Bioresource Technology 172 (2014) 444–448

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Short Communication

A usage of CO2 hydrate: Convenient method to increase CO2 concentration in culturing algae Sho Nakano a, Kwang-Hyeon Chang a, Atsushi Shijima b, Hiroyuki Miyamoto c, Yukio Sato c, Yuji Noto c, Jin-Yong Ha c, Masaki Sakamoto c,⇑ a b c

Department of Applied Environmental Science, Kyung Hee University, 1 Seochon-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea Graduate School of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu-shi, Toyama 939-0398, Japan Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu-shi, Toyama 939-0398, Japan

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

carbon source for algal culture was examined.  Addition of CO2 hydrate to culture medium successfully increased CO2 concentration.  Growth of tested algal species can be enhanced by dissolving CO2 hydrate.  Usage of CO2 hydrate allows quantitative supply of carbon to culture system.

a r t i c l e

i n f o

Article history: Received 14 July 2014 Received in revised form 1 September 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: CO2 hydrate Algal culture Pseudokirchneriella Population growth

107 cells/L

 The applicability of CO2 hydrate as

30 Cell density 25 20 15 10 5 0 24 48 0 Time (h)

1.3 g-hydrate/L 3.2 g-hydrate/L 8.0 g-hydrate/L 20 g-hydrate/L

72

a b s t r a c t The addition of CO2 to algal culture systems can increase algal biomass effectively. Generally, gas bubbling is used to increase CO2 levels in culture systems; however, it is difficult to quantitatively operate to control the concentration using this method. In this study, we tested the usability of CO2 hydrate for phytoplankton culture. Specifically, green algae Pseudokirchneriella subcapitata were cultured in COMBO medium that contained dissolved CO2 hydrate, after which its effects were evaluated. The experiment was conducted according to a general bioassay procedure (OECD TG201). CO2 promoted algae growth effectively (about 2-fold relative to the control), and the decrease in pH due to dissolution of the CO2 in water recovered soon because of photosynthesis. Since the CO2 hydrate method can control a CO2 concentration easily and quantitatively, it is expected to be useful in future applications. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Demand for algae has recently increased in various fields, and algal biomass has been utilized for various applications such as sewage water treatment, capture of atmospheric CO2 and as materials for biofuel production (Verma et al. 2010). In water treatment systems, microalgae are utilized as efficient biocarriers for removal of dissolved nitrogen and phosphorus from sewage (Verma et al., ⇑ Corresponding author. Tel.: +81 766 56 7500; fax: +81 766 56 6182. E-mail address: [email protected] (M. Sakamoto). http://dx.doi.org/10.1016/j.biortech.2014.09.019 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

0 g-hydrate/L

2010). Algae can capture the atmospheric CO2 with ten times higher efficiency than terrestrial plants, and store captured CO2 as lipid in cell. This stored lipid can be utilized to produce the biofuel such as bioethanol, methane and biohydrogen. Thus, recent advancements have led to improved design and operation of photobioreactors to achieve high-efficiency CO2 capture, enabling increased microalgal productivity in cultures grown for the production of biofuel (Verma et al., 2010; Pires et al., 2012). Therefore, efficient methods for culturing various algae in large quantities have become very important (Pires et al., 2012).

S. Nakano et al. / Bioresource Technology 172 (2014) 444–448

445

respectively. As the references, water conditions of CO2 hydrate melted in the laboratory (approximately 23 °C), commercial soda water and non-treated distilled water were also analyzed. CO2 hydrate was generated from distilled water at 4 °C and 3.0– 4.0 MPa by an isothermal method (Makogon and Sloan, 1994). 2.2. Experiment 2. Effects of CO2 hydrate on water conditions

Fig. 1. Schematic illustration of two cavities in gas clathrate hydrates (redrawn from Fig. 2.5 in Sloan and Koh, 2008): (a) pentagonal dodecahedron (512) and (b) tetrakaidecahedron (51262).

