Article pubs.acs.org/est
Profiling Planktonic Biomass Using Element-Specific, Multicomponent Nuclear Magnetic Resonance Spectroscopy Takanori Komatsu,†,‡ Toshiya Kobayashi,‡ Minoru Hatanaka,§ and Jun Kikuchi*,†,‡,∥ †
RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan § Bruker Biospin K. K., 3-9, Moriya-cho, Kanagawa-ku, Yokohama, 221-0022, Japan ∥ Graduate School of Bioagricultural Sciences and School of Agricultural Sciences, Nagoya University, 1 Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan ‡
S Supporting Information *
ABSTRACT: Planktonic metabolism plays crucial roles in Earth’s elemental cycles. Chemical speciation as well as elemental stoichiometry is important for advancing our understanding of planktonic roles in biogeochemical cycles. In this study, a multicomponent solid-state nuclear magnetic resonance (NMR) approach is proposed for chemical speciation of cellular components, using several advanced NMR techniques. Measurements by ssNMR were performed on 13C and 15N-labeled Euglena gracilis, a flagellated protist. 3D dipolar-assisted rotational resonance, double-cross-polarization 1 H−13C correlation spectroscopy, and 1H−13C solid-state heteronuclear single quantum correlation spectroscopy successively allowed characterization of cellular components. These techniques were then applied to E. gracilis cultured in high and low ammonium media to demonstrate the power of this method for profiling and comparing cellular components. Cellular NMR spectra indicated that ammonium induced both paramylon degradation and amination. Arginine was stored as a nitrogen reserve and ammonium replaced by arginine catabolism via the arginine dihydrolase pathway. 15N and 31P cellular ssNMR indicated arginine and polyphosphate accumulation in E. gracilis, respectively. This chemical speciation technique will contribute to environmental research by providing detailed information on environmental chemical properties.
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tures.15,16 Chemical shifts provide chemical speciation and component identification as well as the advantage that NMR spectra can be recorded from solutions, solids, and gases state samples.17 Using high-resolution magic-angle-spinning (HRMAS), cellular metabolites can be analyzed without separation and purification.18−20 Complex mixtures of insoluble molecules can also be analyzed by high-resolution solid-state NMR (ssNMR), in which chemical shift anisotropy and dipole−dipole interactions between 1H and 13C are averaged using magic-anglespinning (MAS) and high-power decoupling, respectively. Also, ssNMR is useful for analyzing dynamics and interactions in complex mixtures of molecules.21,22 A multiphase NMR instrument has been developed that records both HR-MAS and ssNMR spectra.23 Resonance assignments are one of the most challenging issues in ssNMR of complex mixtures of molecules, such as cells and
INTRODUCTION Planktonic metabolism plays important roles in element cycles, such as the cycling of carbon, nitrogen, phosphorus, silicon, and sulfur. The Redfield ratio is a widely accepted empirical rule of elemental ratios (H/C/P = 106/16/1) in marine plankton. Sterner et al. have indicated that the C/N/P ratio varies at different spatial scales.1 N/P ratios in ocean seawater are relatively constant (∼16/1) but varies in freshwater lakes.2 C/N and C/P ratios in oceanic dissolved organic matter are often higher than in the Redfield ratio.3 Aquatic stoichiometry has been actively studied in the field of geosciences, but the mechanism by which the H/C/P elemental ratio is 106/16/1, or varies from this, remains unclear. As elemental ratios are closely related to organismal metabolism, chemical speciation analyses can provide detailed information regarding the underlying mechanism of elemental ratios and their variations. However, the utility of chemical speciation analyses in examining cellular components, compared with stoichiometric analyses, are not well established. NMR has been valuable in studies of environmental samples, including aquatic systems,4−14 and in the field of metabolomics, as it provides much information regarding molecular struc© 2015 American Chemical Society
Received: Revised: Accepted: Published: 7056
