Appl Microbiol Biotechnol (2015) 99:1647–1653 DOI 10.1007/s00253-014-6234-1
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
A simple recovery process for biodegradable plastics accumulated in cyanobacteria treated with ionic liquids Daigo Kobayashi & Kyoko Fujita & Nobuhumi Nakamura & Hiroyuki Ohno
Received: 24 September 2014 / Revised: 13 November 2014 / Accepted: 13 November 2014 / Published online: 29 November 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Here, we proposed a simple recovery process for poly(3-hydroxybutyrate) (PHB) accumulated in cyanobacteria by using ionic liquids (ILs), which dissolve cyanobacteria but not PHB. First, we investigated the effects of IL polarity on hydrogen-bonding receipt ability (β value) and hydrogen-bonding donating ability (α value) and evaluated the subsequent dissolution of cyanobacteria. We found that ILs having α values higher than approximately 0.4 and β values of approximately 0.9 were suitable for dissolution of cyanobacteria. In particular, 1-ethyl-3-methylimidazolium methylphosphonate ([C2mim][MeO(H)PO2]) was found to dissolve cyanobacteria components, but not PHB. Thus, we verified that PHB produced in cyanobacteria could be separated and recovered by simple filtering after dissolution of cyanobacteria in [C2mim][MeO(H)PO2]. Using this technique, more than 98 % of PHB was obtained on the filter as residues separated from cyanobacteria. Furthermore, [C2mim][MeO(H)PO2] maintained the ability to dissolve cyanobacteria after a simple recycling procedure.
Keywords Ionic liquid . Cyanobacteria . Poly(3-hydroxybutyrate) (PHB) . Polarity . Recovery process
D. Kobayashi : K. Fujita (*) : N. Nakamura : H. Ohno Department of Biotechnology, Tokyo University of Agriculture , 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan e-mail: [email protected] D. Kobayashi : K. Fujita : N. Nakamura : H. Ohno Functional Ionic Liquid Laboratories (FILL), 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan K. Fujita : N. Nakamura : H. Ohno Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
Introduction Cyanobacteria produce various useful materials such as lipids, polysaccharides, and biodegradable plastic, using energy from sunlight (Abed et al. 2009; Capon et al. 1983; Melis and Happe 2001; Stanier et al. 1971). Many researchers have investigated the integration of synthesized operons to develop and improve the production of biomaterials from cyanobacteria (Kaneko et al. 2003; Osanai et al. 2014; Muramatsu et al. 2009; Zou et al. 2009). The production of materials and biodiesel by cyanobacteria has been pursued as an alternative and sustainable energy source for the next generation because food crops are inefficient and unsustainable sources of these materials (Mata et al. 2010; Schenk et al. 2008). In order to use biodegradable plastics, lipids, and other useful substrates produced by cyanobacteria, it is important to develop efficient processes for separation and recovery of these materials. Cyanobacteria have cell walls that contain cellulose and peptidoglycans. Because these components are difficult to dissolve in ordinal solvents, including organic solvents, it is difficult to separate useful materials made of organic substances without specific pretreatments, including demineralization, dehydration, and drying or disruption of cell walls by physical or chemical methods (Jacquel et al. 2008). Furthermore, large amounts of organic solvents and energy are required. Therefore, further studies are necessary to realize the appropriate and simple process to recover materials from cyanobacteria. Recently, ionic liquids (ILs) have attracted attention as a potential solvent for a wide area. ILs, which are molten salts having a melting point below 100 °C, possess unique properties, such as high ionic conductivity and high thermal stability, which are difficult to achieve in general organic solvents (Rogers and Seddon 2003; Wilkes 2002; Welton 1999). Furthermore, ILs have negligible vapor pressure and are therefore
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gathering attention as novel green solvents. Several researches had been reported about the collection of useful materials from cyanobacteria because of the effects of IL properties (Kim et al. 2012; Teixeira 2012), though these processes needed heating during treatment and mixing of other solvents. Additionally, many researchers have realized that the properties of ILs can be controlled by designing the structure to treat biomass; indeed, hydrophobic ILs have been developed for the extraction of materials using a liquid-liquid biphasic system (Tang et al. 2012), and highly polar ILs have been shown to successfully dissolve very insoluble polymer, such as cellulose (Fukaya et al. 2008; Swatloski et al. 2002). In previous studies, we have reported the direct dissolution of wet and saliferous cyanobacteria using polar ILs under mild conditions, e.g., without heating, thereby requiring no pretreatment after collection (Fujita et al. 2013). Thus, our previous work supports the hypothesis of using ILs for the simple process to recover the materials in cyanobacteria. Therefore, novel methods for the collection of useful materials produced in cyanobacteria using ILs may be simple and require low energy input if appropriate ILs can be chosen. In this paper, we proposed a concept for a simple process to recover target material produced in cyanobacteria using ILs, which dissolve cyanobacteria but do not dissolve the target material, poly(3-hydroxybutyrate) (PHB), a common biodegradable plastic produced in cyanobacteria. Additionally, we examined parameters of ILs leading to efficient recovery of the target material and discussed the reusability of ILs after PHB recovery. Our data provide insights into novel methods for extraction, purification, and application of materials from cyanobacteria using ILs.
