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PhotoBiobox: A tablet sized, low-cost, high throughput photobioreactor for microalgal screening and culture optimization for growth, lipid content and CO2 sequestration
Biochemical Engineering Journal 103 (2015) 193–197
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
PhotoBiobox: A tablet sized, low-cost, high throughput photobioreactor for microalgal screening and culture optimization for growth, lipid content and CO2 sequestration Jina Heo a,c , Dae-Hyun Cho a , Rishiram Ramanan a , Hee-Mock Oh b,c , Hee-Sik Kim a,c,∗ a Sustainable Bioresource Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon 305-806, Republic of Korea b Bioenergy and Biochemicals Research Center, KRIBB, Yuseong-gu, Daejeon 305-806, Republic of Korea c Green Chemistry and Environmental Biotechnology, University of Science and Technology (UST), Yuseong-gu, Daejeon 305-350, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 April 2015 Received in revised form 17 July 2015 Accepted 19 July 2015 Available online 26 July 2015 Keywords: Algae Screening Photobioreactor Biofuel Optimization Miniaturization
a b s t r a c t Microbial screening and culture optimization is a laborious, multifaceted and expensive procedure often taking years and millions to identify the ideal strain for specific use. In this study, a high throughput, tablet-sized, low cost photobioreactor, herein referred as “PhotoBiobox”, was fabricated and tested for screening and culture optimization of microalgae suitable for biomass and biodiesel production, and CO2 sequestration. PhotoBiobox is equipped with a LED array and photo gradient filter offering different light intensities. PhotoBiobox was fabricated to screen and cultivate several microbial strains in 96-well plate in wide temperature range and CO2 atmosphere. To test its efficacy, a total of 12 microalgal strains were isolated from Korean freshwaters, identified and screened for high growth and lipid accumulation potential. Among the 12 strains, Parachlorella sp., JD076 showed highest growth rate, remarkable stability and tolerance to different temperature regimes. Optimization experiments using PhotoBiobox revealed that this strain had optimum temperature range and light intensity of 24–25 ◦ C and 400 mol m−2 s−1 , respectively, with high growth rate and lipid content in 5% CO2 condition. The screening and optimization of culture conditions using PhotoBiobox took just 15 days, in total, which could radically reduce both screening costs and time. PhotoBiobox can be used as a screening and optimization reactor in several areas of microbiology and cell biology, while serving as a prototype for furthering research on miniaturization in this field. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Algal biotechnology industry is currently a multi-billion dollar global business. The commercial products of this industry are diverse including food, feed, nutraceuticals and cosmetics and, in general, serve high-value niche market [1]. Emergence of use of microalgae for biofuels is partly because of social and environmental issues and partly due to leapfrog in downstream processing of algal biomass in the past decade [2]. Microalgae are highly diverse with described species amounting to 72,536, and an estimated 200,000–1,000,000 species are known to exist [3]. In spite of this diversity, there are only a handful of strains which have been commercialized for the production of high-value products,
∗ Corresponding author at: Sustainable Bioresource Research Center, KRIBB, Daejeon 305-806, Republic of Korea. E-mail address: [email protected] (H.-S. Kim). http://dx.doi.org/10.1016/j.bej.2015.07.013 1369-703X/© 2015 Elsevier B.V. All rights reserved.
and upstream processes like strain selection and improvement for biofuel production has yielded limited results [4]. This is despite the growing use of technology and phylogenetic screening of microalgae compared to conventional taxonomic methods which are cumbersome. Because of the aforesaid reasons, screening for potential super-algae remains one of the most important steps for algal biotechnology industry [5,6]. Screening of algae for high biomass production and for unique applications such as biofuel production has been tedious, consuming enormous amount of time, effort and money [6]. Some large-scale consortium projects in this area are testimony to this predicament [7]. Traditionally, screening process involves cultivation in flasks and since different algae have different optimum light, temperature and carbon requirements, screening for desired microalgae and optimizing culture conditions itself is a research goal. Hence, a rapid method to screen several algae in a single reactor system and optimize culture conditions in one experimental run would be a boon for algal biotechnology research. Only recently, the
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emergence of microfluidics has resulted in relative ease and reduction of time for screening microalgae [6,8]. However, concerns over costs and limitations with respect to screening different microalgae under different conditions remain. Moreover, microfluidic photobioreactor developed does not integrate with existing systems and hence inconvenient to use. Hence, it is imperative to develop a high-throughput, low cost, easy to use, integrated photobioreactor. In this study, we demonstrate the fabrication and use of a tabletsized photobioreactor which can be used to screen for high growth and lipid accumulating microalgae and for optimizing light and temperature conditions for microalgal growth. The photobioreactor, referred herein as “PhotoBiobox”, can also be used to screen for high CO2 sequestering alga. Moreover, PhotoBiobox is a low-cost, integrated system that can be used with laboratory spectrophotometers for growth and lipid measurements and promise it offers for algal biotechnology industry is immense.
