Characterization of oil-extracted residue biomass of Botryococcus braunii as a biofuel feedstock and its pyrolytic behavior

Applied Energy 132 (2014) 475–484

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Applied Energy journal homepage: www.elsevier.com/locate/apenergy

Characterization of oil-extracted residue biomass of Botryococcus braunii as a biofuel feedstock and its pyrolytic behavior Hideo Watanabe a,⇑, Dalin Li b, Yoshinao Nakagawa b, Keiichi Tomishige b, Kunimitsu Kaya c, Makoto M. Watanabe c a b c

Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8573, Japan Department of Applied Chemistry, School of Engineering, Tohoku University, 6-6-07 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan

h i g h l i g h t s  Oil-extracted residue of Botryococcus braunii can be a superior biofuel feedstock.  The residue has higher contents of C, H and volatile matter than other biomass.  Element composition of the residue can be related to original hydrocarbon content.  Pyrolyzed products (char, tar and gases) of the residue were initially characterized.  The residue can be classified as H-rich solid fuel comparable to fossil fuel (coal).

a r t i c l e

i n f o

Article history: Received 8 November 2013 Received in revised form 13 June 2014 Accepted 10 July 2014

Keywords: Botryococcus braunii Algal biomass Oil-extracted residue Biofuel Thermogravimetric analysis Pyrolysis

a b s t r a c t Botryococcus braunii (B. braunii), a green colonial microalga, is one of the most potentially significant renewable energy sources, because of its high productivity of hydrocarbon oils. In order to maximize energy utilization from this alga, it should be important to recover energy from not only the extracted oil but also the biomass residue after oil-extraction. In this work, oil-extracted residue biomass of an identified strain of B. braunii (BOT-22) with different original hydrocarbon content (HC%: from 28 to 51 wt%) has been characterized as a biofuel feedstock in terms of the elemental composition as well as the pyrolytic behavior by thermogravimetric (TG) analysis and steam pyrolysis test. This paper has revealed that the B. braunii with higher HC%, even biomass residue after oil-extraction, is superior in quality of a biofuel feedstock because of higher content of C, H and volatile matter and lower content of N, O, S, P, ash and fixed carbon as compared to that with lower HC% and other algal biomass. Steam pyrolysis of the oil-extracted residue biomass of the B. braunii at 873–973 K can produce highly carbonized char (10%-C), gases of C1–C4 (30%-C) and H2, and tar (60%-C) including mainly heavy component such as polycyclic aromatic hydrocarbons. From the analysis of the pyrolyzed products and its higher heating values from 29 to 36 MJ/kg, the residue biomass of B. braunii can be classified as a hydrogen-rich solid fuel comparable to fossil fuel such as a coal rather than other algal and terrestrial biomass. In summary, not only hydrocarbon oils produced from the B. braunii but also its oil-extracted residue biomass should be a superior feedstock for biofuel production. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae have attracted considerable attention as a renewable energy source, since they have much higher oil productivity as compared to major oil crops [1]. Among a variety of lipid/oil-producing ⇑ Corresponding author. Address: Project Research Bldg. 4F (Rm. 401), University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8573, Japan. Tel./fax: +81 (0)29 853 5898. E-mail address: [email protected] (H. Watanabe). http://dx.doi.org/10.1016/j.apenergy.2014.07.037 0306-2619/Ó 2014 Elsevier Ltd. All rights reserved.

microalgae, Botryococcus braunii (B. braunii), is one of the most promising candidates for renewable production of biofuels, because of its extremely high productivity of hydrocarbon oils [2,3]. For achieving commercial production of hydrocarbons by B. braunii, it is necessary to reduce input energy and costs for a series of processes such as cultivation, harvesting, dewatering/drying and oil extraction, and also to increase the quantity and quality of products which are mainly extracted oils and biomass residue after oil extraction [4].

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For the oil production by the B. braunii, a good number of researches have been addressed in terms of culture conditions such as effects of carbon source [5], a wastewater [6] and light illumination [7], and biosynthesis routes of hydrocarbons [8] as well as gene analysis [9]. The hydrocarbon oils produced by the B. braunii are basically classified into heavy oil fractions, and can be converted into transport fuels such as a gasoline fraction by hydrocracking [10] or catalytic cracking [11]. As above-mentioned, it should be important to recover energy from not only the extracted oil but also the biomass residue after oil-extraction to maximize energy utilization from the B. braunii. Thermochemical conversions of raw or residual algal biomass apart from the B. braunii have been carried out by means of combustion [12], pyrolysis [13–14], gasification [15–17], liquefaction [18–21] and carbonization [22]. For the B. braunii, hydrothermal liquefaction was demonstrated with whole cells of B. braunii including the hydrocarbon oils [23,24], but thermochemical conversion of the residue biomass of B. braunii after oil extraction had hardly been carried out. More recently, pyrolysis of raw and residue biomass of B. braunii has been investigated by means of thermogravimetric (TG) analysis [25]. However, only single batch culture of B. braunii, which was collected from a natural bloom (an unidentified strain), was employed for the TG analysis, and hence no discussion was made on effect of biomass composition, which may be varied significantly by culture conditions, on the pyrolytic behaviors: this should be very important to evaluate the biomass of B. braunii as a feedstock for biofuel production. Moreover, no information was given in the previous work about products from pyrolysis of biomass of B. braunii, whereas it should be very essential from the viewpoint of biofuel feedstock. In this work, oil-extracted residue biomass of an identified strain of B. braunii (called BOT-22), of which growth ability and oil productivity are quite high [6,26], has been characterized as a biofuel feedstock in terms of the elemental composition as well as the pyrolytic behavior by TG analysis and steam pyrolysis test. For this study, five batch cultures of the B. braunii with different hydrocarbon content (HC%) were employed in order to discuss the effect of the biomass composition on the pyrolytic behaviors. Elemental compositions of the B. braunii and its higher heating values (HHV) calculated from the compositions will be described in this paper as a function of HC% for both of raw and residual biomass of B. braunii. Pyrolytic characteristic of the algal biomass samples will be discussed based upon the TG analysis. Furthermore, steam pyrolysis of oil-extracted residue biomass of B. braunii has been carried out in a laboratory-scale continuous feeding reactor with temperature range from 873 K to 973 K. Identification and initial characterization of the pyrolyzed products such as solid (char), condensable liquid (tar) and gas will be described in this paper.

