Simultaneous quantification of cellular lipids and carotenoids inside Chlorella vulgaris using Raman spectrometry

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1 Available online at ScienceDirect Energy Procedia 61 (2014 ) The 6 th International Conference on Applied Energy ICAE2014 Simultaneous quantification of cellular lipids and carotenoids inside Chlorella vulgaris using Raman spectrometry Tsung-Hua Lee a and Hsiang-Yu Wang a-c, * a Department of Chemical Engineering, National Cheng Kung University, No. 1, University Road, Tainan and 701, Taiwan b Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan c University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan Abstract A rapid quantification method for lipids and carotenoids in Chlorella vulgaris is demonstrated in this study. The rapid quantification translates Raman signal intensities into abundances of lipids and carotenoids via calibration curves established by gravimetric method (lipids) and UV spectrometry (carotenoids). The Raman signal intensities and the measured amounts of lipids and carotenoids are highly correlated with R 2 higher than The quantification method is further applied to monitor the actual process of microalgae cultivation. When monitoring the culture of Chlorella vulgaris, we found that the ratio of signals from carotenoids and lipids can change as much as 75% in 1 day when nutrient depletion was induced. Since the signal of total lipids remains relatively stable after the carbon source is depleted, degradation of carotenoids is responsible for the shifting of signal ratio. These results demonstrate the possibility of using Raman spectrometry for rapid quantification of multiple cellular contents and the obtained information is highly valuable for improving cultivation strategies and screening of cells producing valuable products The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( The Authors. Published Elsevier Ltd. Selection Peer-review and/or under responsibility peer-review of under the Organizing responsibility Committee of ICAE of ICAE2014 Keywords: Raman spectrometry, microalgae, lipid quantification, carotenoids quantification, Chlorella vulgaris 1. Introduction Microalgae are considered favorable sources for producing biofuels not only because of their high lipid abundances but also their potential of producing value-added products [1]. The simultaneous production of multiple products is beneficial in improving the economic viability of microalgae biofuel. Main * Corresponding author. Tel.: ext address: hywang@mail.ncku.edu.tw The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( Peer-review under responsibility of the Organizing Committee of ICAE2014 doi: /j.egypro

2 830 Tsung-Hua Lee and Hsiang-Yu Wang / Energy Procedia 61 ( 2014 ) compositions of microalgae include lipids, pigments, proteins and polysaccharides. Among these compositions, highly unsaturated lipids and pigments, such as carotenoids, have a relatively high selling price and a large market size [2]. However, conventional quantification methods for microalgae cellular contents are generally time-consuming, process-demanding and single-targeted [3]. These disadvantages result in tedious screening of microalgae strains producing desired products and unlikely real-time monitor of culture status. Raman spectrometer is an attractive method for the detection of microalgae cellular contents and has been applied to the estimation of iodine value of lipids, amount of betacarotenes, and amount of cellular lipids [4, 5]. The advantages of Raman spectrometry include simple sample pretreatment and less interference from water [6] and these characteristics make the instrument ideal for rapid analysis of microalgae. Nonetheless, simultaneous quantification of multiple microalgae cellular contents has not been demonstrated. In our previously published paper [4], a rapid quantification platform for cellular lipids in Chlorella vulgaris has been demonstrated. This study extends the application of our platform to achieve simultaneous detection of lipids and carotenoids, aiming to expedite the commercialization of microalgae biofuel. 2. Materials and methods 2.1. Chlorella vulgaris culture The microalgae Chlorella vulgaris ESP-31 was cultivated with conditions and nitrogen depletion strategy reported in Yeh [7]. The depletion of carbon source was achieved by discontinuing the aeration Raman signal measurement Microalgae were sampled to the optical density value equal to 4 for 1 ml. After removing suspension and washing by D.I. water at rpm 5 min, 5 l microalgae were sampled from 200 l residual suspension and drop to gold coated slide. Raman spectrum was obtained from microalgae paste according to our previously published paper [4]. The area under the peak at 1157 cm -1 and under the wide peak between cm -1 was calculated and regarded as signal intensity of carotenoids and lipids respectively Lipid and carotenoid quantification by conventional methods Microalgae lipid abundances were determined by conventional gravimetric method [7]. Microalgae lipid was extracted by chloroform/methanol solution (1:2 v/v) at 200 rpm for 30 min, then add chloroform/water (1:2 v/v) at 200 rpm for 30 min. After bottom phase were collected by centrifugation at 1000 rpm for 10 min, the solvent free remnant were weighted as the lipid abundance. Microalgae carotenoids were extracted by DMSO according to a previous report [8]. The microalgae suspension was washed by deionized water twice, and the residual was then extracted by 2ml of DMSO at 60 for 10 min. The solution was centrifuged by rpm for 3 min to remove cell debris. The extract layer was then analyzed by UV spectrometry for the determination of carotenoids abundance [8]. 3. Results and discussion Carotenoids in Chlorella vulgaris produced 3 main peaks at 1009 cm -1, 1157 cm -1 and 1524 cm -1 and lipids produced one peak at 1440cm -1 and a broad peak between 2845 cm -1 and 3107 cm -1 (Chlorella vulgaris spectrum can be found in Lee [4]). However, Raman shifts at 1009 cm -1 and 1524 cm -1 are