Microalgae from these samples were isolated using micropipetting and micropicking [9]. Unialgal strains were transferred to 96-well plates, and genomic DNA was extracted and 18S ribosomal RNA gene was amplified using ch165F and ch1200R primers for green algae [10]. Phylogenetic analysis was performed by aligning the 18S rRNA sequences obtained with reference sequences and subsequent tree construction using maximum likelihood method [11]. The sequences of 18S rRNA gene were aligned by CLUSTAL X [12] and edited by BIOEDIT [13] with published sequences retrieved from NCBI database (http://www.ncbi.nlm.nih.gov). Phylogenetic trees were reconstructed using the neighbor-joining [14], maximum-parsimony [15], and maximum-likelihood [16] algorithms in the MEGA 5 software [17] with bootstrap values based on 1000 replications [16]. 2.4. Comparative analysis of growth and lipid production in isolated microalgae using PhotoBiobox
2. Material and methods 2.1. Design and fabrication of PhotoBiobox The PhotoBiobox composed of four components: a LED light array, a photo gradient filter, a heat diffusion aluminum film, a water cooling block (Supplementary Fig. 1). The LED array constituted 72 LEDs with color temperature of 6000 K in 6 horizontal LED rows consuming 2.88 W per row which were connected to a power input (Yooyoung Tech, Korea). The light intensity was controlled (0–650 mol m−2 s−1 ) using LED dimmer (Topgreener, USA). A light gradient was achieved using a neutral density (ND) filter typically used in camera (Horusbennu, Korea) followed by a 96-well glass plate resulting in different light intensities to each row. Capacity of each well in the plate was 300 L with working volume of 200 L and the plate was sealed with gas and light permeable, water impermeable membrane (Diversified Biotech, USA). The temperature gradient was established using a combination of four water blocks (Skycare, Korea) with dimensions of 4.1 cm × 12.2 cm each, fitted with both hot and cold water inlets and outlets. The water was supplied using two laboratory water circulators operated at 4 ◦ C and 37 ◦ C, creating a gradient for the 96well plate. To enable efficient heat transfer to 96-well glass plate, an aluminum film was attached to the water blocs. Aluminum was selected for heat diffusion because of its excellent thermal conductivity, low-cost, corrosion resistance and relative non-reactivity compared to other metals like copper. Gas (air or CO2 ) inlet was provided using a gas inflow and outflow provided at the bottom of water cooling blocs and would provide the stipulated atmosphere inside the reactor. 2.2. Testing and standardization of PhotoBiobox Before actual experimental run, PhotoBiobox was mock tested using 200 L medium to measure the accuracy of the conditions offered by the system. The temperature gradient offered by the water bloc and heat diffusion was tested using an infrared thermometer (Tecman, China). Subsequently, light intensity gradient was tested for accuracy and stability using light meter (Li-Cor, USA) followed by air circulation with differently regulated partial pressures of CO2 . Each equipment was also tested for accuracy, robustness and durability in three separate mock tests. 2.3. Isolation of microalgae from freshwater, identification and phylogenetic analysis The twelve microalgal strains used in this study were isolated from 15 different sites (fresh water) in Republic of Korea.
Screening of microalgae was performed with PhotoBiobox using a light intensity of 250–300 mol m−2 s−1 at 30 ◦ C in BG11 medium with a steady flow of air and 5% CO2 . All microalgal strains (1 × 104 cells/ml) were inoculated in triplicates in 96 well plates with a working volume of 200 L. The growth rate of Parachlorella sp., was tested in BG11, Bold Basal Medium (BBM) and modified SOT media [18]. After cultivation for 7 days, optical density was measured using a microplate reader at 680 nm (Tecan, Switzerland). After Nile red staining, fluorescence of lipid droplets was measured using a microplate fluorescence reader (BioTek, USA) followed by normalization by optical density (Nile red activity) [5,19]. 2.5. Optimization of culture conditions for Parachlorella sp. using PhotoBiobox PhotoBiobox was further used for optimization of culture conditions for Parachlorella strain. The temperature and light conditions were optimized for this strain using temperature and light gradient provided by PhotoBiobox. The strain was also tested for its ability to grow in high CO2 concentration. 3. Results and discussion 3.1. Testing PhotoBiobox Recently, two microfluidic devices have been used for microalgal screening and optimization. Even with these devices, screening of multiple algal strains is not feasible in one experimental run. Moreover, these devices provide varying light intensities and light/dark cycles but do not provide different temperature regime [6,8]. PhotoBiobox was fabricated keeping these limitations in mind. This system is high-throughput and provides different light regimes like microfluidic systems. In addition, this system provides different temperature regime and can be used for studying the effect of various gaseous composition on microalgae. These advantages in PhotoBiobox was achieved using four low-cost components which include a LED array which provides light in the range of 0–650 mol m−2 s−1 , integrated with LED dimmer and photo gradient filter. The temperature range was achieved using water blocs connected to two water circulators integrated with the system (Fig. 1 & Supplementary Fig. 1). Before screening of algal strains using PhotoBiobox, the instrument was optimized as mentioned before. The optimization experiments proved that PhotoBiobox offered different temperature regime (0–35 ◦ C) and light regime (50–450 mol m−2 s−1 ) which would be used for both screening and culture condition optimization (Supplementary Fig. 2). Since PhotoBiobox provides starkly different conditions within
