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Dispersed air flotation of microalgae using venturi tube type microbubble generator
Biomass and Bioenergy 130 (2019) 105379
Contents lists available at ScienceDirect
Biomass and Bioenergy journal homepage: www.elsevier.com/locate/biombioe
Short communication
Dispersed air flotation of microalgae using venturi tube type microbubble generator
T
Kazuhiro Itoha,∗, Yasuhiro Kashinob, Kentaro Ifukuc, Maeda Koujia, Takuji Yamamotoa, Shogo Taguchia a
Department of Chemical Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo, 671-2201, Japan Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Hyogo, 678-1297, Japan c Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan b
ARTICLE INFO
ABSTRACT
Keywords: Microalgae Flotation separation Microbubble generator Growth phase Triacylglycerol
To evaluate biodiesel production from microalgae, a flotation experiment was conducted using the living cell culture fluid of the diatom Chaetoceros gracilis using a venturi tube type microbubble generator. We compared the separation performance of three different culture periods: 1, 2, and 3 weeks from the start of cultivation. After 1 week, the cells were in the logarithmic growth phase, while after 2 and 3 weeks, cell growth had reached the stationary phase. The amounts of triacylglycerol (TAG) in the foam on the surface of the fluid tank were measured. TAG increased during the first 20 min after the start of circulation without additional coagulants and pH adjustment. The disruption of cells was achieved simultaneously. The amounts of TAG in the culture fluids at weeks 2 and 3 were higher than those at week 1. C. gracilis cells in the stationary phase accumulated large amounts of TAG and were easy to disrupt by pressure fluctuation in the venturi tube. These results provide insight into the fracture strength and buoyance of cells for efficiently separating the cells from large volumes of culture fluid.
1. Introduction An efficient thickening technique is needed to produce biofuel from microalgae. Microalgae have many advantages as a resource for biofuels, such as their worldwide prevalence, simple growth conditions, and high levels of lipids per growing area [1]. To produce carbonneutral and highly profitable fuel, it is necessary to reduce the required fossil fuel energy from the growth stage to the fuel production stage. To achieve a positive energy balance between fuel consumption and production, it is important to develop a highly efficient production system that accounts for the energy required for cell harvest and refinement [2]. Because large-scale culture is often performed in liquid medium, thickening techniques play a particularly important role in reducing the energy required for fuel purification in the downstream process. Gravity and centrifugation sedimentation are the classical methods used to thicken the culture fluid [3,4], which can be enhanced by sonication with ultrasound [5]. However, these methods show low productivity and high costs because the volume of the culture medium is very high compared to the number of cells in the medium. Flotation
separation has attracted attention; in this method, bubbles attached to cells increase the buoyancy of the cells to improve separation efficiency. Dispersed air flotation has a clear advantage over dissolved air flotation in that it does not require energy to pressurize the culture fluid. Hanotu et al. [6] separated microalgal culture fluid by dispersed air flotation to investigate the effect of coagulants and pH conditions on the removal efficiency of solids. Bui et al. [7] evaluated the influence of the shape and size of algae on removal efficiency using the dissolved air flotation process. However, few studies have examined thickening using algal culture fluid, and the influence of cell conditions on thickening performance has not been investigated. In preliminary experiments, a large difference in triacylglycerol (TAG) yield was observed, even when the growth conditions such as temperature, light intensity, medium type, and bubble generation conditions were the same. Therefore, we hypothesized that the cell culture time may affect the removal efficiency. In the present study, we investigated the influence of the culture time of microalgae on the dispersed air flotation. The diatom Chaetoceros gracilis was used in the thickening tests because of its ability to produce large amounts of TAG
Abbreviations:TAG, triacylglycerol (or triglyceride) ∗ Corresponding author. E-mail addresses: [email protected] (K. Itoh), [email protected] (Y. Kashino), [email protected] (K. Ifuku), [email protected] (M. Kouji), [email protected] (T. Yamamoto), [email protected] (S. Taguchi). https://doi.org/10.1016/j.biombioe.2019.105379 Received 7 June 2019; Received in revised form 21 August 2019; Accepted 18 September 2019 0961-9534/ © 2019 Elsevier Ltd. All rights reserved.
Biomass and Bioenergy 130 (2019) 105379
K. Itoh, et al.
Table 1 Initial conditions used in the main cultivation.
