Extractable liquid, its energy and hydrocarbon content in the green alga Botryococcus braunii

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Extractable liquid, its energy and hydrocarbon content in the green alga Botryococcus braunii Y. Li a, R.B. Moore a, J.G. Qin a,*, A. Scott b, A.S. Ball a a b

School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia General Equity Building Society, Level 4, 17 Albert Street, Auckland, New Zealand

article info

abstract

Article history:

Due to sparse sampling across races, studies on various strains of Botryococcus braunii have

Received 12 April 2011

effectively been indiscriminate, and so the target strains for energy production have not

Received in revised form

come clearly into focus. This study compares extractable liquid biofuel content, bioenergy

19 February 2013

content and hydrocarbon content across 16 strains B. braunii (A, B and L races) by direct

Accepted 1 March 2013

combustion of algal biomass using thermogravimetric analysis (TGA), pressure differential

Available online 30 March 2013

scanning calorimetry (PDSC) and gas chromatography/mass spectrometry (GC/MS). All B. braunii strains were cultured in the same environmental conditions in 250 ml flasks, and

Keywords:

were harvested for analysis when algae reached the exponential growth phase. Significant

Botryococcus braunii

differences were detected within and between races A, B and L. The ranges of variation in

Extractable liquid

extractable liquid, biofuel energy and hydrocarbon contents in algal dry biomass were 10

Biofuel energy

e40%, 10e60% and 4e25%, respectively. The race B strains (Ayame 1, Kossou 4, Overjuyo 3

Hydrocarbon production

and Paquemar) had more than 21% of dry weight comprising C31-C36 hydrocarbons, which

Algae

are suitable for biofuel and bioenergy production. The Overjuyo 7 and CCAP 807/2 strains in race A and the Madras 3 and Yamoussoukro 4 strains in race L also showed high biofuel production with extractable liquid biofuel accounting for >30% of dry weight. This study identified particular B. braunii strains that are suitable for biofuel production. The application of TGA and PDSC provides a useful analytical approach for assessing the biodiesel production potential of microalgae. ª 2013 Elsevier Ltd. All rights reserved.

1.

Introduction

Due to concerns over global warming, desirability of CO2 mitigation, and the passing of peak petroleum oil production, research is being conducted into alternative biofuel production using microalgae [1,2]. For example, much attention has been focussed on Botryococcus braunii because of its ability to accumulate unusually high levels of hydrocarbon in the range of 15e35% dry weight [3,4], with up to 76% of the dry weight of cell material being combustible. Therefore, B. braunii has been considered a potential source of renewable carbon-neutral

biofuels. However, studies on various strains of this species have effectively been indiscriminate due to sparse sampling across races, and so the target strains for energy production have not come clearly into focus. The aim of this study was to obtain a comparison of bioenergy and hydrocarbon production across various laboratory strains of B. braunii. The green alga B. braunii is a cosmopolitan species in both temperate and tropical fresh water, and occasionally occurs in brackish water [5e7]. This species is classified into three chemical races according to the type of hydrocarbons found inside the cells: race A contains C21 to C33 odd numbered

* Corresponding author. Tel.: þ61 8 8201 3045; fax: þ61 8 8201 3015. E-mail address: [email protected] (J.G. Qin). 0961-9534/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biombioe.2013.03.002

