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Article

Thinning of Botryococcus braunii Colony Sheath by Pretreatment Enhances Solvent-Based Hydrocarbon Recovery

by
Kenichi Furuhashi
*,
Akinari Magota
,
Yifan Liu
,
Fumio Hasegawa
,
Shigeru Okada
,
Yutaka Kaizu
and
Kenji Imou
Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
*
Author to whom correspondence should be addressed.
Phycology 2022, 2(4), 363-373; https://doi.org/10.3390/phycology2040020
Submission received: 23 August 2022 / Revised: 29 September 2022 / Accepted: 5 October 2022 / Published: 7 October 2022
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Abstract

:
Botryococcus braunii is a green microalga that is attracting attention as an alternative aviation fuel owing to its hydrocarbon production. In this study, we investigated two approaches to reducing the energy required by pretreatment for hydrocarbon recovery by solvent extraction. Saltwater culture has been reported previously only for the B race of Botryococcus braunii; it improved hydrocarbon recovery from the A race too. We developed a hot water rinsing method that reduced the temperature requirement from the 85 °C which was previously reported for the B race. As the salt concentration in the medium increased, the colony sheath that covered the entire colony surface of the Yamanaka strain (race A) became thinner and the hydrocarbon recovery increased. Saltwater culture can be applied to race A without any energy input. Hydrocarbon recovery from the Showa strain (race B) exceeded 90% after nine rinses with 70 °C hot water while maintaining this temperature. Thus, both pretreatments lowered the treatment temperature by at least 15 °C compared to previously reported methods. Both treatments improved hydrocarbon recovery by thinning the colony sheaths.

1. Introduction

Microalgae have recently attracted particular attention as an alternative fuel source for aviation, and research and development on their use are actively underway worldwide [1,2]. The reasons for this include (1) their capacity for high oil production, (2) the potential to build a system for their production that does not compete with food production, and (3) their ability to fix carbon dioxide. However, there are also significant challenges to be overcome, such as reducing input resources and costs so that the carbon dioxide reduction effect associated with biofuel production can be achieved.
When oil is extracted from microalgae for biofuel purposes, the drying process, which requires a significant energy input for latent heat, must be replaced. Alternative methods generally exist for extraction with low-polarity solvents (mainly n-hexane) after cell disruption pretreatment (homogenizing, milling, ultrasonication, hydrothermal treatment) [3,4,5] and with amphiphilic solvents (dimethyl ether, supercritical CO2, ionic liquids) [6,7,8]. While most oil-producing microalgae accumulate oils and fats intracellularly, Botryococcus braunii secretes hydrocarbons extracellularly during metabolism and accumulates them at 25–75% of their dry weight in macromolecular substances that make up the extracellular matrix [9]. More than 95% of the hydrocarbons are found in the extracellular matrix, although there are lipids inside the cell [10,11,12]. Individual cells are held together by this extracellular matrix to form tufted colonies. Therefore, one would expect efficient hydrocarbon extraction from B. braunii using n-hexane, which is a popular and industrially applicable method for the extraction of food oil. However, the hydrocarbon recovery rate did not improve after simply mixing n-hexane and algal slurry [13].
Although physical pretreatment for cell crushing or extraction by amphiphilic solvents is also effective for B. braunii [14,15], n-hexane extraction, which requires a lower energy input for pretreatments, can be applied. The entire surface of the colony formed by cells and intercellular material is covered with the fibrous material called “colony sheath”, which is mainly composed of polysaccharides [16]. The removal of this colony sheath by low-load pretreatment is related to the hydrocarbon recovery by n-hexane. Mechanical pretreatment at a lower load than that which would disrupt cells has been reported to partially crush the colony, expose surfaces not covered by the colony sheath, and increase hydrocarbon recovery [17]. Heat treatment at 80 °C did not improve the hydrocarbon recovery rate. However, more than 95% recovery was obtained at 85 °C, and the colony sheath was lost from the colony surface [18,19]. In addition, the fibril density of the colony sheath of osmotically stressed saltwater-cultured B. braunii was reduced by changes in polysaccharide metabolism, and the hydrocarbon recovery rate was improved significantly without pretreatment [19]. However, all algae used in the reports that discuss the relationship between the presence or absence of colony sheaths and the hydrocarbon recovery rate belong to the B race of B. braunii.
Botryococcusbraunii is divided into at least three chemical races, according to the type of hydrocarbon produced. Among these, races A and B have relatively high hydrocarbon contents and produce straight-chain hydrocarbons, with 23–31 carbons derived from fatty acids and triterpene hydrocarbons and 30–37 carbons derived from farnesyl diphosphate [20]. The A and B races also differ in their polysaccharide metabolism. Although both have a colony sheath and heat treatment improved hydrocarbon extraction efficiency, the effective temperature in race A was 60 °C, which is much lower than the effective temperature of 85 °C of race B [18,21]. Additionally, different polysaccharide metabolisms mean that the saltwater culture that was efficient for hydrocarbon extraction in race B may not be as efficient in race A. The improvement of hydrocarbon extractability from B. braunii leads to milking, which extracts hydrocarbons while keeping algal cells alive. Milking of hydrocarbons from microalgae has been studied in Chlamydomonas, genetically engineered for terpenoid production [22], B. braunii, which produces hydrocarbons, and extracellularly without genetic improvement [23,24].
Polysaccharides eluted from race B of B. braunii by heat treatment have interesting characteristics. First, these polysaccharides are characterized by a reversible sol-gel transition at temperatures (70–80 °C) and are irreversibly solubilized over 85 °C. Once irreversibly solubilized, polysaccharides do not re-transit gel state upon cooling [25]. There is a relationship between the solubilization of polysaccharides and their removal from the colony sheath and a higher hydrocarbon recovery rate, as mentioned above. The removal of this polysaccharide from B. braunii by gel transition is a prerequisite for efficient hydrocarbon extraction using n-hexane. Second, these polysaccharides form an emulsion with n-hexane which reduce the extraction efficiency if they remain in the aqueous phase during extraction [26]. Polysaccharides must be removed by rinsing for efficient hydrocarbon extraction [27]. Using a reversible sol-gel transition and rinsing in a sol state while maintaining a high temperature of 70 °C may enable pretreatment with a heating temperature even lower than 85 °C.
In this study, we aimed to reduce the energy of pretreatment for efficient hydrocarbon recovery and investigated two different approaches. First, we examined the effect of pretreatment by saltwater culture on n-hexane extraction from race A B. braunii. Until now, this pretreatment had only been considered for race B. Second, we clarified the effect of pretreatment by hot water rinsing at lower temperatures than those previously reported for race B. The relationship between changes in the colony sheath and the hydrocarbon recovery rate was determined by varying the above pretreatment loadings in multiple stages then observing the colony sheath.

