Growth characteristics of Botryococcus braunii 765 under high CO2 concentration in photobioreactor

Bioresource Technology 102 (2011) 130–134

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Growth characteristics of Botryococcus braunii 765 under high CO2 concentration in photobioreactor Yaming Ge, Junzhi Liu, Guangming Tian * Department of Environmental Engineering, Zhejiang University, Hangzhou 310029, China

a r t i c l e

i n f o

Article history: Received 6 January 2010 Received in revised form 27 May 2010 Accepted 7 June 2010 Available online 26 June 2010 Keywords: Botryococcus braunii Carbon dioxide Hydrocarbon Sodium hypochlorite Colony

a b s t r a c t To understand the potential of cultivating Botryococcus braunii with flue gas (normally containing high CO2) for biofuel production, growth characteristics of B. braunii 765 with 2–20% CO2 aeration were investigated. The results showed that the strain could grow well without any obvious inhibition under all tested CO2 concentrations with an aeration rate of 0.2 vvm, even without any culture pH adjustment (ranged from 6.0 to 8.0). The maximum biomass among all conditions was 2.31 g L 1 on 25th day at 20% CO2. Hydrocarbon content and algal colony size increased with the increase of CO2 concentration. A negative correlation between algal biomass and culture total phosphorus was observed (from 0.828 to 0.911, P < 0.01). Additionally, 2% sodium hypochlorite solution was used for photobioreactor sterilization to cultivate B. braunii. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae are a group of unicellular or simple multicellular photosynthetic microorganisms that can fix CO2 efficiently from different sources, including the atmosphere, industrial exhaust gases, and soluble carbonate salts (Wang et al., 2008). In recent years, microalgae have attracted more attention for the possibility of being exploited as a renewable source of oil (Metzger and Largeau, 2005; Miao and Wu, 2006; Yoo et al., 2010), whereas its commercial development has been prevented by both technical and economic problems (An et al., 2003). Many reports have suggested using flue gas as carbon origin for microalgal cultivation, which could combine biofuel production with current CO2 mitigation strategies (Vunjak-Novakovic et al., 2005; Wang et al., 2008; Yoo et al., 2010). However, in this case the influences of high CO2 concentration on microalgal growth must be investigated (Ono and Cuello, 2003). Botryococcus braunii—a green colonial microalgae, due to its high hydrocarbon content, is one of the most understudied oil-rich algae. Ranga Rao et al. (2007b) investigated the growth characteristics of B. braunii aerated with 0.5%, 1.0%, and 2.0% CO2, reporting that 2.0% CO2 aeration enhanced algal growth with a twofold increase in biomass and b-carotenoid contents compared with control. Yoo et al. (2010) confirmed that B. braunii could grow with 10% CO2 and flue gas (containing 5.5% CO2) during 14-day cultiva* Corresponding author. Tel.: +86 571 86971975; fax: +86 571 86971898. E-mail address: [email protected] (G. Tian). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.051

tion. In those tested CO2 concentrations, 2.0% was regarded as the best for B. braunii growth. However, since the typical CO2 concentration of flue gas is 10–20% (Ono and Cuello, 2003; Suzuki et al., 1995) by which microalgal growth might be inhibited (Chiu et al., 2008; Yoo et al., 2010), further study on B. braunii growth under higher CO2 must be conducted. This study was aimed to investigate the growth characteristics of B. braunii 765 under high CO2 concentrations ranging from 2% to 20% during 28-day cultivation. To reduce the system design and running cost, 2% sodium hypochlorite solution was used for photobioreactor sterilization, and the culture pH was not controlled. 2. Methods 2.1. Strain and culture The strain B. braunii 765 was obtained from the Culture Collection of Algae, Institute of Hydrobiology, Chinese Academy of Sciences. Algae cells were incubated at 25 °C under continuous light at 120 lmol m 2 s 1 for two weeks before being transported into photobioreactor. The culture for algae cultivation was prepared based on modified BG11 medium, containing the following components (mg L 1): NaNO3 (1500), K2HPO4
3H2O (40), MgSO4
7H2O (75), CaCl2
2H2O (36), C6H8O7
H2O (6), Fe(NH4)3C18H10O14 (6), Na2-EDTA (1), Na2CO3 (20), H3BO3 (2.86), MnCl2
H2O (1.81), ZnSO4
7H2O (0.222), CuSO4
5H2O (0.079), Na2MoO4
2H2O (0.39), Co(NO3)2
6H2O

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(0.049). The medium was autoclaved at 121 °C for 30 min without adjusting pH.

