A microscale approach for simple and rapid monitoring ... - Springer Link

Bioprocess Biosyst Eng (2015) 38:2035–2043 DOI 10.1007/s00449-015-1444-1

ORIGINAL PAPER

A microscale approach for simple and rapid monitoring of cell growth and lipid accumulation in Neochloris oleoabundans Ho Seok Kwak1 • Jaoon Young Hwan Kim1 • Sang Jun Sim1,2

Received: 23 March 2015 / Accepted: 17 July 2015 / Published online: 25 July 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Due to the increasing environmental problems caused by the use of fossil fuels, microalgae have been spotlighted as renewable resources to produce biomass and biofuels. Therefore, the investigation of the optimum culture conditions of microalgae in a short time is one of the important factors for improving growth and lipid productivity. Herein, we developed a PDMS-based high-throughput screening system to rapidly and easily determine the optimum conditions for high-density culture and lipid accumulation of Neochloris oleoabundans. Using the microreactor, we were able to find the optimal culture conditions of N. oleoabundans within 5 days by rapid and parallel monitoring growth and lipid induction under diverse conditions of light intensity, pH, CO2 and nitrate concentration. We found that the maximum growth rate (lmax = 2.13 day-1) achieved in the microreactor was 1.58-fold higher than that in a flask (lmax = 1.34 day-1) at the light intensity of 40 lmol photons m-2 s-1, 5 % CO2 (v/v), pH 7.5 and 7 mM nitrate. In addition, we observed that the accumulation of lipid in the microreactor was 1.5-fold faster than in a flask under optimum culture condition. These results show that the microscale approach has the great potential for improving growth and lipid productivity by high-throughput screening of diverse optimum conditions. Keywords Neochloris oleoabundans PDMS-based microreactor Microscale approach Nile red Microalgal culture & Sang Jun Sim [email protected] 1

Department of Chemical and Biological Engineering, Korea University, Seoul 136-713, South Korea

2

Green School, Korea University, Seoul 136-713, South Korea

Introduction Owing to the soaring energy consumption and the increasing carbon dioxide emission by the use of fossil fuels, diverse environmental issues have been encountered. Therefore, renewable energy resources such as biomass and biofuel are needed. Microalgae have been spotlighted as renewable energy resources due to its potential for producing useful materials such as lipids with reduction of CO2 in the air by photosynthesis. In particular, Neochloris oleoabundans can accumulate the lipid contents up to 54 % of its cell dry weight, and triacylglyceride (TAG) used for biodiesel comprises 80 % of total lipid [1]. The lipid contents in N. oleoabundans depend on the environmental and culturing conditions. Therefore, the optimization of diverse culture conditions is essential for improving yields. In previous studies, N. oleoabundans was investigated for producing biodiesel at a pilot scale and culture conditions were optimized to improve the yield of lipid production [2–5]. Almost all studies require intense labor, time and cost because the cultivation is performed in bulk scale. By reducing the volumetric scale, it is possible to easily and rapidly investigate the optimum conditions for high cell density culture and improving lipid productivity. Moreover, it is easy to determine inherent features of strains under diverse conditions in a microscale environment by monitoring the response at the cellular level. A PDMS-based microreactor is suitable for cultivating photoautotrophic cells such as microalgae which require carbon dioxide and light energy for growth [6], because of the transparent and gas permeable features of PDMS [Poly(dimethylsiloxane)]. Further, PDMS has the advantages of being nontoxic for cultivation of microorganisms in PDMS-based microchip [7, 8]. Moreover, it allows