2. Materials and Methods

2.1. Algal Strain and Culture

Two strains of B. braunii were used in this study, namely the Yamanaka and Showa strains, belonging to races A and B, respectively [28,29]. Table 1 summarizes the pretreatment methods performed on each of these strains. Modified Chu13 medium (Chu13) was used as the standard medium, and saltwater medium was prepared using the method of Furuhashi et al. [30]. The salt concentrations were adjusted to 4 g L−1 (0.4% SM) and 6 g L−1 (0.6% SM). The incubation temperature was 25 °C, and air with a carbon dioxide concentration of 1.5 vol% was supplied from the bottom of the culture bottle with an effective volume of 1.2 L. The photosynthetic photon flux density (PPFD) by fluorescent lamp on the surface of the culture bottle was set at 120 µmol m−2 s−1 and the light/dark cycle was set at 12 h/12 h. Algal samples of the Yamanaka strain were cultured for a total of 40 days. On day 20, all culture sections were subcultured with new medium to an initial concentration of 0.3 g L−1. The Showa strain was used for the extraction test after 25–30 d. Dry weight was measured as follows. First, 40 mL of the algal culture solution was sampled every four days. A glass microfiber filter (GF/A 110-mm diameter, Cytiva, Tokyo, Japan) was used to filter 10 mL of the culture. The remaining algal cells were carefully cleaned with deionized water. After cleaning, algal cells were dried at 105 °C for 24 h. Algal cultures were conducted in triplicate.