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n-alkadienes, and mono-, tri-, tetra-, and pentaenes; race B produces two types of triterpenes called botryococcenes of C30 - C37 with a general formula CnH2n-10 as major hydrocarbons and similar amounts of methyl branched squalene; race L yields a single C40 isoprenoid hydrocarbon, lycopa-14(E),18(E)diene [3,8]. Although intensive research has been conducted to maximize the hydrocarbon content in algal cells via physical optimisation [5,7], the variation in this parameter between races and strains is still a concern when screening B. braunii for biofuel production [2,3]. One outlying dataset claimed that B. braunii produced hydrocarbons in the range of 2e86% of dry weight [2]. For the sake of greater discretion, a need exists to further investigate the differences in hydrocarbon content between strains and races. Hydrocarbon extraction from B. braunii can be accomplished via chloroform, methanol or hexane [3,9,10]. The products obtained by extraction of B. braunii in many studies are raw lipids, rather than hydrocarbons. In fact, ether-lipids, non-polysaccharide biopolymers, polyaldehydes and polyacetals can be dominant in the extracts of some B. braunii strains [3]. Therefore, it is necessary to methodically and equivalently quantify hydrocarbon production between strains and races. Considering energy output, microbially-derived compounds other than hydrocarbons can be utilised, such as biomethane [11], bioethanol [12] and biohydrogen [13]. Similarly, in B. braunii, ether-lipids and cell wall components contribute to the energy yield from biomass. Therefore, the measurement of total bioenergy content in B. braunii can be indicative of energy production potential, rather than only quantifying hydrocarbons. We have undertaken a screen for strains of B. braunii to determine which are most efficient at biofuel (liquid) energy production. The usual way to measure the oil (liquid biofuel) content of algae is by chemical extraction of the algae using solvents. However, oil solubility depends on solvent properties. Therefore, the oil contents of algae are not always fully recovered through solvent extraction. By contrast, the amount of combustible material can be accurately estimated by thermogravimetric analysis (TGA), which measures the change of weight of combustible materials at given temperatures as temperature is increased over time [14], is very accurate. For instance, the current study shows that the accuracy in TGA analysis of combustible algal biomass can reach microgram levels. As combustible weight is quantifiable, the energy capacity can also be measured using differential scanning calorimetry (DSC), which is a thermoanalytical technique allowing precise measurement of the heat capacity [15]. Since biofuels in B. braunii largely exist in the form of hydrocarbons [3], identification of specific hydrocarbons remains central to screening of B. braunii strains for biofuel production. A rapid and sensitive method to identify a range of hydrophobic substances within a test sample is gas chromatography-mass spectrometry (GC/MS). It not only serves in identification of hydrocarbons in B. braunii, but can also assist in and quantification of these. In most previous studies, only the relative content of hydrocarbons with varying carbon chain lengths was reported. Only rarely has quantification of hydrocarbon production been achieved relative to the dry biomass of algae.

In this study, the triple aims were to determine the percentage of dry biomass comprising extractable liquid (by subtracting combustible solids), to quantify the percentage of total combustible energy that comprises extractable liquid energy and to quantify hydrocarbon identities and concentrations across 16 strains of B. braunii comprising three chemical races. We first used TGA to examine the extractable liquid content in algal dry biomass and we then used DSC to determine the percentage of biofuel energy in the total bioenergy of the biomass. Finally, the specific hydrocarbon contents of algal dry weight were analysed by GC/MS. The results of this study provide useful reference points for identifying strains of B. braunii that are optimal for liquid fuel production.

2.

Materials and methods

2.1.

Experimental microalgae

Sixteen strains of Botryococcus braunii used in this study are listed in Table 1. ACOI, UTEX, CCAP and CCAC cultures were obtained from public culture collections. The remaining cultures were obtained from Dr. Pierre Metzger from the Laboratoire de Chimie Bioorganique et Organique Physique, UMR CNRS 7573. The races of all strains except the ACOI and CCAC strains were previously known [16]. The chemical races of ACOI 1248 and CCAC 0121 are demonstrated in the current study Algal stocks were maintained at 23  C in sterilised Jaworski’s Medium (JM) [17]. A 20 ml algal seed stock (approx. 1.0 mg/ml of dry weight) was inoculated into 200 ml of fresh JM medium in 250 ml conical flasks in order to start the culture. Three replicates were used for analysis of each algal strain. The mono-algal cultures were grown at a light intensity of

Table 1 e The sixteen strains of Botryococcus braunii used in this study. Race A

B

L

Strain Names Overjuyo 7 Jillamatong UTEX 572

Origin Bolivia Australia Cambridge

Sources

Pierre Metzger’s collection Pierre Metzger’s collection University of Texas at Austin UTEX 2441 Lake Huaypo University of Texas (Peru) at Austin Lingoult France Pierre Metzger’s collection CCAP 807/1 Cambridge Culture Collection of Algae and Protozoa, CCAP 807/2 Cumbria Scottish Marine institute, UK La Manzo Martinique Pierre Metzger’s collection Kossou 4 Ivory Coast Pierre Metzger’s collection CCAC 0121 Austria University of Cologne (Germany) ACOI 1248 Macedo de University of Coimbra Cavaleiros (Portugal) Overjuyo 3 Bolivia Pierre Metzger’s collection Ayame 1 Ivory Coast Pierre Metzger’s collection Paquemar Martinique Pierre Metzger’s collection Madras 3 India Pierre Metzger’s collection Yamoussoukro 4 Ivory Coast Pierre Metzger’s collection