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integrating multiple functions on a chip, which has been demonstrated as micro-total analysis systems (lTAS) [9, 10]. This microscale approach allows high-throughput screening for growth and analysis of useful products such as lipids, carotenoids, and polymers. The microscale approach has been applied to tissue [11] and various organisms including bacterial and mammalian cells for cultivation and biochemical sensing [12–15]. In case of microalgae, several microscale approaches have been applied to cultivation [16–18], high-throughput screening system for oil production [19–21] and lipid extraction [22]. However, it remains a beginning stage for microalgal cells. In this study, we developed a PDMS-based microreactor for the optimization of culture under diverse conditions and the rapid analysis of lipid production. Herein, we demonstrated improved performance microreactor for culturing and lipid analysis compared to the conventional methods. Using the microsystem, we monitored continuously growth and lipid accumulation of cells under diverse culture conditions by changing into nitrogen-deficient medium. In addition, we easily found diverse optimum conditions for cell growth using microplate reader because of design of microreactor. This microscale approach can be applied to various high-throughput screening systems for rapid determination of biomass and lipid productivity in microalgae.

Bioprocess Biosyst Eng (2015) 38:2035–2043

Materials and methods Fabrication of PDMS-based microreactor The PDMS-based microreactor consists of 16 chambers (Fig. 1b) for the multiple detection of growth in diverse conditions and for lipid analysis. It comprises inlet parts for injecting medium and cell solutions, chambers for the culture and lipid induction and outlet parts for the excretion of medium. The gap of chamber in microchip was designed to be same with the 96-well plates to easily measure cell growth and density. The width of microchannel in inlet is 50 lm (Fig. 1d). In the outlet part, 12 microchannels with widths of 2 lm (Fig. 1e) are connected to the chambers to prevent cell leakage and reduce the pressure in the device during the injection of medium. The device was fabricated in three steps, resulting in the 3.5 mm height of the culturing chambers. First, photoresist (SU-8 50, Microchem) was used to make the mold on silicon wafers via photolithography [23]. PDMS was poured into the SU-8 mold to a height of 3.5 mm and cured thermally at 80 °C. We punched holes in the inlet part (radius 2 mm), chambers (4 mm) and outlet part (2 mm). The urethane mold cast was then made using solutions of urethane and a hardening agent [1:1 (v/v)]. After 5 min, the urethane mold was peeled from the PDMS. Finally, PDMS was again poured into the urethane mold and cured in an 80 °C oven. The

Fig. 1 a Schematic depicting the steps for the fabrication of the PDMS-based microreactor. b Image of the microreactor for multiple detection of cell culture under diverse culture conditions. c Schematic diagram of microreactor and microchannel image of d inlet part and e outlet part

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PDMS and cover glass were treated with plasma and then bonded. The microreactor was immediately filled with TAP-C medium. To prevent blockage of microchannel by the cells in device, the flow rate of injecting solutions was less than 100 ll min-1. Algal strain and seed culture condition Neochloris oleoabundans (UTEX-1185, Algae Culture Collection of the University of Texas) was used for the culture and production of lipids. Cells were grown in 250ml flasks containing 100 ml of TAP-C medium at 130 rpm and 23 °C and were aerated continuously with 5 % CO2enriched air. Light was continuously provided by white fluorescent lamps at 40 lmol photons m-2 s-1. All light intensities were measured with an LI-250 quantum photometer (Lambda Instrument Corp., USA). N-source optimization for growth and lipid accumulation using microreactor The composition of the TAP medium was changed to compare the effect of the N-source on microalgae growth. Nitrate (NO3-) and ammonium (NH4?) were used as the N-source in TAP-C medium. To optimize the concentration of the N-source in the medium, cells were cultivated under six different initial concentrations of nitrate and ammonium: 0, 1, 3, 5, 7 and 10 mM. Other conditions were fixed to analyze only the effect of the Nsource by changing initial concentration of nitrogen. Light was provided continuously by 40 lmol photons m-2 s-1. Temperature was maintained at 23 °C. The pH and concentration of CO2 were 6.5 and 5 % (v/v), respectively. Light intensity optimization for growth and lipid accumulation using microreactor The cells were cultivated under three different light intensities to optimize cell growth over 7 days: 20, 40 and 60 lmol photons m-2 s-1. The temperature, concentration of carbon dioxide and the pH of the medium were maintained at 23 °C, 5 % (v/v) and 6.5, respectively. The initial concentration of nitrate in the medium was 7 mM. At the induction stage for lipid accumulation, we investigated neutral lipid contents in cell body using fluorescent dye Nile red under five different light intensity conditions: 20, 40, 60, 80 and 100 lmol photons m-2 s-1. In addition, we changed to nitrogen-deficient medium (TAP-N,C). CO2 concentration was regulated at 7 % (v/v).

