Effects of different media and nitrogen sources and levels on growth and lipid of green microalga Botryococcus braunii KMITL and its biodiesel properties based on fatty acid composition

Accepted Manuscript Effects of different media and nitrogen sources and levels on growth and lipid of green microalga Botryococcus braunii KMITL and its biodiesel properties based on fatty acid composition Suneerat Ruangsomboon PII: DOI: Reference:

S0960-8524(15)00111-X http://dx.doi.org/10.1016/j.biortech.2015.01.091 BITE 14523

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Bioresource Technology

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30 December 2014 23 January 2015

Please cite this article as: Ruangsomboon, S., Effects of different media and nitrogen sources and levels on growth and lipid of green microalga Botryococcus braunii KMITL and its biodiesel properties based on fatty acid composition, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.01.091

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Effects of different media and nitrogen sources and levels on growth and lipid of green microalga Botryococcus braunii KMITL and its biodiesel properties based on fatty acid composition

Suneerat Ruangsomboon Program in Fisheries Science, Division of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand. Corresponding author’s Tel: 66-2-329-8517, E-mail: [email protected]

ABSTRACT This work aimed to find an optimum culture medium for green microalga Botryococcus braunii KMITL and investigate its biodiesel properties based on fatty acid composition. Four different media were tested. Chlorella medium was the best medium for lipid yield. Among four nitrogen sources tested, KNO3 produced the highest lipid yield. When varied the nitrogen concentrations, this strain gave the highest lipid yield at the highest nitrogen level. When cultivated in the best medium and nitrogen source and level for 30 days, and then cultivated further for 14 days in the medium with no nitrogen, the highest lipid content and yield were 49.94±0.82% and 2.71±0.02 g L-1, respectively. C16:0 fatty acid was the major fatty acid found. Fatty acid profiles of B. braunii KMITL cultivated in Chlorella medium with 1.25 g L-1 KNO3 gave the best biodiesel properties with the lowest iodine value, maximum cetane number, and lowest degree of unsaturation. Keywords: Biodiesel, Chlorella medium, Fatty acid, Lipid productivity, Cetane

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1. Introduction Lipids from microalgae, which can be as much as 20-80% of their weight, are a good alternative source for biodiesel production (Chisti, 2007). Under special cultivation conditions, microalgae can produce more lipid content. Typically, when microalgae are cultivated under a low nutrient or other stressed conditions, their growth is stunted but their lipid production is elevated (Hu, 2004). Green microalga, Botryococcus braunii has a relatively high lipid content, but its growth is slow and its biomass yield is low; therefore, it is not a viable source for commercial biodiesel production. A good algal strain for biodiesel production should grow fast and produce high biomass and lipid yields. Another requirement is that it should be able to adapt and thrive in the local tropical climate of Thailand. Each B. braunii strain thrives and produces lipid content to a different extent when it is cultivated in different kinds of media. Several suitable media for this alga have been reported in the literature, such as modified Chu 13 medium, Bold basal medium and BG-11 medium (Dayananda et al., 2007), modified BG-11 medium (Ge et al., 2011), and Prat medium (Kalacheva et al., 2001). Hence, when a new algal strain is considered to be a good candidate for biodiesel production, an investigation needs to be conducted to find the suitable medium and nutrient level for its rapid growth and lipid production. In addition, it has been reported that nitrogen source and level affect the growth and lipid production of algae (Cheng et al., 2014; Dayananya et al., 2005; Zhila et al., 2005a, 2005b), therefore, these dependencies also need studying.

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Lipid produced from microalgal species usually has a fatty acid profile of mainly C16 and C18 fatty acids similar to that of a vegetable oil; hence, it can be used for biodiesel production (Converti et al., 2009; Francisco et al., 2010). Important biodiesel fuel properties influenced by fatty acid profile are such as cetane number, kinematic viscosity, oxidative stability, cold-flow properties such as cloud point and cold-filter plugging point, and lubricity (Knothe, 2008). For this reason, in selecting an alga for biodiesel production, it is necessary to consider not only its lipid production but also its fatty acid profile. This study aimed to find a suitable medium and nitrogen source and level for cultivating B. braunii KMITL, a new algal strain isolated from a tropical freshwater reservoir in central Thailand that produces high lipid yield and has a suitable fatty acid profile for biodiesel production.