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60 W/m2 with a photoperiod of 12 h light and 12 h dark, at a temperature of 23  C for 34 d. Subsequently each microalgal culture (200 ml) was filtered with a 0.47-mm glass fibre filter (Millipore) facilitated by an air vacuum pump (GE instruments). Each filter, already loaded with algal slurry, was cut in half: one half was lyophilized for thermal energy analyses (vacuum drying at 42  C for 24 h); and the other half was folded into a capped plastic tube and preserved in liquid nitrogen for hydrocarbon determination.

2.2. Solvent-extractable biofuel and thermal energy analyses of microalgae Each filter holding the lyophilized strain of a microalgal species was cut into small pieces each holding 2e3 mg algae, and each piece was weighed before being loaded on a standard aluminium pan (TA Instruments) used for analyses on a thermogravity analyser and a differential scanning calorimeter. A known weight of a glass fibre filter was used as a background control in each measurement. Each algal strain was measured in triplicate for each parameter.

2.3.

Combustible content of dry microalgae

calorimetric analyses. Because of the use of high pressure, the range of combustion temperature in DSC was slightly different from the temperature range in TGA. The instrumental cell constant and temperature calibrations were performed using indium. The cell was then purged for 30 s to establish an oxygen atmosphere before being sealed and pressurised. The microalgal samples were weighed into a standard aluminium pan and placed onto the DSC cell platform, then analysed with continuous heating at a rate of 10  C/min until 500  C was reached, while being held at a constant oxygen pressure of 3500 kPa throughout. The sample was combusted over the temperature range 100  Ce220  C to measure the bioenergy of solvent-extracted liquid, determined by the difference between original cells and oil-extracted cells (J/mg) as shown for the Ayame 1 strain in a preliminary test (Fig. 2). The bioenergy percentage of extractable liquid was calculated as equation (2), corresponding to the measurement of biomass reduction in TGA. All the TGA and DSC data were analysed using Universal Analysis 2000 v3.3B software (TA Instruments). Energy of extractable liquidð%Þ ¼ EC1  BR1 =ðEC1  BR1 þ EC2  BR2 Þ

(2)

where,

The amount of combustible biomass was measured on a thermogravimetric analyser (TGA 2950, TA Instruments). An aluminium pan containing the sample was placed into a tared pan attached to a microbalance and enclosed in a furnace. The furnace and balance were then purged with pure oxygen at a flow rate of 50 ml/min and the furnace was heated with a programmed heating interval of 20  C/min, until 600  C was reached. A quantitative plot of the mass and mass change ( y axis) versus temperature (x axis) was obtained. In a preliminary study, the algal biomass reduction that occurred between 150  C and 250  C represented the major difference between algal cells and hexane-extracted algal cells as demonstrated using the Kossou 4 strain (Fig. 1). The range 150  Ce250  C was therefore selected as the effective flash point for the solvent-extractable biofuels in these strains of microalgae. The mass loss of glass-fibre filter alone was then used to calibrate the microalgal mass loss in the TGA analysis. The biomass loss between 100  C and 150  C was not due to extractable high-energy substances but rather was due to moisture and other non-oil substances, therefore we only included this loss in the denominator (Eq. (1)). The percentage of extractable liquid in microalgal dry biomass was calculated as equation (1): Extractable liquidð%Þ ¼ BR1 =BR2

105

(1)

where, BR1: biomass reduction (mg) from 150  C to 250  C; BR2: biomass reduction (mg) from 100  C to 600  C

2.4. Thermal energy in solvent-extracted microalgal biofuel A differential scanning calorimeter (DSC 2920, TA Instruments) fitted with a high pressure cell was used for all microalgal

EC1: Energy content of algae in DSC from 100 to 220  C (J/mg); EC2: Energy content of algae in DSC from 220 to 500  C (J/mg); BR1: Biomass reduction in TGA from 150 to 250  C (mg); BR2: Biomass reduction in TGA from 250 to 600  C (mg);

2.5.