2.2. Observation of Colony Sheath Using India Ink

Colony sheaths on the surface of algal colonies were observed after mixing with black India ink [31]. The algal slurry was mixed with an equal amount of India ink of 50 nm to 200 nm particle size. Colloids of India ink did not penetrate the colony sheath on the colony surface. A layer (the colony sheath) was observed on the surface of the colony. The mixture was placed on a glass slide and observed under an optical microscope (Olympus, CX-41) after a cover glass was lightly placed on it. Approximation Equation (1) was used to calculate the thickness of the colony sheath.
T ≈ 2 ( S 2 − S 1 ) ( L 1 + L 2 )
where T (mm) is the thickness of the colony sheath, S1 (mm2) is the area enclosed by the inner perimeter (surface of cells and extracellular material without the colony sheath), S2 (mm2) is the area enclosed by the outer perimeter (including the colony sheath), L1 (mm) is the inner perimeter, and L2 (mm) is the inner perimeter. CorelDRAW X7 (Corel) was used to measure the area and perimeter after manually drawing lines on the outer and inner curves on the micrograph with the freehand software tool.

2.3. Hot-Water Rinsing Process

The algal slurry, adjusted to 15 g L−1 and 25 mL by the method described above, was placed into a 50 mL centrifuge sedimentation tube, heated to 60 °C or 70 °C using a water bath, and immediately solid-liquid separated on a 20-µm nylon mesh. All algae on the nylon mesh were collected using deionized water to a make a final volume of 25 mL. One rinsing set consisted of algae collection, heating, and solid-liquid separation cycles. Rinsing was repeated nine times at each temperature.

2.4. Hydrocarbon Recovery Rate by n-Hexane

The hydrocarbon recovery rate after each treatment was defined as the percentage of pure hydrocarbons extracted (with n-hexane and passing through the silica gel column for removing the pigments extracted simultaneously as hydrocarbons) from the wet algal samples to the pure hydrocarbons extracted from freeze-dried samples (hydrocarbon content). Hydrocarbon content (both Yamanaka and Showa strains) and hydrocarbon extraction from the Yamanaka strain without pretreatment were determined using the method described by Magota et al. [18]. Hydrocarbon extraction from the Showa strain without pretreatment was performed differently because the amount of hydrocarbons was too small for the gravimetric method in the hot water rinsing process. Hydrocarbons were extracted from the Showa strain without pretreatment using the following method. First, 1 mL of n-hexane was added to 1 mL of a slurry of algae on nylon mesh collected with deionized water and shaken for 30 s. After collecting the hexane phase to prevent the recovery of the separated aqueous phase, 1 mL of hexane was added to the remaining aqueous phase and shaken again for 30 s. The hexane was collected and diluted in a volumetric flask. The solution was analyzed using gas chromatography with flame ionization detection (GC-FID). The temperature program was set as described in our previous report [32]. Hydrocarbons purified from hydrocarbon content measurements were used as standards. The extracted hydrocarbon content was determined from the sum of the peak areas of the nine hydrocarbons detected at retention times from 33.9 min to 35.7 min, which are reported as hydrocarbons of the Showa strain [28].

3. Results

3.1. Growth Rates of Yamanaka Strain by Salt-Water Culture

Figure 1 shows the growth rates of the Yamanaka strain in Chu13 and saltwater media. There was no difference in the growth rates between 0.4% SM and Chu13 media from 0 to 20 d before subculturing. In contrast, 0.6% SM significantly reduced the growth rate. Immediately after subculturing at 20 days, the growth rate was suppressed in 0.4% SM compared to that in Chu13 medium. However, after 32 days, the growth rate in 0.4% SM recovered and was almost equal to that in Chu13. The culture in 0.6% SM was overstressed by the salt water, with periods of almost no increase in algal concentration during 28–35 days. The 0.6% SM medium 0.6% SM significantly inhibited the growth of the Yamanaka strain of B. braunii.