2. Materials and Methods 2.1. Algal culture and effects of different media on growth, lipid and fatty acid composition Botryococcus braunii strain KMITL was isolated from Klong Boat reservoir, Nakhon Nayok province, Thailand. The isolation process is described in one of our previous works (Ruangsomboon, 2012). The strain was cultured in several media: Modified Chu 13 medium (Largeau et al., 1980), Kratz and Myers medium (Kratz and Myers, 1955), Bold-Basal medium with 3-fold nitrogen and vitamins (3N-BBM+V) (Bischoff and Bold, 1963), and Chlorella medium (Vonshak and Maske, 1982) (Table 1). The microalga was grown in 1-L glass flasks under continuous illumination from

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daylight fluorescent lamps at 200 µE m-2 s-1 with constant air bubbling at 25 oC, in laboratory. The best culture medium was selected for cultivating the microalga outdoor in the next step.

2.2. Effects of different nitrogen sources and levels on growth, lipid and fatty acid composition Microalga was cultivated outdoor in a Chlorella medium (described in section 2.1) in 150 L oval fiberglass pond under natural light with constant air bubbling at an average temperature of 26-35 oC. The effects of nitrogen sources: KNO3 (potassium nitrate), NaNO3 (sodium nitrate), CH4N2O (urea), and NH4 HCO3 (ammonium bicarbonate) were determined with the nitrogen level from each source controlled to be the same at 0.17 g N L-1. The effects of different nitrogen levels were then investigated at the best nitrogen source concentrations of 0.13, 0.31, 0.63, 1.25 and 2.50 g L-1 of KNO3.

2.3. Effects of two-step culture in media with different nitrogen levels on growth, lipid and fatty acid composition The experimental results from section 2.2 showed that the culture medium that offered the highest biomass and lipid yields was the Chlorella medium with KNO3 as nitrogen source at the concentration of 2.5 g L-1, therefore, as a two-step culture experiment, the microalga was cultivated first in this medium up to the late exponential phase of 30 days. It was cultured outdoor in a 150 L oval fiberglass pond under natural light with constant air bubbling at an average temperature of 26-35 oC.

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Then, in the second step, two treatments were separately conducted. For Treatment A, the microalga culture from the first step was further cultivated in the Chlorella medium but with no KNO3 nitrogen source for another 14 days to test whether the lack of nitrogen would stimulate more production of lipid content, as reported in Zhila et al. (2005a, 2005b). As a complementary treatment, Treatment B had the microalga culture further cultivated for 14 days in the same medium in the first step to compare whether the lipid content became more or less than that produced in treatment A. Moreover, since this medium gave the highest biomass and lipid yields in the first place, Treatment B would show whether the lipid yield at the end of the culture period has increased as a result of an increase in biomass or not.

2.4. Determination of algal biomass Cultures (10 ml portions of suspension) were filtered with glass microfiber filter paper (GFC, Whatmann) and washed with distilled water. The paper with the collected algal cells deposited was dried at 105 oC for 24 h, cooled to room temperature in a desiccator, and then the dry weight of the biomass was measured. All experiments were carried out at four replicates. The specific growth rate (µ) was calculated using the method reported by Ceron et al. (2005).

2.5. Extraction and analysis of total lipid and fatty acids Microalga culture was harvested after 30 days of cultivation (late exponential phase), dried at 40 oC, and ground with mortar and pestle. Lipids were extracted from 500 mg of dried biomass with chloroform and methanol (1:2 v/v) (Bligh and Dyer

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1959). The extracted mixture was sonicated at 70 Hz (with Transonic model 460/H, Elma, Singen, Germany) at room temperature. The extraction process was repeated once (a total of two times). The lipid extracts were dried in a rotary evaporator and weighed. Fatty acids were converted into methyl esters by direct transmethylation of the lipid extracts. The fatty acid methyl esters obtained were analyzed by an Agilent Technologies 6890 N Gas Chromatography system (USA) equipped with a flame ionization detector (FID). The details of the methods conducted in this section are already reported in one of our previous works (Ruangsomboon et al., 2013).

2.6. Determination of biodiesel quality properties The following biodiesel quality properties were determined following the equations published by Ramos et al. (2009), Francisco et al. (2010), and Wu and Miao (2014): saponification value (SV), iodine value (IV), cetane number (CN), degree of unsaturation (DU), long-chain saturated factor (LCSF), and cold filter plugging point (CFPP).

2.7. Statistical analysis The average values of four replicates of the microalga’s biomass and lipid content as well as the standard deviations were calculated. Significant differences were determined by using the method of analysis of variance (ANOVA) at 95% confidence interval (probability limit of p < 0.05).

