1. Introduction
Microalgae is well studied in recent years for the promising possibility to be used as new energy stock [1] - [10] . B. braunii is especially expected due to the exceedingly high oil productivity. Shimamura et al. have successfully achieved a fundamental outdoor culture system of B. braunii and an outdoor mass cultivation system of B. braunii has been operated in Algae Biomass and Energy System R & D Center (ABES), University of Tsukuba [11] . Mass culture of microalgae inescapably generates a large amount of biomass residues after process. In these five years, studies considering the efficient use of biomass residues have been gradually reported, such like microalgae residue based carbon solid acid catalyst for biodiesel production [12] , methane production from lipid-extracted biomass residues [13] and kinetic study of microalgae residues in pyrolysis [14] [15] . Since biomass residues might be the environmental problems in the near future, research on biomass residues is necessary with increasing demand of the new energy. From the point of view of material science, biomass residues containing much amount of different components are interesting materials. In this study, we adopted B. braunii residues as a starting material. Carbonization of the bulk sample in a certain condition affords to yield hexagonal crystals. 2D hexagonal crystals with high anisotropy and large surface area are promised to be the building blocks for functional materials.
In our previous study, we reported that different carbonization condition would affect the morphology of the yielded crystals [16] . Polygonal grains were observed in the sample carbonized above 700˚C under argon atmosphere. In this study, we carbonized the B. braunii residue at 700˚C with no argon flow. Thus obtained samples have hexagonal disks on the surface. We observed these structures by scanning electron microscopy. The side length of these hexagonal disks ranges from several hundred nanometers to one micrometers. This is the first paper reporting the hexagonal disk shaped crystals generated during the carbonization of B. braunii residue and any other biomass residues. The crystals were found only in a very limited area of the bulk sample. We performed the infrared absorption spectroscopy, X-ray photoelectron spectroscopy and energy dispersive X-ray spectrometry to analyze the chemical components included inside the sample. Mechanism of the formation of hexagonal disks and role of argon flow are discussed.
2. Experimental
2.1. Preparation of B. braunii Residues Samples
The original sample B. braunii strain (BOT-22) was isolated from the Okinawa prefecture, Japan and is cultivated in University of Tsukuba [17] . This strain is classified race B and produces botryococcene (C34H58) as a main component of hydrocarbons [18] . The B. braunii residue samples were prepared as follows. A mass culture system of B. braunii developed in University of Tsukuba provided culture broth of the BOT-22 strain of B. braunii for this work [11] . The broth of the BOT-22 was concentrated by using PSI as flocculants and dried by sunlight [19] . The dried sample of the B. braunii was soaked into n-hexane for extraction of hydrocarbon oils including a small amount of carotenoids and triacylglycerols. After the extraction, the residual sample was recovered by filtration and then dried again. The obtained materials are plate-like shape with some thickness, and show greenish dark brown color in bulk state.
2.2. Carbonization
B. braunii residues were set in a quartz dish and placed into an Electric Gold Furnace instrument produced by Massachusetts Institute of Technology (MIT) Lincoln-Lab. equipping with an Ishikawa temperature controller and a handmade furnace assembled by Dr. Shinichi Ito (University of Tsukuba). 200 V of voltage was applied and uniformly heated the interior of furnace. Carbonization was performed with no argon flow from room temperature to 700˚C. Then the sample was naturally cooled to room temperature. The carbonized sample was obtained in bulk state with no fragments.
3. Results and Discussion
3.1. SEM Observation
We have mentioned in our previous report that the syngas such like hydrogen gas or carbon monoxide gas would be the trigger of the reduction reaction for the formation of polygonal crystal which consists of Fe3O4 or α-Fe inside [16] . Figure 1(a) shows the very beginning stage of the formation of polygonal crystal, observed in the bulk B. braunii residues carbonized at 700˚C under argon atmosphere. The octahedral shaped crystals imbedded in the sample are clear evidence that reduction reaction occurred. However, the absence of inert gas flow caused a totally different result during the carbonization. In Figure 1(b), hexagonal disk shaped crystals in different sizes were found on the surface and inside of carbonized B. braunii residues. These samples were also carbonized at 700˚C, only the argon flow was stopped initially. A simple diagram summarized as Figure 1(c) shows the difference of the carbonization condition would cause the different crystal shape.