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3.2. Isolation and identification of microalgal strains from freshwater In this study, 12 microalgal strains were isolated from different freshwater sources to identify a strain with high growth under photoautotrophic conditions using CO2 . The strains were identified through 18S rRNA sequencing and morphological analysis. The strains belonged to 2 different phylum comprising 6 families and 10 genera (Supplementary Fig. 4). 3.3. Screening of algae using PhotoBiobox Screening experiments using algae revealed that among the 12 strains screened, Parachlorella sp., JD076 had highest specific growth rate of 0.37 and 0.57 d−1 , followed by Desmodesmus komarekii with specific growth rate of 0.35 and 0.53 d−1 at 28 ◦ C in the presence of air and CO2 , respectively (Table 1), while Monoraphidium dybowskii showed the least tolerance to 5% CO2 with a diminished growth rate [20]. Parachlorella sp., also had higher temperature tolerance compared to other strains exhibiting growth in all tested temperatures (15, 20, 25, 30, 35 ◦ C), while strains like JD069, JD073 and JD075 showed no growth at higher temperature (35 ◦ C) (data not shown). Based on the high specific growth rate in the presence of air and 5% CO2 , temperature tolerance, and relatively high Nile red activity (data not shown) Parachlorella sp., was selected for further studies. 3.4. Optimization of Parachlorella sp. JD076 culture conditions
Fig. 1. (A) Layered representation of different components in PhotoBiobox. (B) Sketch of outside view of PhotoBiobox construct with gas and water inlets and outlets. The size of PhotoBiobox is very similar to that of a modern day tablet enabling easy use. (C) Photograph of inside view of an improved system with LED array and sealed 96-well plate.
the small surface area of 96-well plate, the accuracy of the conditions provided by PhotoBiobox was tested in each well, in three individual experiments. Light intensity and temperature regime provided were very accurate, considering the range of conditions provided. As expected the standard deviation was relatively higher in high light intensities and in crossing point of hot and cold water, which maintain the differential temperature in the PhotoBiobox (Supplementary Fig. 3).
Subsequently, Parachlorella was grown in different temperatures, light regimes and in the presence of air and 5% CO2 . In the presence of air, this strain showed a maximum specific growth rate of 0.4296 d−1 and in 5% CO2 , the strain had more than twice the growth rate of 0.8092 d−1 but under varying optimum conditions (Table 2). The optimum temperature for this strain was 30 ◦ C in the presence of air, but in the presence of CO2 , the strain had different optimum temperature range of 24–25 ◦ C. On the contrary, the strain showed a preference for high light intensity (350–400 mol m−2 s−1 ) in the presence of 5% CO2 , which might also be due to high specific growth rate in this condition (Fig. 2A and B). Analysis of Nile red activity showed that this strain had optimum neutral lipid fluorescence in a narrow temperature range of 23–25 ◦ C and at a light intensity of 250–300 mol m−2 s−1 . While at high CO2 concentration, the strain produced thrice the Nile red fluorescence with an optimum range of 32–35 ◦ C at a light intensity of 350 mol m−2 s−1 (Fig. 2C and D). Hence, the conditions for high growth and high neutral lipid content varied significantly, in this strain [6]. PhotoBiobox could also be used for screening various media for the desired strain in one experimental run. Parachlorella sp., had the highest growth rate in BG11 media, which was used for the optimization experiments, followed by BBM and SOT (Fig. 3A). Thus, it is possible to examine different nutritional strategies including nutrient deficiency in a single experimental run using PhotoBiobox, which could help in identifying the best nutrient requirement for both growth and lipid production for any strain [19]. The growth curve of Parachlorella sp., in PhotoBiobox showed a typical growth pattern seen in cultivation using 96-well plate (Fig. 3B). Screening for microalgae and optimization of culture conditions for high growth rate and Nile red fluorescence was achieved within 15 days using PhotoBiobox (Table 2). Screening and optimization studies with multiple strains may take about 2 years using conventional techniques and tools [8]. PhotoBiobox not only reduces time required for such studies but also reduces the operating costs involved in using a higher scale system. Such advantages
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Table 1 Screening for strains with high specific growth rate using PhotoBiobox. The temperature was maintained at 28 ◦ C and light intensity was 250–300 mol m−2 s−1 during the experimental run. Strain no.