2.2. Experimental system with photobioreactor The photobioreactor (10 cm diameter, 50 cm length, 3 L working volume) used for microalgal cultivation is presented schematically in Fig. 1. It was placed in a cabinet with constant temperature (25 °C) and continuous cool white florescent light (150 ± 10 lmol m 2 s 1). High CO2 concentrations (2%, 5%, 10%, and 20%) were obtained by mixing filtered (0.22 lm) ambient air with pure CO2, and aerated into the photobioreactor at a rate of 600 ml min 1. Since previous studies have reported that the optimal concentration of CO2 aeration for microalgal growth (including B. braunii) was 2% (Chiu et al., 2008, 2009; Ranga Rao et al., 2007b), 2% instead of air was set as control. The photobioreactor was sterilized by 2% (v/v) sodium hypochlorite solution.

2.5. Hydrocarbon estimation For hydrocarbon extraction, 300 mg freeze-dried algal powder was sonicated in 50 ml n-hexane for 30 min after homogenizated for 1 h. Then, the mixture was centrifuged (1000g, 10 min, 4 °C) and the supernatant was collected. The extraction process repeated twice. All the collected supernatant was evaporated under a vacuum to dryness at 30 °C, and the remaining was weighed as hydrocarbon (Sawayama et al., 1992).

2.6. Lipid measurement Three hundred milligrams of freeze-dried samples of B. braunii powder were sonicated for 1 h in 50 ml methanol–chloroform (2:1, v/v). After that, chloroform and 1% NaCl was added to adjust the ratio of methanol, chloroform and water to 2:2:1 (v/v). The chloroform layer was collected, evaporated to dryness under vacuum, and weighted as the total lipid (Takagi and Yoshida, 2006).

2.3. Algal biomass estimation A known volume of B. braunii suspension was centrifuged at 1000g for 10 min at 4 °C. The algal pellet was washed twice with distilled water, freeze dried, and stored in a dry cabinet for later analysis. Algal growth was expressed in terms of dry biomass (g L 1), which was determined gravimetrically (Ranga Rao et al., 2007b).

2.7. Total phosphorus (TP) estimation A sample solution collected from photobioreactor was centrifuged at 1000g for 10 min. Afterwards, the supernatant was filtered through 0.45 lm filter papers, digested (121 °C, 30 min) with potassium persulfate reagent in an autoclave. Finally, the solution was spectrophotometrically analyzed at 700 nm wavelength to calculate TP concentration.

2.4. Chlorophyll and b-carotenoid determination A known volume of B. braunii suspension was centrifuged (1000g, 10 min, 4 °C) and all reside was extracted with 90% (v/v) acetone solution repeatedly. Chlorophyll content in the pooled extract was spectrophotometrically measured at four wavelengths (630, 647, 664 and 730 nm) and quantified using Jeffrey’s (1975) methods. To estimate b-carotenoid content, absorbance of the pooled extract at 450 nm was measured and the content was determined according to Jensen (1978).

Temperature sensor T I-7

Gas outlet

2.8. Microscope observation Exponentially growing B. braunii cells were observed alive with an inverted microscope Leica DM IRB (Leica Mikroscopie, Wetzlar, Germany).

2.9. Statistical analysis The results are expressed as mean ± SE (standard error) of three replicates. All data were performed by SPSS 17.0 for Windows (SPSS Inc., USA). One-way analysis of variance (ANOVA) was used to evaluate differences among the four CO2 concentrations. A value of P < 0.05 was considered statistically significant.

2.5

Fluorescent lamps I-4

2% CO2

5% CO2

10% CO2

20% CO2

I-8

-1

Air

Biomass ( g L )

2.0

E-5

P-4 P-2

I-1

V-1

Gas mixer

Gas filter

CO2 sensor P-1

I-2

Flow meter

Sampling tube

1.5

1.0

0.5

I-6

I-3

E-2

E-4

0.0 0

CO2 Fig. 1. Schematic diagram of the photobioreactor for the experiment.

5

10

15

20

25

Cultivation time (days) Fig. 2. Growth curve of B. braunii 765 under high CO2 aeration.