In algal culture systems that employ carbon dioxide as the primary carbon source, gas bubbling is a common method to supply and control CO2 concentration (Pires et al., 2012). However, when applying this method it is necessary to prepare dedicated equipment and allow sufficient time for dissolution to maintain a high CO2 concentration. In another method, bicarbonate (NaHCO3) has been utilized as a substitute carbon source in algal culture system (Devgoswami et al., 2011). CO2 hydrate which is formed at low-temperature and highpressure, is commonly used for separation and storage of atmospheric carbon dioxide (Linga et al., 2007). The hydrate structures are composed of several polyhedra formed by hydrogen-bonded water molecules (Jeffrey, 1984; Sloan and Koh, 2008). One CO2 Hydrate unit cell is composed with two kinds of cavities, i.e. two pentagonal dodecahedra (512) and six tetrakaidecahedra (51262), and each cavity encapsulate one CO2 molecule (Fig. 1). There are 46 water molecules per unit cell, and the direct evaluation of the hydrate composition is CO2  6.20H2O (Udachin et al., 2001). The generation process and physical property of CO2 hydrate has been studied for CO2 fixation (Linga et al., 2007). However, the usage of CO2 hydrate and application to algal culture has not been considered despite the advantages and applicability of hydrate. CO2 hydrate contains large quantities of carbon dioxide gas and can be used as an efficient material for CO2 supply. Since CO2 hydrate can be kept in a solid state below 0 °C, CO2 concentrations can be controlled quantitatively by weighing and melting CO2 hydrate into a solution directly. Thus, CO2 hydrate can be used as a more efficient source for carbon supply in algal culture systems, while also being used as a quantitative source in various experiments dealing with carbon amounts and their relationship to algal productivity. In this study, we evaluated the capacity and effects of CO2 hydrate added to water as a carbon source in algal culture. We also tested the efficiency of CO2 hydrate applied to algal culture using the green algae (Pseudokirchneriella subcapitata) to investigate the potential for future application of CO2 hydrate to algal cultivation. P. subcapitata is commonly used as a standard test algal species for toxicity testing (OECD, 2011). 2. Methods 2.1. Experiment 1. Comparison with gas bubbling methods Standard CO2 gas (5090 ppm purity, Japan Fine Products Co., Ltd.) or atmospheric air was bubbled into 100 mL distilled water in a 100-mL glass beaker for 30 s without sealing, after which the water temperature (°C), CO2 concentration (mg/L), pH and dissolved oxygen (DO; mg/L) were measured. Water temperature and pH were measured using a pH meter (D-54, Horiba Co., Ltd.), while CO2 and DO were measured with a CO2 meter (CGP-31, DKK-TOA Co., Ltd.) and DO meter (OM51, Horiba Co., Ltd.),

CO2 hydrate prepared from demineralized deep seawater was used to estimate the amount of carbon dioxide that can be dissolved and its effects on culture water conditions. The CO2 hydrate was dissolved into 500 mL distilled water in a 500-mL glass beaker at four concentrations (0, 4, 20 and 100 g-hydrate/L). A solute control was also prepared (100 g-ice/L). Treatments were conducted in triplicate and the beakers were not sealed. Water parameters (water temperature, dissolved CO2, pH and DO) were measured at the start of the experiment (0 h) and 1, 2 and 4 h later. 2.3. Experiment 3. Effect of CO2 hydrate on green algae growth Hydrate prepared from distilled water was used in this experiment. The experiment was conducted according to the general bioassay guidelines (TG-201; OECD, 2011). Briefly, the standard test algae, P. subcapitata, was cultured in hydrate-added medium for 72 h, after which the cell density was measured. A laboratory-cultured single strain of P. subcapitata (NIES-35) obtained from the National Institute for Environmental Studies, Japan, was cultivated in COMBO medium (Kilham et al., 1998). P. subcapitata was cultured in sealed 50-mL Erlenmeyer flasks containing 50 mL COMBO medium adjusted to five CO2 hydrate concentrations (0, 1.3, 3.2, 8.0 and 20 g-hydrate/L). Frozen distilled water was then added to each solution (except for the 20 ghydrate/L treatment) to adjust the total dose (20 g). Samples were subsequently cultivated in an incubator at 21 °C under a 12 h light (120 lmol/m2/s): 12 h dark cycle. During culture, the Erlenmeyer flasks were shaken continuously at 200 rpm using a multi shaker (MMS-210, EYELA). The cell density was then counted under a microscope and water conditions (water temperature, pH, DO and dissolved CO2) were measured before and after the three days. The algal growth rate (day1) from day t0 (=0) to day t (=3) in each treatment was calculated as follows:

Growth rate ¼

lnðX t Þ  lnðX t0 Þ t  t0

ð1Þ

where Xt is the algal cell density (cells/L) at time t (OECD, 2011). 2.4. Statistical analyses The generalized linear model (GLM) was used to estimate the effects of CO2 hydrate dose and time on water conditions (dissolved CO2, pH and DO) in Exp. 2, and the effect of CO2 concentration (at the start of experiment) on algal growth rate in the Exp. 3. Water conditions and algal growth rate were assumed to be gamma (log link) and Poisson distributed, respectively. The best performance model was selected based on AIC (Akaike, 1974; Fukumori et al., 2008). All statistical analyses were conducted using R version R 3.0.3 software (R Development Core Team, 2014). 3. Results and discussion In Exp. 1, the CO2 concentration increased from 4.0 to 22.8 mg/L after CO2 gas bubbling; however, the DO decreased from 7.2 to 3.3 mg/L (Table 1). Similar CO2 levels to those observed in response to gas bubbling were observed when the CO2 hydrate was dissolved into the water at 100 g/L, while no decrease in DO was

446

S. Nakano et al. / Bioresource Technology 172 (2014) 444–448

observed in this treatment. This phenomenon can be explained by the gas–liquid equilibrium in water (Kawasaki et al., 2012). The O2 gas was likely discharged into the air because the gas–liquid interface was filled with CO2 gas due to the bubbling. The melted CO2 hydrate contained high levels of dissolved CO2 (1918 mg/L) and DO (4.8 mg/L), similar to the soda water (dissolved CO2; 1837 mg/L and DO; 5.2 mg/L). These findings indicate that CO2 gas stored in the hydrate diffused in response to melting. However, the pH level of the soda water (5.6) was higher than that of the CO2 hydrate (4.4), suggesting that the pH was influenced by additives in the soda water. Conversely, the increase in CO2 concentration in response to air bubbling was negligible (from 4.0 to 5.4 mg/L). Hanawa et al. (2007) reported that the dissolved CO2 concentration in medium reached 0.07, 1.98, 8.62 and 34.85 mg/L under bubbling with 0.04%, 0.3%, 1.0% and 3.0% (v/v) CO2 gas. In this study, we could increase the CO2 concentration to a similar level by the hydrate method. The CO2 concentration increased to 28.3 mg/L from 1.34 mg/L in response to dissolution of CO2 hydrate (100 g/ L), which was comparable to the effects of 30 s of CO2 gas bubbling (Table 1). In Exp. 2, water temperature decreased slightly in response to dissolution of the CO2 hydrate; however, it was restored to the original condition within 2 h (Fig. 2a). The CO2 concentration showed a rapid increase after dissolution of hydrate, which was maintained until the end of the experiment (Table 2 and Fig. 2b). The CO2