Species/genus
Specific growth rate (, d−1 )
Family
Air JD009 JD022 JD024 JD039 JD040 JD057 JD060 JD069 JD073 JD075 JD076 JD090
Desmodesmus komarekii Acutodesmus obliquus Scenedesmus armatus Desmodesmus communis Scenedesmus deserticola Klebsormidium flaccidum Monoraphidium dybowskii Micractinium sp. Lobosphaeropsis lobophora Choricystis sp. Parachlorella sp. Coelastropsis costata
Scenedesmaceae Scenedesmaceae Scenedesmaceae Scenedesmaceae Scenedesmaceae Klebsormidiaceae Selenastraceae Micractiniaceae Chlorellaceae Coccomyxaceae Chlorellaceae Scenedesmaceae
0.346 0.238 0.258 0.224 0.234 0.191 0.213 0.208 0.224 0.09 0.369 0.324
5% CO2 ± ± ± ± ± ± ± ± ± ± ± ±
0.017 0.036 0.065 0.031 0.014 0.021 0.011 0.025 0.006 0.012 0.034 0.032
0.534 0.515 0.575 0.403 0.498 0.335 0.103 0.240 0.244 0.136 0.572 0.550
± ± ± ± ± ± ± ± ± ± ± ±
0.032 0.049 0.087 0.051 0.030 0.032 0.009 0.01 0.005 0.015 0.046 0.051
Table 2 Optimized culture conditions of Parachlorella sp. JD076 for high growth using PhotoBiobox. Note that specific growth rate of this strain is much higher after optimization compared to screening experiments (Table 1). Culture condition
Specific growth rate (, d−1 )
Optimum temperature (◦ C)
Optimum light intensity (mol m−2 s−1 )
Air CO2
0.4296 ± 0.0075 0.8092 + 0.04863
30 24–25
150–300 400
and culture optimization provided by PhotoBiobox is unmatched as temperature, light and even multiple media and nutrient formulations can be tested. This system also integrates seamlessly with existing systems avoiding the need for any specialized equipment and could be used in any moderately sophisticated microalgal
laboratory compared to few existing high-throughput devices [6,8]. Furthermore, the existing systems are very expensive and difficult to use for screening as they do not allow comparison between two strains, whereas PhotoBiobox is made with readily available lowcost materials [6] and can screen multiple algal strains effectively in
Fig. 2. Matrix of specific growth rate (legend) in different temperature and light regime for Parachlorella culture optimization in the presence of air (A) and 5% CO2 (B). Optimum conditions for high Nile red fluorescence intensity (legend) of Parachlorella were significantly different in the presence of air (C) and 5% CO2 (D).
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Acknowledgements This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Korea Government Ministry of Science, ICT and Future Planning (ABC-2011-0031351), grant from Marine Biotechnology Program funded by Korea Government Ministry of Oceans and Fisheries (No. 20150184), as well as by Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Korea Government Ministry of Health & Welfare (No. HN13C0080), and finally a grant from the KRIBB (Korea Research Institute of Bioscience and Biotechnology) Research Initiative Program (www. kribb.re.kr). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2015.07.013 References
Fig. 3. (A) Specific growth rate of Parachlorella sp., in different media (BG11, SOT & BBM) tested in a single experimental run. The composition of each medium is available in Section 2. (B) Growth curve of Parachlorella sp., in BG11 medium under optimal conditions using PhotoBiobox.
single run, enabling comparison of all tested species. Furthermore, this system could be potentially used for screening and optimization of several microbial strains, other than algae. 4. Conclusions This study demonstrates the fabrication and use of a low-cost, tablet sized photobioreactor which could be used immediately by algal biotechnology industry for screening and optimization of culture conditions for biofuel production. Parachlorella sp., JD076 strain showed high specific growth rate and lipid content compared to 11 other strains screened using PhotoBiobox. Culture condition optimization using PhotoBiobox revealed this strain grows at high CO2 concentration and in a wide temperature range. Moreover, with slight modifications in design, this system could impose wide ranging applications from single cell studies to studying effect of different atmospheres on microbial life in vitro. Overall, PhotoBiobox can be a low-cost and effective photobioreactor which significantly reduces cost and time involved in strain selection and optimization for algal biotechnology.
[1] R.H. Wijffels, M.J. Barbosa, M.H.M. Eppink, Microalgae for the production of bulk chemicals and biofuels, Biofuels Bioprod. Biorefin. 4 (2010) 287–295. [2] Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, A. Darzins, Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances, Plant J. 54 (2008) 621–639. [3] M.D. Guiry, How many species of algae are there? J. Phycol. 48 (2012) 1057–1063. [4] P. Spolaore, C. Joannis-Cassan, E. Duran, A. Isambert, Commercial applications of microalgae, J. Biosci. Bioeng. 101 (2006) 87–96. [5] T.T.Y. Doan, B. Sivaloganathan, J.P. Obbard, Screening of marine microalgae for biodiesel feedstock, Biomass Bioenergy 35 (2011) 2534–2544. [6] H.S. Kim, T.L. Weiss, H.R. Thapa, T.P. Devarenne, A. Han, A microfluidic photobioreactor array demonstrating high-throughput screening for microalgal oil production, Lab Chip 14 (2014) 1415–1425. [7] J. Sheehan, T. Dunahay, J. Benemann, P. Roessler, A Look Back at the US Department of Energy’s Aquatic Species Program: Biodiesel from Algae, National Renewable Energy Laboratory, Golden, 1998. [8] M. Chen, T. Mertiri, T. Holland, A.S. Basu, Optical microplates for highthroughput screening of photosynthesis in lipid-producing algae, Lab Chip 12 (2012) 3870–3874. [9] D.