OD730 [-] Rate of increase in cell density [cell/mL/h]
for 1 week cultivation
for 2 weeks cultivation
for 3 weeks cultivation
6 × 10−2 8.5 × 104
8 × 10−2 8.7 × 104
7 × 10−2 8.6 × 104
Fig. 2. Venturi tube used in the present work.
and because of its high growth rates in large-scale, open-air culture [8]. Because the diatom is generally surrounded by a cell wall composed of silica, known as a frustule, it is necessary to disrupt the silica shell to extract lipids from the culture fluid. Here, we showed that the venturi type microbubble generator can be used not only to disrupt the frustule, but also to disperse air bubbles during flotation operation.
A scheme of the microbubble generator is shown in Fig. 2. The tube was prepared from transparent acrylic resin to observe the inner flow. The pressure of the culture fluid decreases rapidly because the fluid flows through the throat section, which has a small cross-section in the venturi tube. A sudden pressure change downstream of the throat section disperses large air bubbles into the microbubbles. Concurrently, the internal pressure of the cell pushes and breaks the silica shell. These mechanisms of cell disruption occurred when using hydrodynamic cavitation as suggested by Lee et al. [10].
2. Materials and methods 2.1. Cell culture The culture medium consisted of 36 g/L artificial seawater (Marine Art SF-1, Osaka Yakken Co., Ltd., Funabashi City, Japan), 252 mg/L Daigo IMK medium, and 56.84 mg/L sodium metasilicate nonahydrate (Na2SiO3•9H2O) mixed with ion exchange water. The room temperature was maintained at 25 °C. Continuous light was supplied by a fluorescent lamp and the photon irradiance was 50 μmol photons m−2 s−1. The turbidity, i.e., optical density at 730 nm (OD730), and rate of increase in cell density at the start of the main cultivation are listed in Table 1. Cell density was determined by cell counting with a hemocytometer. Lipids from the cell culture fluid or the foam separated by flotation operation were extracted as described by Bligh and Dyer [9]. TAG was measured using a Triglyceride kit (GPO-DAOS method, Wako Pure Chemicals, Osaka, Japan).
3. Results and discussion 3.1. Cell growth Typical photographs of C. gracilis cells are shown in Fig. 3 (a) and (b). Cells at 7 days after the start of culture became larger and rounder than did those at day 3 because of increased lipid storage.
2.2. Microbubble generator A schematic view of the experimental facility is shown in Fig. 1. Six liters of culture fluid were added to the tank (L 320 × W 240 × D 130 mm) and circulated by a pump. Microbubbles were generated by circulating the fluid through the venturi tube. Air was supplied to the narrowest part of the mixing nozzle using a compressor. The mixing nozzle was arranged upstream of the venturi tube to array the bubbles close to the center of the cross-section for stabilizing microbubble generation.
Fig. 1. Schematic view of the test facility.
Fig. 3. Microphotographs of C. gracilis cells at (a) 3 days and (b) 7 days. 2
Biomass and Bioenergy 130 (2019) 105379
K. Itoh, et al.
Fig. 7. TAG levels in the foam.
Fig. 4. Growth curve of C. gracilis. Table 2 Experimental conditions. Cultivation
Liquid flow rate [L/min]
Air flow rate [L/min]
Pressure in venturi tube [kPa]
1 week 2 weeks 3 weeks
22 22 22
0.8 0.8 0.8
−71.5 −70.3 −72.4
Fig. 8. Microphotograph of the foam at 5 min in week 2 culture fluid.
experiments were conducted to evaluate the cell culture fluid of C. gracilis at weeks 1, 2, and 3. The characteristics of TAG have been previously described [8]. Two fatty acids were dominant in C. gracilis: palmitic (16:0) acid and palmitoleic (16:1) acid. The typical particle size of the cells was D10 = 4.1 μm, D50 (median dia.) = 6.4 μm, and D90 = 8.7 μm after two weeks of cultivation.
Fig. 5. Distribution of bubble diameter.