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3. Results and Discussion 3.1. Effects of different media on growth, lipid and fatty acid composition The extents of growth, in terms of biomass concentration at a point in time, of B. braunii KMITL in different media are shown in Fig 1A. The microalga cultured in Chlorella medium grew better than it did in the other tested media. The maximum biomass was obtained in Chlorella medium (1.87±0.05 g L-1), while respectively lesser biomasses were obtained in 3N-BBM+V, Modified Chu 13, and Kratz and Myers medium. B. braunii KMITL also grew the fastest when cultured in Chlorella medium, at a specific growth rate of 0.05 d -1. The highest lipid content, lipid yield, and lipid productivity of 29.22±0.71%, 0.54±0.08 g L-1 and 14.40±0.07 mg L-1 d-1 were obtained in Chlorella medium (Fig. 2). Regarding the fatty acid composition, the microalga cultured in Chlorella medium produced C16:0 fatty acid at the highest percentage of 35.58%, while the combination of C16-C18 fatty acids accounted for 74.38% of the fatty acid composition. The highest percentage of C16-C18 fatty acids combination was obtained from the microalga cultured in Kratz and Meyers medium, at 76.70%. Overall, the fatty acid compositions of the microalga cultured in all media tested were quite similar (Table 2). Table 1 shows the levels of nutrients in the four tested media. It can be seen in the table that the Chlorella medium had higher levels of CaCl2, H3BO3, ZnSO4, MnCl2 CuSO4, Co(NO3)2, EDTA, and Fe2SO4. Even though the Kratz and Myers medium had 3.2 times higher KNO3 and the 3N-BBM+V medium was supplemented with 2 vitamins, still the Chlorella medium supported the growth and lipid yield of the microalga better, indicating that the minor elemental nutrients–B, Zn, Mn, Cu, Co, and Fe–positively influencing the growth and lipid yield of this microalgal strain.

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3.2. Effects of different nitrogen sources and levels on growth, lipid and fatty acid composition B. braunii KMITL cultured in Chlorella medium with KNO3 as the nitrogen source provided a significantly higher biomass (4.84±0.06 g L-1), lipid content (35.24±0.75 %), lipid yield (1.73±0.03 g L-1), specific growth rate (0.045±0.002 d-1) and lipid productivity (16.0±0.61 mg L-1 d -1) than that grown in the medium with the other nitrogen sources (Fig. 1B and Fig. 3). The fatty acid found in the highest percentage in the microalga cultured with this nitrogen source was C16:0 fatty acid, at 43.25%, while the percentage of the combination of C16-C18 fatty acids was 79.84%. This combination was found in the highest percentage, at 83.40%, in the microalga cultured in the medium with NH4 HCO3 as the nitrogen source. The microalga cultured in the medium with KNO3 or NaNO3 as the nitrogen source produced a higher percentage of C16:0 than those with the other nitrogen sources, while the microalga cultured in the medium with Co(NH2)2 or NH4HCO3 as the nitrogen source produced higher percentages of C17:0 and C18:0 than that with KNO3 or NaNO3 (Table 3). After it was discovered that KNO3 was the best nitrogen source for the biomass and lipid yields of B. braunii KMITL, similarly reported for other strains of B. braunii by Cheng et al. (2014) and Dayananda et al. (2005), the best level of KNO3 in the medium was sought, and it was found that the microalga’s highest specific growth rate, biomass yield, lipid yield and lipid productivity of 0.049±0.001 d -1, 5.62±0.06 g L-1, 2.16±0.02 g L-1 and 18.85±0.03 mg L-1 d-1were obtained at the highest KNO3 concentration of 2.50 g L-1 (Fig. 1C and Fig. 4). The C16:0 fatty acid was found at the highest percentage of

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43.00%, while the percentage of the combination of C16-C18 was 79.76%. There was no obvious trend that showed that the microalga’s fatty acid composition depended on the nitrogen level in the medium (Table 4).

3.3. Effects of two-step culture with different levels of nitrogen nutrient in each step on growth, lipid and fatty acid composition As stated in the materials and methods section 2.3, B. braunii KMITL was cultivated in 2 steps and separately for 2 treatments. The first step was the same for both treatments, but in the second step, Treatment A had the microalga further cultivated in the medium with no nitrogen source while Treatment B had the microalga further cultivated in the same medium with the nitrogen source. The results of both treatments show that the microalga culture of Treatment A gave a higher lipid content and lipid yield, at 49.94±0.82% and 2.71±0.02 g L-1 respectively (Fig. 6), than those in Treatment B, but a lower lipid productivity at 4.15±0.33 mg L-1 d -1 (Fig. 6). This confirms the report that an alga produced a higher lipid content under a nitrogen deficient condition (Zhila et al., 2005a). The lower lipid productivity was because of the lower specific growth rate under the stressed condition. Several minor experimental results are discussed below. At 7 days and 14 days into the second step, the lipid content of the microalga culture in Treatment A increased significantly from that at the end of the first step. At 14 days after the start of the second culture step, the end of the experiment, the microalga’s lipid content and lipid yield were the highest. After 7 days into Treatment B, the microalga culture produced the highest biomass yield at 6.60±0.11 g L-1 (Fig. 6), significantly higher than the yields at any time in both