Materials and methods Reagents ILs with different polarities were used to investigate the relationships between solubility of cyanobacteria and polarity of ILs. The commercially available ILs 1-ethyl-3methylimidazolium methylphosphate ([C2mim][MeO(H)PO2]), 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-ethyl-3methylimidazolium bis(trifluoromethanesulfonyl)imide ([C2mim][Tf2N]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([C2mim]BF4), and 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). The other ILs (tetra-n-butylphosphonium methylphosphonate ([TBP][MeO(H)PO 2 ]), tetra-nbutylphosphonium maleate ([TBP][C2H2(COO)2]), tetra-nbutylphosphonium hypophosphorate ([TBP][H2PO2]), tetra-nbutylphosphonium orthosulfuric benzolate ([TBP][Bz-o-SO2]), tetra-n-butylphosphonium benzolate ([TBP]BzCOO), tetra-nbutylphosphonium asparaginate ([TBP]Asp), tetra-n-
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butylphosphonium cysteinate ([TBP]Cys), and tetra-nbutylphosphonium serinate ([TBP]Ser)) were synthesized as previously described (Ando et al. 2013; Kagimoto et al. 2008; Fukaya and Ohno 2013). Tetra-n-butylphosphonium hydroxide solution (TBPH aq.) was purchased from Hokko Chemical Ind. Co., Ltd. (Tokyo, Japan). 1-Ethyl-3-methylimidazolium ethylphosphate ([C 2 mim][EtO(H)PO 2 ]) and 1-ethyl-3butylimidazolium ethylphosphate ([C4mim][EtO(H)PO2]) were synthesized as previously described (Ohno and Fukaya 2009). PHB and other chemicals were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Cyanobacteria cultivation and harvest Synechocystis sp. PCC 6803 (PCC 6803) cells were cultured on BG-11 medium (pH 7.4) for 1 week. PCC 6803 cells that accumulated 5 wt% of PHB were cultured on nitrogen-free BG-11 medium (pH 7.4) for 2 weeks (Panda and Mallick 2007). Cells were cultured with air bubbling. Culture conditions were controlled at 25 °C and photon flux density with 50 mmol m−2 s−1 (cultivation chamber, CLE-303, Tomy Seiko Co., Ltd., Tokyo, Japan). Cells were harvested by centrifugation and lyophilized for 48 h to obtain dry cyanobacteria samples. Wet cyanobacteria samples were harvest by means of centrifuging until the water content was less than 95 wt%. Dissolution of cyanobacteria and PHB in ILs Dried cyanobacteria (1.0 mg) with or without PHB production were added to 1.0 g of ILs having different polarities (Fig. 1), and samples were stirred for 24 h. The solubility of the cyanobacteria was investigated using an optical microscope and observation of the residue after filtration. The solubility was also analyzed by calculation of residues after mixed in IL followed by filtration. The same investigations were also carried out three different conditions below, 10 mg of dried cyanobacteria into 10 g of IL, 1.0 mg of dried cyanobacteria into 1.0 g of IL, and 5 mg of wet sample into 1.0 g of IL. These experiments were carried out several times. The hydrogen-bonding characteristics of ILs have been estimated using solvatochromic probes (Ohno and Fukaya 2009). Kamlet-Taft parameters, hydrogen-bonding acidity (α), hydrogen-bonding basicity (β), and dipolarity/ polarizability (π*) were calculated according to previously reported equations. Three solvatochromic dyes, i.e., 4nitroaniline, N,N-diethyl-4-nitroaniline, and Reichardt’s dye were used to investigate the hydrogen-bonding characteristics described above on cyanobacteria dissolution. The three solubility parameters were calculated from maximum wavelength of UV-vis spectrum of added the solvatochromic dyes. For the analysis of PHB solubility in ILs, purchased PHB was added in the ILs and mixed for 1 h. The solubility was determined by measuring the weight of residue after filtration.
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Fig. 1 Structures of ILs used in this paper
Process of PHB recovery through ILs
Recycling of ILs
In this process, we used cyanobacteria that produced 5 wt% PHB. The PHB production rates were confirmed by highperformance liquid chromatography (HPLC) (Karr et al. 1983). Next, 1.0 mg of the cyanobacteria was added to 1.0 g of ILs, which dissolved the dry cyanobacteria, and samples were then stirred at room temperature. After dissolving cyanobacteria with the polar ILs, the solution was filtered through a PTFE membrane filter (3.0-μm pore size). Residues were collected, washed at least three times with methanol, and applied to HPLC to calculate recovery of PHB after the preparation below. The collected residues were heated in 1 ml H2SO4 at 90 °C for 1 h, then diluted with 1 ml of 0.014 M H 2SO 4. The solution was filtered through polyvinylidene difluoride membrane. The filtrates were then diluted quadruple with 0.014 M H2SO4 and analyzed by HPLC (column: Aminex HPX-87H (300×7.8 mm), flow rate 0.7 ml/min 0.014 M H2SO4, detection: UV absorbance at 214 nm). The standard curves were confirmed to cover the range of the PHB concentration.
After filtering for separation of PHB from the cyanobacteria, a small amount of solvent (i.e., Milli-Q water, methanol, acetone, or ethanol) was added to the polar ILs to precipitate the cyanobacteria component. The precipitated cyanobacteria component was removed by filtration, and the supernatant, i.e., the mixture of IL and solvent, was then evaporated at 40 °C for 6 h to remove the solvent. After vacuum drying, the recovery rate of PHB using recycled ILs was investigated. This process was repeated several times. The structures of the recycled ILs were confirmed by nuclear magnetic resonance (NMR).