-H. Cho, R. Ramanan, B.-H. Kim, J. Lee, S. Kim, Y. Chan, G.-G. Choi, H.-M. Oh, H.-S. Kim, Novel approach for the development of axenic microalgal cultures from environmental samples, J. Phycol. 49 (2013) 802–810. [10] D.-H. Cho, R. Ramanan, J. Heo, J. Lee, B.-H. Kim, H.-M. Oh, H.-S. Kim, Enhancing microalgal biomass productivity by engineering a microalgal–bacterial community, Bioresour. Technol. 175 (2015) 578–585. [11] L. Jin, H.-J. La, H.-G. Lee, J.J. Lee, S. Lee, C.-Y. Ahn, H.-M. Oh, Caulobacter profunda sp. nov., isolated from deep freshwater sediment, Int. J. Syst. Evol. Microbiol. 64 (2014) 762–767. [12] J.D. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. Higgins, The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids 24 (1997) 4876–4882. [13] T.A. Hall, BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT, Nucleic Acids Symp. Ser. 41 (1999) 95–98. [14] N. Saitou, M. Nei, The neighbour-joining method; a new method for reconstructing phylogenetic trees, Mol. Biol. Evol. 4 (1987) 406–425. [15] W.M. Fitch, Toward defining the course of evolution: minimum change for a specific tree topology, Syst. Zool. 20 (1971) 406–416. [16] J. Felsenstein, Confidence limit on phylogenies: an approach using the bootstrap, Evolution 39 (1985) 783–791. [17] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, S. Kumar, MEGA 5 molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods, Mol. Biol. Evol. 28 (2011) 2731–2739. [18] R.A. Andersen, Algal Culturing Techniques, Academic Press, 2005. [19] R. Ramanan, B.-H. Kim, D.-H. Cho, S.-R. Ko, H.-M. Oh, H.-S. Kim, Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii, FEBS Lett. 587 (2013) 370–377. [20] H. Yang, Q. He, J. Rong, L. Xia, C. Hu, Rapid neutral lipid accumulation of the alkali-resistant oleaginous Monoraphidium dybowskii LB50 by NaCl induction, Bioresour. Technol. 172 (2014) 131–137.
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
PhotoBiobox: A tablet sized, low-cost, high throughput photobioreactor for microalgal screening and culture optimization for growth, lipid content and CO2 sequestration Jina Heo a,c , Dae-Hyun Cho a , Rishiram Ramanan a , Hee-Mock Oh b,c , Hee-Sik Kim a,c,∗ a Sustainable Bioresource Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon 305-806, Republic of Korea b Bioenergy and Biochemicals Research Center, KRIBB, Yuseong-gu, Daejeon 305-806, Republic of Korea c Green Chemistry and Environmental Biotechnology, University of Science and Technology (UST), Yuseong-gu, Daejeon 305-350, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 April 2015 Received in revised form 17 July 2015 Accepted 19 July 2015 Available online 26 July 2015 Keywords: Algae Screening Photobioreactor Biofuel Optimization Miniaturization
a b s t r a c t Microbial screening and culture optimization is a laborious, multifaceted and expensive procedure often taking years and millions to identify the ideal strain for specific use. In this study, a high throughput, tablet-sized, low cost photobioreactor, herein referred as “PhotoBiobox”, was fabricated and tested for screening and culture optimization of microalgae suitable for biomass and biodiesel production, and CO2 sequestration. PhotoBiobox is equipped with a LED array and photo gradient filter offering different light intensities. PhotoBiobox was fabricated to screen and cultivate several microbial strains in 96-well plate in wide temperature range and CO2 atmosphere. To test its efficacy, a total of 12 microalgal strains were isolated from Korean freshwaters, identified and screened for high growth and lipid accumulation potential. Among the 12 strains, Parachlorella sp., JD076 showed highest growth rate, remarkable stability and tolerance to different temperature regimes. Optimization experiments using PhotoBiobox revealed that this strain had optimum temperature range and light intensity of 24–25 ◦ C and 400 mol m−2 s−1 , respectively, with high growth rate and lipid content in 5% CO2 condition. The screening and optimization of culture conditions using PhotoBiobox took just 15 days, in total, which could radically reduce both screening costs and time. PhotoBiobox can be used as a screening and optimization reactor in several areas of microbiology and cell biology, while serving as a prototype for furthering research on miniaturization in this field. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Algal biotechnology industry is currently a multi-billion dollar global business. The commercial products of this industry are diverse including food, feed, nutraceuticals and cosmetics and, in general, serve high-value niche market [1]. Emergence of use of microalgae for biofuels is partly because of social and environmental issues and partly due to leapfrog in downstream processing of algal biomass in the past decade [2]. Microalgae are highly diverse with described species amounting to 72,536, and an estimated 200,000–1,000,000 species are known to exist [3]. In spite of this diversity, there are only a handful of strains which have been commercialized for the production of high-value products,
∗ Corresponding author at: Sustainable Bioresource Research Center, KRIBB, Daejeon 305-806, Republic of Korea. E-mail address: [email protected] (H.-S. Kim). http://dx.doi.org/10.1016/j.bej.2015.07.013 1369-703X/© 2015 Elsevier B.V. All rights reserved.