3.2. Changes in Hydrocarbon Recovery Rate and Colony Sheath of Yamanaka Strain

The hydrocarbon recovery rates from algae cultured in Chu13, 0.4% SM, and 0.6% SM were 2.9%, 12.7%, and 60.1%, respectively, averaged over 20 d and 40 d (Table 2). The hydrocarbon recovery rate increased significantly with increasing salt concentration in the medium (p < 0.05, One-way ANOVA). However, no effect on hydrocarbon recovery was observed when the culture was continued for a long period of time. Hydrocarbon recovery did not differ significantly between days 20 and 40 for Chu13, 0.4% SM, and 0.6% SM (p > 0.05, t-test). The hydrocarbon recovery rate tended to increase at 40 days compared to 20 days only in 0.6% SM.
Thick layers (colony sheath) were observed around the colony after mixing the algal slurry cultured in Chu13 with India ink (Figure 2A,B). It was observed that the colony sheaths of algae cultured in 0.4% SM (Figure 2C) and 0.6% SM (Figure 2D) began to thin after four days of culture. In 0.6% SM, further thinning of the colony sheath was observed after 20 days of incubation (Figure 2F), although the colony sheath did not change significantly between day 4 and day 20 in 0.4% SM (Figure 2C,E). In the heat-treated algae cultured in Chu13, colony sheaths were observed at 50 °C, which was almost the same as in the untreated algae (Figure 2G). However, at 60 °C, no colony sheath was observed between the colony and the India ink (Figure 2H). Although the 0.6% cultured algae had a much thinner colony sheath initially, a smaller amount of colony sheath remained when compared to the 60 °C heat-treated algae. The thickness of the colony sheaths covering algae cultured for 20 d in Chu13, 0.4% SM, and 0.6% SM decreased with increasing salinity and hydrocarbon recovery (Table 2). Compared with Chu13, the thickness of the colony sheath was 61% for 0.4% SM and 21% for 0.6% SM.

3.3. Hot-Water Rinsing for Showa Strain

Figure 3 shows the relationship between the number of rinses and the hydrocarbon recovery rates from algal slurries rinsed at 60 °C or 70 °C. Although the recovery rate increased slightly with the number of rinses at 60 °C, the hydrocarbon recovery rate was still less than 5% after nine rinses. However, after rinsing at 70 °C, the hydrocarbon extraction rate increased markedly with an increasing number of rinses, reaching a maximum at the ninth rinsing. There was a high positive correlation (R2 = 0.99, simple regression analysis) between the number of rinses from the first to the seventh rinse at 70 °C and the hydrocarbon extraction efficiency. The extraction efficiency did not improve with rinsing at 60 °C, when polysaccharides began to be released into the tank, but increased markedly with rinsing at 70 °C, when the detected amounts of polysaccharides decreased.
Colony sheaths of algae after hot water rinsing were mixed with India ink and photographed (Figure 4). A layer covering the entire colony was observed in the algae after the ninth rinse at 60 °C (Figure 4B), as was the case with no treatment (Figure 4A). In contrast, in the algae rinsed five times with water at 70 °C (Figure 4C), areas where the boundaries of the colony sheath were not clearly defined were observed in some compartments of the colony surface. As the number of rinses increased from the fifth to the ninth, the layer covering the colony surface could no longer be clearly observed because it became thinner (Figure 4D). Microscopic observations revealed a clear difference in the elution of the colony sheath after hot water rinsing at 60 °C and 70 °C. The conditions of the colony sheath were compared between heat treatment and hot water rinsing. In the heat-treated Showa strain, a colony sheath was observed at 80 °C (Figure 4E), but at 85 °C, no colony sheath was observed between the colony and the India ink (Figure 4F). The colony sheath completely eluted into the aqueous phase after heat treatment at 85 °C, whereas after the ninth hot water rinse at 70 °C a small amount of colony sheath (the boundary between the colony surface and the ink) was observed.