Results Dissolution of cyanobacteria producing PHB The productivity of PHB in cyanobacteria was around 5 % of the total dried weight of cyanobacteria. Although we
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previously showed that ILs having high hydrogen-bonding receipt ability (β value) were able to dissolve wet and saliferous cyanobacteria, dried samples were used in this study to fix the amount of cyanobacteria. As a preliminary analysis for the dissolution of dried cyanobacteria, 0.1 wt% of dried cyanobacteria was added to several ILs, and the solubility of the cyanobacteria was investigated by microscope observation and observance of the residue after filtration. The dissolution of the cyanobacteria component was observed in [C2mim][MeO(H)PO2] and TBPH aq. with stirring at room temperature within 30 min. In both ILs, added cyanobacteria were completely dissolved, and no residues were retained after filtration of the solutions. Even when the solubility was investigated with different amounts of cyanobacteria and ILs, similar results were observed. However, the viscosity of the sample became high and was hard to deal with, when over 0.1 wt% of cyanobacteria was added into IL. The increase of viscosity makes difficult to deal with and to evaluate the solubility of cyanobacteria, so further investigations were taken: 1.0 mg of dried cyanobacteria into 1.0 g of ILs or 5.0 mg of wet cyanobacteria into 1.0 g of ILs. In the case of [C2mim]Cl, a similar dissolution of cyanobacteria was observed after 24 h of stirring at 80 °C, although the sample required heating at 80 °C to achieve the liquid state. On the other hand, no dissolution was observed in [C2mim]BF4 after stirring for 24 h, and the sample remained dispersed. The solubility of cyanobacteria in these ILs was similar between cyanobacteria exhibiting PHB production and cyanobacteria that did not produce PHB. Although [C2mim][MeO(H)PO2] and TBPH aq. yielded highly soluble cyanobacteria at room temperature, the viscosities of the two IL solutions differed; the viscosity of TBPH aq. was much higher than that of [C2mim][MeO(H)PO2]. Because the viscosity of the IL solution could easily affect the solubility of the cyanobacteria, we next investigated Fig. 2 Photographs of the dissolution of dried cyanobacteria in [C2mim][Me(H)PO2] and [C2mim]BF4 at room temperature without stirring
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solubility without stirring of the solution. A few drops of each IL were placed on dried cyanobacteria on glass slide, and solubility was monitored under a microscope. In the case of [C 2 mim][MeO(H)PO 2 ], the apparent dissolution of cyanobacteria was observed after 4 h (Fig. 2), and dissolution increased over time. Similar observations were made for cyanobacteria in TBPH aq., indicating that the viscosity did not affect cyanobacteria solubility. These results confirmed that [C2mim][MeO(H)PO2] and TBPH aq. were able to dissolve 0.1 wt% of dried cyanobacteria without stirring or heating. To clarify the properties of ILs that facilitated efficient dissolution of cyanobacteria, we performed subsequent analyses of the hydrogen-bonding receipt and donor values. In a previous study, we had reported that the hydrogen-bonding receipt ability (β value) of IL affected the solubility of cyanobacteria. Interestingly, some reports have demonstrated that both the β values and the α values are important for determining the ability of the IL to dissolve cellulose (Ohno and Fukaya 2009). Indeed, these values have been suggested to be important for breaking the strong interaction between cellulose molecules. A similar mechanism based on the polarity of IL is expected to be involved in the dissolution of the cell wall in cyanobacteria. Therefore, we next investigated the relationships between the solubility of cyanobacteria and the polarity (both α and β values) of ILs (Fig. 3). Though it was revealed in preliminary analysis that the dissolution of cyanobacteria into ILs, e.g., [C2mim][MeO(H)PO2] and TBPH aq., is possible without stirring and heating, the solubility was analyzed with mild stirring at room temperature for the evaluation of relationships with polarity of ILs, to wipe out the effect of viscosity depending on the ILs and change in the viscosity through the progress of the dissolution of cyanobacteria in ILs. The β and α values of [C2mim][MeO(H)PO2], which was associated
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Fig. 3 Relationships between the solubility of cyanobacteria and Kamlet-Taft parameters (hydrogen bonding receipt ability [β value] and hydrogen-bonding donating ability [α value])
with high solubility of cyanobacteria, were 1.00 and 0.52, respectively. However, [TBP]Asp had a higher β value than [C2mim][MeO(H)PO2] and a low α value of 0.1; no dissolution of cyanobacteria was observed with this IL. Additionally, no dissolution was observed in [C2mim]BF4, which had a higher α value than [C2mim][MeO(H)PO2] and a low β value of 0.79. Solubility of PHB As described in the “Introduction,” we sought to achieve simple recovery of materials (PHB) from cyanobacteria by filtration after mixing cyanobacteria in ILs, which dissolves the cyanobacteria component but does not dissolve PHB. To investigate the solubility of PHB in ILs that effectively dissolve cyanobacteria after only a short incubation without heating, TBPH aq. and [C2mim][MeO(H)PO2] were chosen and evaluated. A 10-mg amount of purchased PHB was added to 1.0 g of both IL samples and was stirred for 24 h without heating. After addition of PHB, the TBPH aq.-containing solution became clear, and no residue was observed after stirring for 24 h. In the case of [C2mim][MeO(H)PO2], almost no dissolution of PHB was observed, as suggested by the remaining white turbidity that did not change after addition of PHB. The cloudy [C2mim][MeO(H)PO2] solution was filtered, and the white powder obtained by filtration was freeze-dried after washing with methanol. The total weight of the freeze-dried white powder was consistent with that of the PHB added to [C2mim][MeO(H)PO2]. Recovery of PHB in cyanobacteria As a preliminary examination, wet cyanobacteria (0.5 wt%) that did not produce PHB were dissolved in [C2mim][MeO(H)PO2] and then filtered through a filter with a 3-μm pore size. No residues representing cyanobacteria or components were retained in the filter. However, when commercial PHB was mixed in [C2mim][MeO(H)PO2] and then filtered, all of the added PHB was retained on the filter. These results suggested that our method did indeed provide a simple,