February 18, 2015 April 23, 2015 April 30, 2015 May 14, 2015 DOI: 10.1021/acs.est.5b00837 Environ. Sci. Technol. 2015, 49, 7056−7062
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Environmental Science & Technology
constant in NMR) were recorded using 90 deg pulse under 12kHz MAS to obtain quantitative profiles of cellular carbons. 31P DP-MAS spectra with 5 s tr were recorded using 60-degree pulse (T1 estimated at ∼7.2 s). Therefore, the 60-degree pulse was the Ernst angle when tr/T1 was 0.693, under 40-kHz MAS, for obtaining quantitative cellular phosphate profiles. 15N crosspolarization (CP) -MAS spectra with 1 ms contact time were also recorded to obtain cellular nitrogen profiles (15N DP-MAS was not used because sensitivity was far less than in 15N CP-MAS). Small phase incremental alteration (SPINAL) decoupling38 (∼70 kHz) was employed during acquisition time, with 1H, 13C, 31 P, and 15N chemical shifts calibrated using adamantane (δ1H, 1.91 ppm), carbonyl carbon in glycine (δ1H, 176.46 ppm), ammonium phosphate dibasic (δ31P, 1.33 ppm), and glycine (δ15N, 33.4 ppm) as references.39 Multidimensional spectroscopy, including J- incredible natural abundance double quantum transfer experiment40 (INADEQUATE), second-order Hamiltonian among analogous nuclei plus (SHA+),41 double CP 1 H−13C correlation spectroscopy (double-CP),42,43 1H−13C solid-state heteronuclear single quantum correlation spectroscopy (ssHSQC),44 and 3D dipolar-assisted rotational resonance (DARR)45 spectra were also recorded for signal assignments. The parameters used in multidimensional NMR experiments are described in detail in the Supporting Information. HR-MAS for Cellular Metabolic Analysis. HR-MAS was employed to analyze cellular metabolism without extraction and separation. Spectra were recorded using an Avance III HD-500 instrument equipped with a triple-resonance 4.0 mm HR-MAS probe. Lyophilized cells (5 mg) were suspended in 55 mL of deuterated water containing 100 mM potassium phosphate buffer (pH 7.0) and then transferred into a HR-MAS rotor (55 μL vol). 13C NMR spectra with a 15 s recycling delay were recorded under 5 kHz MAS. Waltz decoupling (2.5 kHz) was employed during acquisition time. 1H−13C-HSQC46 and 1 H−13C HSQC-total correlation spectroscopy47 spectra were also recorded for signal assignments.
environmental samples, because their numerous component nuclei cause severe spectral resonance overlaps. Various spectral editing techniques have been introduced for resonance assignment in environmental NMR studies.24−27 Multivariate analysis techniques have also been applied to NMR spectra to resolve and assign resonances.4,8,28 Chemoselective isotopic tags have been introduced to improve resonance assignment capability of small molecules in metabolomics studies.29 In protein NMR, uniform labeling with stable isotopes and multidimensional NMR allow resonances to be assigned.30 Stable isotopic labeling has also contributed to NMR analyses of complex mixtures in biomass examined in environmental studies.27,31,32 Stable isotopic labeling is a powerful technique for confirming metabolic processes in laboratory-scale experiments, using collected samples from natural sources.17,33−35 Finally, recent technical developments in ssNMR have also contributed to resonance assignments, for example, in fast MAS techniques that have made it possible to use 1H NMR in ssNMR without requiring homonuclear decoupling.36,37 Here, the practical aspects of ssNMR for complex mixtures of molecules, referred to as “multicomponent NMR”, will be described. In the current study, Euglena gracilis, a flagellated freshwater protist, was selected as a test case because this organism shows promise for use as a biosource for producing nutrients and oils. Advanced multidimensional ssNMR, including three-dimensional 13C correlation spectroscopy and fast MAS techniques, were introduced to resolve and assign resonances. Then, ammonium-induced cellular component replacements were analyzed as an application of multicomponent ssNMR, and HR-MAS and multinuclear ssNMR employed to support this method. These spectra serve well for explaining metabolism in ammonium-induced cellular component replacements and, in addition, demonstrate that multicomponent ssNMR is a powerful analytical method for cellular as well as environmental biochemical studies.