4. Discussion

4.1. Relationship between Colony Sheath and Hydrocarbon Recovery in Race A

In our previous study, the hydrocarbon recovery rate from the Yamanaka strain (race A) increased sharply at 50 °C and 60 °C, from 5.8% to 90.8% [18]. The colony sheath observed at 50 °C was completely lost at 60 °C (Figure 2G,H). This phenomenon was identical to that of the B race Showa strain, as revealed by optical microscopy in this study and electron microscopy [19], although the elution temperatures differed by 25 °C and the structure of the colony sheath was different. In saltwater cultures, increased hydrocarbon recovery and thinner colony sheaths were observed with increasing salt concentrations. These results indicate that the race A colony sheath, like that of the B race, inhibited contact between hydrocarbons and n-hexane in the extracellular matrix. Removal of the colony sheath was necessary to increase hydrocarbon recovery, even in race A.
In the Yamanaka strain, hydrocarbon recovery only increased to approximately 12%, even in the 0.4% SM culture, which decreased the growth rate. The 0.4% and 0.6% salinities used in this study are brackish compared to the salinity of seawater, which is around 3.5%. Especially in oceanfront areas, this salt concentration can be adjusted by simply mixing 11–17% seawater with freshwater, thus saving valuable freshwater resources compared to seawater. A thinning of the colony sheath was observed after only four days of incubation in 0.4% SM or 0.6% SM. Therefore, it is possible to increase the hydrocarbon recovery rate by culturing in saltwater medium for several days after culturing in freshwater medium. In the Showa strain, hydrocarbon recovery was improved after culturing seven days, although salinity was different (0.3% SM) [30]. It is reported that hydrocarbon recovery from Showa strain exceeded 30% in the 0.3% SM culture, with no change in the growth rate within the same literature. The effect of saltwater culture on hydrocarbon recovery was less for the Yamanaka strain (race A) than for the Showa strain (race B).
A side effect of saltwater culture, namely, an enlargement of colony size, has been reported for the Showa strain [30]. In the B race, other factors reported to increase colony size include increased hydraulic retention time (HRT) in continuous culture, addition of glucose for mixotrophy, and increased light intensity [33,34,35]. Colonies in the saltwater cultures were observed to be enlarged even in race A (Figure 2A–C). As colony size enlarges, light transmission increases significantly. In outdoor culture, increasing the water depth helps maintain the water temperature [36]. Smaller particles absorb all the light near the surface of the water and can only breathe in deeper areas. When the water depth is increased in outdoor culture ponds, the reduced growth rate of race A in saltwater culture may be recovered by increased colony size. The relationship between water depth, colony size, and actual photosynthetic rate should be examined in the future.

4.2. Rinsing of Colony Sheath in Race B at Lower Temperatures

The polysaccharides that make up the colony sheath change to the sol state at 70 °C and are irreversibly solubilized at 85 °C (Figure 5) [25]. The difference between the presence or absence of a colony sheath at 80 °C and 85 °C (Figure 4E,F) is related to the gel-sol transition of polysaccharides. Figure 2E shows a colony that was heat treated at 80 °C and cooled to room temperature before microscopic observation. These observations suggest that once the colony sheath transits to the sol state, it reversibly re-transitions to the gel state around B. braunii colonies during cooling to room temperature. In our previous study, there was a clear difference in the hydrocarbon recovery rates between 80 °C (4.9%) and 85 °C (94.4%) [18]. The removal of the colony sheath and the improvement in hydrocarbon recovery require pretreatment by heating to a temperature above the irreversible point.
However, the hydrocarbon recovery rate may be improved if the colony sheath is removed by hot water rinsing using the temperature range of the solubilization point (70 °C). It was clear that the colony sheath could be removed by rinsing with warm water at 70 °C and then filtering while maintaining the temperature (Figure 5). Quantitative evaluation of thickness was not possible because the colony sheath of the Showa strain was thinner than that of the Yamanaka strain. Although only qualitatively evaluated by the figure, it could be seen that the colony sheath was removed as the number of washes increased. A clear colony sheath was observed even after rinsing nine times with warm water at 60 °C, below the solubilization point, and the hydrocarbon recovery was only 5.3%. These observations suggest that almost no colony sheaths were removed after rinsing at 60 °C.
The hydrocarbon recovery rate in this study was the ratio with the amounts of hydrocarbons extracted by n-hexane from freeze-dried algae as denominator. B. braunii accumulates more than 95% of the hydrocarbons in the extracellular matrix and extracellular hydrocarbons are recovered by solvent extraction from dried algae [10,11,12]. In freeze-dried Chlorella sp. and Scenedesmus sp., some intracellular lipids are extracted by n-hexane without crushing the cell wall [37]. Some of the intracellular hydrocarbons, less than 5% of the total hydrocarbon content, are also assumed to have been extracted from the freeze-dried B. braunii by n-hexane. The hydrocarbon recovery of the algae after nine rinses at 70 °C was nearly equal (100% hydrocarbon recovery rate) to that from the freeze-dried algae. It is possible that not only extracellular hydrocarbons but also some intracellular hydrocarbons, which account for only less than 5% of the total hydrocarbon content, were extracted due to cell wall damage by hot water. This study proposes a pretreatment method that lowers the heating temperature by 15 °C (from 85 °C to 70 °C) compared to previously used processes. Compared to other pretreatments that require the use of electricity, the proposed hot water rinsing treatment has a year-round demand below 100 °C and has the potential to utilize waste heat from factories and biomass power plants [38]. The larger the temperature difference, the higher the heat exchange rate and the lower the energy input. An efficient hydrocarbon recovery process was examined for an algal slurry (92% water content) heated at 85–90 °C. In the future, we will derive the optimal parameters for the process of washing the concentrated slurry at 70 °C and clarify the effect of reducing the energy input to the process. Polysaccharides of the colony sheath recovered in aqueous phase by hot water rinsing are composed primarily of galactose and arabinose and the sheath maintains the gel-sol transition ability due to recovering at temperatures below 85 °C [26]. As with the mucilaginous polysaccharides of seaweed, their use as a thickening or gelling material in food products is also expected in the future.
B. braunii secretes most of the hydrocarbons extracellularly. Taking advantage of this feature, a process called milking has been studied in B. braunii to extract hydrocarbons while keeping algal cells alive and re-culturing the extracted residues [23,24]. The saltwater culture used for race A is expected to be effective in reducing the contact time between the solvent and the algae and is useful for milking. On the other hand, hot water rinsing treatment is not expected to be useful for milking. Although it is a lower temperature heat treatment than previously reported, protein denaturation in B. braunii occurs at about 60 °C [26]. It is difficult to use the hot water rinsing treated algae in re-cultures. However, hot water rinsing can improve hydrocarbon recovery rates to extremely high levels and it is assumed that combining with the saltwater culture would provide significant advantages for process automation. Saltwater culture enlarges the colony size of B. braunii and allows gravity filtration with wire screens [32]. For example, the same effect as hot water rinsing process can be easily achieved by simply applying warm water to the algae on a tilting wire screen. The combined process may reduce the amount (load) of hot water rinsing because saltwater culture thins the colony sheath physiologically. Furthermore, when considering the use of low-grade factory waste heat as described above, the fusion of the two methods proposed in this study is an efficient process that can be used on a large scale.