low-energy procedure for recovery of PHB by filtration after dissolution in IL. Next, untreated wet cyanobacteria (0.5 wt%) that produced PHB were mixed, stirred for 24 h at room temperature in [C2mim][MeO(H)PO2], and then filtered. The PHB recovery rate was calculated by using the residue on the filter. PHB was analyzed with HPLC as crotonic acid after treatment with sulfuric acid, which yields crotonic acid after acid decomposition of PHB (Karr et al. 1983). The PHB recovery rate was over 98 %. Most of the PHB produced in cyanobacteria was collected by filtration after dissolution of cyanobacteria in ILs. Although almost all PHB produced in cyanobacteria had been collected, the purity of the residue suggested that the ratio of PHB to other components on the filter was about 30 %. Recycling of [C2mim][MeO(H)PO2] After wet cyanobacteria (0.5 wt%) was stirred in [C2mim][MeO(H)PO2] for 24 h at room temperature, the residue on the filter was collected, and the recovery rate of PHB was calculated. The collected filtrate was examined to determine the recyclability of the IL. As a first step, the components isolated after dissolution of the cyanobacteria in [C2mim][MeO(H)PO2] were segregated by addition of water as a poor solvent. Next, the precipitate was removed by filtration and vacuum-dried for 6 h at 40 °C. After these steps, recycled [C2mim][MeO(H)PO2] was obtained as a colorless liquid. No structural changes were observed by NMR analysis before and after the recycling procedure. When organic solvents, such as methanol, were added to the filtrate, the recycled [C2mim][MeO(H)PO2] was colored with chlorophyll and contained several other components, suggesting that water was suitable as a poor solvent for the recycling of [C2mim][MeO(H)PO2] to achieve relatively good purity of the recycled [C2mim][MeO(H)PO2]. When the solubility of cyanobacteria and the recovery rate of PHB produced in cyanobacteria were investigated using the recycled [C2mim][MeO(H)PO2] sample, the capacity of recycled [C 2 mim][MeO(H)PO 2 ] for cyanobacteria dissolution remained unchanged. Furthermore, the recovery rate of
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[C2mim][MeO(H)PO2] and yield of PHB remained over 98 % using the recovered [C2mim][MeO(H)PO2], even after several cycles (Table 1), further supporting the use of water as a suitable solvent for [C2mim][MeO(H)PO2] recycling. The standard deviation of the PHB recovery rate using [C2mim][MeO(H)PO2] was observed as 0.64.
Discussion The solubility of dried cyanobacteria in ILs was different depending on the particular IL used. To clarify the properties of ILs, both α and β values which indicate the polarity of solvent were measured and the effect on the solubility of cyanobacteria was investigated. By comparing the solubility of cyanobacteria and the polarity of ILs, we found that ILs having α and β values over approximately 0.4 and 0.9, respectively, were appropriate for the dissolution of cyanobacteria (Fig. 3). Therefore, it is possible to improve the dissolution of cyanobacteria by altering the structure of the IL, which controls polarity, as measured through α and β values. To achieve simple recovery of PHB from cyanobacteria by filtration after mixing cyanobacteria in IL, the solubility of PHB was analyzed in polar ILs, TBPH aq., and [C2mim][MeO(H)PO2], which dissolve cyanobacteria after only a short incubation without heating. The results suggested that 0.1 wt% PHB was completely dissolved in TBPH aq. After dissolution of PHB in TBPH aq., no precipitation of PHB was observed when water was added, suggesting that PHB was degraded. Hydrolysis of PHB was predicted in TBPH aq., which is a strongly basic solution. On the other hand, PHB was not dissolved in [C2mim][MeO(H)PO2] after stirring for 24 h, suggesting that [C2mim][MeO(H)PO2] was an appropriate IL for our method (i.e., it dissolved cyanobacteria but not PHB). Therefore, we proposed a procedure to recover PHB accumulated in cyanobacteria simply by filtration after dissolution of cyanobacteria in [C2mim][MeO(H)PO2]. The recovery of PHB in cyanobacteria was evaluated after dissolution of cyanobacteria in [C 2mim][MeO(H)PO2]. HPLC analysis suggested the filtered residue contained components of cyanobacteria other than PHB. When the cyanobacteria that did not produce PHB were dissolve in [C2mim][MeO(H)PO2], no precipitates were observed on the Table 1 IL recovery rates and PHB yields from cyanobacteria after repeated processing
Scheme 1 Process for the recovery of PHB accumulated in cyanobacteria using ILs
filter after filtration as mentioned above. There were two possible reasons for the observed differences in dissolution. First, the cyanobacteria component may be changed during the time that the organism was cultured under nitrogen-free conditions for 2 weeks (which was required to produce PHB). Second, the existing PHB in the cell may have made the cyanobacteria component difficult to dissolve in IL. Further studies are needed to determine the identities of the other components isolated with PHB and to determine the optimal conditions for improving the purity of PHB. For example, when the residue obtained by filtration after mixing cyanobacteria in [C2mim][MeO(H)PO2] was dissolved in chloroform, the purity of PHB dissolved in chloroform was improved to approximately 96 % (data not shown). From the results observed in this study, we proposed a simple process for recovery of PHB produced in cyanobacteria using [C2mim][MeO(H)PO2] as shown in Scheme 1. [C2mim][MeO(H)PO2] proved to be a useful, recyclable solvent for this process, and we expect that this process can be adapted for the isolation of other materials produced in cyanobacteria. We developed a simple process to recover useful material produced in cyanobacteria using ILs. Our analysis of the polarity of ILs and effects of the polarity on solubility of cyanobacteria revealed that polar ILs having high α and β values were able to dissolve cyanobacteria effectively. Especially, we identified [C2mim][MeO(H)PO2] and achieved simple, low-energy recovery of PHB by filtration after dissolution of cyanobacteria. The recovery rate of PHB was over 98 %. The IL was also highly recyclable and could be reused without loss of recovery rates. This method shows great promise and potential applicability for isolation of biomaterials from cyanobacteria.
Recycle number
Recovery of IL (%) Yield of PHB (%)
1
2
3
4
5
99 98
98 98
98 98
98 99
98 98
Acknowledgments This research was supported JST through the CREST program. It was also supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 21225007 and 26810066). K.F. is grateful to the funds from the Women’s Future Development Organization of Tokyo University of Agriculture and Technology. We would like to dedicate this article to celebrate the
Appl Microbiol Biotechnol (2015) 99:1647–1653 20th anniversary of the Department of Biotechnology and Life Science at Tokyo University of Agriculture and Technology.