and upstream processes like strain selection and improvement for biofuel production has yielded limited results [4]. This is despite the growing use of technology and phylogenetic screening of microalgae compared to conventional taxonomic methods which are cumbersome. Because of the aforesaid reasons, screening for potential super-algae remains one of the most important steps for algal biotechnology industry [5,6]. Screening of algae for high biomass production and for unique applications such as biofuel production has been tedious, consuming enormous amount of time, effort and money [6]. Some large-scale consortium projects in this area are testimony to this predicament [7]. Traditionally, screening process involves cultivation in flasks and since different algae have different optimum light, temperature and carbon requirements, screening for desired microalgae and optimizing culture conditions itself is a research goal. Hence, a rapid method to screen several algae in a single reactor system and optimize culture conditions in one experimental run would be a boon for algal biotechnology research. Only recently, the
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J. Heo et al. / Biochemical Engineering Journal 103 (2015) 193–197
emergence of microfluidics has resulted in relative ease and reduction of time for screening microalgae [6,8]. However, concerns over costs and limitations with respect to screening different microalgae under different conditions remain. Moreover, microfluidic photobioreactor developed does not integrate with existing systems and hence inconvenient to use. Hence, it is imperative to develop a high-throughput, low cost, easy to use, integrated photobioreactor. In this study, we demonstrate the fabrication and use of a tabletsized photobioreactor which can be used to screen for high growth and lipid accumulating microalgae and for optimizing light and temperature conditions for microalgal growth. The photobioreactor, referred herein as “PhotoBiobox”, can also be used to screen for high CO2 sequestering alga. Moreover, PhotoBiobox is a low-cost, integrated system that can be used with laboratory spectrophotometers for growth and lipid measurements and promise it offers for algal biotechnology industry is immense.
Microalgae from these samples were isolated using micropipetting and micropicking [9]. Unialgal strains were transferred to 96-well plates, and genomic DNA was extracted and 18S ribosomal RNA gene was amplified using ch165F and ch1200R primers for green algae [10]. Phylogenetic analysis was performed by aligning the 18S rRNA sequences obtained with reference sequences and subsequent tree construction using maximum likelihood method [11]. The sequences of 18S rRNA gene were aligned by CLUSTAL X [12] and edited by BIOEDIT [13] with published sequences retrieved from NCBI database (http://www.ncbi.nlm.nih.gov). Phylogenetic trees were reconstructed using the neighbor-joining [14], maximum-parsimony [15], and maximum-likelihood [16] algorithms in the MEGA 5 software [17] with bootstrap values based on 1000 replications [16]. 2.4. Comparative analysis of growth and lipid production in isolated microalgae using PhotoBiobox
2. Material and methods 2.1. Design and fabrication of PhotoBiobox The PhotoBiobox composed of four components: a LED light array, a photo gradient filter, a heat diffusion aluminum film, a water cooling block (Supplementary Fig. 1). The LED array constituted 72 LEDs with color temperature of 6000 K in 6 horizontal LED rows consuming 2.88 W per row which were connected to a power input (Yooyoung Tech, Korea). The light intensity was controlled (0–650 mol m−2 s−1 ) using LED dimmer (Topgreener, USA). A light gradient was achieved using a neutral density (ND) filter typically used in camera (Horusbennu, Korea) followed by a 96-well glass plate resulting in different light intensities to each row. Capacity of each well in the plate was 300 L with working volume of 200 L and the plate was sealed with gas and light permeable, water impermeable membrane (Diversified Biotech, USA). The temperature gradient was established using a combination of four water blocks (Skycare, Korea) with dimensions of 4.1 cm × 12.2 cm each, fitted with both hot and cold water inlets and outlets. The water was supplied using two laboratory water circulators operated at 4 ◦ C and 37 ◦ C, creating a gradient for the 96well plate. To enable efficient heat transfer to 96-well glass plate, an aluminum film was attached to the water blocs. Aluminum was selected for heat diffusion because of its excellent thermal conductivity, low-cost, corrosion resistance and relative non-reactivity compared to other metals like copper. Gas (air or CO2 ) inlet was provided using a gas inflow and outflow provided at the bottom of water cooling blocs and would provide the stipulated atmosphere inside the reactor. 2.2. Testing and standardization of PhotoBiobox Before actual experimental run, PhotoBiobox was mock tested using 200 L medium to measure the accuracy of the conditions offered by the system. The temperature gradient offered by the water bloc and heat diffusion was tested using an infrared thermometer (Tecman, China). Subsequently, light intensity gradient was tested for accuracy and stability using light meter (Li-Cor, USA) followed by air circulation with differently regulated partial pressures of CO2 . Each equipment was also tested for accuracy, robustness and durability in three separate mock tests. 2.3. Isolation of microalgae from freshwater, identification and phylogenetic analysis The twelve microalgal strains used in this study were isolated from 15 different sites (fresh water) in Republic of Korea.