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treatments. Due to this high biomass yield, the lipid yield was the highest at the end of the second step, though still lower than that in Treatment A. On the other hand, its lipid productivity decreased because its specific growth rate also decreased probably from the numerous algal cells already present having to compete for nutrients. Regarding the fatty acid composition of B. braunii KMITL, the highest percentage of fatty acid was C16:0 fatty acid in the range of 37.62-43.07%. The percentage of the combination of C16-C18 was in the range of 81.19-87.41%. In all experimental treatments, the microalga’s fatty acid compositions were quite similar (Table 5). It was found that indoor cultivation in the laboratory (section 3.1) gave a low biomass yield, between 1.16-1.87 g L-1, whereas outdoor cultivation gave the highest biomass yield of 4.84-6.60 g L-1. This might be because the indoor temperature was kept fixed at 25 ºC while the outdoor temperature was higher, between 26-35 ºC. The outdoor natural light illumination was also stronger at 814-1,478 µE m-2 s-1. It seems that this B. braunii KMITL algal strain thrives, yielding more biomass, under high temperature and illumination of the outdoor environment. To summarize, it was found that a way to stimulate the microalga to produce more lipid content and lipid yield is to culture it in two steps, in which the medium with a nitrogen source that gives the highest lipid yield is used up to the late exponential phase for 30 days in the first step and then the medium with no nitrogen source is used instead for the next 14 days in the second step. In addition, regarding the fatty acid composition of B. braunii KMITL, the highest percentage fatty acid was C16:0 fatty acid. Dayananda et al. (2007) also reported that the highest percentage fatty acid found in B. braunii was C16:0 fatty acid.

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Furthermore, the fatty acid composition of B. braunii KMITL found in this study agreed closely with that reported in the previous work of Ashokkumar and Rengasamy (2012).

3.4. Biodiesel properties of B. braunii KMITL Generally, cetane number, heat of combustion and viscosity increase with increasing fatty acid chain length, meaning that long chain fatty acids (C16–18) are more preferable as a biodiesel fuel (Francisco et al., 2010; Miao et al., 2009). This study found that the percentages of the combination of C16-C18 fatty acids found in B. braunii KMITL in all experimental treatments were in the range of 69.84-87.41% of the total fatty acids; hence, the fatty acids from this algal strain are favorable for biodiesel production. The percentages of saturated and unsaturated fatty acids were in the ranges of 62.10-82.48% and 17.52-37.90% of the total fatty acids, respectively (Table 2-5). Biodiesel properties of B. braunii KMITL are shown in Table 6. Saponification value (SV) is a measure of the average molecular weight (or chain length) of all fatty acids present. The SV values of B. braunii KMITL cultures in all experimental treatments were in the range of 207.18-223.75. Iodine value (IV) is a crude measure of the total unsaturation of a biodiesel which is related to its oxidative stability: a high IV biodiesel is less oxidatively stable than a low IV one (Knothe, 2009). The maximum IV value of the European standard is 120 g I2 100 g−1. The IV values of B. braunii KMITL were in the range of 19.65-52.49 g I2 100 g−1, which are lower than those of many kinds of algae reported by Francisco et al. (2010) and Wu and Miao (2014).

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Cetane number (CN) is a prime indicator of biodiesel quality related to ignition delay time and combustion quality. A higher CN value means that the biodiesel has better ignition properties and gives better engine performance. Two worldwide standards of biodiesel quality stipulate that a minimum CN should be 47 or 51 (ASTM D6751, 2012 and Fuel Standard (Biodiesel) Determination, 2003). The CN numbers of this microalgal strain in all experimental treatments, in the range of 58.30-66.82, were higher than those stated in these standards. Also, the cetane number of this B. braunii KMITL strain was higher than that of the B. braunii strain reported by Ashokkumar et al. (2014), at 55.4, and Nascimento et al. (2013), at 52.67. Degree of unsaturation (DU) indicates the oxidative stability of a biodiesel pertaining to the stability of its long-term storage. A low DU means that the biodiesel is more stable for long-term storage. The DU values of this algal strain varied from 20.32% to 48.62%. These values are lower than those of green microalgae Scenedesmus obliquus and Chlorella pyrenoidosa which were in the range of 76.53132.08 % (Wu and Miao, 2014). Cold filter plugging point (CFPP) is another important biodiesel quality parameter typically used to predict the flow performance of a biodiesel at low temperatures. Lowtemperature properties depend mostly on the saturated fatty acids content; the effect of unsaturated fatty acid composition can be considered negligible (Ramos et al., 2009). CFPP is correlated with long chain saturated factor (LCSF). The B. braunii KMITL in this study had LCSF values in the range of 4.75-11.30 % and CFPP values in the range of -1.56-19.03 oC. A higher CFPP value indicates worse low-temperature biodiesel properties (Wu et al., 2005), signifying a higher tendency that the biodiesel will precipitate and clog the filter (Mittelbach and Remschmidt, 2004).