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BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
A simple recovery process for biodegradable plastics accumulated in cyanobacteria treated with ionic liquids Daigo Kobayashi & Kyoko Fujita & Nobuhumi Nakamura & Hiroyuki Ohno
Received: 24 September 2014 / Revised: 13 November 2014 / Accepted: 13 November 2014 / Published online: 29 November 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Here, we proposed a simple recovery process for poly(3-hydroxybutyrate) (PHB) accumulated in cyanobacteria by using ionic liquids (ILs), which dissolve cyanobacteria but not PHB. First, we investigated the effects of IL polarity on hydrogen-bonding receipt ability (β value) and hydrogen-bonding donating ability (α value) and evaluated the subsequent dissolution of cyanobacteria. We found that ILs having α values higher than approximately 0.4 and β values of approximately 0.9 were suitable for dissolution of cyanobacteria. In particular, 1-ethyl-3-methylimidazolium methylphosphonate ([C2mim][MeO(H)PO2]) was found to dissolve cyanobacteria components, but not PHB. Thus, we verified that PHB produced in cyanobacteria could be separated and recovered by simple filtering after dissolution of cyanobacteria in [C2mim][MeO(H)PO2]. Using this technique, more than 98 % of PHB was obtained on the filter as residues separated from cyanobacteria. Furthermore, [C2mim][MeO(H)PO2] maintained the ability to dissolve cyanobacteria after a simple recycling procedure.
Keywords Ionic liquid . Cyanobacteria . Poly(3-hydroxybutyrate) (PHB) . Polarity . Recovery process
D. Kobayashi : K. Fujita (*) : N. Nakamura : H. Ohno Department of Biotechnology, Tokyo University of Agriculture , 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan e-mail: [email protected] D. Kobayashi : K. Fujita : N. Nakamura : H. Ohno Functional Ionic Liquid Laboratories (FILL), 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan K. Fujita : N. Nakamura : H. Ohno Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
Introduction Cyanobacteria produce various useful materials such as lipids, polysaccharides, and biodegradable plastic, using energy from sunlight (Abed et al. 2009; Capon et al. 1983; Melis and Happe 2001; Stanier et al. 1971). Many researchers have investigated the integration of synthesized operons to develop and improve the production of biomaterials from cyanobacteria (Kaneko et al. 2003; Osanai et al. 2014; Muramatsu et al. 2009; Zou et al. 2009). The production of materials and biodiesel by cyanobacteria has been pursued as an alternative and sustainable energy source for the next generation because food crops are inefficient and unsustainable sources of these materials (Mata et al. 2010; Schenk et al. 2008). In order to use biodegradable plastics, lipids, and other useful substrates produced by cyanobacteria, it is important to develop efficient processes for separation and recovery of these materials. Cyanobacteria have cell walls that contain cellulose and peptidoglycans. Because these components are difficult to dissolve in ordinal solvents, including organic solvents, it is difficult to separate useful materials made of organic substances without specific pretreatments, including demineralization, dehydration, and drying or disruption of cell walls by physical or chemical methods (Jacquel et al. 2008). Furthermore, large amounts of organic solvents and energy are required. Therefore, further studies are necessary to realize the appropriate and simple process to recover materials from cyanobacteria. Recently, ionic liquids (ILs) have attracted attention as a potential solvent for a wide area. ILs, which are molten salts having a melting point below 100 °C, possess unique properties, such as high ionic conductivity and high thermal stability, which are difficult to achieve in general organic solvents (Rogers and Seddon 2003; Wilkes 2002; Welton 1999). Furthermore, ILs have negligible vapor pressure and are therefore
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gathering attention as novel green solvents. Several researches had been reported about the collection of useful materials from cyanobacteria because of the effects of IL properties (Kim et al. 2012; Teixeira 2012), though these processes needed heating during treatment and mixing of other solvents. Additionally, many researchers have realized that the properties of ILs can be controlled by designing the structure to treat biomass; indeed, hydrophobic ILs have been developed for the extraction of materials using a liquid-liquid biphasic system (Tang et al. 2012), and highly polar ILs have been shown to successfully dissolve very insoluble polymer, such as cellulose (Fukaya et al. 2008; Swatloski et al. 2002). In previous studies, we have reported the direct dissolution of wet and saliferous cyanobacteria using polar ILs under mild conditions, e.g., without heating, thereby requiring no pretreatment after collection (Fujita et al. 2013). Thus, our previous work supports the hypothesis of using ILs for the simple process to recover the materials in cyanobacteria. Therefore, novel methods for the collection of useful materials produced in cyanobacteria using ILs may be simple and require low energy input if appropriate ILs can be chosen. In this paper, we proposed a concept for a simple process to recover target material produced in cyanobacteria using ILs, which dissolve cyanobacteria but do not dissolve the target material, poly(3-hydroxybutyrate) (PHB), a common biodegradable plastic produced in cyanobacteria. Additionally, we examined parameters of ILs leading to efficient recovery of the target material and discussed the reusability of ILs after PHB recovery. Our data provide insights into novel methods for extraction, purification, and application of materials from cyanobacteria using ILs.