Screening of microalgae was performed with PhotoBiobox using a light intensity of 250–300 mol m−2 s−1 at 30 ◦ C in BG11 medium with a steady flow of air and 5% CO2 . All microalgal strains (1 × 104 cells/ml) were inoculated in triplicates in 96 well plates with a working volume of 200 L. The growth rate of Parachlorella sp., was tested in BG11, Bold Basal Medium (BBM) and modified SOT media [18]. After cultivation for 7 days, optical density was measured using a microplate reader at 680 nm (Tecan, Switzerland). After Nile red staining, fluorescence of lipid droplets was measured using a microplate fluorescence reader (BioTek, USA) followed by normalization by optical density (Nile red activity) [5,19]. 2.5. Optimization of culture conditions for Parachlorella sp. using PhotoBiobox PhotoBiobox was further used for optimization of culture conditions for Parachlorella strain. The temperature and light conditions were optimized for this strain using temperature and light gradient provided by PhotoBiobox. The strain was also tested for its ability to grow in high CO2 concentration. 3. Results and discussion 3.1. Testing PhotoBiobox Recently, two microfluidic devices have been used for microalgal screening and optimization. Even with these devices, screening of multiple algal strains is not feasible in one experimental run. Moreover, these devices provide varying light intensities and light/dark cycles but do not provide different temperature regime [6,8]. PhotoBiobox was fabricated keeping these limitations in mind. This system is high-throughput and provides different light regimes like microfluidic systems. In addition, this system provides different temperature regime and can be used for studying the effect of various gaseous composition on microalgae. These advantages in PhotoBiobox was achieved using four low-cost components which include a LED array which provides light in the range of 0–650 mol m−2 s−1 , integrated with LED dimmer and photo gradient filter. The temperature range was achieved using water blocs connected to two water circulators integrated with the system (Fig. 1 & Supplementary Fig. 1). Before screening of algal strains using PhotoBiobox, the instrument was optimized as mentioned before. The optimization experiments proved that PhotoBiobox offered different temperature regime (0–35 ◦ C) and light regime (50–450 mol m−2 s−1 ) which would be used for both screening and culture condition optimization (Supplementary Fig. 2). Since PhotoBiobox provides starkly different conditions within
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3.2. Isolation and identification of microalgal strains from freshwater In this study, 12 microalgal strains were isolated from different freshwater sources to identify a strain with high growth under photoautotrophic conditions using CO2 . The strains were identified through 18S rRNA sequencing and morphological analysis. The strains belonged to 2 different phylum comprising 6 families and 10 genera (Supplementary Fig. 4). 3.3. Screening of algae using PhotoBiobox Screening experiments using algae revealed that among the 12 strains screened, Parachlorella sp., JD076 had highest specific growth rate of 0.37 and 0.57 d−1 , followed by Desmodesmus komarekii with specific growth rate of 0.35 and 0.53 d−1 at 28 ◦ C in the presence of air and CO2 , respectively (Table 1), while Monoraphidium dybowskii showed the least tolerance to 5% CO2 with a diminished growth rate [20]. Parachlorella sp., also had higher temperature tolerance compared to other strains exhibiting growth in all tested temperatures (15, 20, 25, 30, 35 ◦ C), while strains like JD069, JD073 and JD075 showed no growth at higher temperature (35 ◦ C) (data not shown). Based on the high specific growth rate in the presence of air and 5% CO2 , temperature tolerance, and relatively high Nile red activity (data not shown) Parachlorella sp., was selected for further studies. 3.4. Optimization of Parachlorella sp. JD076 culture conditions
Fig. 1. (A) Layered representation of different components in PhotoBiobox. (B) Sketch of outside view of PhotoBiobox construct with gas and water inlets and outlets. The size of PhotoBiobox is very similar to that of a modern day tablet enabling easy use. (C) Photograph of inside view of an improved system with LED array and sealed 96-well plate.
the small surface area of 96-well plate, the accuracy of the conditions provided by PhotoBiobox was tested in each well, in three individual experiments. Light intensity and temperature regime provided were very accurate, considering the range of conditions provided. As expected the standard deviation was relatively higher in high light intensities and in crossing point of hot and cold water, which maintain the differential temperature in the PhotoBiobox (Supplementary Fig. 3).
Subsequently, Parachlorella was grown in different temperatures, light regimes and in the presence of air and 5% CO2 . In the presence of air, this strain showed a maximum specific growth rate of 0.4296 d−1 and in 5% CO2 , the strain had more than twice the growth rate of 0.8092 d−1 but under varying optimum conditions (Table 2). The optimum temperature for this strain was 30 ◦ C in the presence of air, but in the presence of CO2 , the strain had different optimum temperature range of 24–25 ◦ C. On the contrary, the strain showed a preference for high light intensity (350–400 mol m−2 s−1 ) in the presence of 5% CO2 , which might also be due to high specific growth rate in this condition (Fig. 2A and B). Analysis of Nile red activity showed that this strain had optimum neutral lipid fluorescence in a narrow temperature range of 23–25 ◦ C and at a light intensity of 250–300 mol m−2 s−1 . While at high CO2 concentration, the strain produced thrice the Nile red fluorescence with an optimum range of 32–35 ◦ C at a light intensity of 350 mol m−2 s−1 (Fig. 2C and D). Hence, the conditions for high growth and high neutral lipid content varied significantly, in this strain [6]. PhotoBiobox could also be used for screening various media for the desired strain in one experimental run. Parachlorella sp., had the highest growth rate in BG11 media, which was used for the optimization experiments, followed by BBM and SOT (Fig. 3A). Thus, it is possible to examine different nutritional strategies including nutrient deficiency in a single experimental run using PhotoBiobox, which could help in identifying the best nutrient requirement for both growth and lipid production for any strain [19]. The growth curve of Parachlorella sp., in PhotoBiobox showed a typical growth pattern seen in cultivation using 96-well plate (Fig. 3B). Screening for microalgae and optimization of culture conditions for high growth rate and Nile red fluorescence was achieved within 15 days using PhotoBiobox (Table 2). Screening and optimization studies with multiple strains may take about 2 years using conventional techniques and tools [8]. PhotoBiobox not only reduces time required for such studies but also reduces the operating costs involved in using a higher scale system. Such advantages
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Table 1 Screening for strains with high specific growth rate using PhotoBiobox. The temperature was maintained at 28 ◦ C and light intensity was 250–300 mol m−2 s−1 during the experimental run. Strain no.