Hydrocarbon analyses

Frozen filters loaded with algal cells in the capped plastic tubes were thawed in hot water (60  C), and then refrozen again in liquid nitrogen. After repeatedly being quick frozen and thawed 4e5 times, the thawed filter was transferred to a glass bottle containing 10 ml of hexane (SigmaeAldrich). After 5 min sonication, 10 ml of extract was decanted and saved. The algal filter was repeatedly extracted until the solvent became colourless. The extractions were pooled and refiltered on a 0.45-mm glass fibre filter, then concentrated to 3 ml using a rotary evaporator (Bu¨chi Rotavapor R-114, 40  C, 335 mbar) in preparation for gas chromatography/mass spectrometry (GC/MS) analysis. Analysis of microalgal hydrocarbons was carried out on a GC/MS (comprising Agilent Technologies 5975C mass spectrometer, 7890A gas chromatograph, and 7683B autosampler) equipped with a capillary column (Phenomonex EC-5, 15 m  0.25 mm using a 0.25 mm film). The carrier gas used was helium, which was applied in constant flow mode at a rate of 1.8 ml/min. Each sample (1 ml) was injected into the capillary column at an initial temperature of 40  C for 4 min, followed by a linear ramp of 20  C per min until 350  C was reached, and then the sample was held steady at 350  C for 4 min. The GC/MS interface temperature was set to 280  C and the FID was 325  C. The mass spectrometer was scanned from 35 m/z to 550 m/z, with 70 eV of electron ionisation. The MS quadrupole was set to 150  C. In this study, 1-chlorooctadecane (Supelco, 44-2260, 1000 mg) was used as the internal standard (IS). More than 20 B. braunii hydrocarbons of known structure and concentration

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Fig. 1 e The difference of TGA analyses between algal cells and solvent extracted cells in strain of Kossou 4.

(obtained from Dr. P. Metzger) were used to calibrate the response factor of hydrocarbons to the IS for the quantification of extracted hydrocarbons.

data presented in the figures are non-transformed values (means  SE). A significance level of 0.05 was used for all tests.

2.6.

3.

Results

3.1.

Extractable liquid biofuel content of dry biomass

Statistical analyses

Data analyses were performed with SPSS (ver. 18.0 for Windows). Each parameter was tested using one-way ANOVA by strain and race, respectively. Data were transformed by square root or logarithm to meet assumptions of normality and homogeneity of variances when necessary. However, the

The extractable liquid biofuel content in 16 experimental strains of B. braunii accounted for 10e40% of algal dry mass (Fig. 3). Extractable liquid biofuel is here defined using the

Fig. 2 e The difference of DSC analyses between algal cells and solvent extracted cells in strain of Ayame 1.

107

def

g efg efg

fg

g efg def

ade

ad

a b

Race B (B)

CCAC 0121

La Manzo

Paquemar

Overjuyo 3

Kossou 4

Ayame 1

CCAP 807/2

Overjuyo 7

UTEX 2441

UTEX 572

Jillamatong

Race A (A)

Yamoussoukro

bc

c

Madra 3

b

ACOI 1284

a

CCAP 807/1

45 40 35 30 25 20 15 10 5 0

Lingoult

Oil content (% of Dry weight)

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Race L (AB)

Strains of Botryococcus braunii Fig. 3 e The percent of organic-solvent-extractable oil content in dry weight of Botryococcus braunii examined by thermogravity analyser (TGA). The different small letters indicate the significant difference among strains; the different capital letters in the brackets indicate the difference among races (P < 0.05).

simple definition combustible apolar liquid, and was in practise defined as organic-solvent-extractable (OSE) oil which is the portion of cell contents dissolvable in hexane. Oils from B. braunii contained not only hydrocarbons but also ether-lipids which are apolar. In race A, the OSE contents of Overjuyo 7 and CCAP 807/2 were higher (>31%) than other strains (P < 0.05), and these two strains had similar high levels as race B and L strains. The OSE content of the UTEX 572 strain was the lowest (w10%, P < 0.05), while no significant differences were detected between the Lingoult and Jillamatong strains, or between CCAP 807/1 and UTEX2441 strains (P > 0.05) of race A. Unlike race A, race B strains consistently contained high OSE levels (>30% of dry mass), except the ACOI 1284 strain which contained w10% (P < 0.05). In race L, there was no significant difference between the Madras 3 and Yamoussoukro 4 strains (P > 0.05) both having OSE contents of about 21%e27%. When averaged, the OSE contents in races B and L were higher than those in race A (P < 0.05).