Materials and methods Reagents ILs with different polarities were used to investigate the relationships between solubility of cyanobacteria and polarity of ILs. The commercially available ILs 1-ethyl-3methylimidazolium methylphosphate ([C2mim][MeO(H)PO2]), 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-ethyl-3methylimidazolium bis(trifluoromethanesulfonyl)imide ([C2mim][Tf2N]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([C2mim]BF4), and 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). The other ILs (tetra-n-butylphosphonium methylphosphonate ([TBP][MeO(H)PO 2 ]), tetra-nbutylphosphonium maleate ([TBP][C2H2(COO)2]), tetra-nbutylphosphonium hypophosphorate ([TBP][H2PO2]), tetra-nbutylphosphonium orthosulfuric benzolate ([TBP][Bz-o-SO2]), tetra-n-butylphosphonium benzolate ([TBP]BzCOO), tetra-nbutylphosphonium asparaginate ([TBP]Asp), tetra-n-
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butylphosphonium cysteinate ([TBP]Cys), and tetra-nbutylphosphonium serinate ([TBP]Ser)) were synthesized as previously described (Ando et al. 2013; Kagimoto et al. 2008; Fukaya and Ohno 2013). Tetra-n-butylphosphonium hydroxide solution (TBPH aq.) was purchased from Hokko Chemical Ind. Co., Ltd. (Tokyo, Japan). 1-Ethyl-3-methylimidazolium ethylphosphate ([C 2 mim][EtO(H)PO 2 ]) and 1-ethyl-3butylimidazolium ethylphosphate ([C4mim][EtO(H)PO2]) were synthesized as previously described (Ohno and Fukaya 2009). PHB and other chemicals were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Cyanobacteria cultivation and harvest Synechocystis sp. PCC 6803 (PCC 6803) cells were cultured on BG-11 medium (pH 7.4) for 1 week. PCC 6803 cells that accumulated 5 wt% of PHB were cultured on nitrogen-free BG-11 medium (pH 7.4) for 2 weeks (Panda and Mallick 2007). Cells were cultured with air bubbling. Culture conditions were controlled at 25 °C and photon flux density with 50 mmol m−2 s−1 (cultivation chamber, CLE-303, Tomy Seiko Co., Ltd., Tokyo, Japan). Cells were harvested by centrifugation and lyophilized for 48 h to obtain dry cyanobacteria samples. Wet cyanobacteria samples were harvest by means of centrifuging until the water content was less than 95 wt%. Dissolution of cyanobacteria and PHB in ILs Dried cyanobacteria (1.0 mg) with or without PHB production were added to 1.0 g of ILs having different polarities (Fig. 1), and samples were stirred for 24 h. The solubility of the cyanobacteria was investigated using an optical microscope and observation of the residue after filtration. The solubility was also analyzed by calculation of residues after mixed in IL followed by filtration. The same investigations were also carried out three different conditions below, 10 mg of dried cyanobacteria into 10 g of IL, 1.0 mg of dried cyanobacteria into 1.0 g of IL, and 5 mg of wet sample into 1.0 g of IL. These experiments were carried out several times. The hydrogen-bonding characteristics of ILs have been estimated using solvatochromic probes (Ohno and Fukaya 2009). Kamlet-Taft parameters, hydrogen-bonding acidity (α), hydrogen-bonding basicity (β), and dipolarity/ polarizability (π*) were calculated according to previously reported equations. Three solvatochromic dyes, i.e., 4nitroaniline, N,N-diethyl-4-nitroaniline, and Reichardt’s dye were used to investigate the hydrogen-bonding characteristics described above on cyanobacteria dissolution. The three solubility parameters were calculated from maximum wavelength of UV-vis spectrum of added the solvatochromic dyes. For the analysis of PHB solubility in ILs, purchased PHB was added in the ILs and mixed for 1 h. The solubility was determined by measuring the weight of residue after filtration.
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Fig. 1 Structures of ILs used in this paper
Process of PHB recovery through ILs
Recycling of ILs
In this process, we used cyanobacteria that produced 5 wt% PHB. The PHB production rates were confirmed by highperformance liquid chromatography (HPLC) (Karr et al. 1983). Next, 1.0 mg of the cyanobacteria was added to 1.0 g of ILs, which dissolved the dry cyanobacteria, and samples were then stirred at room temperature. After dissolving cyanobacteria with the polar ILs, the solution was filtered through a PTFE membrane filter (3.0-μm pore size). Residues were collected, washed at least three times with methanol, and applied to HPLC to calculate recovery of PHB after the preparation below. The collected residues were heated in 1 ml H2SO4 at 90 °C for 1 h, then diluted with 1 ml of 0.014 M H 2SO 4. The solution was filtered through polyvinylidene difluoride membrane. The filtrates were then diluted quadruple with 0.014 M H2SO4 and analyzed by HPLC (column: Aminex HPX-87H (300×7.8 mm), flow rate 0.7 ml/min 0.014 M H2SO4, detection: UV absorbance at 214 nm). The standard curves were confirmed to cover the range of the PHB concentration.
After filtering for separation of PHB from the cyanobacteria, a small amount of solvent (i.e., Milli-Q water, methanol, acetone, or ethanol) was added to the polar ILs to precipitate the cyanobacteria component. The precipitated cyanobacteria component was removed by filtration, and the supernatant, i.e., the mixture of IL and solvent, was then evaporated at 40 °C for 6 h to remove the solvent. After vacuum drying, the recovery rate of PHB using recycled ILs was investigated. This process was repeated several times. The structures of the recycled ILs were confirmed by nuclear magnetic resonance (NMR).