Species/genus
Specific growth rate (, d−1 )
Family
Air JD009 JD022 JD024 JD039 JD040 JD057 JD060 JD069 JD073 JD075 JD076 JD090
Desmodesmus komarekii Acutodesmus obliquus Scenedesmus armatus Desmodesmus communis Scenedesmus deserticola Klebsormidium flaccidum Monoraphidium dybowskii Micractinium sp. Lobosphaeropsis lobophora Choricystis sp. Parachlorella sp. Coelastropsis costata
Scenedesmaceae Scenedesmaceae Scenedesmaceae Scenedesmaceae Scenedesmaceae Klebsormidiaceae Selenastraceae Micractiniaceae Chlorellaceae Coccomyxaceae Chlorellaceae Scenedesmaceae
0.346 0.238 0.258 0.224 0.234 0.191 0.213 0.208 0.224 0.09 0.369 0.324
5% CO2 ± ± ± ± ± ± ± ± ± ± ± ±
0.017 0.036 0.065 0.031 0.014 0.021 0.011 0.025 0.006 0.012 0.034 0.032
0.534 0.515 0.575 0.403 0.498 0.335 0.103 0.240 0.244 0.136 0.572 0.550
± ± ± ± ± ± ± ± ± ± ± ±
0.032 0.049 0.087 0.051 0.030 0.032 0.009 0.01 0.005 0.015 0.046 0.051
Table 2 Optimized culture conditions of Parachlorella sp. JD076 for high growth using PhotoBiobox. Note that specific growth rate of this strain is much higher after optimization compared to screening experiments (Table 1). Culture condition
Specific growth rate (, d−1 )
Optimum temperature (◦ C)
Optimum light intensity (mol m−2 s−1 )
Air CO2
0.4296 ± 0.0075 0.8092 + 0.04863
30 24–25
150–300 400
and culture optimization provided by PhotoBiobox is unmatched as temperature, light and even multiple media and nutrient formulations can be tested. This system also integrates seamlessly with existing systems avoiding the need for any specialized equipment and could be used in any moderately sophisticated microalgal
laboratory compared to few existing high-throughput devices [6,8]. Furthermore, the existing systems are very expensive and difficult to use for screening as they do not allow comparison between two strains, whereas PhotoBiobox is made with readily available lowcost materials [6] and can screen multiple algal strains effectively in
Fig. 2. Matrix of specific growth rate (legend) in different temperature and light regime for Parachlorella culture optimization in the presence of air (A) and 5% CO2 (B). Optimum conditions for high Nile red fluorescence intensity (legend) of Parachlorella were significantly different in the presence of air (C) and 5% CO2 (D).
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Acknowledgements This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Korea Government Ministry of Science, ICT and Future Planning (ABC-2011-0031351), grant from Marine Biotechnology Program funded by Korea Government Ministry of Oceans and Fisheries (No. 20150184), as well as by Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Korea Government Ministry of Health & Welfare (No. HN13C0080), and finally a grant from the KRIBB (Korea Research Institute of Bioscience and Biotechnology) Research Initiative Program (www. kribb.re.kr). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2015.07.013 References
Fig. 3. (A) Specific growth rate of Parachlorella sp., in different media (BG11, SOT & BBM) tested in a single experimental run. The composition of each medium is available in Section 2. (B) Growth curve of Parachlorella sp., in BG11 medium under optimal conditions using PhotoBiobox.
single run, enabling comparison of all tested species. Furthermore, this system could be potentially used for screening and optimization of several microbial strains, other than algae. 4. Conclusions This study demonstrates the fabrication and use of a low-cost, tablet sized photobioreactor which could be used immediately by algal biotechnology industry for screening and optimization of culture conditions for biofuel production. Parachlorella sp., JD076 strain showed high specific growth rate and lipid content compared to 11 other strains screened using PhotoBiobox. Culture condition optimization using PhotoBiobox revealed this strain grows at high CO2 concentration and in a wide temperature range. Moreover, with slight modifications in design, this system could impose wide ranging applications from single cell studies to studying effect of different atmospheres on microbial life in vitro. Overall, PhotoBiobox can be a low-cost and effective photobioreactor which significantly reduces cost and time involved in strain selection and optimization for algal biotechnology.
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