ARTICLE Live Cell Imaging Compatible Immobilization of Chlamydomonas reinhardtii in Microfluidic Platform for Biodiesel Research Jae Woo Park,1 Sang Cheol Na,1 Thanh Qua Nguyen,2 Sang-Min Paik,3 Myeongwoo Kang,1 Daewha Hong,4 Insung S. Choi,4 Jae-Hyeok Lee,5 Noo Li Jeon1,2,3,6 1
Division of WCU (World Class University) Multiscale Mechanical Design, School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, Korea 2 School of Mechanical and Aerospace Engineering, Seoul National University, 151-744, Seoul, Korea 3 Interdisciplinary Program for Bioengineering, Seoul National University, Seoul, Korea 4 Department of Chemistry, KAIST, Daejeon, Korea 5 Department of Botany, University of British Columbia, Vancouver, BC, Canada 6 Institute of Advanced Machinery and Design, Seoul National University, Seoul, Korea; telephone: þ82-2-880-7111; fax: þ82-2-880-7119; e-mail: [email protected]
ABSTRACT: This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the “biological glue.” We take advantage of hydroxyproline (Hyp)based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm2. Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. Biotechnol. Bioeng. 2015;112: 494–501. ß 2014 Wiley Periodicals, Inc. Correspondence to: N.L. Jeon Contract grant sponsor: Korean CCS R&D Center (KCRC) Contract grant number: NRF-2013M1A8A1035962, 2013M1A8A1056300 Contract grant sponsor: National Research Foundation of Korea Contract grant sponsor: Ministry of Science, ICT and Future Planning Received 1 July 2014; Revision received 19 August 2014; Accepted 5 September 2014 Accepted manuscript online 12 September 2014; Article first published online 21 October 2014 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25453/abstract). DOI 10.1002/bit.25453
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Biotechnology and Bioengineering, Vol. 112, No. 3, March, 2015
KEYWORDS: Chlamydomonas reinhardtii; biodiesel; microfluidics; immobilization
Introduction Finding alternatives to fossil fuels is an urgent priority in global energy research. Recent energy shortage and climate crisis brought renewed attention to microalgae as the next frontier of green biotechnology. Microalgae have attracted increasing interest as they are a renewable source of sustainable biofuels (Akkerman et al., 2002; Banerjee et al., 2002; Ghirardi et al., 2000; Melis, 2002; Metzger and Largeau, 2005). Lipid (feedstock for green diesel production) accumulation in microalgae is dependent on environmental stress (i.e., nutrient deprivation) and culture conditions (i.e., light, pH, and temperature). Current methods for culture optimization and strain development utilize slow and laborintensive off-line gravimetric and chromatographic analysis. Conventional agar plate and batch culture system typically observe cells on agar plate and microscope coverslip which is limited to acquire the quality of long-term continuous monitoring, as cells tend to grow and migrate out of the focal plane of microscope. Microfluidics-based experimental methods have expanded our knowledge of various microorganisms by offering well-controlled microenvironmental experimental conditions that could not be produced with conventional macroscale methods (Ingham and Vlieg, 2008). Microfluidic platforms can generate precise spatially and temporally controlled microenvironments at single cell level. Understanding the mechanism of lipid droplet formation, ß 2014 Wiley Periodicals, Inc.
