Evaluation of Cell-Viability, Intracellular Lipid-Component and Efficiency of Lipid-Extraction of Chlamydomonas reinhardtii Cells Treated by UV-C Irradiation Aiming to Use Cell Directly

Research Article

Austin J Biotechnol Bioeng. 2021; 8(1): 1108.

Evaluation of Cell-Viability, Intracellular Lipid-Component and Efficiency of Lipid-Extraction of Chlamydomonas reinhardtii Cells Treated by UV-C Irradiation Aiming to Use Cell Directly

Nakanishi A1,2*, Ozawa N†1, Watanabe M2 and Sakihama Y3

¹Graduate School of Bionics, Tokyo University of Technology, Hachioji, Japan

²School of Bioscience and Biotechnology, Tokyo University of Technology, Hachioji, Japan

³Tokyo University of Technology, Hachioji, Japan

Co-first author

*Corresponding author: Nakanishi A, Graduate School of Bionics, Tokyo University of Technology, 1404-1 Katakuramachi, Hachioji, Tokyo, Japan; School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1, Katakuramachi, Hachioji, Tokyo, Japan

Received: March 20, 2021; Accepted: April 20, 2021; Published: April 27, 2021


Aim: We aimed finally to construct a system to utilize intracellular lipids of Chlamydomonas reinhardtii by direct cell-use. To realize the system, a system of simple cell-sterilization to avoid environmental contamination without degradation of intracellular lipids was required. Industrially, a simple collecting system of internal lipids was also required.

Methods and results: C. reinhardtii cultured in a photo bioreactor under autotrophic condition was irradiated by UV-C. After the irradiation with different time to cells under different culturing conditions, those cells were evaluated for cell-viability by staining with neutral red, intracellular cell components and efficiency of lipid-extraction with GC/FID, respectively. By UV-C irradiation for 10 min, C. reinhardtii cells after N-depletion were sterilized. Additionally, although the cells were morphologically crumbled under an optical microscope, the contents and the components of intracellular lipids showed few differences.

Conclusion: C. reinhardtii cells were efficiently sterilized by UV-C irradiation and few treated cells leaked the intracellular lipids, indicating that the lipids could not be simply collected by centrifuging direct cell collection.

Significance and impact of study:

In this study, the sterilized cells could gradually leak the intracellular contents, indicating the possibility of direct-use of the cells to utilize lipids produced by C. reinhardtii.

Keywords: Green alga; Algal lipids; UV-C treatment; Direct cell-use


GC/FID: Gas chromatography/flame ionization detector; N-depletion: Nitrogen depletion; OD: Optical Density; PBR: Photo- Bioreactor; SEM: Scanning Electron Microscope; UV: Ultraviolet; vvm: Volume per Volume per Minute