2. Materials and methods 2.1. Preparation of algal biomass samples The BOT-22 strain of B. braunii [6,26] used in this work was originally isolated from the Okinawa prefecture, Japan [8]. This strain is classified race B and produces botryococcene (C34H58) as a main component of hydrocarbons [8]. Botryococcus seed culture system developed in University of Tsukuba [27] provided culture broth of the BOT-22 strain of B. braunii for this work. The broth was concentrated by a centrifuge, and followed by drying at 333 K for overnight. A part of the dried sample of the B. braunii was soaked into n-hexane for extraction of hydrocarbon oils including a small amount of carotenoids. After the extraction, the residual sample was recovered by filtration and

then dried again. Hydrocarbon content, HC%, was defined in this paper as a mass fraction extractable by the n-hexane to dried alga mass. Note that almost all of hydrocarbon fractions were able to be extracted by only n-hexane from the dried samples, whereas mixed solvents such as chloroform and methanol were needed for the extraction of total lipids including triglycerides and others. Both of raw (without n-hexane extraction) and residue (after the oil-extraction) samples of the dried B. braunii were milled and sieved, and then the size fraction from 106 to 500 lm was used for following analyses and pyrolysis tests. Dry powders of Chlorella sp. (commercially available from Yaeyama Shokusan Co., Ltd., Japan) and cedar wood (used in previous work [28]) were also employed as references of algal and lignocellulosic biomass, respectively. 2.2. Proximate and ultimate analysis Raw and residue samples of B. braunii underwent compositional analyses. Proximate analysis such as moisture and ash contents was carried out using test methods based upon Japanese Industrial Standards (JIS) for densified refuse derived fuel. Moisture content was measured as weight loss percentage of biomass sample by heating at 380 K for one hour (JIS Z7302-3) and ash content was measured as percentage of residual mass (ash) after heating the samples at 1088 K under ambient air atmosphere for one hour (JIS Z7302-4). C, H and N contents in the biomass were measured using a CHN Elemental Analyzer (Perkin–Elmer 2400), where O content was deduced by difference: thus, O = 100  (C + H + N). Analysis on the other elements was carried out by an Energy Dispersive X-ray (EDX) Fluorescence Spectrometer (Shimadzu, EDX-800HS). Higher heating value (HHV) of the biomass samples was calculated according to following Dulong’s equation:

Q ¼ 0:3383C þ 1:442ðH  O=8Þ

ð1Þ

where Q is in MJ/kg unit and C, H and O are the percentages by mass of carbon, hydrogen and oxygen [23]. 2.3. Thermogravimetry (TG) analysis TG/DTA (differential thermal analysis) analysis of the biomass samples was carried out with using Shimadzu DTG-60. The sample of around 10 mg in a Pt pan was heated with a ramp rate 10 K/min up to 1073 K under nitrogen atmosphere with flow rate of 50 mL/ min. Air flow with 50 mL/min was employed for analyzing combustion characteristic of solid product (char + ash) from pyrolysis test of the algal biomass. 2.4. Steam pyrolysis test and characterization of pyrolyzed products Pyrolysis of oil-extracted residue biomass of B. braunii in presence of steam was demonstrated in a laboratory-scale continuous feeding reactor, which was originally designed for catalyst activity test of steam gasification of biomass [29]. The reactor consisted basically of co-axial double cylindrical tubes made of quartz glass, which was equipped with electrical furnace and connected with ports for introduction and effluent of gas. The algal biomass powder was continuously fed from a mechanically vibrating nozzle with nitrogen gas flow (A) into top part of the outer tube, where pyrolysis took place in presence of steam supplied by the evaporation of water fed by a microsyringe pump. The biomass powder was vertically falling within heating zone and pyrolyzed to form products such as solid and gas. The solid product, that is char (fixed carbon) and ash, accumulated onto a sintered glass plate on the bottom of the outer tube. Another carrier gas flow (B) was supplied from below, and passing through the sintered glass plate to

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transport the gaseous product from biomass pyrolysis into the inner tube, of which lower end was set just above the glass plate. (Catalyst bed was set in the inner tube end for steam gasification of biomass [29], but no catalyst was employed in the range of this paper.) The gaseous product flew upward within the inner tube and went out from heating zone of the reactor. Compounds with relatively higher boiling point were condensed together with unreacted steam (water) around outlet part of the reactor, and eventually those with relatively lower boiling point was collected by a bubbling trap into ice-cold 2-poropanol. Such condensable components produced from the pyrolysis of the biomass were named tar in this paper, whereas sometimes called bio-oil in literatures. A soap-film flow meter and gas-sampling ports for a syringe and a bag were set after the bubbling trap. Feeding rate of the biomass and steam was 60 mg/min and 50 lL/min, respectively. The pyrolysis tests were performed under atmospheric pressure at temperature of 873, 923 or 973 K. Flow rate of nitrogen carrier gas was 60 mL/min which was sum of 30 mL/min for both ports of A and B described above. Effluent gas was collected from the sampling port by a syringe and analyzed by gas chromatograph (GC). The concentration of CO, CO2 and CH4 was determined by FID–GC equipped with a methanator and that of H2 was determined by TCD–GC. During continuous biomass feeding for test time 15 min, the gas samplings were conducted at predetermined interval to see change in the concentrations, and flow rate of the gas was measured by the soap-film flow meter. The formation rate of the gaseous products was based on the concentration and the flow rate of the effluent gas. Gaseous compounds with carbon number more than two (>C2) in the gas-bag sampling was analyzed qualitatively by GC–MS and their concentrations were determined by GC-FID. The yield of carboncontaining gaseous products (CO, CO2, CH4 and >C2) was calculated by the formation rate and reaction time normalized by the total carbon supplying rate of the biomass. The amount of char formed from the pyrolysis of the algal biomass, which was accumulated in the sintered glass plate in the bottom of the reactor, can be determined after the pyrolysis test. Oxygen gas mixed with nitrogen was supplied from the port B (below the glass plate) for combustion of char, and the amount of formed CO2 measured by FID-GC made an estimate of char amount. As a result, we can measure the yield of gaseous products and solid product (char). On the other hand, it is difficult to determine the amount of residual tar precisely. This is because tar is easily condensable in the reactor system and a part of tar cannot be collected. Therefore, the yield of tar is obtained by the subtraction of carbon-based yield of gaseous and solid products from the total. Tar component collected by washing around outlet of the reactor with 2-propanol was analyzed qualitatively by GC–MS.