concentration varied greatly within replicates that received 100 g-hydrate/L (6.1–63 mg/L), which might be attributed to the inhomogeneous distribution of CO2 gas in hydrate. Therefore, it is necessary to verify the distribution of CO2 gas in hydrate. In the 100 g-ice/L treatment, the pH was slightly lower than that of the 0 g-hydrate/L treatment; nevertheless, both treatments had equal CO2 concentrations. The pH decreased considerably due to the addition of CO2 hydrate (Table 2 and Fig. 2c). This phenomenon is explained by the chemical equilibrium of inorganic carbon species (Baba and Shiraiwa, 2012). The DO increased with time, but was not influenced by CO2 hydrate (Table 2 and Fig. 2d). In Exp. 3, treatment with 20 g-hydrate/L caused the cell density of P. subcapitata to increase by approximately 2-fold relative to that of the 0 g-hydrate/L treatment within 72 h (Fig. 3a). The growth rate was positively influenced by elevated CO2 concentration (Fig. 3b). Although the initial CO2 concentration varied depending on the hydrate dose, the values at the end did not differ greatly owing to algal consumption (Fig. 4a). The pH values in the 8.0 and 20 g-hydrate/L treatments were lower at the start of the experiment, as with the water experiment (Fig. 4b). However, the values of pH and DO increased with time due to the algal photosynthesis (Fig. 4c and d). Devgoswami et al. (2011) reported the algae growth was enhanced by using bicarbonate salt (NaHCO3) with maintenance of high pH level because, much HCO 3 dissolved in solution with high pH by the chemical equilibrium of inorganic

Table 1 Water conditions in the Exp. 1 and 2. Treatment

Temperature (°C)

CO2 (mg/L)

pH

DO (mg/L)

1

Distilled water CO2 gas bubbling Melted CO2 hydrate Soda water Air bubbling

23.0 21.0 23.6 22.1 21.0

4.0 22.8 1918.0 1837.0 5.4

7.5 6.3 4.4 5.6 6.5

7.2 3.3 4.8 5.2 7.3

2⁄

100 g-ice/L 0 g-hydate/L 4 g-hydate/L 20 g-hydate/L 100 g-hydate/L

20.3 20.8 20.6 21.0 20.5

1.4 (±0.1) 1.3 (±0.1) 2.0 (±0.3) 7.3 (±3.3) 28.3 (±25.0)

6.2 7.5 5.3 5.0 5.1

(±0.4) (±0.0) (±0.1) (±0.1) (±0.1)

(±0.1) (±0.0) (±0.1) (±0.1) (±0.2)

8.7 9.1 9.2 9.3 8.9

Water conditions at the start of the experiment (mean ± SE).

80

(b) CO2 concentration

(a) Water temperature

22

60

20

40 20

18

0

16 10

mg/L

OC

24

(c) pH

8 6 4 0

1

2

3

4

0

1

2

3

mg/L

11.0 10.5 10.0 9.5 9.0 8.5 8.0

(d) DO

pH

⁄

Experiment

4

Hour

Hour 0 g-hydrate/L 100 g-ice/L

4 g-hydrate/L 20 g-hydrate/L 100 g-hydrate/L

Fig. 2. Effect of CO2 hydrate on water conditions (mean ± SE). (a) Water temperature, (b) pH, (c) DO and (d) dissolved CO2 concentration.

(±0.1) (±0.0) (±0.0) (±0.0) (±0.1)

447

S. Nakano et al. / Bioresource Technology 172 (2014) 444–448 Table 2 GLM used to estimate the effects of CO2 hydrate (g/L) and time (h) on water conditions in the Exp. 2. Variables

Parameter coefficient in the best model (± SE)

CO2 pH DO

AIC

Intercept

Hydrate

Time

Hydrate  Time

Best

Full

1.40(±0.13) 1.69 (±0.01) 2.20 (±0.005)

0.04 (±0.005) 0.001 (±0.0004) –

– – 0.01 (±0.002)

– – –

285.89 28.74 16.61

287.64 32.34 13.19

× 107 cells/L

Selection of the best model was based on AIC.