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EXPERIMENTAL SECTION Cell Culture and Sample Preparation. E. gracilis cells were provided by Euglena Co., Ltd. (Tokyo, Japan) and cultivated in modified Cramer-Myers medium, containing 10 g/L uniformly-13C-labeled D-glucose (99% 13C, Sigma-Aldrich, Inc., St. Louis, MO), 0.4 or 1.7 g/L 15N-labeled ammonium chloride (>99% 15N, Cambridge Isotope Laboratories, Inc., Tewksbury, MA), 1 g/L KH2PO4, 0.2 g/L MgSO4·7H2O, 0.02 g/L CaCl2, 0.8 g/L sodium citrate, 3 mg/L Fe2(SO4)3·H2O, 1.8 mg/L MnCl2· 4H2O, 1.5 mg/L CoSO4·7H2O, 0.4 mg/L ZnSO4·7H2O, 0.2 mg/ L Na2MoO4·2H2O, 0.02 mg/L CuSO4·5H2O, 2.48 mg/L H3BO3, 0.5 mg/L thiamine HCl, and 0.02 mg/L cyanocobalamin, at a temperature of 300 K under artificial lighting with 12 h/ 12 h light/dark cycles. Continuously cultivated cells were inoculated into media and cultured for 3, 5, or 7 days. Then, the cultures were centrifuged at 3000 rpm for 5 min and the cells were collected and washed twice with distilled water, lyophilized, and submitted to NMR analyses. Solid-State NMR for Cellular Component Profiling. The ssNMR spectra were recorded using an Avance III HD-500 instrument equipped with a double-resonance 1.3 and 4.0 mm MAS probe and an Avance III-HD 400 WB instrument equipped with double-resonance 1.3 and 2.5 mm MAS probes (Bruker Corp., Billerica, MA). Lyophilized cells were directly packed into ssNMR rotors and spectra recorded over 12−60 kHz of magicangle-spinning. 13C direct-polarization (DP) -MAS spectra with 60 s recycling delay (tr, > 5T1, T1, longitudinal relaxation time
RESULTS AND DISCUSSION The 13C DP-MAS spectra (Figure 1a) allowed total carbon profiling of E. gracilis. CP-MAS and multiple-CP48 spectra were also recorded, whereas DP-MAS spectra provided better quantitative profiles of cellular carbons (Supporting Information Figure S1). Analyses described below revealed that the predominant resonances in the spectra were lipids, proteins, and paramylon, the last being a crystallized and triple-helical ß1,3-glucan found uniquely in Euglenoids.49 This was quite different from 13C NMR spectra from HR-MAS, in which there were only resonances from mobile molecules (Supporting Information Figure S3). Because high power SPINAL decoupling eliminated 1H−13C dipolar−dipolar interactions, 13C DPMAS spectra became a detector of cellular macromolecular 13C. Multiple-CP spectra did not provide quantitative spectra because 1 H longitudinal relaxation times in paramylon were far larger than those in lipid (Supporting Information Figure S2). Signal Assignments in Cellular Biomacromolecules Using 13C−13C Correlation ssNMR Spectra. First, the total carbon profile from 13C DP-MAS was interpreted by tackling resonance assignments in ssNMR spectra, using 13C−13C and 1 H−13C correlation multidimensional ssNMR spectra. Signal assignments in a J-INADEQUATE spectrum are shown in Figure 1(b). 13C resonance of D-glucopyranosyl residues in paramylon and lipid fatty acids were assigned according to their chemical 7057
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Figure 2. Signal assignments in 3D-DARR spectra for macromolecules in E. gracilis cultured in high ammonium medium (31 mM). (a) Cubic view of 3D-DARR spectrum of E. gracilis cells. (b) Three selected F1− F3 slices shown as overlays. Signals in blue, red, and green (174.0, 128.9, and 80.4 ppm, respectively) slices predominantly from proteins, lipids, and paramylon, respectively. Three selected F1−F3 slices of (a) at δ13C, (c−e) 85.5, 128.2, and 158.2 ppm (F2). In slice (c), correlations between C3 and other carbons in paramylon glucopyranosyl residue, with four, magnetically different, glucopyranosyl residues in spectrum. (d) Signal assignments of phenylalanine and tyrosine residues in proteins and unsaturated lipid fatty acids. (e) Signal assignments of protein arginine residues. 13C resonance frequency, MAS frequency, and rotor diameter, 100 MHz, 15 kHz, and 2.5 mm, respectively.