3 Tsung-Hua Lee and Hsiang-Yu Wang / Energy Procedia 61 ( 2014 ) shadowed by signals from chlorophyll and they are unsuitable for the quantification [9]. Therefore, signal at 1157 cm -1 was chosen as the marker for quantification of carotenoids. The signal intensity under the wide peak between cm -1 was used as the marker for lipid quantification [4]. The calibration curve between Raman signal and carotenoids abundance (measured by UV spectrometry) is shown in Fig.1(a). The correlation coefficient was high (R 2 =0.96) in the range from 0.93 to 3.13 mg carotenoids/g dry biomass. Fig 1(b) shows the calibration curve for Raman signal intensity and lipid amount measured by gravimetric method. Similarly to the calibration curve of carotenoids, it is also highly linear. Figure 1(a) and (b) were further applied to the real-time monitoring of Chlorella vulgaris culture. Chlorella vulgaris were first cultivated under nitrogen starvation period to enhance the accumulation of lipids and then carbon depletion was induced to observe the change of cellular contents (Fig 2). The ratio of signals from carotenoids and lipids at the 61 st day of cultivation was around 0.002; however, this ratio decreased to in the next day and remained constant for three days. Since the signal of total lipids remains relatively stable during this period, degradation of carotenoids is responsible for the shifting of cellular compositions. The timing for harvest can be decided according to different purposes. Microalgae can be harvested at the highest signal ratio for simultaneous production of two valuable metabolic products or at the lowest signal ratio to reduce the cost for removing pigments from biodiesel. Raman signal intensity (a.u.) R 2 =0.96 Slope=68213 Intercept= Raman signal intensity (a.u.) R 2 =0.94 Slope=16607 Intercept= Total carotenoids (mg/g) Total Lipids (mg/g) (a) (b) Fig. 1. (a) Calibration curve of Raman signal (1157 cm -1 ) v.s. carotenoid amount measured by UV spectrometry. (b) Calibration curve of Raman signal ( cm -1 ) v.s. lipid amount measured by gravimetric method. Abundance (mg/g) Lipid Carotenoids Carotenoids/Lipids Ratio (Carotenoids/Lipids) Cultivation period (days) 0.000

4 832 Tsung-Hua Lee and Hsiang-Yu Wang / Energy Procedia 61 ( 2014 ) Fig. 2. Raman signal intensity of lipids and carotenoids along long period of starvation. 4. Conclusions This study demonstrates a rapid and noninvasive quantification technique for multiple microalgae cellular contents. The Raman spectrometry platform is validated with gravimetric method (lipid) and UV spectrometry (carotenoids). The obtained information from the rapid quantification is highly valuable for expediting the improvement of cultivation strategies and screening of cells producing valuable products. Furthermore, Raman spectrometry platform can serve as a monitor system in the commercial production of biodiesel for determining optimal harvest timing and controlling the quality of products. Acknowledgements The authors thank the National Science Council of Taiwan for funding this work via NSC P and NSC B MY3. The support from NCKU s top university grants is also appreciated. References [1] J. Singh, S. Gu, Commercialization potential of microalgae for biofuels production, Renewable and Sustainable Energy Reviews 2010;14: [2] P. Spolaore, C. Joannis-Cassan, E. Duran, A. Isambert, Commercial applications of microalgae, Journal of Bioscience and Bioengineering 2006;101: [3] R. Halim, B. Gladman, M.K. Danquah, P.A. Webley, Oil extraction from microalgae for biodiesel production, Bioresource Technol 2011;102: [4] T.H. Lee, J.S. Chang, H.Y. Wang, Rapid and in vivo quantification of cellular lipids in chlorella vulgaris using near-infrared raman spectrometry, Anal Chem 2013;85: [5] T.H. Lee, J.S. Chang, H.Y. Wang, Current developments in high-throughput analysis for microalgae cellular contents, Biotechnology Journal 2013;8: [6] E. Smith, G. Dent, Modern raman spectroscopy: A practical approach, Wiley. com; [7] K.L. Yeh, J.S. Chang, Nitrogen starvation strategies and photobioreactor design for enhancing lipid production of a newly isolated microalga chlorella vulgaris ESP-31: Implications for biofuels, Biotechnology Journal 2011;6: [8] M.J. Griffiths, C. Garcin, R.P. van Hille, S.T.L. Harrison, Interference by pigment in the estimation of microalgal biomass concentration by optical density, J. Microbiol. Methods 2011;85: [9] P. Heraud, J. Beardall, D. McNaughton, B. Wood, In vivo prediction of the nutrient status of individual microalgal cells using raman microspectroscopy, FEMS microbiology letters 2007;275: Tsung Hua Lee is a PhD student in the Department of Chemical Engineering in National Cheng Kung University, Taiwan. His research experience includes cantilever sensing system and the quantitative/qualitative analysis by Raman spectrometry. His current research focuses on developing rapid/high-throughput analysis systems for microflora cellular contents.

5 Tsung-Hua Lee and Hsiang-Yu Wang / Energy Procedia 61 ( 2014 ) Hsiang-Yu Wang, Ph.D. is an assistant professor in the Department of Chemical Engineering at National Cheng Kung University, Taiwan. Dr. Wang is devoted to the integration of microfluidics, biology, and engineering. Her current research interests focus on the development of optical and microfluidic techniques for rapid detection and selection of energy-generating microorganisms.