In recent years, bio-production by photosynthetic plants has attracted attention because of the effectiveness of plant-derived components such as vitamins and oils, carbon recyclability and low environmental burden [1,2]. Regarding bio-production by plants, the material productivity is calculated by the product of biomass productivity and content of intracellular material [3], resulting in enhanced biomaterial productivity with high biomass productivity. Relating to biomass productivity, several green algal strains show superior biomass productivity with higher carbon fixation efficiency more 10-50 times than popular terrestrial plants [4] for instance, Chlamydomonas sp. JSC4 and Dunaliella salina exhibited high biomass productivity as 915mgL-1d-1 [5] and 540mgL-1d-1 [6], respectively. Depending on the high biomass productivity, green algae Coccomyxa subellipsoidea and Haematococcus pluvialis demonstrated high material productivity of 232.37mgL-1d-1 of lipid [7] and 17.1mgL-1d-1 of astaxanthin [8]. Among the green algae, the unicellular green alga Chlamydomonas reinhardtii is especially paid attention in industrial use in recent years [9] because of accumulation of the information and technology of culturing condition [10], metabolic flow [11] and safety [12] regarding to this strain so far as a model green algae. Particularly, the proven safety of C. reinhardtii is an advantageous property for the use of its metabolites as value-added products in the industrial fields of cosmetics and foods. C. reinhardtii is able to accumulate approximate 10~20 % lipid (e.g., linoleic acid, lauric acid, stearic acid) as its cell component [13,14] so those lipids are expected to be used for moisturizing in the cosmetic field [15]. The use of lipids as the cell component, however, faces the problem of cost to extract lipids from the cells because of the robustness of the cell [16]. To extract lipids from the cell, C. reinhardtii needs strong crashing systems such as a bead-beater system [17]. Thus, research and development simply to use intracellular lipids could lead to an increase of the value of lipids derived from C. reinhardtii as a commercial use. Then, our study aimed to construct the system directly to use the cells for simple utilizing the intracellular lipids without the process of lipid purification based on non-toxicity [12]. The system, however, could be useful for industrial use; the direct use of living cells as the protector of intracellular metabolites could possibly cause contamination in the environment after releasing those cells to the environment [18], requesting a treatment simultaneously for supporting the efficient lipid-extraction and killing the cell. So far, the treatments using Ultraviolet (UV) irradiation have be considered [19], and the report revealed that the treatment using UV-B (280~320 nm) triggered the easy lipid-extraction because of necrosis and apoptosis of C. reinhardtii. However, the report focused on the extraction of intracellular lipids not aiming for complete cell death and does not evaluate the extraction efficiency relating nitrogen depletion in detail. Therefore, the treatment using UV irradiation needs more evaluations and improvements simply to use intracellular lipids by direct cell-use. On the other hand, although there are the reports relating the efficiency of sterilizing C. reinhardtii cells by the UV-C treatment, those did not refer to the efficiency of lipidextraction [20,21]. Therefore, the system-construction to use cells directly for the efficient use of intracellular lipids by UV treatment could be drastically meaningful on industrial use.

In this study, C. reinhardtii containing lipids was treated by UV-C irradiation, and the cell viability was evaluated by using the staining method with neutral red. Simultaneously, the production and composition of lipids in the treated cells were analyzed to evaluate the effect of UV-C treatment using GC/FID. The use of lipids from the UV-C treated C. reinhardtii cells could lead the progression of industrially green-algal use.

Materials and Methods

Microalgal strain and PBR operation

Chlamydomanas reinhardtii strain C-9: NIES-2235 was cultivated in a photobioreactor filled with Modified Bold 3 N medium (MB3N) as described in a previous study [22]. The photobioreactor was equipped with white fluorescent lamps (100μmol photons·m-2·s-1) and a bubbling system of 0.8% CO2 gas at an aeration rate of 0.05vvm and worked at room temperature (23°C).

Evaluation of culturing condition

Cell growth was evaluated as cell numbers using value of Optical Density (OD) of 750nm with a spectro-photometer U-2900 (Hitachi, Tokyo, Japan) via appropriate calibration curve for OD750 versus cell numbers. The pH of the broth was measured with pH meter FEP20 (Mettler Toledo, Tokyo, Japan) after centrifugation at 5000×g for 1min at 23°C. Nitrate concentration was measured using an optical method. The broth was centrifuged at 5,000×g for 1 min at 23°C, and the supernatant was collected and filtered with a 0.45μm filter (Millex®-LCR 13mm, Millipore, Carrigtwohill, Ireland). The flow through was diluted 50-fold with distilled water, and the absorbance of the diluted supernatant was measured at 220nm (i.e., Abs220) using a spectro-photometer U-2900. The residual nitrate content was evaluated using an appropriate calibration curve for Abs220 versus nitrate concentration.

Evaluation of cell viability after UV-C treatment

Cells (5.0×105 cells) were irradiated in 2mL of phosphate buffered saline (PBS) (pH=7.4) on a 33mm diameter glass share by using a UV irradiator Etbella (SEVEN BEAUTY Co., Ltd., Tokyo, Japan) equipped with a UV-C lamp (the ultraviolet radiation output: 3.49mWcm-2). One hundred eighty μL of the UV-treated suspension (4.5×104 cells) were mixed with 20μL of neutral red (red pigment) (Tokyo Kasei Co., Ltd., Tokyo, Japan) for 5 minutes [23]. The neutral red solution (1.5mgmL-1) was prepared as below, 1.5mg of neutral red was dissolved in 1ml of PBS (pH=7.4); the solution was filtered with 0.22μm filter (Nylon Syringe Filter, Membrane-Solutions). After the staining process, the cells were washed with PBS, and the viability was evaluated by observing the stain-treated cells on cell counter plates (Fukae Kasei Co., Ltd, Kobe, Japan). The life/death of the cells was decided with the criterion: staining/not staining with neutral red.