3. Results and discussion 3.1. Proximate and ultimate analysis of B. braunii biomass before and after oil-extraction Moisture and ash contents as well as C, H, N and O contents of B. braunii biomass before and after oil-extraction are summarized in Table 1 with the data of Chlorella sp. and cedar wood. Differentconditioned batch cultures of B. braunii (strain BOT-22) resulted in a wide range of HC% from 28% to 51% in the range of this work, as shown in Table 1, which were named as Bot. #1–#5 in this paper. Elemental composition data of other algae and B. braunii from literatures are given in Table 2 for comparison. Raw samples (without n-hexane extraction) of the B. braunii have much higher C and H contents and lower O content as compared to other algal (Chlorella sp. and Table 2) and lignocellulosic

477

biomass (cedar wood). Data of B. braunii in previous work [25] shown in Table 2 are similar to that of Bot. #4-Raw in this work shown in Table 2, whereas values of HC% are quite different (23.9% in previous work [25] and 51.1% for the Bot. #4 in this work). As shown in Table 1, oil extraction by n-hexane obviously makes a decrease in C and H contents, and an increase in N and O contents. This is because the component extractable by n-hexane is mainly hydrocarbons such as botryococcene (C34H58) with high C and H contents. Even after the extraction, residue biomass samples of the B. braunii have slightly higher C and H contents than those of other algal and lignocellulosic biomass. This might be because of existence of algaenan, which is chemically resistant bio-polymers presumably synthesized from the hydrocarbons and hence have high C and H contents [32]. As for moisture and ash contents shown in Table 1, ash contents of the B. braunii are slightly lower than those of other algae (Table 2) and increase with decreasing of HC%. Thus, they can be described as a decreasing function of the HC%. The same trend can be observed for the moisture content. Fig. 1 shows contents of C (1-a) and H, N, O (1-b) as a function of HC% of the B. braunii. Higher HC% results in higher C and H contents and lower N and O contents. It should be noted that this trend is observed for not only the raw samples but also the oil-extracted residue samples. This is also attributed to existence of the algaenan: the B. braunii with higher HC% might have higher content of the algaenan. This point will be further discussed in Section 3.4 by means of TG analysis. From practical viewpoints, it is useful that the elemental contents can be described as a function of HC%: only the extraction by n-hexane may give some indications of C and H contents. 3.2. Van Krevelen diagram and calculated HHV for B. braunii biomass before and after oil-extraction Fig. 2 indicates the classification of the B. braunii biomass as a solid fuel based upon Van Krevelen diagram, which is plot of H/C versus O/C atomic ratios on dry and ash free basis [33]. In addition to the data of the B. braunii (raw and residue, shown in Table 1), data of other algal (Table 2) and terrestrial biomass [34,35] as well as various solid fuels [36] are also shown in this figure. The B. braunii biomass has quite lower O/C ratio and slightly higher H/C ratio as compared to other algal and terrestrial biomass. Moreover, the B. braunii biomass even after oil-extraction can be classified as H-rich solid fuel in comparison to coal which has similar O/C ratio but much lower H/C ratio than the B. braunii biomass. As shown in Fig. 2, higher heating values (HHV) of B. braunii biomass were expected to be much higher than other biomass, and actually calculated by using Eq. (1) for the biomass before and after oil-extraction with the composition data shown in Table 1. Fig. 3 shows the calculated HHV plotted against HC% of the B. braunii biomass. The HHV in this work can be described as a simple increasing function of HC% for each raw and oil-extracted residue sample of B. braunii. HHV of raw sample with high HC% of 55.1% is 40.6 MJ/kg, which is nearly as high as that of fossil oil 42 MJ/kg [36]. HHV of residue samples are lower than that of raw ones, but even higher than that of other algal biomass shown in Table 2. The calculated HHV of the residue sample in this work are in good agreement with actual measured values ranging from 31 to 34 MJ/kg in previous work (BOT-22 strain) [4] as well as 35 MJ/kg for an unidentified strain [25], which are comparable to HHV of solid fossil fuel such as coal [36]. 3.3. Analysis on the other elements of B. braunii biomass after oilextraction An EDX type fluorescence spectrometer was able to detect the other elements such as S, P, Cl, Ca, K, Na, Mg, Si and Fe for B. braunii

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Table 1 Proximate and ultimate analysis of B. braunii (Bot.) biomass before and after oil-extraction. Biomass sample #

Raw (before oil-extraction) (wt%) HC%

Bot. Bot. Bot. Bot. Bot.

a b c d e f

a

b

Residue (after oil-extraction) c

(wt%)

(wt%)b

(d.a.f.-wt%)