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3. Results and discussion 3.1. Algal growth Overall, the algal strain was confirmed to grow well with 2–20% CO2 aeration without obvious inhabitation (Fig. 2). B. braunii cells showed different initial lag periods with CO2 aeration of different concentration, short (1–2 days) with 10% and 20% while long (3–4 days) with 2% and 5%. After the lag periods, algal cells grew rapidly at all CO2 aeration levels till the TP in culture was almost completely consumed (data would be shown later). Among all the conditions, the maximum algal biomass was 2.31 g L 1 on day 25 with 20% CO2. When aerated with 10% CO2, the algal biomass first increased to 1.61 g L 1 on day 24 without obvious logarithmic period (Fig. 2). Till day 24, the algal cells under 5% CO2 kept the smallest biomass among the four treatments and it reached 1.69 g L 1 on day 26. Differing from the others, the B. braunii under 2% CO2 aeration kept growing thoroughly and its biomass reached 2.18 g L 1 on day 28. These results were similar to the conclusion of Yoo et al. (2010). However, we observed neither the long adaptation period Yoo et al. (2010) investigated nor the inhibition Chiu et al. (2008) reported under 10% CO2 aeration. This might be due to the difference of algae strains, and the strain used in our study could tolerate or adapt itself to high CO2 concentration with a high initial cell density (about 0.13 g L 1) (Chiu et al., 2008; Lee et al., 2002; Yun et al., 1997). Moreover, B. braunii blooms have appeared in variety waters, including freshwater, brackish and saline lakes (Chiang et al., 2004; Dayananda et al., 2007; Papa et al., 2009; Wake and Hillen, 1980), which could clearly show the potential competitive advantage of B. braunii in nature. In this study, 2% sodium hypochlorite was successfully used to sterilize the photobioreactors for B. braunii cultivation, which also could indirectly verify the competitive advantage of this algal species. Furthermore, by using sodium hypochlorite for photobioreactor sterilization instead of using autoclaved photobioreactor, the cost of cultivating B. braunii for biofuel production could be greatly reduced. 3.2. Phosphorus consumption With 2%, 5%, 10%, and 20% CO2 aeration, TP in culture was almost completely consumed on day 24, 25, 19, and 20, followed by the decrease of algal biomass after 3–5 days (Figs. 2 and 3). Statistical analysis showed significantly negative correlation between culture TP and algal biomass, with correlation coefficients respectively being 0.828**, 0.911**, 0.898** and 0.829** (**P < 0.01)

4.0

3.3. Culture pH Average culture pH decreased from about 7.5–7.0, 6.8, 6.5, and 6.3 with 2%, 5%, 10%, and 20% CO2 aeration, respectively (Fig. 4). Obviously, the culture pH just slightly reduced with increase of CO2 concentration from 2% to 20%. Similar results were also found in Chlorella sp. with 2–15% CO2 (Chiu et al., 2008). This might be because most of the influent CO2 would flow out of photobioreactor directly when the CO2 concentration was more than 2% (Chiu et al., 2008). It was worth noting that the culture pH was not controlled in the present study. Though the culture pH changed from 6.0 to 8.0 (Fig. 4), B. braunii grew well without obvious inhibition. Similarly, Dayananda et al. (2007) reported that culture pH had no significant effects on the biomass yield and hydrocarbons production of B. braunii when it ranged from 6.0 to 8.5. Dunaliella tertiolecta was also found to be able to grow sufficiently without controlling culture pH, even under flushing with 24% CO2-enriched air (Suzuki et al., 1995). This ability of algae might be related to osmoregulation which was achieved biochemically by synthesis or dissimilation of intracellular glycerol (Suzuki et al., 1995). Goyal and Gimmler (1989) had reported that D. tertiolecta could maintain a constant intracellular pH over a wide range of external pH values (6.5–8.5). The present algal strain might also have such adjustment mechanisms which would be of significance when the strain was cultivated with aerated flue gas containing 10–20% CO2 (Suzuki et al., 1995). 3.4. Chlorophyll In the beginning, as the algae cells in each culture increased with the growth of B. braunii, the chlorophyll contents in cultures accordingly increased. Under 2%, 5%, 10% and 20% CO2 aeration, chlorophyll content reached to 35.04, 21.16, 20.78 and 27.18 mg L 1 on day 26, 24, 20 and 14, respectively (Fig. 5a). Then, the chlorophyll content began to decrease, and it decreased to 25.54, 12.82, 9.99 and 11.4 mg L 1 under 2%, 5%, 10% and 20% CO2 aeration in the end (Fig. 5a). This might be resulted from a pro-

2% CO2

5% CO2

8.0

2% CO2

5% CO2

10% CO2

20% CO2

7.5

10% CO2

20% CO2

3.0

7.0

pH

-1

Total Phosphorus (mg L )

5.0

under 2%, 5%, 10% and 20% CO2. Additionally, no significant nitrogen reduction was found (data not shown) compared with its initial concentration (NaNO3, 1.5 g L 1). Therefore, it might be phosphorus deficiency that limited the B. braunii growth in later cultivation stage. This is consistent with previous conclusion that phosphorus was one of the most important limitation elements in eutrophication, whose most objectionable symptom was the appearance of floating algal blooms (Schindler, 1977).