30 25 20 15 10 5 0

(a) Cell density

0 g-hydrate/L 1.3 g-hydrate/L 3.2 g-hydrate/L 8.0 g-hydrate/L 20 g-hydrate/L

0

24

48

72

Time (h)

Day-1

(b) Growth rate

CO2 (mg/L) Fig. 3. Effect of CO2 hydrate on cell density of P. subcapitata. (a) Time dependent changes in cell density (mean ± SE). (b) Relationship between initial CO2 concentration and algal growth rate; solid and dashed lines indicate the regression curve estimated by GLM and 95% confidence interval, respectively. The estimated coefficient and AIC were 0.01 ± 0.0008 and 87.0, respectively.

mg/L

(a) CO2 Concentration

40 20 0

10 mg/L

9

(b) pH Acknowledgements

8 7 6

mg/L

4. Conclusions In this study, the CO2 concentration in water increased by CO2 hydrate dissolving to similar CO2 levels of gas bubbling method. DO level decreased by gas bubbling, however higher DO was maintained by usage of hydrate. In the algal culture system, CO2 hydrate positively affected the algae growth. Therefore, CO2 hydrate can be applied as supplement of carbon source for algal culture. However, it is necessary to consider the cost performance and availability of CO2 hydrate in future study.

80 60

carbon species. In this study, the pH level decreased by using CO2 gas and CO2 hydrate indeed. However, pH decrease can be absorbed by using pH buffers. In large scale algal culture system, the large amount (maximum 330 L) of algae which included freshwater and ocean species have been produced by reactor system or open pond with CO2 gas supplement (Pires et al., 2012). Low-Decarie et al. (2013) reported that P. subcapitata grows well under high CO2 concentrations (ambient CO2 concentration 1000 ppm). We did not supply the hydrate repeatedly; therefore, the CO2 was exhausted at the end of the experiment, indicating that repeated application will be needed for the long-term culture of P. subcapitata. The optimum CO2 concentration for growth varies depending on the species of algae. The growth rate of Chlorella vulgaris increases under high CO2 conditions (e.g., 243 mg/L, Jeong et al., 2003). Conversely, Chiu et al. (2009) reported that the marine algae Nannochloropsis oculata grows well when incubated in the presence of 2% (v/v) CO2 in air, but that its growth was prevented at higher CO2 concentrations (5–15%). Chlamydomonas reinhardtii was found to have a CO2-concentrating mechanism that altered the metabolic pathway depending on CO2 concentration (Hanawa et al., 2007). The specific growth rate of C. reinhardtii was 1.7-fold higher in aeration with 1.0% (v/v) CO2 air than the 0.04% CO2 (Baba and Shiraiwa, 2012). Accordingly, it is necessary to determine suitable CO2 concentrations for targeted algae to enable more effective cultivation.

9.5 (c) DO 9.0 8.5 8.0 7.5 0

24

48

72

We thank Mr. S. Yoneshima and Mrs. N. Watanabe for their assistance during the laboratory work and insightful suggestions. The first author also acknowledges the scholarship from the Department of Applied Environmental Science, Kyung Hee University. This study was partly supported by Grants-in-Aid to M. Sakamoto (No. 23510031) from Japan Society for the Promotion of Science, and a research fund (Collaborative Research Project for Environmentally Conscious Advanced Technology) from Toyama Prefectural University.

Time (h) Control (0 g-hydrate/L) 1.3 g-hydrate/L 3.2 g-hydrate/L

References 8.0 g-hydrate/L 20 g-hydrate/L

Fig. 4. Effect of CO2 hydrate on water conditions (mean ± SE). (a) Dissolved CO2, (b) pH and (c) DO.

Akaike, H., 1974. A new look at the statistical model identification. IEEE Trans. Autom. Control 19, 716–723. Baba, M., Shiraiwa, Y., 2012. High-CO2 response mechanisms in microalgae. In: Najafpour, M.M. (Ed.), Advances in Photosynthesis – Fundamental Aspects. InTech, Croatia, pp. 300–320.