Results Dissolution of cyanobacteria producing PHB The productivity of PHB in cyanobacteria was around 5 % of the total dried weight of cyanobacteria. Although we
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previously showed that ILs having high hydrogen-bonding receipt ability (β value) were able to dissolve wet and saliferous cyanobacteria, dried samples were used in this study to fix the amount of cyanobacteria. As a preliminary analysis for the dissolution of dried cyanobacteria, 0.1 wt% of dried cyanobacteria was added to several ILs, and the solubility of the cyanobacteria was investigated by microscope observation and observance of the residue after filtration. The dissolution of the cyanobacteria component was observed in [C2mim][MeO(H)PO2] and TBPH aq. with stirring at room temperature within 30 min. In both ILs, added cyanobacteria were completely dissolved, and no residues were retained after filtration of the solutions. Even when the solubility was investigated with different amounts of cyanobacteria and ILs, similar results were observed. However, the viscosity of the sample became high and was hard to deal with, when over 0.1 wt% of cyanobacteria was added into IL. The increase of viscosity makes difficult to deal with and to evaluate the solubility of cyanobacteria, so further investigations were taken: 1.0 mg of dried cyanobacteria into 1.0 g of ILs or 5.0 mg of wet cyanobacteria into 1.0 g of ILs. In the case of [C2mim]Cl, a similar dissolution of cyanobacteria was observed after 24 h of stirring at 80 °C, although the sample required heating at 80 °C to achieve the liquid state. On the other hand, no dissolution was observed in [C2mim]BF4 after stirring for 24 h, and the sample remained dispersed. The solubility of cyanobacteria in these ILs was similar between cyanobacteria exhibiting PHB production and cyanobacteria that did not produce PHB. Although [C2mim][MeO(H)PO2] and TBPH aq. yielded highly soluble cyanobacteria at room temperature, the viscosities of the two IL solutions differed; the viscosity of TBPH aq. was much higher than that of [C2mim][MeO(H)PO2]. Because the viscosity of the IL solution could easily affect the solubility of the cyanobacteria, we next investigated Fig. 2 Photographs of the dissolution of dried cyanobacteria in [C2mim][Me(H)PO2] and [C2mim]BF4 at room temperature without stirring
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solubility without stirring of the solution. A few drops of each IL were placed on dried cyanobacteria on glass slide, and solubility was monitored under a microscope. In the case of [C 2 mim][MeO(H)PO 2 ], the apparent dissolution of cyanobacteria was observed after 4 h (Fig. 2), and dissolution increased over time. Similar observations were made for cyanobacteria in TBPH aq., indicating that the viscosity did not affect cyanobacteria solubility. These results confirmed that [C2mim][MeO(H)PO2] and TBPH aq. were able to dissolve 0.1 wt% of dried cyanobacteria without stirring or heating. To clarify the properties of ILs that facilitated efficient dissolution of cyanobacteria, we performed subsequent analyses of the hydrogen-bonding receipt and donor values. In a previous study, we had reported that the hydrogen-bonding receipt ability (β value) of IL affected the solubility of cyanobacteria. Interestingly, some reports have demonstrated that both the β values and the α values are important for determining the ability of the IL to dissolve cellulose (Ohno and Fukaya 2009). Indeed, these values have been suggested to be important for breaking the strong interaction between cellulose molecules. A similar mechanism based on the polarity of IL is expected to be involved in the dissolution of the cell wall in cyanobacteria. Therefore, we next investigated the relationships between the solubility of cyanobacteria and the polarity (both α and β values) of ILs (Fig. 3). Though it was revealed in preliminary analysis that the dissolution of cyanobacteria into ILs, e.g., [C2mim][MeO(H)PO2] and TBPH aq., is possible without stirring and heating, the solubility was analyzed with mild stirring at room temperature for the evaluation of relationships with polarity of ILs, to wipe out the effect of viscosity depending on the ILs and change in the viscosity through the progress of the dissolution of cyanobacteria in ILs. The β and α values of [C2mim][MeO(H)PO2], which was associated
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Fig. 3 Relationships between the solubility of cyanobacteria and Kamlet-Taft parameters (hydrogen bonding receipt ability [β value] and hydrogen-bonding donating ability [α value])
with high solubility of cyanobacteria, were 1.00 and 0.52, respectively. However, [TBP]Asp had a higher β value than [C2mim][MeO(H)PO2] and a low α value of 0.1; no dissolution of cyanobacteria was observed with this IL. Additionally, no dissolution was observed in [C2mim]BF4, which had a higher α value than [C2mim][MeO(H)PO2] and a low β value of 0.79. Solubility of PHB As described in the “Introduction,” we sought to achieve simple recovery of materials (PHB) from cyanobacteria by filtration after mixing cyanobacteria in ILs, which dissolves the cyanobacteria component but does not dissolve PHB. To investigate the solubility of PHB in ILs that effectively dissolve cyanobacteria after only a short incubation without heating, TBPH aq. and [C2mim][MeO(H)PO2] were chosen and evaluated. A 10-mg amount of purchased PHB was added to 1.0 g of both IL samples and was stirred for 24 h without heating. After addition of PHB, the TBPH aq.-containing solution became clear, and no residue was observed after stirring for 24 h. In the case of [C2mim][MeO(H)PO2], almost no dissolution of PHB was observed, as suggested by the remaining white turbidity that did not change after addition of PHB. The cloudy [C2mim][MeO(H)PO2] solution was filtered, and the white powder obtained by filtration was freeze-dried after washing with methanol. The total weight of the freeze-dried white powder was consistent with that of the PHB added to [C2mim][MeO(H)PO2]. Recovery of PHB in cyanobacteria As a preliminary examination, wet cyanobacteria (0.5 wt%) that did not produce PHB were dissolved in [C2mim][MeO(H)PO2] and then filtered through a filter with a 3-μm pore size. No residues representing cyanobacteria or components were retained in the filter. However, when commercial PHB was mixed in [C2mim][MeO(H)PO2] and then filtered, all of the added PHB was retained on the filter. These results suggested that our method did indeed provide a simple,
low-energy procedure for recovery of PHB by filtration after dissolution in IL. Next, untreated wet cyanobacteria (0.5 wt%) that produced PHB were mixed, stirred for 24 h at room temperature in [C2mim][MeO(H)PO2], and then filtered. The PHB recovery rate was calculated by using the residue on the filter. PHB was analyzed with HPLC as crotonic acid after treatment with sulfuric acid, which yields crotonic acid after acid decomposition of PHB (Karr et al. 1983). The PHB recovery rate was over 98 %. Most of the PHB produced in cyanobacteria was collected by filtration after dissolution of cyanobacteria in ILs. Although almost all PHB produced in cyanobacteria had been collected, the purity of the residue suggested that the ratio of PHB to other components on the filter was about 30 %. Recycling of [C2mim][MeO(H)PO2] After wet cyanobacteria (0.5 wt%) was stirred in [C2mim][MeO(H)PO2] for 24 h at room temperature, the residue on the filter was collected, and the recovery rate of PHB was calculated. The collected filtrate was examined to determine the recyclability of the IL. As a first step, the components isolated after dissolution of the cyanobacteria in [C2mim][MeO(H)PO2] were segregated by addition of water as a poor solvent. Next, the precipitate was removed by filtration and vacuum-dried for 6 h at 40 °C. After these steps, recycled [C2mim][MeO(H)PO2] was obtained as a colorless liquid. No structural changes were observed by NMR analysis before and after the recycling procedure. When organic solvents, such as methanol, were added to the filtrate, the recycled [C2mim][MeO(H)PO2] was colored with chlorophyll and contained several other components, suggesting that water was suitable as a poor solvent for the recycling of [C2mim][MeO(H)PO2] to achieve relatively good purity of the recycled [C2mim][MeO(H)PO2]. When the solubility of cyanobacteria and the recovery rate of PHB produced in cyanobacteria were investigated using the recycled [C2mim][MeO(H)PO2] sample, the capacity of recycled [C 2 mim][MeO(H)PO 2 ] for cyanobacteria dissolution remained unchanged. Furthermore, the recovery rate of
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[C2mim][MeO(H)PO2] and yield of PHB remained over 98 % using the recovered [C2mim][MeO(H)PO2], even after several cycles (Table 1), further supporting the use of water as a suitable solvent for [C2mim][MeO(H)PO2] recycling. The standard deviation of the PHB recovery rate using [C2mim][MeO(H)PO2] was observed as 0.64.