5. Conclusions

Two pretreatment methods (saltwater culture for race A and reduced heat treatment temperature for race B) were tested for the efficiency of hydrocarbon extraction and reduction in the energy used for pretreatment. The pretreatment effect of saltwater culture, which had previously been reported only for race B, was also obtained for race A. As the salt concentration increased, the colony sheath became thinner, and hydrocarbon recovery increased. For race B, rinsing with hot water at a temperature 15 °C, lower than the previously reported effective heat treatment temperature of 85 °C, significantly increased hydrocarbon recovery. As the number of rinses increased, the colony sheath became thinner, and hydrocarbon recovery exceeded 90% at the ninth wash. Both treatments increased hydrocarbon recovery by thinning the colony sheath. It is important to clarify the impacts of these two novel methods on reducing the energy input to the extraction process through a detailed study of the extraction from concentrated algal slurry in future.

Author Contributions

Conceptualization, K.F., A.M., F.H., Y.K. and K.I.; methodology, K.F., A.M. and S.O.; validation, K.F., A.M. and Y.L.; investigation, A.M. and Y.L.; resources, K.F. and K.I.; writing—review and editing, K.F., S.O., Y.K. and K.I.; visualization, K.F., A.M. and Y.L.; supervision, K.F. and K.I.; project administration, K.F. and K.I. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by JSPS KAKENHI (Grant Number JP22K14975).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are contained within the article.

Acknowledgments

We would like to express our sincere gratitude to Sueko Atobe and Syoko Miyagi for their technical support.

Conflicts of Interest

The authors declare no conflict of interest.