Figure 1. Multicomponent NMR spectra of E. gracilis cultured in high ammonium medium (31 mM). (a) Quantitative DP-MAS spectrum of E. gracilis. Spectra allowed total carbon profiling of E. gracilis. (b) Signals assigned in J-INADEQUATE spectra according to chemical structure. Blue, red, and green assignment labels, proteins, lipids, and paramylon, respectively.
chemical shifts for each glucopyranosyl residue, whereas magnetically nonequivalent, glucoside residues in dehydrated paramylon have been previously reported.50,51 As shown here, 3D-DARR spectra provided structural information on polysaccharides as well as proteins and lipids (Figures 2d and 2e) in the cellular multicomponent environment. Signal Assignment in Cellular Biomacromolecules Using 1H−13C Correlation ssNMR Spectra under Fast MAS. Profiling of cellular components was attempted using 1H NMR spectroscopy under fast MAS. 1H 1D-single-pulse spectra at MAS frequency of 20−60 kHz are shown in Supporting Information Figure S5a. Sharp doublet signals were considered to indicate the presence of mobile lipid fatty acid chains and signal splitting assumed to have been caused by 1JCH. Broad signals were considered to indicate the presence of more rigid molecules, including proteins and paramylon. Fast MAS at 60 kHz was used to make broad signals sharper than when using conventional MAS at 20 kHz (Supporting Information Figure S5b) because fast MAS partly averaged out 1H−1H dipole− dipole interactions. However, these signals remained too ambiguous to be identified. Notably, resolution in 1H NMR spectra would be improved when spectra are recorded with a more high-field magnet under faster MAS conditions.52 Analysis of E. gracilis cells was attempted using 1H-detected 2D 1 H−13C correlation spectroscopy, with two 1H−13C inversed pulse sequences. One of these was ssHSQC (Figure 3a), in which insensitive nuclei enhanced by polarization transfer (INEPT),
structures (Table S1). Amino acid residues in cellular proteins were difficult to assign because of the numerous cellular proteins. The present proposed strategy for assigning signals in a cellular ssNMR spectrum involved using a 3D-DARR technique. The cubic view and three selected F1−F3 slices (overlaid) from a 3DDARR spectrum are shown in Figures 2a and 2b, respectively. The blue slice shown in Figure 2b, in which the predominant correlations were for protein carbon atoms, was the F1−F3 slice at 174.0 ppm (F2). The red slice, in which the predominant correlations were between alkene and alkane lipid carbon atoms, was the F1−F3 slice at 128.9 ppm (F2). The green slice, in which the predominant correlations were from paramylon carbon atoms, was the F1−F3 slice at 80.4 ppm (F2). Compared with 2D 13C−13C correlation spectra (Figure S4), compounds were separated using their δ13C values in the F2 (or F1 or F3) dimension in the 3D-DARR spectrum, in contrast with separation by retention time, as is used in various types of chromatography. Detailed resonance assignments were performed in 3D-DARR spectra. Interestingly, in the slice of δ13C (F2) at 85.5 ppm, there were more than four, magnetically nonequivalent, glucopyranosyl residues (Figure 2(c)). 13C chemical shifts assigned in 3DDARR as well as in J-INADEQUATE spectra are listed in Table S1. To our knowledge, this is the first report that assigns 13C 7058
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Figure 3. 1H-detected solid-state NMR spectra of E. gracilis cultured in high ammonium medium (31 mM). In ssHSQC spectroscopy, a pulse sequence (a) and spectra (c). In double-CP, a pulse sequence (b) and spectra (d). In pulse sequence diagrams, sharper and broader filled boxes represent 90° and 180° hard pulses, respectively. The phase cycling in (a) ϕ1 = (+y, −y), ϕ2 = (+y, +y, −y, −y), ϕ3 = (+x)8(−x)8, ϕ4 = (+x)4(−x)4, and ϕrec = (+y, −y, −y, +y, −y, +y, +y, −y, −y, +y, +y, −y, +y, −y, −y, +y). The phase cycling in (b) ϕ1 = (+y, −y), ϕ2 = (+x), ϕ3 = (+x, +x, +y, +y, +y, +y, +x, +x), ϕ4 = (+x)8(−x)8, ϕ5 = (+y)8(−y)8, ϕ6 = (+x), ϕ7 = (−y)8(+y)8, and ϕrec = (+y, −y, −y, +y, −y, +y, +y, −y, −y, +y, +y, −y, +y, −y, −y, +y). 13C resonance frequency, MAS frequency, and rotor diameter were 100 MHz, 60 kHz, and 1.3 mm, respectively.