3.2.

OSE energy of dry biomass

The amount of organic solvent extractable energy, or oil energy, in races B and L was greater than that in race A (P < 0.05, Fig. 4), though care should be taken with this generalisation, as strain ACOI 1284 of race B was much lower in energy than the others. Among race A strains, the highest OSE energy was found in the strain CCAP 807/2, followed by the Overjuyo 7 strain (P < 0.05). The OSE energy content of UTEX 572 was the lowest of all race A strains (P < 0.05). In race B strains, the Kossou 4 and Paquemar strains contained the highest OSE energy (>55%). Other race B strains were also of generous OSE energy

levels (across a gradient from 40% to 50%), except the ACOI 1284 strain which contained the lowest OSE energy of all the race B strains (at approx. 10%, with P < 0.05). Among race L strains, the Madras 3 strain contained higher OSE energy than the Yamoussoukro 4 strain (P < 0.05). Cross race comparisons show that CCAP 807/2 (race A), Madras 3 (race L) and several race B strains (Ayame 1, Overjuyo 3, and La Manzo) contained similar amounts of OSE energy (P > 0.05) at close to or above 50%. Among the 16 B. braunii strains, Paquemar and Kossou 4 contained the highest OSE energy levels.

3.3.

Hydrocarbon determination

Even though hydrocarbon standards were obtained from Dr. Metzger, a few compounds still remained unidentified in our chromatographs. Furthermore, hydrocarbon information in the GC/MS library of the Agilent MS was sparse, and poor resolution of GC peaks occurred in some cases. Nevertheless, the dominant hydrocarbons were quantified. In this study, hydrocarbon content was calculated as a percentage of total algal dry biomass for each alga, based on TGA measurement. Hydrocarbons with odd numbered carbon chains from C23 to C31 were evident in race A, while C31-C36 hydrocarbons were evident in race B, and C40 was the only hydrocarbon evident in race L (Table 2). The hydrocarbons present varied markedly among B. braunii strains, both with respect to chain length obtained and concentration in cells. The hydrocarbon content in race B was relatively more than in other races. Among the race B strains, the maximum hydrocarbon content was around 24% of dry biomass which occurred in the Ayame 1, Overjuyo 3 and Paquemar B strains. In race A, the highest hydrocarbon content obtained was in the Jillamatong strain at

108

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gh ef

50 40

i

hi

60 a

30

ac

gh

efg

efg

fg e

d

20

e

bc

b

d

Race A (A)

Race B (B)

Yamoussoukro

Madra 3

ACOI 1284

CCAC 0121

La Manzo

Paquemar

Overjuyo 3

Kossou 4

Ayame 1

CCAP 807/2

Overjuyo 7

UTEX 2441

UTEX 572

Jillamatong

0

CCAP 807/1

10 Lingoult

Oil energy content (% of algal total energy)

70

Race L (B)

Strains of Botryococcus braunii Fig. 4 e The solvent-extracted oil energy percentage of total thermal value in various Botryococcus braunii strains. The different small letters indicate the significant difference among strains; the different capital letters in the brackets indicate the difference among races (P < 0.05).

19.29%. Although the lowest hydrocarbon content among the race A strains studied was in UTEX 572 (6.67%), this was still slightly greater than in the lowest producing race L strain Yamoussoukro 4 at 4.09%. The highest hydrocarbon content produced in the race L strains studied was by the Madras 3 strain at 7.48%, and so overall, the race L strains produced less hydrocarbon than other races.

4.