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CO2 concentration optimization for growth and lipid accumulation using microreactor To optimize the carbon dioxide concentration for growth, cells were cultivated for 7 days under five different conditions of carbon dioxide concentration: 0.03 (air), 3, 5, 7 and 10 % CO2 (v/v). The temperature and pH of the medium were maintained at 23 °C and 6.5. The light intensity and initial concentration of nitrate were 40 lmol photons m-2 s-1 and 7 mM, respectively. For inducing lipid accumulation of cells in microreactor, we switched over to TAP-N medium. The pH condition and light intensity were maintained at 7.0 and 60 lmol photons m-2 s-1, respectively. Optimization of initial pH in medium using microreactor To optimize the media pH, cell growth was measured under five different pH conditions: 6.5, 7.0, 7.5, 8.0 and 8.5. The different conditions of initial pH in the medium were adjusted using 1 M HCl and 1 M NaOH. The 5 % CO2-enriched air was supplied to the TAP-C culture medium. The light intensity was 40 lmol photons m-2 s-1 and the temperature was maintained at 23 °C. The nitrate (7 mM) was used for the N-source in the TAP-C medium. To analyze the effect of initial pH condition for lipid accumulation, we changed to TAP-N,C medium in microreactor. In addition, CO2 concentration and light intensity were regulated at 7 % (v/v) and 60 lmol photons m-2 s-1. Effect of different reactor system Three different bioreactors (PDMS-based microreactor, 6-well plates and flask) were used to compare the maximum growth and lipid accumulation of N. oleoabundans in optimum culture conditions. The initial concentration of seed culture was adjusted to an optical density (OD680) of 0.1. The light intensity was 40 lmol photons m-2 s-1, and 5 % CO2-enriched air was supplied to the TAP-C medium. All cultures are conducted under high humidity condition in CO2 incubator to prevent evaporation of moisture. The temperature and pH of culture medium were maintained at 23 °C and 7.5, respectively, for 7 days. To induce lipid accumulation, we switched over to nitrogen-deficient medium (TAP-N,C). In induction phase, the light intensity was 60 lmol photons m-2 s-1, and 7 % CO2 was supplied continuously. The initial pH of medium was 7.0. The lipid content was monitored by fluorescence microscope every 24 h during 5 days.

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Analysis for growth and lipid contents Cell growth was monitored every 24 h by measuring optical density at a 680 nm using a microplate reader (BioTek, USA). The shaking duration and amplitude were 10 s and 5 mm, respectively. The specific growth rate was calculated using the Gompertz function [24]. For lipid analysis, the medium of the microreactor was changed to nitrogen-deficient medium (TAP-C,N) using a syringe pump. To analyze lipid content, Nile red stock solution (0.5 mg ml-1 in dimethyl sulfoxide) was put into the chamber using the syringe pump at a flow rate of 50 ll min-1 to avoid cell leakage in cell chamber. Lipid was detected by staining the cells using Nile red. To minimize the Nile red staining of PDMS, we diluted Nile red stock solution to 0.5 lg ml-1 which is possible to lipid staining of cells. After 10 min, the fluorescence intensity was measured at 575 nm using a fluorescence microscope. Spot intensity of lipid in cells was calculated with image analysis software (i-solution, Korea). The intensity denoted the average intensity value of each pixel, where 0 was the minimum and 255 was the maximum as determined by analysis of digital image. Lipid contents were calculated by

Fig. 2 a Growth curve, b maximum growth rate and growth value of cells under different initial nitrate (NO3-) concentrations. c Growth curve, d maximum growth rate and growth value of cells under

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the average of fluorescence intensities of 100 cells in culture. The resulting fluorescence signal data were obtained by subtracting the background signal of the buffer, including Nile red, from the signal of the stained cells.