2.0

6.5

1.0

6.0

0.0

5.5 0

5

10

15

20

25

30

Cultivation time (days) Fig. 3. Phosphorus consumption by B. braunii 765 under high CO2 aeration.

0

5

10

15

20

25

Cultivation time (days) Fig. 4. Culture pH change under high CO2 aeration.

30

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a

40

2% CO2

5% CO2

10% CO2

20% CO2

Table 1 Hydrocarbon and lipid contents in B. braunii 765 under high CO2 aeration.

-1

Chlorophyll (mg L )

CO2 aeration (%)

30

2 5 10 20

20

*

10

0 0

5

10

15

20

25

30

Cultivation time (days)

-1

β−carotenoid (mg L )

b

25

2% CO2

5% CO2

20

10% CO2

20% CO2

15

Total hydrocarbon content (%) 16.43 ± 0.24 18.25 ± 0.76 21.03 ± 0.30 24.45 ± 0.91

*

a b c d

Total lipid content (%) 10.41 ± 0.89 11.21 ± 0.64 12.44 ± 0.38 12.71 ± 0.83

a ab ab b

Different letters mean statistically significant differences (P < 0.05).

over, judging from Figs. 2 and 5, b-carotenoid might play an important role in B. braunii photosynthesis, especially when the chlorophyll was deficient under 20% CO2 aeration (Owens et al., 1987). Algal photosynthesis could produce much dissolved O2 which could cause feedback inhibition to algal photosynthesis (Marquez et al., 1995). b-carotenoid could protect chlorophyll from photodestruction by inactivating oxygen species (Anderson and Robertson, 1960; Krinsky, 1988). Additionally, Ranga Rao et al. (2007b) found that carotenoid content increased with increase of CO2 aeration level, which was only consistent with the phase I in this study. This might be due to the different CO2 concentrations in the two studies.

3.6. Hydrocarbon and lipid

10 5 0 0

5

10

15

20

25

30

Cultivation time (days) Fig. 5. Chlorophyll (a) and b-carotenoid (b) contents of B. braunii 765 under high CO2 aeration.

gressively inhibition on polyketides biosynthesis by low oxygen partial pressures under high CO2 concentration (Dufossé et al., 2005). Accordingly, the chlorophyll content under 2% CO2 was generally higher than that under the other three CO2 concentrations. Moreover, as shown in Figs. 2 and 5a, the algal growth did not cease immediately after the chlorophyll content decreased. These results indicated that there might be other photosynthesis pathways of B. braunii that did not depend completely on chlorophyll (normally considered as main photosynthesis pigments) under high CO2 aeration (Owens et al., 1987). 3.5. b-carotenoid b-carotenoid content in this study was higher than previous reports of B. braunii (Ranga Rao et al., 2007a,b), but lower than Dunaliella sp. which was used for b-carotenoid production. Four phases of b-carotenoid content changes were observed (Fig. 5b): I increase, in which b-carotenoid content increased to 15.62, 13.65, 16.68, and 15.48 mg L 1 on day 14, 12, 8, and 10 with 2%, 5%, 10%, and 20% CO2, respectively; II decrease, in which the b-carotenoid content decreased to 8.54, 6.26, 7.43 mg L 1 with 2%, 5%, 10% CO2 on day 22, and 11.17 mg L 1 with 20% CO2 on day 16; III rapid increase, which happened after phase II in 1–2 days with 2%, 5%, 10% CO2 and 12 days with 20% CO2; and IV rapid decrease, which appeared in all cultures in the final stage of the experiment. Similar dynamic results were also reported in several previous studies (e.g. Ranga Rao et al., 2007b), but no reasonable explanation could be referred. As carotenoid biosynthesis mechanism is very complicated, the explanation to these dynamic results needs further study. More-

As shown in Table 1, the hydrocarbon content of B. braunii 765 increased with increase of CO2 concentration, being 16.43%, 18.25%, 21.03% and 24.45% (w/w) with 2%, 5%, 10% and 20% CO2 aeration, respectively. This is consistent with previous results of Ranga Rao et al. (2007a) and Metzger et al. (1985). Lipid content was 10.41%, 11.21%, 12.44% and 12.71% (w/w) with 2%, 5%, 10% and 20% CO2 aeration, respectively. Muradyan et al. (2004) have reported that one-day long increase in CO2 from 2% to 10% could provoke a 30% increase in total fatty acids amount of dunaliella salina, while a 7-day long increase could result 2.7-fold higher lipid content. Compared to their report, both the algal lipid content and the CO2-induced increase in our study were lower. This might be mainly due to the difference of algal strains. Besides, as nitrogen deprivation would generally increase algal lipid content (Hsieh and Wu, 2009; Li et al., 2010), the relative lower lipid content might be resulted from the much higher nitrogen content of culture in our study compared to other researches (e.g. Ranga Rao et al., 2007a).