3.2. Flotation test The negative pressures measured downstream of the throat of the venturi tube are shown in Table 2. The measurement accuracies of the flow rate and pressure were ± 0.03 L/min in air, ± 1 L/min in liquid, and ± 3.0 kPa in pressure. The bubble diameter distribution measured by laser scattering (NP-6000T, Nippon Denshoku Co., Ltd., Bunkyo-ku, Japan) is shown in Fig. 5. Changes in the cell density of the culture fluid in the tank during the flotation experiment are shown in Fig. 6. The origin of the horizontal axis reflects the start of circulation. The cell density decreased significantly during the first 20 min for all samples. This tendency in Fig. 6 agrees well with the concentration of the dispersed air flotation in Ref. [6]. The statistical error in cell density at 20 min was less than 0.4 × 106 [cells/mL]. The cell density in the fluid was kept uniform because the fluid in the tank was circulated during the flotation test. The rate of decrease of the cell density from 0 to 20 min was 53%, 56%, and 62% for weeks 1, 2, and 3, respectively. Additional experiments for weeks 2 and 3 indicated that the rates of decrease during 20 min were 67% and 54% for 2 weeks and 62% for 3 weeks. Therefore, for all experimental conditions, more than 50% of the cells were separated and thickened into a foam. The rate of decrease of the cell density appeared to be independent of the cultivation time under the
Fig. 6. Cell density in the culture fluid.
Changes in cell density over time during growth are indicated in Fig. 4. As demonstrated previously [8], cell density increases rapidly during week 1, which is the “logarithmic phase”. The growth rate decreases between weeks 2 and 3. In this period, which is the “stationary phase”, cells tend to accumulate TAG [8]. After 3 weeks, cell density decreases during the “senescent phase”. In the present study, flotation 3
Biomass and Bioenergy 130 (2019) 105379
K. Itoh, et al.
Fig. 9. Photographs of generated foam on the culture fluid.
in the weeks 2 and 3 culture fluid, when the cells had a round shape and high lipid levels during the stationary phase. The surface of the weeks 2 and 3 culture fluid after 5 min bubbled well, as shown in Fig. 9. As the negative pressure in the venturi tube increased (−40 kPa) with maintaining the liquid flow rate, the TAG levels in the foam decreased drastically. The negative pressure in the venturi tube, rather than the pump, had a dominant influence on the cell disruption in the present experiment; details of this will be provided in our next report. According to the above results, microalgae can be thickened by using a venturi tube type microbubble generator without using any coagulants or frothers and by adjusting the acidic conditions. However, it is necessary to investigate the removal efficiency again when the type of microbubble generator (particularly, the magnitude of internal pressure fluctuation) and type of cells (amounts of lipid and breakability) change.
present experimental conditions. The difference in the initial cell density was maintained until 50 min. The amounts of TAG present in the foam are shown in Fig. 7. To evaluate the value of TAG, 2 mL foam was sampled from the surface of the culture fluid at each time point. In the week 1 culture fluid, TAG levels increased rapidly until 5 min, reaching a maximum at 20 min. This indicates that the cells (or lipids extracted from the cells) moved from the culture fluid to the foam by air flotation. After 20 min, the TAG level decreased gradually. The amount of lipid being supplied to the foam decreased to below that being removed for sampling. In the weeks 2 and 3 culture fluid, TAG levels in the foam increased rapidly until 5 min similar to the observation in the week 1 fluid. In this culture fluid, higher levels of TAG were obtained between 10 and 40 min; all samples contained the same initial levels of TAG. Large fluctuations were observed in the data between 20 and 30 min in the week 2 culture fluid. This may be because the foam was sampled from different locations because the foam rotated on the free surface during circulation. It is noted that the depth (~70 mm) of culture fluid in the tank was very shallow in comparison with that in many previous flotation tests because of the scarcity of test fluid. As a result, the uniformity of the TAG in the foam may have been affected by the flow under the free surface. The separation efficiency was also limited because a part of the cells was sucked into the test loop before floating. The higher TAG levels in the weeks 2 and 3 culture fluid may be related to cell disruption in the venturi tube. The cell culture fluid was passed through the venturi tube approximately 67 times from 0 to 20 min according to estimation based on the fluid flow rate and fluid volume. Exposure to negative pressure in the venturi tube caused gradual cell disruption during circulation. As shown in Fig. 3, the cells had a round shape, even after 1 week in the present cultivation. If the thickness of the silica shell is kept constant, the proof stress of the spherical shell to the internal pressure decreases generally with increase in curvature radius. The difference in TAG levels in the foam may be related to the dependence of the disruption rate of cells on the culture time. Although the disruption rate was not quantitatively evaluated, cell debris was observed, as shown in the photograph in Fig. 8. This debris increased after 5 min and the number of intact cells decreased with increasing circulation time. The debris and lipids extracted from cells condensed faster than living cells. Therefore, TAG levels increased
4. Conclusions A venturi type microbubble generator was used to efficiently thicken a large amount of culture fluid containing microalgae. More than 50% of the cells in the culture fluid were separated and thickened into a foam in the first 20 min. The negative pressure in the generator disrupted the silica shells. In the present case using C. gracilis, a thickening effect was obtained with live culture solution without any additional coagulants and pH adjustment. The obtained TAG levels after week 2 or 3 of cultivation during the stationary phase were higher than those after one week of cultivation during the logarithmic phase. This result indicates that cells in the stationary phase accumulated large amounts of TAG and were easy to disrupt by pressure fluctuation in the venturi tube. The dependence of the cell type or negative pressure level on the removal efficiency should be further investigated. Declaration of interest None. Acknowledgements by 4
This work is based on results obtained from a project commissioned the New Energy and Industrial Technology Development