Mid

Ash

C

H

N

O

(d.a.f.-wt%)c

e

Mid

Ash

C

H

N

Oe

4.8 5.4 2.7 2.2 1.9

3.3 2.3 2.1 2.0 1.3

61.5 61.8 67.7 66.3 71.1

8.8 8.8 10.0 9.6 10.5

5.3 5.7 2.2 4.0 1.8

22.4 23.7 20.1 20.1 16.6

#1 #2 #3 #4 #5

28.4 30.7 37.4 45.0 51.1

3.4 3.0 1.8

2.5 1.8 1.3

68.5 67.5 75.0

9.7 9.7 10.9

3.8 3.9 1.3

18.0 18.9 12.8

1.2

0.85

77.3

11.3

1.1

10.3

Chlorella sp. Cedar woodf

– –

3.2 9.2

5.4 0.3

53.5 51.3

7.6 5.9

10.3 0.1

28.6 42.7

Hydrocarbon content is defined here as a mass fraction extractable by n-hexane to dried alga mass. Air-dried based weight%. Dried and ash free based weight%. Moisture content. By difference: 100  (C + H + N). Li et al. [28].

Table 2 Elemental composition data of algal biomass from literatures. Algae

Dunaliella tertiolecta Chlorella vulgaris Chlorella vulgaris Chlorella vulgaris Spirulina Spirulina Nannochloropsis oculata Botryococcus braunii a b c d e

(wt%)

(d.a.f.-wt%)b

Asha

C

H

N

Oc

S

HHV

23.6 7.0 6.1 6.8 7.6 7.2 26.4 0.69

53.3 52.6 51.8 53.6 54.4 47.6 57.8 77.04

5.2 7.1 7.2 7.3 7.6 7.4 8.0 12.40

9.8 8.2 7.5 9.2 7.5 7.1 8.6 1.23

31.7 32.2 33.5 29.4 33.5 37.4 25.7 9.86

– 0.5 – 0.5 0.83 0.5 – 0.18

(19.8)d 23.2 21.3 23.2 21.2 16.9 17.9 35.6e

(MJ/kg)

Reference

[18] [30] [19] [31] [31] [15] [30] [25]

Air-dried based weight%. Dried and ash free based weight%. By difference. Calculated by Dulong’s equation (HHV = 0.3383C + 1.442(H  O/8)). Lower heating value.

biomass samples. Among these elements, S, P and Cl could be a catalytic poison and thereby cause problem in catalytic conversions from biomass to liquid and/or gaseous fuels [37,38]. Fig. 4 shows contents of these three elements for B. braunii samples after oil-extraction with different original HC% (no data for Bot. #4 here), cedar wood and Chlorella sp. as references. Low contents of these three elements in cedar wood can be attributed to its main components (cellulose, hemi-cellulose and lignin) which do not contain these elements. S content for residue samples of B. braunii in this work ranging from 0.1 to 0.3 wt% is comparable to data of Chlorella sp., other algae and B. braunii in previous work [25] (some of which shown in Table 2), where sulfur exists likely in form of amino-acid residues such as cysteine and methionine in proteins of algae, and occasionally contains in sulfonium compounds [39]. Content of P in Chlorella sp. is quite higher than that of the residue samples of B. braunii. Phosphorus is one of the major nutrient elements required for normal growth of algae [40], and is distributed to major cell constituents such as protein, lipid, nucleic acids, nucleotides and polyphosphates [41]. Luxury uptake of phosphorus by microalgae may take place under certain cultivation conditions, which leads to accumulation of polyphosphates presumably for energy storage [42]. This could be a reason of the higher S content in the Chlorella sp. in this work, whereas details of this algal sample were unknown as it is a commercial product. Contents of S and P for Chlorella sp. reported recently by Soares et al. [43] are 0.64 and 1.3 wt%, which are comparable to data shown in Fig. 4. Apart from the discussion on the Chlorella sp., the contents of S and P for residue sample of B. braunii decrease

basically with increase in the HC%. This means that residue biomass of B. braunii with higher HC% is also preferable as biomass feedstock for catalytic conversion to biofuels in terms of low contents of possible catalytic poisons. 3.4. TG analysis of B. braunii biomass before and after oil-extraction Pyrolytic behavior of B. braunii biomass has been characterized by employing thermogravimetry (TG) analysis, since it is basic and important features as a biomass feedstock for biofuel production by the thermochemical conversions. Fig. 5(a) shows TG curves of raw and residue biomass of B. braunii (Bot. #5), Bot.-Oil (a fraction extractable by n-hexane), and cedar wood and Chlorella sp. as references. Weight loss shown in Fig. 5(a) is basically due to pyrolysis under nitrogen atmosphere. TG curve of Bot. #5-Raw is shifted to lower temperature as compared to that of Bot. #5-Residue. This is because the raw sample contains Bot.-Oil with 51.1% that can be pyrolyzed at low temperature around 563 K as shown in Fig. 5(a). Weigh loss of Bot. #5-Residue with respect to temperature is much gradual as compared to that of Bot. #5-Raw, although both samples have been almost completely pyrolyzed by 773 K. This temperature for the complete pyrolysis is same for all raw and residue samples in the range of this paper (see Supporting Information, Fig. S1), which is common in the previous work [25]. This is also preferable feature of the B. braunii as the feedstock, since lignocellulosic biomass is usually required for higher temperature (up to 973 K) due to content of persistent lignin: the weight loss above 640 K is even

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Fig. 3. Calculated HHV of B. braunii biomass before and after oil-extraction as a function of HC% (Chlorella sp. 23.9 MJ/kg, cedar wood 18.2 MJ/kg).

Fig. 1. Elemental composition of B. braunii biomass before and after oil-extraction as a function of HC%: C (a) and others (b).

Fig. 4. Contents of S, P and Cl for B. braunii biomass after oil-extraction, and cedar wood and Chlorella sp. as references.