Discussion The solubility of dried cyanobacteria in ILs was different depending on the particular IL used. To clarify the properties of ILs, both α and β values which indicate the polarity of solvent were measured and the effect on the solubility of cyanobacteria was investigated. By comparing the solubility of cyanobacteria and the polarity of ILs, we found that ILs having α and β values over approximately 0.4 and 0.9, respectively, were appropriate for the dissolution of cyanobacteria (Fig. 3). Therefore, it is possible to improve the dissolution of cyanobacteria by altering the structure of the IL, which controls polarity, as measured through α and β values. To achieve simple recovery of PHB from cyanobacteria by filtration after mixing cyanobacteria in IL, the solubility of PHB was analyzed in polar ILs, TBPH aq., and [C2mim][MeO(H)PO2], which dissolve cyanobacteria after only a short incubation without heating. The results suggested that 0.1 wt% PHB was completely dissolved in TBPH aq. After dissolution of PHB in TBPH aq., no precipitation of PHB was observed when water was added, suggesting that PHB was degraded. Hydrolysis of PHB was predicted in TBPH aq., which is a strongly basic solution. On the other hand, PHB was not dissolved in [C2mim][MeO(H)PO2] after stirring for 24 h, suggesting that [C2mim][MeO(H)PO2] was an appropriate IL for our method (i.e., it dissolved cyanobacteria but not PHB). Therefore, we proposed a procedure to recover PHB accumulated in cyanobacteria simply by filtration after dissolution of cyanobacteria in [C2mim][MeO(H)PO2]. The recovery of PHB in cyanobacteria was evaluated after dissolution of cyanobacteria in [C 2mim][MeO(H)PO2]. HPLC analysis suggested the filtered residue contained components of cyanobacteria other than PHB. When the cyanobacteria that did not produce PHB were dissolve in [C2mim][MeO(H)PO2], no precipitates were observed on the Table 1 IL recovery rates and PHB yields from cyanobacteria after repeated processing
Scheme 1 Process for the recovery of PHB accumulated in cyanobacteria using ILs
filter after filtration as mentioned above. There were two possible reasons for the observed differences in dissolution. First, the cyanobacteria component may be changed during the time that the organism was cultured under nitrogen-free conditions for 2 weeks (which was required to produce PHB). Second, the existing PHB in the cell may have made the cyanobacteria component difficult to dissolve in IL. Further studies are needed to determine the identities of the other components isolated with PHB and to determine the optimal conditions for improving the purity of PHB. For example, when the residue obtained by filtration after mixing cyanobacteria in [C2mim][MeO(H)PO2] was dissolved in chloroform, the purity of PHB dissolved in chloroform was improved to approximately 96 % (data not shown). From the results observed in this study, we proposed a simple process for recovery of PHB produced in cyanobacteria using [C2mim][MeO(H)PO2] as shown in Scheme 1. [C2mim][MeO(H)PO2] proved to be a useful, recyclable solvent for this process, and we expect that this process can be adapted for the isolation of other materials produced in cyanobacteria. We developed a simple process to recover useful material produced in cyanobacteria using ILs. Our analysis of the polarity of ILs and effects of the polarity on solubility of cyanobacteria revealed that polar ILs having high α and β values were able to dissolve cyanobacteria effectively. Especially, we identified [C2mim][MeO(H)PO2] and achieved simple, low-energy recovery of PHB by filtration after dissolution of cyanobacteria. The recovery rate of PHB was over 98 %. The IL was also highly recyclable and could be reused without loss of recovery rates. This method shows great promise and potential applicability for isolation of biomaterials from cyanobacteria.
Recycle number
Recovery of IL (%) Yield of PHB (%)
1
2
3
4
5
99 98
98 98
98 98
98 99
98 98
Acknowledgments This research was supported JST through the CREST program. It was also supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 21225007 and 26810066). K.F. is grateful to the funds from the Women’s Future Development Organization of Tokyo University of Agriculture and Technology. We would like to dedicate this article to celebrate the
Appl Microbiol Biotechnol (2015) 99:1647–1653 20th anniversary of the Department of Biotechnology and Life Science at Tokyo University of Agriculture and Technology.
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