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Among all of the B. braunii KMITL cultivated in various media and nitrogen sources and levels, the microalga cultured in Chlorella medium with KNO3 as the nitrogen source at the concentration of 1.25 g L-1 was found to be the best direct biodiesel feedstock because of these desirable properties–the maximum content of saturated fatty acid of 82.48%, the lowest content of unsaturated fatty acid of 17.52%, the lowest monounsaturated fatty acid content of 14.72% (Table 4), the lowest IV of 19.65 g I2 100 g−1, the maximum CN of 66.82, and the lowest DU of 20.32% (Table 6). However, its CPFF value calculated to be at 19.03 OC is not very good. Further investigation and development are needed to overcome this deficiency.

4. Conclusions B. braunii KMITL gave higher biomass and lipid yields when cultured outdoor than in the laboratory. Its lipid content was as high as 49.94±0.82%. Its fatty acid composition was suitable for biodiesel production, having a cetane value in the range of 58.30-66.82. Therefore, this algal strain is a good source for biodiesel production.

Acknowledgement This study was supported by the National Research Council of Thailand (NRTC). The author wishes to thank the undergraduate students in the 2013 Program in Fisheries Science, Faculty of Agricultural Technology, KMITL for collecting the experimental data.

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FIGURE CAPTIONS Fig. 1. Biomass concentration of B. braunii KMITL in the medium: (A) cultured in various media (under laboratory condition); (B) various nitrogen sources and (C) nitrogen concentrations (g L-1) (under outdoor condition).

Fig. 2. Biomass yield, lipid yield, lipid content, specific growth rate, and lipid productivity of B. braunii KMITL2 cultured in different media. Different small letters on the lines and bars indicate a significant difference (p<0.05). Error bars represent ± S.D. of four replicates.

Fig. 3. Biomass yield, lipid yield, lipid content, specific growth rate, and lipid productivity of B. braunii KMITL2 cultured with various nitrogen sources. Different small letters on the lines and bars indicate a significant difference (p<0.05). Error bars represent ± S.D. of four replicates.

Fig. 4. Biomass yield, lipid yield, lipid content, specific growth rate, and lipid productivity of B. braunii KMITL cultured with different nitrogen levels. Different small letters on the lines and bars indicate a significant difference (p<0.05). Error bars represent ± S.D. of four replicates.

Fig. 5. Biomass of B. braunii KMITL cultured in 2 steps: with then without a nitrogen source. N2.5 (30d)+N0 (14d) is cultivation in the Chlorella medium with KNO3 at 2.5 g L-1 for 30 days and then without KNO3 for another 14 days; N2.5 (30d)+N2.5 (14d) is

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cultivation in the Chlorella medium with KNO3 at 2.5 g L-1 for 30 days and then still in the same medium for another 14 days. Error bars represent ± S.D. of four replicates.

Fig. 6. Biomass yield, lipid yield, lipid content, specific growth rate, and lipid productivity of B. braunii KMITL cultured in 2 steps, with and then without a nitrogen source. N2.5 (30d)+N0 (7d)/(14d) is cultivation in the Chlorella medium with KNO3 at 2.5 g L-1 for 30 days and then without KNO3 for another 7 days/14 days; N2.5 (30d)+N2.5 (7d)/(14d) is cultivation in the Chlorella medium with KNO3 at 2.5 g L-1 for 30 days and then still in the same medium for another 7 days/14 days. Different small letters on the lines and bars indicate a significant difference (p<0.05). Error bars represent ± S.D. of four replicates.

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0

Specific growth rate (d-1) 0.05

0.04

a

0.03

0.00 a b

b

Specific growth rate Lipid productivity

c

b

b

15

a 10

0.02

0.01 5

0

Lipid productivity (mg L-1 d-1)

Chlorella medium

a 40

3N-BBM+V

a c

0.06

Kratz and Myers medium

4

50

Modified Chu 13 medium

Biomass yield Lipid yield Lipid content

Lipid content (%)

5

Chlorella medium

6

3N-BBM+V

1

Kratz and Myers medium

Modified Chu 13 medium

Biomass and Lipid yield (g L-1)

21

20

5

a

40

b

4

c

30

d

3 20

a

2

b c

1 a 0

b

c

c

c

KNO3 NaNO3 KNO NaNO 3 3 Co(NH2)2 NH4HCO3

Fig. 3.