Fig. 2. Van Krevelen diagram for raw and residue biomass of B. braunii, other algal and terrestrial biomass as well as various solid fuels.

gradual against temperature, as shown in Fig. 5(a) for cedar and in previous work [34] for other lignocellulosic biomass. Temperature-derivative of the TG curves shown in Fig. 5(a) is presented in Fig. 5(b), which is named as DTG curves. Peak I around 563 K appearing in the DTG curves of Bot.-Oil and Bot.-Raw corresponds to botryococcene as main component of hydrocarbon produced by the BOT-22 strain [8,44]. For the DTG curves of Bot.-Residue, Peak I0 around 578 K appears instead of the Peak I,

which means removal of the hydrocarbon oil by n-hexane extraction. The Peak I0 might correspond to proteins and/or polysaccharides, because broad peaks of Chlorella sp. (553–593 K) should derive from proteins as main component (about 70 wt%) and a DTG peak of starch is around 603 K [45]. Peak II around 738 K appears in DTG curves of both raw and residue of the B. braunii biomass. This peak is much higher temperature than Peak III around 623 K, which obviously corresponds to cellulose, and should derive from algaenan which is chemically resistant bio-polymers presumably synthesized from the hydrocarbons [32]. Another unidentified strain of B. braunii presented in previous work [25] has a DTG strong peak around similar temperature to the Peak II in this paper, but another DTG peak around 653 K is obviously different from the Peak I and I0 in this paper. This could be attributed to difference in biomass compositions between different strains even the same species. Based upon the discussion above on DTG curves of raw and residue of the B. braunii, contents of volatile matter (V.M.) and fixed carbon (F.C.) were deduced by analysis of TG data (see Fig. S1) with assumptions as follows: (i) Light component of V.M. (V.M.L) corresponds to weight fraction from 423 to 613 K, which would be botryococcene for raw samples and others for residue samples.

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Fig. 5. (a) TG curves of raw and residue biomass of B. braunii (Bot. #5), Bot. -Oil (fraction extractable by n-hexane), and cedar wood and Chlorella sp. as reference. (ramp rate 10 K/min, under nitrogen atmosphere with flow rate of 50 mL/min) and (b) temperature-derivative of TG curves (DTG): note that baselines for cedar wood and Chlorella sp. are shifted to 1.0%/K for discrimination of these references to the others.

(ii) Heavy component of V.M. (V.M.H) corresponds to weight fraction from 613 K to 873 K, which would be mainly algaenan. (iii) Residual mass at 873 K is sum of F.C. and ash. The results are shown in Fig. 6, where the dry-wt% is calculated by deducting water content which is assumed to be weight loss up to 423 K. Raw samples have higher V.M.L and lower V.M.H as compared to residue samples, which is obviously due to removal of the hydrocarbon oil by extraction with n-hexane. V.M.H for residue sample would relate to the content of algaenan, and increases with increase in HC%. This is consistent with the increasing trend of C and H with respective to HC% owing to existence of the algaenan, as already discussed in Section 2.1. Raw and even oil-extracted residue of B. braunii have lower F.C. as compared other biomass (Chlorella sp. 24.7 dry-wt%, cedar wood 18.7 dry-wt%). Higher HC% results in higher total V.M. (V.M.L + V.M.H) as well as lower F.C. and hence is also preferable as biomass feedstock for catalytic conversion to biofuels. 3.5. Pyrolysis of oil-extracted residue biomass of B. braunii Pyrolysis of oil-extracted residue biomass of B. braunii (Bot. #2-Residue) in presence of steam was demonstrated in order to

find out actual pyrolyzed products of the algal biomass. Pyrolysis tests were generally carried out in an inert environment (nitrogen or helium), whereas steam can be used as pyrolyzing or gasifying agent alone or in mixture with carbon dioxide. For example, presence of steam in pyrolysis of lignocellulosic biomass resulted in significant increase in the yield of liquid products [46]. In this work, steam was supplied to the pyrolysis tests in order to facilitate thermal decomposition reactions of (residual) biomass of B. braunii, which was quite high-caloric as shown in Figs. 2 and 3, and therefore should be more persistent as compared to other algal and lignocellulosic biomass. In the range of this paper, feeding rates of the biomass and steam were fixed to 60 mg/min and 50 lL/min, respectively. This combination of the feeding rates led to atomic ratio of O/C as 1.32, where O was sum of oxygen from steam supplied, moisture content of the biomass itself and oxygen content of the biomass (Table 1) and C was carbon from the biomass fed: this was same level of the O/C in our previous studies in steam reforming of tar derived from pyrolysis of cedar wood [28,29]. Fig. 7 shows carbon-based yield of solid (char), liquid (tar: condensable at ambient temperature) and gaseous products from the pyrolysis tests as a function of pyrolysis temperature. Briefly, yields of char, tar and gas are around 10%, 60% and 30%, respectively, which are lower char and gas, and much higher tar yields as compared to steam pyrolysis of lignocellulosic biomass: for example, 20% char, 25% tar and 55% gas for pyrolysis of cedar wood biomass at 873 K [29]. Identification and initial characterization of each product will be described in detail as following. In Fig. 7, char yield is about 8–10% and not so sensitive to a change in the temperature, which is actually comparable to F.C. component shown in Fig. 6 (Bot. #2-Residue). This means char formation is not strongly dependent on pyrolysis mode, where the pyrolysis mode is slow one in the TG experiments and that is fast in the pyrolysis tests. It is also implied that the reactivity of char with respect to steam is not so high, because presence of steam in the pyrolysis test does not reduce char amount as compared to that in TG where no steam is supplied apart from moisture content of the biomass itself. Table 3 shows proximate and ultimate analysis of char produced by steam pyrolysis at 973 K of the Bot. #2-Residue biomass. Contents of moisture and ash shown in this table were estimated from TG/DTA of the as-formed char including ash under air flow (find its result in Fig. S2). Contents of C, H, N (and O by difference) were measured by the CHN analyzer, and could be converted to those contents of char itself (dry and ash free %) by considering the contents of moisture and ash. The char has much higher C content, lower contents of H and O, and hence is considered to be highly carbonized. N content in the char is comparable to that in original biomass (Bot. #2-Residue in Table 1) in terms of C:N ratio. Distribution of nitrogen at pyrolysis into solid and volatile matters with same level is interesting from the viewpoint of nitrogen-recycling. The C:N ratio in the char composition is in fairly good agreement with that of algal chars in literature [47], and therefore the char could be potentially used for soil amendment. As already shown in Fig. 7, in addition to C1 gases such as CO, CO2 and CH4, components of C2–C4 gas can be determined by GC–MS analysis (shown in Fig. S3). Unsaturated hydrocarbons such as ethylene (C2H4), propylene (C3H6) and C4s (butene C4H8 and butadiene C4H6) rather than alkanes were produced by the pyrolysis of the algal biomass residue. Carbon-based total gas conversion (C1–C4) increases gradually with increase in temperature: from 30% at 873 K to 35% at 973 K. Fig. 8 shows yield of flammable gas including hydrogen as a function of the temperature. The yield of all the gases apart from C3 to C4s increases with increase in temperature, implying higher thermal decomposition activity at higher temperature. Total HHV of all the flammable gases shown in right