The hexagonal disks were carefully observed under SEM, as shown in Figure 2. The length of one side of the hexagon ranges from several nanometers to one micrometer. Figure 2(a) is a hexagonal disk with bottom part broken. It is interesting to find the crack of this defect is composed of two straight lines; both are almost parallel to the sides of the original hexagonal disk. In Figure 2(b), the hexagonal disk imbedded perpendicular to the surface of the bulk sample, only half of the hexagon could be observed. We assume this figure indicates that the hexagonal crystal is still under growing process. Most of the hexagonal disks were found like in Figure 2(c) and Figure 2(d), which hexagonal disks solitarily attach on the carbonized sample. Figure SI1 shows a SEM image of the bulk sample with some hexagonal disks adhering on the surface. Some of the hexagonal disks have thickness more than 100 nm (See Figure SI2(a), Figure SI2(b)). Magnification shows a part of the hexagonal disk was separated (Figure SI2(c)). Two hexagonal disks attached to each other were also observed under SEM, as shown in Figure SI2(d) Figure SI2(e). In Figure SI2(d), two hexagonal disks with a very small hexagon were found on the edge of bulk sample, indicating that the crystals are on the process of growth. A similar pattern was also found in Figure SI2(e) that two hexagons with one side length of 500 nm attach to each other. Figure SI2(f) shows a zigzag edge resembling the external form of honeycomb structure and that of double hexagon disks observed in Figure SI2(e). In Figure SI2(g), a hexagonal disk with a defect that three sides are parallel to the side of original hexagonal disk was observed. A crystal with a round hole was found (See Figure SI2(h)). In Figure SI2(i), a hexagon shape-like hole was found on the surface of
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Figure 1. (a) Bulk B. braunii residues carbonized at 700˚C under argon flow observed under SEM. (b) Bulk B. braunii residues carbonized at 700˚C with no argon flow observed under SEM. (c) Different carbonization conditions afford to yield different crystal morphology.
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Figure 2. Hexagonal disk structures of carbonized B. braunii residues observed under SEM. (a) ×40000; (b) ×50000; (c) ×55000; (d) ×55000.
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Figure SI1.SEM images B. braunii residue carbonized at 700˚C with no argon flow. Many hexagonal disk pointed by arrows could be observed (×4500).
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Figure SI2.SEM images of hexagaonal disks obtained by carbonization of B. braunii residue at 700˚C with no argon flow (a) ×43000; (b) ×40000; (c) ×65000; (d) ×65000; (e) ×45000; (f) ×40000; (g) ×16000; (h) ×22000; (i) ×3000.
the bulk sample.
3.2. Infrared Absorption Measurement
Figure 3 shows the infrared absorption (IR) spectra of B. braunii residue samples carbonized at 700˚C with argon and no argon flow, respectively. The absorption peak appears around 1559 cm−1 and 1573 cm−1 in the sample treated with argon flow and no argon flow, respectively. This characteristic peak implies the partially formation of graphite inside of the bulk sample. Another prominent peak found around 1041 cm−1 and 1573 cm−1 in the sample carbonized with argon flow and no argon flow, respectively, ascribes to the vibration of Si-O stretching. These results suggest that carbonization at 700˚C with no argon flow can also afford to yield graphite structure and the flocculant polysilicato-iron works as a “glue” in the bulk sample.
3.3. X-Ray Photoelectron Spectroscopy
X-ray photoelectron spectroscopy (XPS) measurement was performed to reveal the components that consists the carbonized samples. As shown in Figure 4(a), both samples mainly consists of carbon and oxygen which peaks appear around 285 eV (C1s), 531 eV (O1s) and 747 eV (OKLL). A slight amount of potassium was detected for the sample treated with argon flow. Significant differences were found in Figure 4(b). Peak at 56 eV, which due to Fe3p, could only be found in the sample treated with argon flow. Other peaks (Fe2p1/2 and Fe2p3/2, 724 eV and 712 eV) of Fe also could only be found in the sample carbonized under argon atmosphere. Peak of magnesium appears at 51 eV in both samples. However, peaks at 153 eV and 102 eV, each representing Si2s and Si2p, respectively, only appeared in the sample carbonized with no argon flow.
3.4. Energy Dispersive X-Ray Spectrometry
The hexagonal disks were analyzed by energy dispersive X-ray spectroscopy to detect the chemical components inside. Figure 5 shows the results measured for entire region of two samples treated in different conditions. In the previous study, we revealed that carbonization under argon atmosphere would provide an environment for reduction reaction, that, a large amount of Fe would be generated, as shown in Figure 5(a). We can obviously find the difference in Figure 5(b) that no significant peak due to Fe could be found in the sample treated with no argon flow. Instead, a large ration of potassium and silicon exist in the sample. These results coincide with the IR and XPS measurement results very well.
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Figure 3. Infrared absorption spectra of B. braunii residue carbonized at 700˚C in different carbonization conditions.
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Figure 4. X-ray photoelectron spectra of B. braunii residue carbonized at 700˚C in different carbonization conditions.