0.06

50

10 0

Specific growth rate Lipid productivity

0.05

a

20

15

0.04 b 0.03

b

b

10

0.02 5 0.01 0.00

a

b

b

b

0

KNO3 NaNO NaNO3 Co(NH ) NH HCO KNO 3 3 2 2 4 3

Lipid productivity (mg L-1 d-1)

Biomass yield Lipid yield Lipid content

Specific growth rate (d-1)

6

Lipid content (%)

Biomass and Lipid yield (g L-1)

22

c

b

20 10

1

c 0.31

0.13

c

d

e

0

2.50

b

1.25

a

Nitrogen concentration (mg L-1) Biomass yield

Lipid yield

Lipid content

20

0.05 15 0.04 0.03

10

0.02 5 0.01 a

a

b

0.00

b

b

0

Nitrogen concentration (mg L-1) Specific growth rate

Lipid productivity

Lipid productivity (mg L-1 d-1)

b

a

2

d

c

2.50

30 3

c

1.25

4

b

0.63

40

a

0.31

b

ab

0.13

b

b

Specific growth rate (d-1)

ab

5

0

Fig. 4.

0.06

50 a

Lipid content (%)

6

0.63

Biomass and Lipid yield (g L-1)

23

24

8

Biomass (g L-1)

7 6 5 4 3 2

N2.5 (30d)+N0 (14d)

1

N2.5 (30d)+N2.5 (14d)

0 0

10

20 Time (day)

Fig. 5.

30

40

0

Fig. 6. a

a d b

4

b c b d

1

a b c d 40

30

20

10

0

0.00

a b c d c

N2.5(30d) +N2.5 (14d)

a

50

N2.5(30d) +N2.5 (7d)

b

Specific growth rate (d-1)

c

N2.5(30d) +N0 (14d)

3

60

N2.5(30d) +N0 (7d)

6

Lipid content (%)

Biomass yield Lipid yield Lipid content

N2.5(30d)

2

N2.5(30d) +N2.5 (14d)

5

N2.5(30d) +N2.5 (7d)

7

N2.5(30d) +N0 (14d)

8

N2.5(30d) +N0 (7d)

N2.5(30d)

Biomass and Lipid yield (g L-1)

0.06

a

0.05

0.02

Specific growth rate Lipid productivity

0.04 15

0.03

d 10

b

0.01

c c 5

0

Lipid productivity (mg L-1 d-1)

25

20

26

Table 1 Levels of chemical nutrients in the culture media. Concentration (mg L-1)

Modified

Kratz and

3N-

Chlorella

Chu13 medium

Myers medium

BBM+V

medium

KNO3

200

4000

-

1250

NaNO3

-

-

750

-

KH2PO4

-

-

175

1250

K2HPO4

40

1000

75

-

MgSO4.7H2O

100

250

75

1000

CaCl2

-

-

-

84

CaCl2. 2H2O

54

-

25

-

-

25

-

-

H3BO3

2.85

2.86

-

114

ZnSO4.7H2O

0.02

0.22

-

88

ZnCl2.6H2O

-

-

0.03

-

MnCl2.4H2O

1.80

1.81

0.246

14

-

0.02

-

7

0.08

0.08

-

16

-

-

-

5

0.08

-

0.012

-

-

165

-

-

0.05

-

0.024

NaCl

-

-

5

-

EDTA

-

-

4.5

500

Fe(SO4)3. 6H2O

-

4

-

-

Fe2SO4.7H2O

-

-

-

50

0.01

-

-

-

-

-

0.582

-

Citric acid

100

-

-

-

Vitamin B1

-

-

1.2

-

Vitamin B12

-

-

0.01

-

Ca(NO3)2.4H2O

MoO3 CuSO4.5H2O Co(NO3)2.6H2O CoCl2.6H2O Na citrate Na2MoO4.2H2O

Fe citrate FeCl3.6H2O

27

Table 2 Fatty acid profiles of Botryococcus braunii KMITL cultivated in various media.

Fatty acid (%) C4:0 C6:0 C8:0 C10:0 C11:0 C12:0 C13:0 C14:0 C14:1 C15:0 C15:1 C16:0 C16:1 C17:0 C18:0 C18:1n9t C18:1n9c C18:2n6t C18:2n6c C18:3n6 C18:3n3 C20:0 C20:1 C20:2 C21:0 C20:3n6 C20:3n3 C20:4n6 C22:0 C22:1n9 C:20:5n3 C24:0 C16-C18 Saturated fatty acid Unsaturated fatty acid Monounsaturated fatty acid Polyunsaturated fatty acid Total fatty acid nd – not detected