Biomass and Bioenergy 130 (2019) 105379
K. Itoh, et al.
Organization (NEDO, 14102439-0) and Advanced Low Carbon Technology Research and Development Program (ALCA, JPMJAL1105). We would like to thank Mr. Haruki Tanaka, and Mr. Tomoki Nakasuji, the students who carefully performed the experiments. We would like to thank Editage (www.editage.jp) for English language editing.
[4] J. Patra, G. Das, H. Shin, Microbial Biotechnology: Volume 2. Application in Food and Pharmacology, Springer, 2018, p. 133 Sec. 6.6.4. [5] M. Mubarak, A. Shaija, T.V. Suchithra, Ultrasonication: an effective pre-treatment method for extracting lipid from Salvinia molesta for biodiesel production, Resource-Efficient Technol. 2 (2016) 126–132. [6] J. Hanotu, H. Bandulasena, W. Zimmerman, Microflotation performance for algal separation, Bioengineering 109 (7) (2012) 1663–1673. [7] T. Bui, S. Nam, M. Han, Micro-bubble flotation of freshwater algae: a comparative study of differing shapes and sizes, Separ. Sci. Technol. 50 (2015) 1066–1072. [8] H. Tokushima, N. Kashino, Y. Nakazato, A. Masuda, K. Ifuku, Y. Kashino, Advantageous characteristics of the diatom Chaetoceros gracilis as a sustainable biofuel producer, Biotechnol. Biofuels 9 (2016) 235. [9] E.G. Bligh, W.J. Dyer, A rapid method for total lipid extraction and purification, Can. J. Biochem. Physiol. 37 (1959) 911–917. [10] A.K. Lee, D.M. Lewis, P.J. Ashman, Microalgal cell disruption by hydrodynamic cavitation for the production of biofuels, J. Appl. Phycol. 27 (2015) 1881–1889.
References [1] A.E. Abomohra, W. Jin, R. Tu, S. Han, M. Eid, H. Eladel, Microalgal biomass production as a sustainable feedstock for biodiesel, Renew. Sustain. Energy Rev. 64 (2016) 596–606. [2] R. Slade, A. Bauen, Micro-algae cultivation for biofuels: cost, energy balance, environmental impacts and future prospects, Biomass Bioenergy 53 (2013) 29–38. [3] M. Singh, R. Shukla, K. Das, Harvesting of Microalgal Biomass, Biotechnological Applications of Microalgae, CRC Press, London, 2013, pp. 77–87.
5
Contents lists available at ScienceDirect
Biomass and Bioenergy journal homepage: www.elsevier.com/locate/biombioe
Short communication
Dispersed air flotation of microalgae using venturi tube type microbubble generator
T
Kazuhiro Itoha,∗, Yasuhiro Kashinob, Kentaro Ifukuc, Maeda Koujia, Takuji Yamamotoa, Shogo Taguchia a
Department of Chemical Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo, 671-2201, Japan Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Hyogo, 678-1297, Japan c Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan b
ARTICLE INFO
ABSTRACT
Keywords: Microalgae Flotation separation Microbubble generator Growth phase Triacylglycerol
To evaluate biodiesel production from microalgae, a flotation experiment was conducted using the living cell culture fluid of the diatom Chaetoceros gracilis using a venturi tube type microbubble generator. We compared the separation performance of three different culture periods: 1, 2, and 3 weeks from the start of cultivation. After 1 week, the cells were in the logarithmic growth phase, while after 2 and 3 weeks, cell growth had reached the stationary phase. The amounts of triacylglycerol (TAG) in the foam on the surface of the fluid tank were measured. TAG increased during the first 20 min after the start of circulation without additional coagulants and pH adjustment. The disruption of cells was achieved simultaneously. The amounts of TAG in the culture fluids at weeks 2 and 3 were higher than those at week 1. C. gracilis cells in the stationary phase accumulated large amounts of TAG and were easy to disrupt by pressure fluctuation in the venturi tube. These results provide insight into the fracture strength and buoyance of cells for efficiently separating the cells from large volumes of culture fluid.