Results and discussion Effect of N-source and concentration for growth We investigated the effect of N-sources and their concentrations on cell growth. In Fig. 2a and c, the cell growth showed the maximum at the same concentration (7 mM) of nitrate and ammonium. In addition, the lmax and DOD680 were also maximized at a 7 mM concentration (Fig. 2b, d). In particular, the 7 mM nitrate condition of medium induced the maximum lmax (2.13 day-1) and cell density (DOD680 = 4.39) of N. oleoabundans. These results were 1.06-fold and 1.25-fold higher than those of 7 mM ammonium, respectively. Moreover, the maximum growth rate (lmax) and cell density (DOD680) of culture under the nitrate condition for the N-source in medium increased up to the 7 mM concentration of nitrate. Under the nitrate

different initial ammonium (NH4?) concentrations. Data are represented as the mean ± standard deviation (SD) (n = 3)


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culture conditions, both lmax and DOD680 rapidly decreased at nitrate conditions over 7 mM, such as 10 mM of nitrate, as shown in Fig. 2b, d. In the ammonium-based culture case, both the maximum growth rate (lmax) and cell density (DOD680) under the TAP medium with a 10-mM concentration of ammonium were higher than those under the medium with a 5-mM concentration of ammonium. These results show that N. oleoabundans can more easily use nitrate than ammonium for high-density cultivation, which are in agreement with those of previous studies [25]. Effect of light intensity for growth The optimum light intensity was investigated for high-density cultivation. Figure 3a shows that N. oleoabundans reached a maximum growth rate and cell density under 40 lmol photons m-2 s-1. The maximum growth rate (lmax = 2.017 day-1) is 1.76 and 1.63 times higher than those obtained at 20 and 60 lmol photons m-2 s-1, respectively (Table 1). In addition, the maximum cell density (DOD680 = 4.34) of the culture was 1.56-fold and 1.14-fold higher than that under 20 and 60 lmol photons m-2 s-1, respectively. Under light intensity of less than 40 lmol photons m-2 s-1, the light energy was insufficient for cell growth. On the contrary, the light energy was oversupplied at 60 lmol photons m-2 s-1, where 20 % of the cells died from the strong light. Effect of CO2 concentration for growth To optimize the supplied carbon dioxide concentration for cell growth, N. oleoabundans was cultured under five different concentrations of CO2. In the air condition [0.03 % (v/v)] with close to 0 % (v/v) CO2, the growth was lower than in other conditions (Fig. 3b). Up to 5 % (v/v) CO2, the maximum growth rate and cell density were increased to 2.04 day-1 and 4.39 (DOD680), respectively, as shown in Table 1. The lmax and DOD680 reached maximum values at the CO2 concentration of 5 % (v/v). Both values were approximately three times higher than those for cultures under the air condition. These indicated that the viability of N. oleoabundans was hindered at CO2 concentrations above 7 % (v/v) because the pH value of the medium decreased at the excessive CO2 concentration. These results showed that cell growth was inhibited by low pH conditions.