Discussion

This study was novel in that a thermogravimetric analyser, a pressure differential scanning calorimeter (DSC) and a GC/MS were used together to provide quantification of organicsolvent-extracted oil, its energy, and the hydrocarbon profiles for a wide range of B. braunii isolates (16 strains across three races). The application of TGA and DSC provides a new approach in quantifying the OSE-oil content and OSE energy in microalgae. Although wide variation existed in hydrocarbon concentrations between B. braunii strains and races, race B seemed to contain the highest relative levels, with the exception of the ACOI 1284 strain. Given the 1e4 fold variation in solventextractable oil contents measured by TGA, the importance of strain selection for biofuel production is clear. Compared to previous reports on the strains selected in this study, the oil content measured by TGA was less than in the previous reports, in different degrees. For example, the total OSE oil contents in strains of La Manzo, Yamoussoukro 4 and Overjuyo 7 reported by Metzger and Largeau [18] were 53%, 35% and 62% respectively, with hydrocarbon contents of 32%, 3% and 0.4% respectively. However, our study showed that the

organic-solvent-extractable oils of these strains were 32%, 21% and 26%, and their hydrocarbon contents were 16%, 4% and 7.8%, respectively. There are three possible reasons for the differences in total oil content in the same strains between studies. Firstly, the range of 150  Ce250  C in TGA may not cover the flash points for all solvent-extracted compounds (Fig. 1); secondly, some macromolecular lipids and polar compounds (such as carbohydrates and algaenans) may not be extracted by the apolar solvent hexane [18] (these compounds may correspond to the second peak in TGA chromatographs between 250  C and 300  C, but further research is needed to align thermochemical analyses as these compounds could not be extracted by hexane, and so they were not considered a part of combustible liquid in this study); thirdly, different culture conditions could result in differences in lipid content [3,19]. Meanwhile, a quantification method used by Brown et al. [20] may be questionable as no other studies have reported similarly high values for biofuel liquid content as they did, even though there have been four decades of study since on this same algal species. While B. braunii performance in oil production across chemical races and strains depends on culture conditions [21], the comparative oil content of a range of B. braunii strains under a fixed set of culture conditions, such as used in this study, can potentially provide a reliable indication of comparative suitability for biofuel production. Further, the weight loss and heat production of algae with and without solvent extraction indicates that thermogravimetric determination can be a reliable measure of the readily extractable liquid fraction of algal dry weight. In other studies, a range of hydrocarbons and many ether lipids have been identified in the solvent extracts of B. braunii

Table 2 e The hydrocarbon percentage (%) of B. braunii dry weight. C Noa

Race A

Race B

Race L

Lingoult Overjuyo 7 Jillamatong UTEX UTEX CCAP CCAP Ayame 1 Kossou 4 Overjuyo 3 Paquemar La CCAC ACOI Madras 3 Yamoussoukro 4 572 2441 807/1b 807/2b Manzo 0121 1284 1.11 2.98 2.64 2.79

0.72 2.06 1.40 0.51 0.68 1.40

7.01

3.29 3.38 3.77 1.66 3.73 2.02 1.44

0.60 0.67

3.70 2.69

2.96 0.39

1.30

1.44 0.61

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23 25 27 27 27 29 29 31 31 31 31 32 32 32 33 33 33 34 34 34 36 40 Total (% of dry weight)

2.98 2.69 4.85 7.22

3.40 9.10

5.50

4.98 7.61 6.67

5.65 4.44 1.61

5.92 2.48

2.19 3.52

4.79 2.08 7.26 0.83 6.16 2.22 1.30

2.42

2.14

2.51 6.32

3.55

3.66 1.16

3.23

2.07 1.54

1.85

2.63 16.53

7.81

19.29

6.67

11.36

24.2

21.12

24.97

24.64

16.07

13.77

8.09

7.48 7.48

4.09 4.09

a The details of hydrocarbon structure and identification are referred to the studies of Metzger et al. [3,8]. b The data of shading cultures were lost during sample processing.