Figure 4. Multicomponent NMR to profile total biomass in E. gracilis cultured in media containing different ammonium concentrations (6 or 31 mM). 13 C DP-MAS spectrum of E. gracilis cultured in low (a) and high ammonium (b) for 7 days. 13C HR-MAS spectrum of E. gracilis cultured in low (c) and high ammonium (d) for 3 days. Asterisks, resonance from tetrafluoroethylene used as spacer in HR-MAS rotor. 15N CP-MAS spectrum of E. gracilis cultured in low (e) and high (f) ammonium for 3 days; (e) and (f) recorded at same receiver gain. Large amounts of paramylon observed in E. gracilis were grown in low ammonium, but large amounts of proteins observed in E. gracilis were grown in high ammonium.
particularly in proteins and lipids, in which spectral resonances were considerably overlapped. Acquiring detectable signals in 1 H-detected 2D 1H−13C correlation experiments depended on molecular motion. The dipole−dipole interaction-based heteronuclear magnetization transfer technique, including CP in double-CP, was less effective at detecting mobile molecules, such as lipids, because mobile molecular motion averaged out the effects of dipole−dipole interactions to some extent in a spectrum. However, the spin−spin-coupling-based heteronuclear magnetization transfer technique, including INEPT in
including spin−spin-coupling-based heteronuclear magnetization transfer, was used to cause heteronuclear magnetization transfer.44 The other pulse sequence was double-CP, with CP used to cause heteronuclear magnetization transfer (Figure 3b).42,43 In an ssHSQC spectrum, lipid signals were detected, but protein and paramylon signals were barely detectable (Figure 3c). However, in a double-CP spectrum, broad paramylon and protein signals were indeed observed (Figure 3d). The differences of detectable molecules in these two 2D 1 H−13C correlation spectra assisted in resonance assignments, 7059
DOI: 10.1021/acs.est.5b00837 Environ. Sci. Technol. 2015, 49, 7056−7062
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gracilis cultured in high ammonium medium, is shown in Figure 5. Incorporated ammonium was conjectured to be anabolized by
ssHSQC, was ineffective at detecting rigid molecules, such as paramylon, because the transverse magnetization coherence lifetime was far shorter than the heteronuclear magnetization transfer period. From this, the dipole−dipole interactions were not averaged out, compared with mobile molecules. Holland et al. have stated that signals from rigid molecules can be detected in ssHSQC spectra recorded using fast MAS (60 kHz) when delays caused by 1JCH (τ) are optimized, such that they are shorter than 1/41JCH and carefully synchronized with the rotor period (delays were also synchronized with the rotor period in this study).44 However, here, proteins and paramylon were barely detected in ssHSQC analysis of E. gracilis cells. Several modifications will be required to use the inverse INEPT technique to analyze rigid molecules. For example, rigid molecules can be detected when homonuclear decoupling is employed over heteronuclear through-bond magnetization transfer periods. This occurs in the MAS-J-HMQC53 and MAS-J-HSQC54 techniques as well as the previously reported 1H-detected through-bond heteronuclear correlation spectroscopy.55 Application: Profiling Cellular Components in E. gracilis Cultured in High and Low Ammonium Media. The multicomponent NMR approach was applied to E. gracilis cultured in high and low ammonium media to demonstrate its power as a method for profiling biomass. 13C DP-MAS and 15N CP-MAS spectra of E. gracilis harvested at 7 and 3 days, respectively, after inoculation are shown in Figures 4a, 4b, 4e, and 4f (See also Supporting Information Figures S7 and S8 and Tables S1 and S2, showing 13C DP-MAS and 15N CP-MAS spectra of E. gracilis harvested at 3, 5, and 7 days after inoculation and their annotations). 13C HR-MAS spectra were also recorded to evaluate E. gracilis metabolism in high and low ammonium (Figures 4c and 4d). In 13C DP-MAS spectra, protein resonances were greater in E. gracilis cultured in high ammonium medium than in low ammonium. In 15N CP-MAS spectra, total intensity of these spectra was greater in E. gracilis cultured in high than in low ammonium (recorded at same spectrometer receiver digital gain). These results indicated that protein amounts in E. gracilis cultured in high ammonium were larger than in low. Ammonium-induced paramylon degradation has been reported by Sumida et al.,56 showing that concentrations of enzymes involved in degrading paramylon, such as β-1,3-glucanphosphorylase and laminarinase, increase when a culture is fed ammonia.56 The increased amino acid pool in the current work indicated that high ammonium concentrations activated amination and transamination, leading to high amounts of amino acids and proteins. In 13C HR-MAS spectra, resonances from amino acids, especially ornithine, were greater in E. gracilis cultured in high ammonium medium than in low. 1H−13C HSQC spectra with assignments and a chemical shift list of assigned metabolites are shown in Supporting Information Figure S10 and Table S4, respectively. E. gracilis possesses an arginine dihydrolase pathway, in which arginine is catabolized to ornithine via citrulline, by successive action of arginine deiminase, ornithine carbamoyl transferase, and carbamate kinase, with concurrent ammonia release.57 Therefore, abundant ornithine in E. gracilis cultured in high ammonium indicated strong activity in the arginine dihydrolase pathway. 15N CP-MAS spectra explained the abundance of guanidium nitrogens of cellular arginine.58 Free or peptide forms of arginine have also been reported to accumulate in E. gracilis as a major cellular nitrogen reserve.57 A schematic summary of the proposed mechanism, in which paramylon was degraded and cellular proteins accumulated in E.