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Fig. 6. Composition of volatile matters, fixed carbon deduced from TG analysis, and ash for raw and residue biomass of the B. braunii.: V.M.L is presumed as LIGHT Volatile Matter (weight loss from 423 K to 613 K); V.M.H is presumed as HEAVY V.M. (weight loss from 623 K to 873 K); residual weight at 873 K is presumed as Fixed Carbon (F.C.) with Ash, and F.C. is calculated using Ash contents in Table 1 with converted dry-weight%.

Fig. 7. Distribution of carbon-containing products from steam pyrolysis of oilextracted residue biomass of B. braunii (Bot. #2-Residue): feeding rate of the biomass 60 mg/min, steam 50 lL/min. Yield of solid (char) and gaseous product is expressed in unit of carbon-based yield, and tar is calculated by difference: 100 – yields of gases and char. H2/CO shown above the figure is in molar ratio.

Table 3 Proximate and ultimate analysis of solid product (char including ash) from steam pyrolysis of Bot. #2-Residue biomass at 973 K. Samplea Char + Ash Char (calculated)

(wt%)b (d.a.f.-wt%)c

Mid

Ashe

C

H

N

Of

1.4 –

38.1 –

53.2 88.0

1.7 2.7

5.1 8.5

0.5 0.8

a Solid pyrolysate of Bot. #2-Residue at 973 K was char including ash (Char + Ash). Elemental composition of char itself (Char) was calculated by subtracting contents of ash and moisture from (Char + Ash). b Air-dried based weight%. c Dried and ash free based weight%. d Moisture content was defined as weight loss from ambient temperature to 423 K for TG analysis under air flow (Fig. S2). e Ash content was defined as residual mass at 1073 K for TG analysis under air flow (Fig. S2). f By difference.

vertical axis in Fig. 8 was calculated using heat of combustion for each gas from the literature [48]. The total HHV increases with increase in temperature: 13.1 kJ/g-biomass at 973 K corresponding

Fig. 8. Effect of temperature on formation of flammable gases from steam pyrolysis of oil-extracted residue biomass of B. braunii (Bot. #2-Residue): feeding rate of the biomass 60 mg/min, steam 50 lL/min. Calculated total HHV of all the gases is shown in the right vertical axis in this figure.

to about 45% of original biomass residue, 29.4 MJ/kg-biomass (shown in Fig. 3). Tar yield shown in Fig. 7 is about 60% for the pyrolysis temperatures in this paper, which is obviously major component among pyrolyzed products. A part of tar especially with high boiling point was not able to reach the ice-cold bubbling trap with 2-propanol, as it was easily condensed around part just after outlet from the reactor. Therefore, such condensed tar was collected by washing with 2-propanol after pyrolysis test. The 2-propanol solution of the tar was analyzed by GC–MS. A typical GC–MS chart (total ion current chromatogram) of tar derived from pyrolysis of the algal biomass residue at 923 K is shown in Fig. 9. Identification of peaks numbered in the figure was carried out by taking account of similarity between obtained MS spectrum and reference one supplied by NIST (National Institute of Standards and Technology) attaching to the GC–MS system. List of chemical species identified by the GS–MS analysis are summarized in Table 4. Chemical structure of representative peaks is also shown in Fig. 9. In the region of retention time (R.T.) less than 10 min, smaller peaks are identified as nitrogen-containing hetero-cyclic compound (N-cyc) as pyridine,

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Fig. 9. GC–MS analysis of tar derived from the steam pyrolysis of oil-extracted residue biomass of B. braunii (Bot. #2-Residue) at 973 K. Total ion current chromatogram is displayed in unit of relative intensity to the solvent peak. Numbers indicating peaks corresponds to the list of chemical species identified (see Table 4).