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[3,18]. Besides ether lipids, many other polar compounds have also been identified in B. braunii, such as polysaccharides and algaenans [18], which are also potentially convertible to biofuels [11e13]. From the point of view of energy content, lipids contain a very high energy content, on average 38.9 kJ/g [22], which is much higher than the ignite coals (15e21 kJ/g) for instance. Similarly, the analysis of heat capacity of biofuel energy (e.g. hydrocarbon energy) as a fraction of dry biomass is beneficial when screening microalgae for biofuel production. The combined results from TGA and DSC measurements show that the race A strains CCAP 807/2 and Overjuyo 7, all race B strains except ACOI 1284, and both of the race L strains, Madras 3 and Yamoussoukro 4, are most productive in extractable liquid energy compared to the other B. braunii strains studied. Although the organic-solvent-extractable oil content measured was between 10% and 40% of algal dry biomass, thermal energy of the extractable liquids comprised between 10% and 60% of the total bioenergy. The extractable bioenergy in Overjuyo 7 and CCAP 807/2 race A strains, most of the race B strains (excl. ACOI 1284) and two race L strains, was more than 40% of the total bioenergy, but of special interest were the Kossou 4 and Paquemar strains of race B, in which the extractable bioenergy was close to 60% of the total bioenergy of the dry algal biomass. Although the proportion of extractable bioenergy in race A was on average less than that of races B and L, it was strain-specific to a large extent. In view of these results, the analysis of oil thermal energy via DSC, when expressed as a percentage of the dry residual biomass after extraction, can provide reliable information on biofuel energy content across strains of B. braunii. In this study, hydrocarbon contents are in some cases substantially different from those arrived at in previous studies of the same strain. For example, Metzger et al. [23] reported that the Jillamatong strain contained 5%e15% hydrocarbon while we obtained about 19% hydrocarbon for this strain as a percentage of dry weight. The hydrocarbon content of Overjuyo 7 was 7.8% in this study, which is much higher than the value of 0.4%e2% determined by Metzger et al. [24]. The Lingoult strain was reported to have 32% hydrocarbon as a percentage of dry biomass [25], which is much higher than the 16% obtained in this study. The hydrocarbon content of La Manzo was 32e43% in another study [25], which is much higher than the 16% determined in this study. On the other hand, we found that the Ayame 1 strain contained 24% hydrocarbon, which is only slightly less than the 27% reported before [18], and the hydrocarbon content of the Yamoussoukro 4 strain was 3% of dry biomass in a previous study [8], which is quite similar to 4% in this study. There being no trend, the unpredictable discrepancies and inconsistencies across studies could be due to the different solvents being used for extraction, the particular settings used in TGA analysis, the GC/MS quantification method, the algal culture conditions [5,26], or some combination of these. Overall the results from the current study have very practical value because these sixteen strains were compared under the same environmental conditions. Similar to findings by Metzger and Largeau [3], the content and composition of hydrocarbons in this study varied significantly between strains. In race B strains, a tenfold difference in

hydrocarbons was reported to exist between B. braunii strains [18]. Among race L strains, the range of hydrocarbon content varied from 0.1% to 8% [23,27], again a tenfold range or greater. By contrast, in the current study, in race A for example, the lowest hydrocarbon content obtained was 6.67% (UTEX 572), and a tenfold range of hydrocarbon concentrations was not seen within any one race. As such our methods of quantifying hydrocarbon were internally well controlled. Other notable cases within our dataset underscore the value of the methods. Even though the Jillamatong strain had relatively less solventextractable oil content and lower bioenergy than othere race A strains, the hydrocarbon content was more than the other race A strains (excl. CCAP strains) at up to 19.29% of dry biomass. Conversely, the hydrocarbon content in Overjuyo 7 was 11.36%, which was not correlated to its high level of solvent-extractable oil content and biofuel energy. It is concluded that Overjuyo 7 may contain relatively high levels of other extractable liquids, while Jillamatong’s extractable liquids are mainly hydrocarbons. Within race B, the hydrocarbon content of each strain corresponded well to the solventextractable oil content and biofuel energy level. Race B strains Ayame 1, Kossou 4, Overjuyo 3 and Paquemar all produced > 21% hydrocarbons relative to dry biomass. Although there were only 11 botryococcenes quantified in this study, a large variation in the botryococcene composition was noticed. In a specific example, the Ayame 1 strain displayed C33-C34 botryococcene patterns similar to those in a previous report [28]. In the two race L strains, Madras 3 and Yamoussoukro 4, C40 lycopadiene accounted for 7.48% and 4.09% of dry biomass, respectively. Race L strains had high solvent-extractable oil energy content and it seems unlikely that this could be solely due to the hydrocarbons contained. In all, hydrocarbon contents and solvent-extractable oil energy contents of B. braunii did not vary by orders of magnitude within or across races, when grown under identical conditions. A key point of this study is that the hydrocarbon portion of algal biomass in each strain was less than the organic-solventextractable oil content measured by TGA. As extreme examples, hydrocarbon in Overjuyo 7 of race A was 7.81% of dry biomass (Table 2), but extractable oil content was 31% by TGA (Fig. 3); and the hydrocarbon content in strain Madras 3 of race L was 7.48% (Fig. 4) and its solvent-extractable oil content was 30% by TGA (Fig. 4). As an example of a lesser but still clear difference, the hydrocarbon percentage of Paquemar (race B) was 24.64% (Table 2), and its extractable oil content was about 38% (Fig. 4). Metzger and Largeau (1999) also reported discrepancies between hydrocarbon and extractable oil contents in B. braunii. For the purposes of clear comparison, only the hydrocarbon contents identified from the GC/MS library, constructed for this study based on known B. braunii hydrocarbon standards were included here. The combined amounts of hydrocarbons with known structures identified here were lower than the total extractable oil content measured by TGA, and this was in some ways surprising given the long years dedicated by others to elucidating both the hydrocarbons and the other combustible liquid compounds of B. braunii. While there are two major apolar components (hydrocarbons and ether lipids) responsible for liquid bioenergy in B. braunii [3,18], it is conceivable that other types of combustible compounds may exist in the extraction solvents.