Figure 5. Schematic summary of estimated paramylon degradation mechanism and cellular protein accumulation in E. gracilis cultured in high ammonium medium. Incorporated ammonium immediately involved in amination with constitutive carbons provided by paramylon degradation. Arginine synthesized and stored as a major nitrogen reserve. Ammonium also replaced by arginine degradation via arginine dihydrolase pathway and, finally, cellular proteins synthesized.
the glutamate dehydrogenase (GDH) pathway or the glutamate dehydrogenase-glutamine 2-oxoglutarate amidotransferase (GS/ GOGAT) cycle. NADH-dependent GOGAT has been observed in E. gracilis.59 Some studies have pointed out that the primary role of the GDH pathway is glutamate anabolism.60 The presence of these pathways was not proven by the present results. Arginine might have been generated from glutamate, if the proposed pathways were active, and accumulated as a major nitrogen reserve. Ammonium was released from arginine catabolism via the arginine dihydrolase pathway. 31 P DP-MAS for Phosphate Chemical Speciation. Phosphate is one of the limiting growth factors, along with carbon and nitrogen, for microalgae in aqueous environments. Therefore, profiling techniques for cellular or environmental phosphate are important. Various absorption photometry and inductively coupled plasma-optical emission spectrometry (ICPOES, or mass spectrometry, ICP-MS) have been used to quantify phosphates. However, these analyses did not allow chemical speciation evaluation. NMR is one of the most prospective chemical speciation methods for environmental samples.61 31P DP-MAS spectra of E. gracilis are shown in Supporting Information Figure S9. Interestingly, the resulting spectra showed that E. gracilis accumulated polyphosphate molecules (δ31P = −18 − −26 ppm). Organisms that accumulate polyphosphate have been successfully employed to remove phosphate from wastewater, and E. gracilis is expected to be useful in treating some wastewaters.62 From the present results, cellular 31P ssNMR could be used to contribute in determination of cellular polyphosphate accumulation. Takeda et al. have reported nondestructive, quantitative, elemental analysis using magnetic field-variable NMR.63 Also, an approach to improve magnetic field homogeneity has been reported to develop high-resolution magnetic field-variable NMR, which employs nondestructive quantitative elemental analysis and chemical speciation simultaneously.64 Such elemental analysis with chemical speciation would contribute to advancing our understanding of biogeochemical cycles. 7060
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ASSOCIATED CONTENT
S Supporting Information *
Supporting Information is available and includes several 13C-1D ssNMR spectra, relaxation experiments, MAS frequency dependence in the 1H NMR spectra, all 13C DP-MAS, 15N CP-MAS, and 13 C HR-MAS spectra, signal assignments of metabolites in HRMAS HSQC spectra, and 31P NMR spectra of cells. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b00837.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-45-503-9490; fax: +81-45-503-9489; e-mail: jun.
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Euglena Co., Ltd. for providing E. gracilis cells. The authors also thank Yuri Tsuboi (RIKEN CSRS) for her technical assistance during this study. This research was supported in part by Grants-in-Aid for Scientific Research (Grant No. 25513012, to J.K.), and also partially supported by the Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry and fisheries” funded from Bio-oriented Technology Research Advancement Institution (NARO). The RIKEN Junior Research Associate Program also supported this study.
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DOI: 10.1021/acs.est.5b00837 Environ. Sci. Technol. 2015, 49, 7056−7062