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Phycology 02 00020 g001 550
Figure 1. Growth rates of B. braunii Yamanaka strain cultured in different media. Black arrow indicates day 20 when algae were subcultured into new medium (n = 3, mean ± SD).
Figure 1. Growth rates of B. braunii Yamanaka strain cultured in different media. Black arrow indicates day 20 when algae were subcultured into new medium (n = 3, mean ± SD).
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Figure 2. Optical microscope image of algal slurry of Yamanaka strain mixed with India ink. White bars in the lower right corner of the image indicate 20 µm. (A,B), Chu13; (C), 0.4% SM at day 4; (D), 0.4% SM at day 20; (E), 0.6% SM at day 4; (F), 0.6% SM at day 20; (G), heat treatment at 55 °C; (H), heat treatment at 60 °C.
Figure 2. Optical microscope image of algal slurry of Yamanaka strain mixed with India ink. White bars in the lower right corner of the image indicate 20 µm. (A,B), Chu13; (C), 0.4% SM at day 4; (D), 0.4% SM at day 20; (E), 0.6% SM at day 4; (F), 0.6% SM at day 20; (G), heat treatment at 55 °C; (H), heat treatment at 60 °C.
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Figure 3. Effect of the number of rinses with hot-water at 60 °C and 70 °C on hydrocarbon recovery rate (n = 3, mean ± SD).
Figure 3. Effect of the number of rinses with hot-water at 60 °C and 70 °C on hydrocarbon recovery rate (n = 3, mean ± SD).
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Figure 4. Optical microscope image of algal slurry of Showa strain mixed with India ink. (A), no treatment; (B), 9th hot-water rinsing at 60 °C; (C), 5th hot-water rinsing at 70 °C; (D), 9th hot-water rinsing at 70 °C; (E), heat treatment at 80 °C; (F), heat treatment at 85 °C.
Figure 4. Optical microscope image of algal slurry of Showa strain mixed with India ink. (A), no treatment; (B), 9th hot-water rinsing at 60 °C; (C), 5th hot-water rinsing at 70 °C; (D), 9th hot-water rinsing at 70 °C; (E), heat treatment at 80 °C; (F), heat treatment at 85 °C.
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Figure 5. Schematic illustration of the effect of heating temperature and hot-water rinsing on colony sheath.
Figure 5. Schematic illustration of the effect of heating temperature and hot-water rinsing on colony sheath.
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Table
Table 1. Pretreatment methods for solvent extraction conducted on each strain of B. braunii.
Table 1. Pretreatment methods for solvent extraction conducted on each strain of B. braunii.
StrainRacePretreatment
YamanakaA raceSalt-water culture (0.4, 0.6%)
ShowaB raceHot-water rinsing (60, 70 °C)
Table
Table 2. Hydrocarbon recovery rate and thickness of colony sheath of B. braunii Yamanaka strain cultured in three different media.
Table 2. Hydrocarbon recovery rate and thickness of colony sheath of B. braunii Yamanaka strain cultured in three different media.
MediumHydrocarbon Recovery Rate (%)Thickness (µm)
20 d40 d20 d
Chu133.9 ± 1.61.8 ± 0.62.00 ± 0.15
0.4%SM12.0 ± 3.613.3 ± 3.61.21 ± 0.18
0.6%SM54.0 ± 4.266.1 ± 6.60.42 ± 0.13
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Furuhashi, K.; Magota, A.; Liu, Y.; Hasegawa, F.; Okada, S.; Kaizu, Y.; Imou, K. Thinning of Botryococcus braunii Colony Sheath by Pretreatment Enhances Solvent-Based Hydrocarbon Recovery. Phycology 2022, 2, 363-373. https://doi.org/10.3390/phycology2040020

AMA Style

Furuhashi K, Magota A, Liu Y, Hasegawa F, Okada S, Kaizu Y, Imou K. Thinning of Botryococcus braunii Colony Sheath by Pretreatment Enhances Solvent-Based Hydrocarbon Recovery. Phycology. 2022; 2(4):363-373. https://doi.org/10.3390/phycology2040020

Chicago/Turabian Style

Furuhashi, Kenichi, Akinari Magota, Yifan Liu, Fumio Hasegawa, Shigeru Okada, Yutaka Kaizu, and Kenji Imou. 2022. "Thinning of Botryococcus braunii Colony Sheath by Pretreatment Enhances Solvent-Based Hydrocarbon Recovery" Phycology 2, no. 4: 363-373. https://doi.org/10.3390/phycology2040020

APA Style

Furuhashi, K., Magota, A., Liu, Y., Hasegawa, F., Okada, S., Kaizu, Y., & Imou, K. (2022). Thinning of Botryococcus braunii Colony Sheath by Pretreatment Enhances Solvent-Based Hydrocarbon Recovery. Phycology, 2(4), 363-373. https://doi.org/10.3390/phycology2040020

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