pyrazine and their derivatives, as well as aromatic hydrocarbon (AH). In the region of R.T. from 10 to 20 min, there are larger peaks identified as polycyclic aromatic hydrocarbon (PAH) with two-ring such as naphthalene and its derivatives, nitrogen-containing PAH (N-PAH) such as quinolone and indole, and acetamide (N-amd). For R.T. more than 20 min, there is a peak assigned as three-ring PAH of anthracene or phenanthrene, and an unidentified peak with a higher molecular weight. These compounds of tar identified here is quite different from those of tar derived from cedar wood [38], which are mainly single-ring AH (benzene, toluene, phenol and so on) and oxygenates (ketone, carboxylic acid and so on). This would be because cedar wood has contents of higher oxygen and lower nitrogen, like a typical lignocellulosic biomass. Tar derived from spent malt [49] with relatively high nitrogen content about 5 wt% actually contained nitrogen-containing compounds such as pyrrole, pyridine and acetonitrile, but not N-PAHs such as quinolone and indole found in this work. Typical composition of biomass tar produced by pyrolysis at relatively high temperature (more than 923 K) included PAHs with two or three rings to some extent but single-ring AHs as major component as around 65 wt% [50], which is hardly detected

in the GC–MS analysis in Fig. 9 and Table 4. Tar composition with PAH such naphthalene as the major component found in this work is somewhat similar with that of coal tar [51], rather than that of biomass described above. This would be related to an acceptedtheory that the algaenan produced by B. braunii can be a major source of petroleum oils [52]. In summary, tar derived from steam pyrolysis of the oil-extracted residue biomass of B. braunii includes mainly heavy components such as PAHs and N-PAHs, and therefore would have a high caloric value. Catalytic gasification of the tar to hydrogen or syngas (CO + H2) [28,29,34–38,49] or catalytic cracking to high-value lighter fractions [53] should be useful in order to recovery energy efficiently from the pyrolyzed tar. 3.6. Future tasks for algal biofuel production The results and discussion presented in this paper so far has revealed that the B. braunii, not only the extracted oil but also the residue biomass after oil-extraction, should have very high potentiality as a biofuel feedstock. Future tasks that should be addressed for practical biofuel production from algae (including B. braunii) will be briefly reviewed in this section for future

Table 4 List of chemical species identified by GC–MS analysis for tar derived from pyrolysis of Bot. #2-Residue biomass at 973 K.

a

Peak #

RT (min)

Relative intensitya (%)

Chemical species identified by GC–MSb

Chemical formula

Typec

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

4.04 4.92 4.99 5.52 7.71 9.28 10.98 11.07 12.29 12.65 12.71 13.27 13.78 13.84 14.65 14.94 16.06 17.44 18.33 21.32 28.09

3.06 3.78 2.84 2.26 3.74 3.78 25.97 10.11 12.51 4.89 8.96 13.76 8.24 16.04 6.53 7.22 18.76 5.89 21.79 8.71 21.84

Pyridine Pyrazine x-methyl pyrazine y-methyl pyrazine x-ethynyl y-methyl benzene x-ethynyl pyridine Naphthalene Acetamide Methyl-naphthalene x-aminopyridine Benzocycloheptatriene Quinoline Biphenyl x-methyl pyridazine x-methyl phenol Acenaphthene Acenaphthylene Fluorene Indole Anthracene or phenanthrene not determined

C5H5N C4H4N2 C5H6N2 C5H6N2 C9H8 C7H5N C10H8 C2H5NO C11H10 C5H6N2 C11H10 C9H7N C12H10 C5H6N2 C7H8O C12H10 C12H8 C13H10 C8H7N C14H10 –

N-cyc N-cyc N-cyc N-cyc AH N-cyc PAH N-amd PAH N-cyc PAH N-PAH AH N-cyc AH PAH PAH PAH N-PAH PAH –

GC–MS peak intensity shown here is relative value to the intensity of solvent (2-propanol) peak. ‘‘x-’’ and ‘‘y-’’ in this column mean the position of substitution groups cannot be determined by GC–MS analysis. c AH: aromatic hydrocarbons, PAH: polycyclic aromatic hydrocarbons, N-cyc: nitrogen-containing hetero-cyclic compound, N-amd: amide compound, N-PAH: nitrogencontaining PAH. b

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reference. The tasks are basically distilled into the reduction of costs and energy consumption for a series of processes such as cultivation, harvesting, dewatering/drying, oil/lipid-extraction, and conversions of the extracted-oil/lipid and the residue biomass into high-value biofuels [54–57]. Application of wastewaters or swages (domestic, municipal or industrial one) to cultivation process of algae for biomass production as well as wastewater treatment (nutrient removal) [58–60] should be crucially important for reducing the cost. Actually, cultivation of the B. braunii (BOT-22 strain) with use of soybean curd wastewater as a part of culture medium had been carried out by a part of the authors of this paper [6]. Moreover, the technology to exploit oil-yielding heterotrophic algae which is capable of using the organic matter in wastewater, together with the nitrogen/ phosphorus nutrient-utilizing oil-yielding autotrophic algae (B. braunii) has been being developed as a part of an on-going project ‘‘Next-generation Energies for Tohoku Recovery’’ in Japan [61], in which some authors of this paper are currently involved. Another important process in terms of reducing energy consumption shall be oil-extraction including pretreatment process. Drying out concentrated culture broth of algae prior to extraction by an organic solvent (n-hexane in this study) might not be realistic in practical biofuel production because of high latent heat of water evaporation, unless an efficient heat exchanger and/or an energy-saving dryer using natural heat source such as sunlight irradiation were to be employed. For this reason, ‘‘wet extraction’’ process, meaning the extraction of oil/lipids from concentrated culture broth of algae without drying, should be preferable. Wet extraction of hydrocarbons from B. braunii has been recently carried out by means of heating treatment of the alga prior to n-hexane extraction [62], using a water-miscible solvent of 1,2dimethoxyethane [63], or using liquid dimethyl ether (DME) [64]. More recently, repeated non-destructive hydrocarbon extraction from B. braunii (BOT-22 strain), has been demonstrated with use of n-heptane solvent by a part of the authors of this paper [65]. For conversion methods of raw and residual algal biomass after oil-extraction, examples of combustion, gasification, pyrolysis, liquefaction and carbonization were cited in Introduction section of this paper. In addition to them, anaerobic digestion (AD) of algal biomass (residue) is another potential candidate, and not only recycles nutrients (nitrogen and phosphorous) but also produces biogas (methane) [66]. AD process is appropriate for algal residue with high moisture content (80–90%), which might be provided from the wet extraction described above if successful. Actually, methane production by the AD of B. braunii has been reported, indicating same level of methane productivity with other algae [67]. Hydrothermal liquefaction/carbonization can be used for converting wet algal biomass into a liquid/solid fuel. It should be noted that wet algal biomass (slurry) can be also applied for ‘‘steam’’ pyrolysis or gasification by means of catalytic ‘‘steam’’ reforming of biomass tar. As briefly described in this section, all the efforts to maximize efficiency in terms of costs and energy to each process would enable us to produce and use algal biofuel as a sustainable resource in practical scale in the future. 4. Conclusions From elemental composition analyses and TG analysis, the B. braunii (strain BOT-22) with higher HC%, even biomass residue after oil-extraction, is found to be superior in quality of a biofuel feedstock because of higher contents of C, H and volatile matter and lower contents of N, O, S, P, ash and fixed carbon as compared to that with lower HC% and other algal biomass. Steam pyrolysis of the oil-extracted residue biomass of the B. braunii at 873–973 K can produce highly carbonized char (10%-C), gases of C1–C4 (30%-C) and H2, and tar (60%-C) including mainly heavy component such