448

S. Nakano et al. / Bioresource Technology 172 (2014) 444–448

Chiu, S.Y., Kao, C.Y., Tsai, M.T., Ong, S.C., Chen, C.H., Lin, C.S., 2009. Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration. Bioresour. Technol. 100, 833–838. Devgoswami, C.R., Kalita, M.C., Talukdar, J., Bora, R., Sharma, P., 2011. Studies on the growth behavior of Chlorella, Haematococcus and Scenedesmus sp. in culture media with different concentrations of sodium bicarbonate and carbon dioxide gas. Afr. J. Biotechnol. 10, 13128–13138. Fukumori, K., Oi, M., Doi, H., Takahashi, D., Okuda, N., Miller, W.T., Kuwae, M., Miyasaka, H., Genkai-Kato, M., Koizumi, Y., Omori, K., Takeoka, H., 2008. Bivalve tissue as a carbon and nitrogen isotope baseline indicator in coastal ecosystems. Coastal Shelf Sci. 79, 45–50. Hanawa, Y., Watanabe, M., Karatsu, Y., Fukuzawa, H., Shiraiwa, Y., 2007. Induction of a high-CO2-inducible, periplasmic protein, H43, and its application as a highCO2-responsive marker for study of the high-CO2-sensing mechanism in Chlamydomonas reinhardtii. Plant Cell Physiol. 48, 299–309. Jeffrey, G.A., 1984. Hydrate inclusion compounds. In: Atwood, J.L., Davies, J.E.D., MacNichol, D.D. (Eds.), Inclusion compounds, vol. 1. Academic Press, London, p. 135. Jeong, M.L., Gillis, J.M., Hwang, J.Y., 2003. Carbon dioxide mitigation by microalgal photosynthesis. Bull. Korean Chem. Soc. 24, 1763–1766. Kawasaki, T., Ohara, H., Nakamura, N., Xu, Q., Matsumoto, H., Kanegae, M., Shiina, T., 2012. Stabilization of anthocyanin in blackcurrant beverages using CO2 microbubbles (in Japanese). Nippon Shokuhin Kagaku Kogaku Kaishi 59, 611– 615.

Kilham, S.S., Keeger, A.D., Lynn, G.S., Goulden, E.C., Herrera, L., 1998. COMBO: a defined freshwater culture medium for algae and zooplankton. Hydrobiologia 377, 47–159. Linga, P., Kumar, R., Englezos, P., 2007. The clathrate hydrate process for post and pre-combustion capture of carbon dioxide. J. Hazard. Mater. 149, 625–629. Low-Decarie, E., Jewell, M.D., Fussmann, G.F., Bell, G., 2013. Long-term culture at elevated atmospheric CO2 fails to evoke specific adaptation in seven freshwater phytoplankton species. Proc. R. Soc. Biol. Sci., 280, 20122598. Makogon, T.Y., Sloan, E.D., 1994. Phase equilibrium for methane hydrate from 190 to 262 K. J. Chem. Eng. Data 39, 351–353. OECD, 2011. Freshwater alga and cyanobacteria, growth inhibition test no. 202. In: OECD Guidelines for Testing of Chemicals. Pires, J.C.M., Alvim-Ferraz, M.C.M., Martins, F.G., Simoes, M., 2012. Carbon dioxide capture from flue gases using microalgae: engineering aspects and biorefinery concept. Renew. Sustainable Energy Rev. 16, 3043–3053. R Development Core Team, 2014. R: a language and environment for statistical computing. In: R Foundation for Statistical Computing. Vienna, Austria. Sloan, E.D., Koh, C.A., 2008. Clathrate Hydrates of Natural Gases, third ed. CRC Press, New York, p. 54. Udachin, K.A., Ratcliffe, C.I., Ripmeester, J.A., 2001. Structure, composition, and thermal expansion of CO2 hydrate from single crystal X-ray measurements. J. Phys. Chem. B 105, 4200–4204. Verma, N.M., Mehrotra, S., Shukla, A., Mishra, N.B., 2010. Prospective of biodiesel production utilizing microalgae as the cell factories: a comprehensive discussion. Afr. J. Biotechnol. 9, 1402–1411.