1. Introduction An efficient thickening technique is needed to produce biofuel from microalgae. Microalgae have many advantages as a resource for biofuels, such as their worldwide prevalence, simple growth conditions, and high levels of lipids per growing area [1]. To produce carbonneutral and highly profitable fuel, it is necessary to reduce the required fossil fuel energy from the growth stage to the fuel production stage. To achieve a positive energy balance between fuel consumption and production, it is important to develop a highly efficient production system that accounts for the energy required for cell harvest and refinement [2]. Because large-scale culture is often performed in liquid medium, thickening techniques play a particularly important role in reducing the energy required for fuel purification in the downstream process. Gravity and centrifugation sedimentation are the classical methods used to thicken the culture fluid [3,4], which can be enhanced by sonication with ultrasound [5]. However, these methods show low productivity and high costs because the volume of the culture medium is very high compared to the number of cells in the medium. Flotation
separation has attracted attention; in this method, bubbles attached to cells increase the buoyancy of the cells to improve separation efficiency. Dispersed air flotation has a clear advantage over dissolved air flotation in that it does not require energy to pressurize the culture fluid. Hanotu et al. [6] separated microalgal culture fluid by dispersed air flotation to investigate the effect of coagulants and pH conditions on the removal efficiency of solids. Bui et al. [7] evaluated the influence of the shape and size of algae on removal efficiency using the dissolved air flotation process. However, few studies have examined thickening using algal culture fluid, and the influence of cell conditions on thickening performance has not been investigated. In preliminary experiments, a large difference in triacylglycerol (TAG) yield was observed, even when the growth conditions such as temperature, light intensity, medium type, and bubble generation conditions were the same. Therefore, we hypothesized that the cell culture time may affect the removal efficiency. In the present study, we investigated the influence of the culture time of microalgae on the dispersed air flotation. The diatom Chaetoceros gracilis was used in the thickening tests because of its ability to produce large amounts of TAG
Abbreviations:TAG, triacylglycerol (or triglyceride) ∗ Corresponding author. E-mail addresses: [email protected] (K. Itoh), [email protected] (Y. Kashino), [email protected] (K. Ifuku), [email protected] (M. Kouji), [email protected] (T. Yamamoto), [email protected] (S. Taguchi). https://doi.org/10.1016/j.biombioe.2019.105379 Received 7 June 2019; Received in revised form 21 August 2019; Accepted 18 September 2019 0961-9534/ © 2019 Elsevier Ltd. All rights reserved.
Biomass and Bioenergy 130 (2019) 105379
K. Itoh, et al.
Table 1 Initial conditions used in the main cultivation.
OD730 [-] Rate of increase in cell density [cell/mL/h]
for 1 week cultivation
for 2 weeks cultivation
for 3 weeks cultivation
6 × 10−2 8.5 × 104
8 × 10−2 8.7 × 104
7 × 10−2 8.6 × 104
Fig. 2. Venturi tube used in the present work.
and because of its high growth rates in large-scale, open-air culture [8]. Because the diatom is generally surrounded by a cell wall composed of silica, known as a frustule, it is necessary to disrupt the silica shell to extract lipids from the culture fluid. Here, we showed that the venturi type microbubble generator can be used not only to disrupt the frustule, but also to disperse air bubbles during flotation operation.
A scheme of the microbubble generator is shown in Fig. 2. The tube was prepared from transparent acrylic resin to observe the inner flow. The pressure of the culture fluid decreases rapidly because the fluid flows through the throat section, which has a small cross-section in the venturi tube. A sudden pressure change downstream of the throat section disperses large air bubbles into the microbubbles. Concurrently, the internal pressure of the cell pushes and breaks the silica shell. These mechanisms of cell disruption occurred when using hydrodynamic cavitation as suggested by Lee et al. [10].
