical day in a tropical outdoor environment. Fig. 4. Dissolved CO2 concentration under different CO2 concentrations (v/v) in C.

vulgaris culture and 3N-BBM+V medium. Fig. 5. FAMEs, carbohydrates and protein yields of C. vulgaris under different CO2 input conditions (means ± SD): Effect of different CO2 concentrations (a), Effect of different CO2 input conditions (b). * - shows statistical significance (p

Control of CO₂ input conditions during outdoor culture of Chlorella vulgaris in bubble column photobioreactors.

A study on the optimization of CO2 usage during outdoor microalgae cultivation in order to further maximize the CO2 to biomass conversion efficiency i...
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