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Botryococcenes (C30-C37) are the hydrocarbons most often reported to be produced by B. braunii strains, in other words emphasis in the literature is predominantly on the race B strains [3,8]. Our study has confirmed that C31-C36 hydrocarbons (triterpenoid derivatives) were present and at high combined concentrations in each of the race B strains studied. Furthermore, hydrocarbon production was greater on average in race B strains than in race A or race L strains (Table 2). In view of the combined solvent-extracted biofuel content and energy analyses, certain race B strains Ayame 1, Kossou 4, Overjuyo 3 and Paquemar had the best potential for biofuel production. The influence of various culture parameters on hydrocarbon production is now well known. This leaves the best candidate strains to be chosen based on inherent genetic potential, using parameters such as those measured in this study. Even though culture conditions (such as nutrients) have been established, to date no large scale culture of B. braunii strains has proven financially worthwhile. On the other hand, the cloning of the squalene synthase gene homologues from a strain of B. braunii race B, and their expression in Escherichia coli and Saccharomyces cerevisiae [29,30] offers promising prospects for the production of hydrocarbons by moving the gene to a fast-growing micro-organism [3,30]. Other genes of the triterpenoid synthesis pathway, and its upstream feeder the isoprenoid biosynthesis pathway, can also benefit these analyses if and when these are cloned and sequenced. The race B strains recommended in this study can extend options for research on hydrocarbon biosynthesis pathways, by identifying optimal strains from which to mine genetic content. Additionally strains analysed here may have potential as native sources of biofuels: botryococcenes, alkadienes, lycopadiene and ether lipids, or as models for biochemical and genetic analyses to determine which enzymes and pathways produce these combustible compounds, not just which enzymes produce botryococcenes.

5.

Conclusion

Through comparison of extractable liquid biofuel content, bioenergy content and hydrocarbon content across 16 strains, this study provides useful information on B. braunii strains for further biofuel research. The selection of suitable strains is an essential step towards successful production of biofuel from microalgae. Just as the present study identified particular B. braunii strains, the type of TGA and DSC analyses used can well aid in other algal species selection. For example, this study may serve as model for how such studies would be conducted across algal genera that produce triglycerides as their main combustible compounds.

Acknowledgements We are grateful to Dr Pierre Metzger from Laboratory of Organic and Bioorganic Chemistry Physics, Ecole Nationale Supe´rieure de Chimie de Paris (France) for supplying most of the B. braunii strains and all of the hydrocarbon standards used. We also thank Dr Rachel Pilar and Dr Milena Ginic-Markovic of the

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Nanomaterials Lab at Flinders University for TGA and DSC assistance, Dr Stephen Clarke of Flinders University for suggesting the use of TGA and DSC, and Dr Daniel Jardine of the Advanced Analytical Laboratory at Flinders University for GC/MS technical assistance. Dr Jardine’s previous experience with B. braunii hydrocarbon analysis and attention to detail greatly facilitated this work. This project is supported by General Equity Building Society (New Zealand) acting as financier for WWCC Limited of Hong Kong.

references

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