2. Materials and methods 2.1. Cell culture The culture medium consisted of 36 g/L artificial seawater (Marine Art SF-1, Osaka Yakken Co., Ltd., Funabashi City, Japan), 252 mg/L Daigo IMK medium, and 56.84 mg/L sodium metasilicate nonahydrate (Na2SiO3•9H2O) mixed with ion exchange water. The room temperature was maintained at 25 °C. Continuous light was supplied by a fluorescent lamp and the photon irradiance was 50 μmol photons m−2 s−1. The turbidity, i.e., optical density at 730 nm (OD730), and rate of increase in cell density at the start of the main cultivation are listed in Table 1. Cell density was determined by cell counting with a hemocytometer. Lipids from the cell culture fluid or the foam separated by flotation operation were extracted as described by Bligh and Dyer [9]. TAG was measured using a Triglyceride kit (GPO-DAOS method, Wako Pure Chemicals, Osaka, Japan).
3. Results and discussion 3.1. Cell growth Typical photographs of C. gracilis cells are shown in Fig. 3 (a) and (b). Cells at 7 days after the start of culture became larger and rounder than did those at day 3 because of increased lipid storage.
2.2. Microbubble generator A schematic view of the experimental facility is shown in Fig. 1. Six liters of culture fluid were added to the tank (L 320 × W 240 × D 130 mm) and circulated by a pump. Microbubbles were generated by circulating the fluid through the venturi tube. Air was supplied to the narrowest part of the mixing nozzle using a compressor. The mixing nozzle was arranged upstream of the venturi tube to array the bubbles close to the center of the cross-section for stabilizing microbubble generation.
Fig. 1. Schematic view of the test facility.
Fig. 3. Microphotographs of C. gracilis cells at (a) 3 days and (b) 7 days. 2
Biomass and Bioenergy 130 (2019) 105379
K. Itoh, et al.
Fig. 7. TAG levels in the foam.
Fig. 4. Growth curve of C. gracilis. Table 2 Experimental conditions. Cultivation
Liquid flow rate [L/min]
Air flow rate [L/min]
Pressure in venturi tube [kPa]
1 week 2 weeks 3 weeks
22 22 22
0.8 0.8 0.8
−71.5 −70.3 −72.4
Fig. 8. Microphotograph of the foam at 5 min in week 2 culture fluid.
experiments were conducted to evaluate the cell culture fluid of C. gracilis at weeks 1, 2, and 3. The characteristics of TAG have been previously described [8]. Two fatty acids were dominant in C. gracilis: palmitic (16:0) acid and palmitoleic (16:1) acid. The typical particle size of the cells was D10 = 4.1 μm, D50 (median dia.) = 6.4 μm, and D90 = 8.7 μm after two weeks of cultivation.
Fig. 5. Distribution of bubble diameter.
3.2. Flotation test The negative pressures measured downstream of the throat of the venturi tube are shown in Table 2. The measurement accuracies of the flow rate and pressure were ± 0.03 L/min in air, ± 1 L/min in liquid, and ± 3.0 kPa in pressure. The bubble diameter distribution measured by laser scattering (NP-6000T, Nippon Denshoku Co., Ltd., Bunkyo-ku, Japan) is shown in Fig. 5. Changes in the cell density of the culture fluid in the tank during the flotation experiment are shown in Fig. 6. The origin of the horizontal axis reflects the start of circulation. The cell density decreased significantly during the first 20 min for all samples. This tendency in Fig. 6 agrees well with the concentration of the dispersed air flotation in Ref. [6]. The statistical error in cell density at 20 min was less than 0.4 × 106 [cells/mL]. The cell density in the fluid was kept uniform because the fluid in the tank was circulated during the flotation test. The rate of decrease of the cell density from 0 to 20 min was 53%, 56%, and 62% for weeks 1, 2, and 3, respectively. Additional experiments for weeks 2 and 3 indicated that the rates of decrease during 20 min were 67% and 54% for 2 weeks and 62% for 3 weeks. Therefore, for all experimental conditions, more than 50% of the cells were separated and thickened into a foam. The rate of decrease of the cell density appeared to be independent of the cultivation time under the
Fig. 6. Cell density in the culture fluid.