Modified Chu 13 0.06 0.21 0.94 0.62 6.73 6.34 3.83 2.69 4.20 1.07 1.22 34.84 9.53 5.01 4.14 0.96 11.75 2.24 0.20 2.50 nd nd 0.20 0.06 0.01 0.26 0.01 0.02 0.20 0.05 0.07 0.06 71.17 66.75 33.25 27.90 5.35 100

Kratz and Myers Nd 0.18 0.48 1.09 5.44 4.86 2.89 2.36 2.95 0.94 1.07 37.70 9.41 4.55 4.00 0.97 13.48 1.52 0.23 2.57 2.27 0.08 0.31 0.21 Nd Nd 0.02 0.02 0.25 Nd 0.06 0.08 76.70 64.91 35.09 28.19 6.90 100

3NBBM+V 0.05 0.14 0.48 0.94 0.49 6.61 7.11 2.06 2.84 0.87 0.92 38.37 9.21 4.81 4.62 1.47 11.51 1.41 0.14 2.04 2.80 0.09 nd 0.24 0.05 0.19 0.02 0.02 0.25 0.13 0.01 0.10 76.38 67.04 32.96 26.08 6.87 100

Chlorella nd 0.08 0.18 0.51 5.82 6.68 3.32 1.84 3.08 0.87 1.14 35.58 9.64 4.86 4.86 1.74 11.03 1.41 0.17 2.16 2.92 0.03 0.51 0.37 0.11 0.16 0.03 0.09 0.35 0.19 0.07 0.17 74.38 65.27 34.73 27.34 7.40 100

28

Table 3 Fatty acid profiles of Botryococcus braunii KMITL cultivated with various nitrogen sources. Fatty acid (%)

KNO3

NaNO3

Co(NH2)2

NH4 HCO3

C4:0

0.20

0.63

0.48

0.29

C6:0

0.06

0.49

0.29

0.50

C8:0

0.72

1.01

0.64

0.53

C10:0

1.36

1.00

0.83

0.72

C11:0

0.06

0.20

0.12

0.09

C12:0

6.96

6.87

6.18

4.56

C13:0

6.71

5.30

5.97

3.44

C14:0

1.41

1.45

1.24

1.81

C14:1

1.92

3.31

2.42

3.37

C15:0

0.50

0.53

0.42

0.67

C15:1

0.24

0.41

1.14

0.62

C16:0

43.25

43.48

32.88

39.44

C16:1

4.17

6.54

4.78

8.77

C17:0

3.62

0.03

9.93

8.44

C17:1

0.12

0.03

nd

nd

C18:0

0.80

1.02

13.23

5.81

C18:1n9t

0.10

0.25

0.17

0.14

C18:1n9c C18:3n6

17.80 3.52

16.64 3.56

10.30 3.13

11.18 3.89

6.45 0.01 79.84 65.67 34.32 24.36 9.97 100

7.16 0.09 78.72 62.10 37.90 27.18 10.72 100

5.82 0.04 80.24 72.24 27.76 18.81 8.95 100

5.74 nd 83.40 66.29 33.71 24.08 9.62 100

C18:3n3 C22:0 C16-C18 Saturated fatty acid Unsaturated fatty acid Monounsaturated fatty acid Polyunsaturated fatty acid Total fatty acid nd – not detected

29

Table 4 Fatty acid profiles of Botryococcus braunii KMITL cultivated at various nitrogen levels. Nitrogen concentration (g L-1)

Fatty acid (%) 0.13

0.31

0.63

1.25

2.50

C4:0

1.16

0.32

0.11

0.25

0.15

C6:0 C8:0 C10:0

2.13 1.91 3.74

0.43 1.18 1.29

0.09 0.27 1.04

0.14 0.85 1.09

0.08 0.39 0.77

C11:0

0.41

0.41

0.22

0.27

0.17

C12:0

3.83

1.49

6.81

7.50

5.89

C13:0

1.46

1.93

3.29

4.19

6.17

C14:0

7.05

5.75

1.14

2.96

2.36

C14:1

3.95

8.68

1.32

3.02

2.26

C15:0

2.55

1.50

0.40

0.58

0.49

C15:1

1.97

2.11

0.93

1.73

1.50

C16:0

39.69

49.47

43.97

52.83

43.00

C16:1

2.74

3.54

3.77

1.48

1.37

C17:0

1.48

0.20

6.65

2.58

3.50

C17:1

3.74

2.39

1.84

1.45

1.84

C18:0

9.13

6.86

2.61

6.48

5.46

C18:1n9t

3.70

5.89

4.96

nd

7.83

C18:1n9c

5.23

3.86

13.69

7.04

10.75

C18:2n6t

1.02

1.37

0.05

nd

0.26

3.12 nd nd 69.84 74.53 25.47 21.34 4.13 100

1.34 nd nd 74.90 70.83 29.17 26.47 2.71 100

6.82 nd nd 84.37 66.61 33.39 26.51 6.88 100

2.80 nd 2.78 74.65 82.48 17.52 14.72 2.80 100

5.30 0.46 nd 79.76 68.43 31.57 25.55 6.02 100

C18:3n6 C18:3n3 C20:0 C16-C18 Saturated fatty acid Unsaturated fatty acid Monounsaturated fatty acid Polyunsaturated fatty acid Total fatty acid nd – not detected

30

Table 5 Fatty acid profiles of Botryococcus braunii KMITL cultivated with a nitrogen source and then without it.