Fig. 3 Growth curves of cells under different a light intensities, b CO2 concentrations and c initial pH conditions of medium. Data are represented as the mean ± standard deviation (SD) (n = 3)

Effect of initial pH in medium for growth The culture results at five different initial pH conditions are shown in Fig. 3c. In the growth phase, N. oleoabundans shows improved growth rate and cell density under alkaline medium conditions compared to those under acidic culture conditions (Table 1). The growth increased with an increasing pH from 6.5 to 7.5, followed by decrease at pH 8.0

(Fig. 3c). These results are consistent with those of earlier researches. The growth of N. oleoabundans shows the optimum growth rate and lipid productivity in an alkaline condition (pH 8.0–9.0) in bulk-scale cultivation by Santos et al. [26, 27]. However, our result indicates that the growth of N. oleoabundans was inhibited by alkalinity of pH 8.0 in microscale, because the cells were easily affected by external

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2040 Table 1 Maximum growth rate and growth value under various light intensities, CO2 concentrations and initial pH conditions of medium


Diverse culture conditions

a

b

lmax

DOD680

Light intensity (lE m-2 s-1) 20

2.8962 ± 0.0442

0.9282 ± 0.0142

1.1440 ± 0.0175

2.7833 ± 0.1102

40

4.4103 ± 0.0674

0.8018 ± 0.0122

2.0168 ± 0.0309

4.3397 ± 0.1593

60

3.9800 ± 0.0608

1.1850 ± 0.0181

1.2315 ± 0.0189

3.7867 ± 0.0866

CO2 concentration (%, v/v) 0.03 (air)

1.5799 ± 0.0241

0.8646 ± 0.0132

0.6700 ± 0.0103

1.4830 ± 0.0587

3

2.4719 ± 0.0378

0.8318 ± 0.0127

1.0896 ± 0.0167

2.3863 ± 0.0936

5

4.4603 ± 0.0681

0.8031 ± 0.0123

2.0363 ± 0.0312

4.3897 ± 0.1593

7

4.4297 ± 0.0677

0.8620 ± 0.0132

1.8842 ± 0.0289

4.3400 ± 0.1656

10

2.8095 ± 0.0429

0.8169 ± 0.0125

1.2610 ± 0.0193

2.7267 ± 0.1050

pH conditions 6.0

1.4495 ± 0.0221

0.7572 ± 0.0116

0.7019 ± 0.0108

1.3500 ± 0.0462

6.5

2.2105 ± 0.0338

0.6141 ± 0.0094

1.3198 ± 0.0202

2.1633 ± 0.1470

7.0

3.3418 ± 0.0510

0.8318 ± 0.0127

1.4730 ± 0.0226

3.2633 ± 0.1650

7.5 8.0

4.4613 ± 0.0681 3.6545 ± 0.0558

0.8031 ± 0.0123 0.7254 ± 0.0111

2.0368 ± 0.0312 1.8472 ± 0.0283

4.3897 ± 0.1593 3.6233 ± 0.0709

Values are the mean ± standard deviation (SD) (n = 3)

Fig. 5 Culture and fluorescence images of N. oleoabundans in growth and induction phase

stimulation by large surface area, permeability and dimension of microreactor compared to bulk scale [28–30]. Fig. 4 a Growth curve, b maximum growth rate and growth value of cells under different volumetric conditions: flask (100 ml), 6-well plate (10 ml) and PDMS-based microreactor (100 ll) at the light intensity of 40 lmol photons m-1 s-2, 5 % CO2 (v/v), pH 7.5 and 7 Mm nitrate. Data are represented as the mean ± standard deviation (SD) (n = 3)

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Growth performance in different reactor system under optimum condition To compare the performance of the microreactor with the conventional culture systems including microplate and


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Fig. 7 Fluorescence intensity of lipid stained using Nile red in a microreactor and a flask at 60 lmol photons m-1 s-2, 7 % CO2 (v/v), pH 7.0