Changes in cell density over time during growth are indicated in Fig. 4. As demonstrated previously [8], cell density increases rapidly during week 1, which is the “logarithmic phase”. The growth rate decreases between weeks 2 and 3. In this period, which is the “stationary phase”, cells tend to accumulate TAG [8]. After 3 weeks, cell density decreases during the “senescent phase”. In the present study, flotation 3
Biomass and Bioenergy 130 (2019) 105379
K. Itoh, et al.
Fig. 9. Photographs of generated foam on the culture fluid.
in the weeks 2 and 3 culture fluid, when the cells had a round shape and high lipid levels during the stationary phase. The surface of the weeks 2 and 3 culture fluid after 5 min bubbled well, as shown in Fig. 9. As the negative pressure in the venturi tube increased (−40 kPa) with maintaining the liquid flow rate, the TAG levels in the foam decreased drastically. The negative pressure in the venturi tube, rather than the pump, had a dominant influence on the cell disruption in the present experiment; details of this will be provided in our next report. According to the above results, microalgae can be thickened by using a venturi tube type microbubble generator without using any coagulants or frothers and by adjusting the acidic conditions. However, it is necessary to investigate the removal efficiency again when the type of microbubble generator (particularly, the magnitude of internal pressure fluctuation) and type of cells (amounts of lipid and breakability) change.
present experimental conditions. The difference in the initial cell density was maintained until 50 min. The amounts of TAG present in the foam are shown in Fig. 7. To evaluate the value of TAG, 2 mL foam was sampled from the surface of the culture fluid at each time point. In the week 1 culture fluid, TAG levels increased rapidly until 5 min, reaching a maximum at 20 min. This indicates that the cells (or lipids extracted from the cells) moved from the culture fluid to the foam by air flotation. After 20 min, the TAG level decreased gradually. The amount of lipid being supplied to the foam decreased to below that being removed for sampling. In the weeks 2 and 3 culture fluid, TAG levels in the foam increased rapidly until 5 min similar to the observation in the week 1 fluid. In this culture fluid, higher levels of TAG were obtained between 10 and 40 min; all samples contained the same initial levels of TAG. Large fluctuations were observed in the data between 20 and 30 min in the week 2 culture fluid. This may be because the foam was sampled from different locations because the foam rotated on the free surface during circulation. It is noted that the depth (~70 mm) of culture fluid in the tank was very shallow in comparison with that in many previous flotation tests because of the scarcity of test fluid. As a result, the uniformity of the TAG in the foam may have been affected by the flow under the free surface. The separation efficiency was also limited because a part of the cells was sucked into the test loop before floating. The higher TAG levels in the weeks 2 and 3 culture fluid may be related to cell disruption in the venturi tube. The cell culture fluid was passed through the venturi tube approximately 67 times from 0 to 20 min according to estimation based on the fluid flow rate and fluid volume. Exposure to negative pressure in the venturi tube caused gradual cell disruption during circulation. As shown in Fig. 3, the cells had a round shape, even after 1 week in the present cultivation. If the thickness of the silica shell is kept constant, the proof stress of the spherical shell to the internal pressure decreases generally with increase in curvature radius. The difference in TAG levels in the foam may be related to the dependence of the disruption rate of cells on the culture time. Although the disruption rate was not quantitatively evaluated, cell debris was observed, as shown in the photograph in Fig. 8. This debris increased after 5 min and the number of intact cells decreased with increasing circulation time. The debris and lipids extracted from cells condensed faster than living cells. Therefore, TAG levels increased
4. Conclusions A venturi type microbubble generator was used to efficiently thicken a large amount of culture fluid containing microalgae. More than 50% of the cells in the culture fluid were separated and thickened into a foam in the first 20 min. The negative pressure in the generator disrupted the silica shells. In the present case using C. gracilis, a thickening effect was obtained with live culture solution without any additional coagulants and pH adjustment. The obtained TAG levels after week 2 or 3 of cultivation during the stationary phase were higher than those after one week of cultivation during the logarithmic phase. This result indicates that cells in the stationary phase accumulated large amounts of TAG and were easy to disrupt by pressure fluctuation in the venturi tube. The dependence of the cell type or negative pressure level on the removal efficiency should be further investigated. Declaration of interest None. Acknowledgements by 4
This work is based on results obtained from a project commissioned the New Energy and Industrial Technology Development
Biomass and Bioenergy 130 (2019) 105379
K. Itoh, et al.
Organization (NEDO, 14102439-0) and Advanced Low Carbon Technology Research and Development Program (ALCA, JPMJAL1105). We would like to thank Mr. Haruki Tanaka, and Mr. Tomoki Nakasuji, the students who carefully performed the experiments. We would like to thank Editage (www.editage.jp) for English language editing.
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