Fatty acid (%) N2.5

N2.5+ N2.5+N0 N2.5+N0 N2.5 (7d) (14d) (7d)

N2.5+ N2.5 (14d)

C4:0

0.07

0.16

0.14

nd

0.28

C6:0

0.18

0.31

0.29

0.08

0.05

C8:0

1.11

1.11

0.85

0.52

0.45

C10:0

0.80

1.25

2.05

0.49

0.82

C11:0

0.15

0.37

0.98

0.11

0.07

C12:0

3.27

4.70

1.66

4.30

2.97

C13:0

4.29

5.08

5.37

5.41

3.15

C14:0

1.55

1.98

3.58

1.38

1.30

C14:1

2.07

2.06

2.32

2.50

1.82

C15:0

0.47

0.57

0.95

2.63

1.54

C15:1

0.46

0.18

0.62

0.62

0.45

C16:0

41.11

43.07

39.90

41.44

37.62

C16:1

6.63

4.93

5.23

6.40

5.83

C17:0

10.33

7.10

6.34

4.58

10.41

C17:1

2.14

2.56

2.93

3.38

2.11

C18:0

6.93

7.75

8.09

6.73

8.51

C18:1n9c

14.00

12.70

14.63

15.13

18.62

C18:2n6t

2.02

1.51

1.93

1.34

1.44

C18:3n3 C16-C18 Saturated fatty acid

2.42

2.36

2.14

2.96

2.87

85.58 70.26

81.98 73.45

81.19 70.20

81.96 67.67

87.41 67.17

29.74 25.30 4.44 100

26.30 22.43 3.87 100

29.80 25.73 4.07 100

32.33 28.03 4.30 100

33.14 28.83 4.31 100

Unsaturated fatty acid Monounsaturated fatty acid Polyunsaturated fatty acid Total fatty acid nd – not detected

31

Table 6 Biodiesel properties of B. braunii KMITL under various cultivation conditions. IV (g

LCSF

I2 100 SV

g-1)

CN

DU

(wt.

CFPP

(wt.%)

%)

(oC)

Medium Modified Chu 13 medium

216.81

37.51 61.91

38.60

5.98

2.31

Kratz and Myers medium

213.27

42.01 61.18

41.98

6.39

3.59

3N-BBM+V

212.16

40.12 61.80

39.83

6.80

4.89

Chlorella medium

213.04

42.59 61.06

42.13

6.88

5.15

KNO3

212.22

47.51 59.90

44.29

4.75

-1.56

NaNO3

215.04

52.49 58.30

48.62

4.99

-0.79

Co(NH2)2

211.06

40.50 61.83

36.71

9.97

14.83

NH4 HCO3

210.50

47.25 60.18

43.33

6.85

5.05

0.13

223.75

26.26 64.00

29.60

8.53

10.33

0.31

214.25

28.83 64.42

31.88

8.38

9.84

0.63

209.13

39.74 62.27

40.26

5.70

1.44

1.25

213.77

19.65 66.82

20.32

11.30

19.03

2.50

210.21

36.54 62.95

37.59

7.03

5.61

N 2.5

208.86

30.70 64.60

34.18

7.58

7.32

N 2.5+N0 (7d)

211.11

26.65 65.36

30.17

8.18

9.23

N 2.5+N0 (14d)

210.93

29.45 64.66

33.87

8.04

8.77

N 2.5+N2.5 (7d)

208.87

32.30 64.20

36.63

7.51

7.11

N 2.5+N2.5 (14d)

207.18

33.80 64.03

37.45

8.02

8.71

Nitrogen source

Nitrogen concentration (g L-1)

Two-step cultivation

SV–saponification value, IV–iodine value, CN–cetane number, DU–degree of unsaturation, LCSF–long chain saturated factor, CFPP–cold filter plugging point.

32

Highlights

• B. braunii KMITL is a good source of biodiesel • Outdoor cultivation yielded higher biomass and lipids • Cultivation in a nitrogen-rich then poor medium gave the highest lipid yield • The lipids showed the highest cetane number reported in the literature