Mapping was carried out for a hexagonal disk. The results in Figure 6 indicate that components (C, O, Fe, Si, Mg and K) are uniformly dispersed in the sample. Quantitative analysis results of the hexagonal disk are summarized in Table 1. The average calculated ration of atoms number of Fe consisted in hexagonal disk is higher than those in entire region. The ration of carbon and oxygen has no significant change in the hexagonal disk and entire region. We found that in each point analysis for the hexagonal disks, no matter with the place, the ratio of number of atoms for Mg, Si and Fe, are nearly fixed as 2:3:6. The hexagonal disk consists of more than 52% of carbon atoms and 25% of oxygen atoms. The carbon atoms basically come from the B. braunii cells and oxygen atoms come from B. braunii cells, flocculant, and metallic oxide contained in the culture medium.
3.5. Hypothesis of the Formation of Hexagonal Disk
The hexagonal disks are not “regular” hexagon, if we look carefully. Hexagonal disks were randomly selected to investigate the morphology. We recorded the lengths of each side of the hexagonal disk, as summarized in Table SI1. Six sides of each hexagonal disk are referred as to “a-f”, respectively, where “a” represents the longest side and others are assigned in the counterclockwise direction. It is worth mentioning that the shortest side of each hexagonal disk can be found only in side “b” and “f”. Moreover, the ratio of the length between the longest and shortest side as calculated, is almost close to 1.5. Three sides “c-e” show almost the same length in each hexagonal disk. The ratio between the longest side and the average length of side “c-e” were calculated and summarized in Table SI1. Surprisingly, this ratio is almost fixed to 1.2 and is unrelated to the size or thickness of the hexagonal disks. Combining the knowledge we learned from all the results, we would like to build a hypothesis that this anisotropic crystal,
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Figure 5. Energy dispersive X-ray spectroscopy of B. braunii residue carbonized in different carbonization conditions. (a) For the entire region of sample carbonized at 900˚C with argon flow; (b) For the entire region of sample carbonized at 700˚C with no argon flow.
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Figure 6. Mapping results of energy dispersive X-ray analysis performed for B. braunii residue carbonized at 700˚C with no argon flow.
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Table 1. Quantitative analysis of B. braunii residue carbonized at 700˚C with no argon flow.
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Table SI1.Summary of the side length of hexagonal disks.
consisting of large amount of carbon and oxygen, and small amount of Si, Mg, and Fe, is a carbon material with metals and silicon slightly incorporated. Crystal nucleus was formed at the initial stage during the cooling process. EDS line analysis were performed for a hexagonal disk, as shown in Figure SI3. Carbon, magnesium, silicon and potassium are uniformly dispersed. However, the oxygen and iron dramatically increase the intensity in the hexagonal shape area. We assume this indicates the existence of iron oxide. Fu et al. reported as-synthe- sized α-Fe2O3 microflakes in microscale with hexagonal shape by a hydrothermal method [20] .
We assume the micro sized hexagonal disk start from a very small size metal oxide resembling the hexagonal disk. The plausible mechanism is as follows. The 700˚C carbonization temperature is high enough to generate a large amount of syngas such as CO, CO2 and CH4 during the carbonization process of B. braunii residues. Absence of argon flow then creates an environment that mixed gases stagnate in the gold furnace. Subsequent cooling process affords metal oxides deposition; meanwhile syngas act as a carbon source and grow in the order resembling the hexagonal disk shape of metal oxide. The deposited metal oxides may play the role like a substrate for the graphite growth just like the role metal substrates play in chemical vapor deposition method for graphene or any other carbon materials growth [21] [22] [23] . Since more than half of the components in the hexagonal disk are carbon, we refer to this novel 2D hexagonal disk as “Carbon Hexagon”. It is worth mentioning that absence of argon flow prevents the reduction reaction of Fe2O3, thus, instead of polygonal structure, hexagonal disk were obtained.
4. Conclusion
Microscale 2D hexagonal disks were found in the B. braunii residues carbonized at 700˚C with no argon flow. These hexagonal disks have self-similarity which is not related to size or thickness. In this study, we emphasized the importance of
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Figure SI3.EDS line analysis of a hexagonal disk obtained by carbonization of B. braunii residue at 700˚C with no argon flow.
argon flow that would affect the properties of final carbon material. 2D hexagonal shape has high anisotropy and large surface area, thus is promised to be the building blocks for functional materials. Recently, self-assembled structures of graphene or graphite are studied in the carbon material science because the dimensional anisotropy can significantly affect the thermal, optical, and electronic properties of the material [24] . The hexagonal disk can be a good candidate for the micro-size electronic devices. This report is the first discovery of the hexagonal disks in the biomass residues treated by carbonization. This study not only opens a new avenue for the discharged biomass material from the point of view of materials science, but also consummates the new energy system based on microalgae.
Acknowledgements
We appreciate the nice glasswork from Engineering Workshop, Research Facility Center for Science and Technology, Open Facility Network Office, University of Tsukuba. We are especially grateful to Dr. Shinichi Ito for his great support of assembling the furnace. A part of this work was supported by NIMS microstructural characterization platform as a program of “Nanotechnology Platform” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We also thank Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, Japan.