7 days. It shows the advantage of microreactor to shorten the period of growth phase, which is essential for highthroughput screening of optimum culture conditions. In Fig. 4b, the maximum cell density in the microreactor was 4.49 (DOD680). This result was 1.2 and 1.02 times higher than those obtained in the 6-well plates and the flask, respectively. In addition, N. oleoabundans reached a maximum value of lmax of 2.13 day-1 in the microreactor, followed by 1.6 day-1 in the 6-well plates and 1.34 day-1 in the flask. The growth rate of N. oleoabundans in microreactor was 1.58-fold faster than the flask, because the available surface area, mass transfer and permeability of the PDMS-based microreactor are larger and higher than those of the 6-well plates and the flask [28, 29, 31, 32]. The 6-well plates showed faster growth (lmax = 1.60 day-1) in the early phase than the flask (lmax = 1.34 day-1) due to the large surface area and low shading effect (Fig. 4b). However, the maximum cell density (DOD680) of the flask was higher than that of the 6-well plates, because the aeration of the well plate was less than that of the flask. These results showed the advantage of the PDMS-based microdevice for culturing microalgae and the analysis of growth in a short time period. Lipid analysis Fig. 6 Maximum fluorescence intensity of neutral lipid contents using Nile red under diverse culture conditions: a light intensities, b CO2 concentrations and c initial pH conditions of medium. Data are represented as the mean ± standard deviation (SD) (n = 3)

flask, cells were cultivated in three different culture devices including flask, 6-well plate and a PDMS-based microreactor for 7 days. The cell growth reached a maximum value in the PDMS-based microreactor (Fig. 4a). The cells were reached to the maximum growth at 5 days as shown in Fig. 4a. In case of flask, the steady state was reached at

Lipid in the cell body was induced by changing the nitrogendeficient media in the microreactor using a syringe pump. Figure 5 displays the difference in color and fluorescence of the cells during the growth and induction phases. The lipid was detected using fluorescent dye, Nile red, 5 days after changing the media. In addition, we found the optimum conditions for lipid accumulation of N. oleoabundans in a short time using microreactor. In Fig. 6a, the maximum lipid content of cells was reached at 60 lmol photons m-2 s-1. Under the lower

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light intensity less than 40 lmol photon m-2 s-1, the light stress was not strong enough to produce lipid. On the contrary, the light stress at more than 80 lmol photons m-2 s-1 was so strong to affect viability of N. oleoabundans. It was observed 20 % of total cells died by strong light (100 lmol photons m-2 s-1). In Fig. 6b, we observed that lipid accumulation increased as the CO2 concentration increased at CO2 concentration less than 7 %. On the contrary, under more than 7 % CO2 (v/v) concentration, lipid accumulation was reduced by lower pH of medium caused by excessive CO2 supply. Finally, we found that the maximum value of lipid content was achieved at pH 7.0, 2.8-fold and 1.4-fold higher than those obtained at pH 6.5 and pH 8.5 (Fig. 6c). Figure 7 shows that the lipid contents of the different culture system (microreactor and flask) increased linearly as time progressed at the optimum culture conditions; 60 lmol photons m-1 s-2, 7 % CO2 (v/v), pH 7.0. In the microreactor cultivation, we found that the accumulation rate of neutral lipid content was 1.5-fold faster than that in the flask at the same.

Conclusions We developed a PDMS-based microreactor for the rapid and efficient investigation of optimum conditions for cell growth and lipid production continuously by changing into the nitrogen-deficient medium in microchip. With the large surface area and high permeability, we confirmed that microreactor has strong advantages for research of microalgae by significantly shortening culture time and allowing simple and rapid monitoring of cell growth and accumulation of lipid comparing the other culture systems; 6-well plates and flask. With these advantages, we optimized culture conditions for improving growth rate and accumulation of lipid in considerably less time. These results show the great potential of microreactor system for improving growth and lipid accumulation by highthroughput screening of diverse optimum conditions. Acknowledgments This study was supported by grants (2014M1A8A1049278) from Korea CCS R&D Center of the NRF funded by the Ministry of Science, ICT, and Future Planning of Korea, the National Research Foundation of Korea (NRF) grants (Grant No. NRF-2013R1A2A1A01015644/2010-0027955), the Korea Institute of Energy Technology Evaluation and Planning and Ministry of Trade, Industry and Energy of ‘‘Energy Efficiency and Resources Technology R&D’’ project Korea (20152010201900) and UniversityInstitute Cooperation Program (2013).

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