Evaluation of intercellular contents of chlorophyll a/b after UV-C treatment

Cell-weights were evaluated for constant irradiation of UV-C to the cells. The cell-concentration in the broth was measured by using a calibration curve of OD750-weight, and then cells (0.1-0.5 mg) were collected from the broth by centrifugation at 5,000×g for 3min at 23°C. After discarding the supernatant, 1mL of methanol was added and vigorously stirred using putiburu MODEL 2330 (Waken B tech Co., Ltd, Kyoto, Japan) at 2,000rpm for 5min at 23°C. The solution containing chlorophyll a/b was collected by centrifugation at 5,000×g for 3min at 23°C. To evaluate the concentration of chlorophyll a/b, the values of A665 and A652 of the collected solution were measured after drawing a baseline with A750 using methanol with a spectrophotometer U-2900 and substituted in the formula as below, chlorophyll a (μgmL-1): 16.72 × A665 - 9.16 × A652; chlorophyll b (μgmL-1): 34.09 × A652 - 15.28 × A665 [24]. The contents of chlorophyll a/b in the cells were finally shown using the unit μgmg-1.

Preparation for analysis of lipid-extraction efficiency

One mg of cells was harvested from broth after 3 days of nitrogen depletion by centrifugation (5000×g, 5min, 23°C) and treated by following 3 steps of cell-breaking processes as below: 1) UVtreatment step, 2) glass beads-adding step, and 3) shaking step. In the 1) step, the cells were UV-irradiated for 60min on a 33mm diameter glass share with a UV irradiator Etbella (SEVEN BEAUTY Co., Ltd.). In the 2) step, the glass beads of 0.5mm were added into the cells. In the 3) step, the cells were vortexed at 23°C under the condition (2,500rpm, 60min) with putiburu MODEL 2330 (WakenBtech Co., Ltd, Kyoto, Japan). After those processes, the treated cell-components were dried with an evaporator and weighted in detail.

Evaluation of lipid-quantity and profile

The total lipids were esterified and methylated with fatty acid methylation kit (Nacalai Tesque, Kyoto, Japan). The fatty acid methyl esters were identified and quantified using a capillary gas chromatograph GC-2025 (Shimadzu, Kyoto, Japan) equipped with a DB-23 capillary column (60m, 0.25mm internal diameter, 0.15μm film thickness) (Agilent Technologies, CA, United States) with method previously described by Nakanishi et al. [13]. Heptadecanoic acid (Sigma-Aldrich Co., MO, US) was used as an internal standard, and rapeseed oil (Merck KGaA, Darmstadt, Germany) was used as a quantitative standard.

Results and Discussion

This study aimed to construct a practical system that could easily use intracellular lipids by directly using the cells of C. reinhardtii. First of all, the time-course profile of C. reinhardtii under each culture condition was evaluated (Figure 1). In this study, C. reinhardtii was cultured in autotrophic MBBM, resulting that the biomass production was 248 ± 20 mgL-1 at 96h (4d) of culture. So far, the abundant information of biomass production of C. reinhardtii cultured in various media has shown various data: around 700mgL- 1 in 4d of culture in a well-growing heterotrophic TAP medium; around 150mg in an autotrophic HSM medium [25], indicating that the autotrophic culturing system in this study works well. Although the nitrogen depletion was shown at 4d, the biomass production constantly increased and finally reached 1.8 ± 0.3gL-1 in 13d (9d of nitrogen depletion (9d N-depletion)). Additionally, the results also indicated that the cultural conditions at 4d (0 d N-depletion) and 13d (9 d N-depletion) showed initial and late log-phase in this study, respectively (i.e., log-phase: 4 ~ 13 d).