3.7. Microscope observation of B. braunii colony The algal cells under different concentration of CO2 formed colonies of different size, color and compact degree. At 2% CO2, the colony (10–20 lm in diameter) was of green compact structure. When the algal strain was cultivated at 5% CO2, the colony (20– 30 lm in diameter) turned into yellow–green and showed incompact structure margined with unicell. The algal colony at 10% and 20% CO2 aeration showed similar color and compact degree to that of 5% CO2, whereas the colony diameter increased to 30–40 lm. Zhang and Kojima (1998) reported that the average size of B. braunii colony was determined by the average light intensity: as cell concentration increased, the average light intensity within the photobioreactor decreased due to mutual shading, leading to decrease in the colony size. However, in this study, the algal colony size under 20% CO2 was the biggest while the cell intensity was also the highest, even though the highest cell intensity might cause the most serious mutual shading. This might because B. braunii colony size was also related to many other factors including extracellular polysaccharides production rate, mechanical strength of colony, turbulence in reactors (Zhang and Kojima, 1998).

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Furthermore, it might be easier for bigger colony to form flocculation, which could decrease the cost of algal harvest. Therefore, it is valuable to make clear how the bigger colony formed when aerated with high CO2. The relative work would be ongoing in our lab. 4. Conclusion This study suggested that when using flue gas to cultivate B. braunii 765 for biofuel production, the effects of high CO2 might not be a problem. Moreover, the cost of algal cultivation and harvest could be reduced by several means, such as photobioreactor sterilization using sodium hypochlorite, no pH adjustment, as well as algal colony flocculation. Also considering its competitive advantage in nature, B. braunii could be one of the most promising microalgae for future biofuel production, and further process control and optimization is worth studying. Acknowledgements This research was supported by the National Natural Science Foundation of China (40871101). We gratefully acknowledge all the reviewers for their great contribution to the improvement of this article. References An, J.Y., Sim, S.J., Lee, J.S., Kim, B.W., 2003. Hydrocarbon production from secondarily treated piggery wastewater by the green alga Botryococcus braunii. J. Appl. Phycol. 15, 185–191. Anderson, I.C., Robertson, D.S., 1960. Role of carotenoids in protecting chlorophyll from photodestruction. Plant Physiol. 35, 531. Chiang, I.Z., Huang, W.Y., Wu, J.T., 2004. Allelochemicals of Botryococcus braunii (chlorophyceae). J. Phycol. 40, 474–480. Chiu, S.Y., Kao, C.Y., Chen, C.H., Kuan, T.C., Ong, S.C., Lin, C.S., 2008. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Biores. Technol. 99, 3389–3396. 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. Dayananda, C., Sarada, R., Kumar, V., Ravishankar, G.A., 2007. Isolation and characterization of hydrocarbon producing green alga Botryococcus braunii from Indian freshwater bodies. Electron. J. Biotechnol. 10, 1–14. Dufossé, L., Galaup, P., Yaron, A., Arad, S.M., Blanc, P., Chidambara Murthy, K.N., Ravishankar, G.A., 2005. Microorganisms and microalgae as sources of pigments for food use: a scientific oddity or an industrial reality? Trends Food Sci. Technol. 16, 389–406. Goyal, A., Gimmler, H., 1989. Osmoregulation in Dunaliella tertiolecta: effects of salt stress, and the external pH on the internal pH. Arch. Microbiol. 152, 138–142. Hsieh, C.H., Wu, W.T., 2009. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour. Technol. 100, 3921–3926. Jeffrey, S.W., Sielicki, M., Haxo, F.T., 1975. Chloroplast pigment patterns in dinoflagellates. J. Phycol. 11, 374–384. Jensen, A., 1978. Chlorphylls and carotenoids. In: Hellebust, J.A., Craigie, J.S. (Eds.), Handbook of Phycological Methods. Cabbridge University Press, London, pp. 69–70.

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