optimization of culture conditions and screening new strains that have high lipid productivity would benefit greatly from implementation of microfluidic techniques. Unlike mammalian cells, investigation of microalgae in microfluidic system has been limited due to their inherent motility and small size. Moreover, motile microalgae respond to light stimuli also known as phototaxis (Witman, 1993). Due to their small size and motility, it is hard to capture on conventional physically modified microstructures as well as track single cellular behavior with conventional microscopic system. Given these properties, long-term continuous monitoring under different culture conditions has not been possible. Several approach have been explored to capture microalgae, micropillar-based cell trap (Kim et al., 2010) was successfully used to culture Botryococcus braunii. Cells trapped in a single device were exposed to different light conditions to examine their effect on biomass formation. However, B. braunii cells were captured at a single-colony resolution (>50 mm, not as single-cell, cells). This system cannot be applied to other microalgal species such as Chlamydomonas reinhardtii, most widely studied model microalgae whose genome is fully sequenced and annotated (Merchant et al., 2007), as the vegetative cells of C. reinhardtii are much smaller in size (
Division of WCU (World Class University) Multiscale Mechanical Design, School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, Korea 2 School of Mechanical and Aerospace Engineering, Seoul National University, 151-744, Seoul, Korea 3 Interdisciplinary Program for Bioengineering, Seoul National University, Seoul, Korea 4 Department of Chemistry, KAIST, Daejeon, Korea 5 Department of Botany, University of British Columbia, Vancouver, BC, Canada 6 Institute of Advanced Machinery and Design, Seoul National University, Seoul, Korea; telephone: þ82-2-880-7111; fax: þ82-2-880-7119; e-mail: [email protected]
ABSTRACT: This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the “biological glue.” We take advantage of hydroxyproline (Hyp)based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm2. Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. Biotechnol. Bioeng. 2015;112: 494–501. ß 2014 Wiley Periodicals, Inc. Correspondence to: N.L. Jeon Contract grant sponsor: Korean CCS R&D Center (KCRC) Contract grant number: NRF-2013M1A8A1035962, 2013M1A8A1056300 Contract grant sponsor: National Research Foundation of Korea Contract grant sponsor: Ministry of Science, ICT and Future Planning Received 1 July 2014; Revision received 19 August 2014; Accepted 5 September 2014 Accepted manuscript online 12 September 2014; Article first published online 21 October 2014 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25453/abstract). DOI 10.1002/bit.25453
494
Biotechnology and Bioengineering, Vol. 112, No. 3, March, 2015
KEYWORDS: Chlamydomonas reinhardtii; biodiesel; microfluidics; immobilization
Introduction Finding alternatives to fossil fuels is an urgent priority in global energy research. Recent energy shortage and climate crisis brought renewed attention to microalgae as the next frontier of green biotechnology. Microalgae have attracted increasing interest as they are a renewable source of sustainable biofuels (Akkerman et al., 2002; Banerjee et al., 2002; Ghirardi et al., 2000; Melis, 2002; Metzger and Largeau, 2005). Lipid (feedstock for green diesel production) accumulation in microalgae is dependent on environmental stress (i.e., nutrient deprivation) and culture conditions (i.e., light, pH, and temperature). Current methods for culture optimization and strain development utilize slow and laborintensive off-line gravimetric and chromatographic analysis. Conventional agar plate and batch culture system typically observe cells on agar plate and microscope coverslip which is limited to acquire the quality of long-term continuous monitoring, as cells tend to grow and migrate out of the focal plane of microscope. Microfluidics-based experimental methods have expanded our knowledge of various microorganisms by offering well-controlled microenvironmental experimental conditions that could not be produced with conventional macroscale methods (Ingham and Vlieg, 2008). Microfluidic platforms can generate precise spatially and temporally controlled microenvironments at single cell level. Understanding the mechanism of lipid droplet formation, ß 2014 Wiley Periodicals, Inc.
optimization of culture conditions and screening new strains that have high lipid productivity would benefit greatly from implementation of microfluidic techniques. Unlike mammalian cells, investigation of microalgae in microfluidic system has been limited due to their inherent motility and small size. Moreover, motile microalgae respond to light stimuli also known as phototaxis (Witman, 1993). Due to their small size and motility, it is hard to capture on conventional physically modified microstructures as well as track single cellular behavior with conventional microscopic system. Given these properties, long-term continuous monitoring under different culture conditions has not been possible. Several approach have been explored to capture microalgae, micropillar-based cell trap (Kim et al., 2010) was successfully used to culture Botryococcus braunii. Cells trapped in a single device were exposed to different light conditions to examine their effect on biomass formation. However, B. braunii cells were captured at a single-colony resolution (>50 mm, not as single-cell, cells). This system cannot be applied to other microalgal species such as Chlamydomonas reinhardtii, most widely studied model microalgae whose genome is fully sequenced and annotated (Merchant et al., 2007), as the vegetative cells of C. reinhardtii are much smaller in size (