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as PAHs and N-PAHs. Based upon the analysis of the pyrolyzed products and calculated HHV from 29 to 36 MJ/kg, the residue biomass of B. braunii can be classified as H-rich solid fuel which is comparable to fossil fuel such as a coal rather than other algal and terrestrial biomass. These characteristics of the residue biomass can be related to existence of the algaenan, which is C–H rich bio-polymers presumably synthesized from the hydrocarbons from the B. braunii. We can conclude that not only hydrocarbon oils produced from the B. braunii but also its oil-extracted residue biomass should be superior feedstock for biofuel production. Acknowledgements This study was supported by the Special Fund for the Next Generation Energy Development Project, MEXT, Japan. The authors are grateful to the Chemical Analysis Center, University of Tsukuba, for conducting CHN elemental analysis. Elemental analysis was carried out with EDX-820HS in Shimadzu Corporation. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apenergy.2014. 07.037. References [1] Chisti Y. Biodiesel from microalge. Biotechnol Adv 2007;25:294–306. [2] Banerjee A, Sharma R, Chisti Y, Banerjee UC. Botryococcus braunii: a renewable source of hydrocarbons and other chemicals. Crit Rev Biotechnol 2002;22:245–79. [3] Metzger P, Largeau C. Botryococcus braunii: a rich source for hydrocarbons and related ether lipids. Appl Microbiol Biotechnol 2005;66:486–96. [4] Shiho M, Kawachi M, Horioka K, Nishida Y, Ohashi K, Kaya K, et al. Business evaluation of a green microalgae Botryococcus braunii oil production system. Proc Environ Sci 2012;15:90–109. [5] Tanoi T, Kawachi M, Watanabe MM. Effects of carbon source on growth and morphology of Botryococcus braunii. J Appl Phycol 2011;23:25–33. [6] Yonezawa N, Matsuura H, Shiho M, Kaya K, Watanabe MM. Effects of soybean curd wastewater on the growth and hydrocarbon production of Botryococcus braunii strain BOT-22. Bioresour Technol 2012;109:304–7. [7] Sakamoto K, Baba M, Suzuki I, Watanabe MM, Shiraiwa Y. Optimization of light for growth, photosynthesis, and hydrocarbon production by the colonial microalga Botryococcus braunii BOT-22. Bioresour Technol 2012;110:474–9. [8] Ishimatsu A, Matsuura H, Sano T, Kaya K, Watanabe MM. Biosynthesis of isoprene units in the C34 botryococcene molecule produced by Botryococcus braunii strain Bot-22. Proc Environ Sci 2012;15:56–65. [9] Ioki M, Baba M, Bidadi H, Suzuki I, Shiraiwa Y, Watanabe MM, et al. Modes of hydrocarbon oil biosynthesis revealed by comparative gene expression analysis for race A and race B strains of Botryococcus braunii. Bioresour Technol 2012;109:271–6. [10] Hillen LW, Pollard G, Wake LV, White N. Hydrocracking of the oils of Botryococcus braunii to transport fuels. Biotechnol Bioeng 1982;24:193–205. [11] Kitazato H, Asaoka S, Iwamoto H. Catalytic cracking of hydrocarbons from microalgae. J Jpn Pet Inst 1989;32:28–34. [12] Chen C, Ma X, Liu K. Thermogravimetric analysis of microalgae under different oxygen supply concentrations. Appl Energy 2011;88:3189–96. [13] Phukan MM, Chutia RS, Konwar BK, Kataki R. Microalgae Chlorella as a potential bio-energy feedstock. Appl Energy 2011;88:3307–12. [14] Rizzo AM, Prussi M, Bettucci L, Libelli IM, Chiaramonti D. Characterization of microalga Chlorella as fuel and its thermogravimetric behavior. Appl Energy 2013;102:24–31. [15] Hirano A, Hon-Nami K, Kunito S, Hada M, Ogushi Y. Temperature effect on continuous gasification of microalgal biomass: theoretical yield of methanol production and its energy balance. Catal Today 1998;45:399–404. [16] Demirbas A. Thermochemical conversion of mosses and algae to gaseous products. Energy Sources A 2009;31:746–53. [17] Demirbas A. Hydrogen from mosses and algae via pyrolysis and steam gasification. Energy Sources A 2009;32:172–9. [18] Minowa T, Yokoyama S, Kishimoto M, Okakura T. Oil production from algal cells of Dunaliella teriolecta by direct thermochemical liquefaction. Fuel 1995;74:1735–8. [19] Minowa T, Sawayama S. A novel microalgal system for energy production with nitrogen cycling. Fuel 1999;78:1213–5. [20] Jane U, Das KC, Kastner JR. Comparison of the effects of Na2CO3, Ca(PO4)2, and NiO catalysts on the thermochemical liquefaction of microalga Spirulina platensis. Appl Energy 2012;98:368–75.

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