RIKEN Accel. Prog. Rep. 48 (2015)

Ⅲ-4. Radiation Chemistry & Biology

Effects of Ar-ion beam irradiation on survival rate of Aurantiochytrium sp.
K. Ikeda,*1 R. Kose,*1 T. Okuda,*1 Y. Sakakura,*1 K. Kaya,*1 Y. Kazama,*2 and T. Abe*2

Growth rate ( hour⁻¹ ) = ln( X₁ / X₀ ) / ( t₁ - t₀ )where X₁ and X₀ are cell densities ( cells/mL ) at times t₁and t₀, respectively. During the primary screening, 22 and12 mutants were selected from the SYLR6#3 and NB6-3strains, respectively. However, the characteristic features ofthe selected mutants in terms of their colors and highgrowth rates completely disappeared.
The effects of other heavy-ion beams on cells are being
investigated.

1501005004060
Dose ( Gy )Fig.1 Survival curves of Aurantiochytrium sp.

*1*2

Sea Act Co. Ltd.
RIKEN Nishina Center

Strains

Survival rate (%) = (number of colonies) / (cellnumber before irradiation) x 100
The survival rates of both strains rapidly decreased with adose of 20 Gy (Fig.1). The colony-forming cells wereisolated from the agar plates and suspended in the sterilized
GTY medium. The cell suspension was transferred to thecell-culture plates with 96 wells for primary screening. Celldensity was measured using the iMark Microplate Reader(BIO RAD) at 650 nm. During the primary screening, basedon the colony color and a higher growth rate, mutant cells
were selected and transferred to test tubes for secondary
screening. The colonies of Aurantiochytrium with high lipid
contents showed cells also show orange to brown color.
Therefore, to select mutants with high lipid contents, darkorange to brown colonies were picked out from the various
mutated colonies.
During the secondary screening, the selected cells werecultured in test tubes with reciprocal shaking at 25˚C for 48
h. The cell growth rate was measured using packed cellvolume (PCV, %) and was converted to cell number.
Growth rate was calculated using the following equation:SYLR6#3
NB6-3

Survival rate（％）

The organisms that belong to the Aurantiochytrium
sp. are heterotrophic marine thraustochytrids with a highgrowth rate. They produce many bioactive compounds
such as squalene, docosahexanoic acid ( DHA, C22:6n-3 ) andodd-numbered fatty acids, and they are being improved interms of their cellular cell functions for industrial
utilization1 , 2).
To introduce novel cell functions, irradiation with a swift,
heavy-ion beam to induce mutations is an effective
method3). At RIKEN RI Beam Factory (RIBF), microbial
cells and plant seeds are irradiated with heavy-ion beams
such as of carbon and argon (Ar).
In this study, Aurantiochytrium spp.NB6-3 and SYLR6#3
were selected because of their short lag times and industrialusage. The strains were exposed to accelerated Ar ions witha dose range of 20 Gy to 80 Gy. After irradiation, cells werecultured on GTY medium (2% glucose, 1% Tryptone, 0.5%
yeast extract and 1.8% Red Sea Salt)-containing agar plates(90mm) at 25˚C for 48 h. The colonies were counted
using the colony forming unit (CFU) to estimate theirsurvival rates and were isolated for mutant screening.
Values were expressed as averages of duplicate CFU
experiments. Survival rate was obtained using the formula:80-4980-3680-2660-56
60-48
60-46
60-44
60-43
60-42
60-41
60-13
40-54
40-53
40-50
40-39
40-30
40-21
40-20
40-17
20-32
20-14
20-7
Wild

020

(SYLR6#3)

0.00

80

(NB6-3)
80-45

48h
80-37
96h
80-30

48h
96h

80-22
80-21
80-14
60-51
60-50
60-37
60-24
60-7
Wild

0.05
0.10
Growth rate (hour⁻¹)

0.00

0.05
0.10
Growth rate (hour⁻¹)

Fig.2 Growth rates of isolated strains estimated during
the secondary screening
References1) A. N. Jakobsen et al. : Appl Microbiol Biotechnol. 80 297-306(2008).
2) Q. Li et al. : Agric. Food Chem., 57, 4267-4272 (2009)3) A. Tanaka et al. : J. Radiat. Res., 51, 223-233 (2010)- 310 -完全版2014_本文.indd 310

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