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Selection and characterization of Euglena anabaena

var. minor as a new candidate Euglena species for
industrial application
ab a a bc d
Kengo Suzuki , Sharbanee Mitra , Osamu Iwata , Takahiro Ishikawa , Sueo Kato & Koji
ab
Yamada
a
Research & Development Department, euglena Co., Ltd., Tokyo, Japan
b
JST, CREST, Tokyo, Japan
c
Life and Environmental Science, Shimane University, Shimane, Japan
d
Faculty of Human Development, Kokugakuin University, Tokyo, Japan
Published online: 19 May 2015.

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To cite this article: Kengo Suzuki, Sharbanee Mitra, Osamu Iwata, Takahiro Ishikawa, Sueo Kato & Koji Yamada (2015):
Selection and characterization of Euglena anabaena var. minor as a new candidate Euglena species for industrial
application, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1045828

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 Bioscience, Biotechnology, and Biochemistry, 2015
Selection and characterization of Euglena anabaena var. minor as a new
candidate Euglena species for industrial application
Kengo Suzuki1,2, Sharbanee Mitra1, Osamu Iwata1, Takahiro Ishikawa2,3, Sueo Kato4 and
Koji Yamada1,2,*
1
Research & Development Department, euglena Co., Ltd., Tokyo, Japan; 2JST, CREST, Tokyo, Japan; 3Life and
Environmental Science, Shimane University, Shimane, Japan; 4Faculty of Human Development, Kokugakuin
University, Tokyo, Japan
Received February 20, 2015; accepted April 13, 2015
http://dx.doi.org/10.1080/09168451.2015.1045828
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Euglena gracilis is a microalgae used as a model
organism. Recently, mass cultivation of this species
has been achieved for industrial applications. The
genus Euglena includes more than 200 species that
share common useful features, but the potential
industrial applications of other Euglena species have
not been evaluated. Thus, we conducted a pilot
screening study to identify other species that
proliferate at a sufficiently rapid rate to be used for
mass cultivation; we found that Euglena anabaena
var. minor had a rapid growth rate. In addition, its
cells accumulated more than 40% weight of carbo-
hydrate, most of which is considered to be a eugle-
noid specific type of beta-1-3-glucan, paramylon.
Carbohydrate is stored in E. anabaena var. minor
cells during normal culture, whereas E. gracilis
requires nitrogen limitation to facilitate paramylon
accumulation. These results suggest the potential
industrial application of E. anabaena var. minor.
Key words: Euglena anabaena var. minor; Euglena
gracilis; paramylon
In general, microalgae exhibit more efficient photo-
synthesis than terrestrial plants, thereby facilitating the
culture of microalgae as alternative sources of food,
biomass, and materials for producing fuel.1,2) Many
microalgae, such as Chlorella, Spirulina, and
Dunaliella, are cultivated on a large scale and used in
industrial applications.3) A photosynthetic protist spe-
cies, Euglena gracilis, is also employed in the indus-
trial applications of microalgae. E. gracilis was
originally used as a model organism in basic research,
especially investigations of photosynthesis, but studies
showed that E. gracilis stores a good balance of nutri-
ents.4–6) E. gracilis cells are rich in protein, which
includes high rate of methionine, a characteristic of ani-
mal proteins, and all essential vitamins are synthesized
or incorporated in the cells. In addition, the E. gracilis
cells include nutritionally important fatty acids, DHA,
*Corresponding author. Email: yamada@euglena.jp
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
docosahexaenoic acid, and EPA, eicosapentaenoic acid.
Therefore, mass cultured cells were considered to be
suitable for use as a nutritional food source. In addi-
tion, Euglena species are known to store a specific type
of beta-1-3-glucan called paramylon.7) Paramylon
possesses a multiple functionality represented by
immunostimulatory activity (unpublished); its produc-
tion has increased the suitability of Euglena for
industrial applications.
E. gracilis has been used as a model organism for
decades, so its culture system is well established. The
mass cultivation of E. gracilis can be achieved using
the basic information available. In contrast, little is
known about the culture of other Euglena species. The
genus Euglena includes more than 200 species, and
many appear to have specific characteristics.8) For
example, E. sanguinea produces a highly valuable
carotenoid compound called astaxanthin,9) while
E. mutabilis is known to tolerate very low pH and
arsenic stress,10) and some Euglena species are known
to propagate in salt water.11) Thus, each Euglena spe-
cies has specific characteristics, but paramylon particles
can be observed in the cells of each species, and some
of them should also possess the capacity to produce a
wax ester, which is a fascinating characteristic of
E. gracilis.7)
The prerequisites for the growth of E. gracilis were
studied comprehensively decades ago. E. gracilis
assimilates ammonia nitrogen rather than nitrate nitro-
gen.12) It can utilize various carbon sources for growth
enhancement, and it requires vitamin B1 and B12 for
proliferation.13) Based on this information, the culture
media were optimized intensively for E. gracilis. As a
result, a basic autotrophic culture medium, i.e. Cramer–
Myers (CM) medium,12) and a high-yield heterotrophic
culture medium, i.e. Koren–Hutner (KH) medium,14)
were formulated and they have been used widely. In
contrast to the specialized culture media used for E.
gracilis, other Euglena species are maintained in versa-
tile media, which support the proliferation of a wide
range of algae. To broaden the scope of industrial
 2 K. Suzuki et al.
applications of Euglena, it would be beneficial to con-
duct screenings to identify the valuable species and to
optimize their culture conditions. In this study, we
screened some of the available Euglena strains to iden-
tify the species that can proliferate at a sufficiently
rapid rate, and we evaluated more appropriate culture
conditions for the screened strain.
Materials and methods
Euglenoid strains and maintenance. The strains
used in this study were isolated from ponds and rivers
in various regions of Japan, except for the E. gracilis
strain NIES-48, which was provided by NIES, JAPAN.
The isolated strains were identified as E. anabaena var.
minor, E. clara, E. deses, E. granulata, E. schmitzii,
and E. stellata based on careful observations using dye
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staining. Although the details will be reported else-
where, E. anabaena var. minor was identified by the
consistency with the original description, in which E.
anabaena var. minor was described as fusiform cells
under natural condition, 36–43 μm long, 9–12 μm
wide, eight chloroplasts, and pyrenoids covered with
saucer-shaped paramylon sheath.15) The cell size of our
strain was 35–46 μm long and 10–14 μm wide with 5–
8 chloroplasts in the log growth phase. The other vari-
ant species of E. anabaena could be distinguished by
the cell size. Under natural condition, E. anabaena var.
anabaena is 88–94 μm long, 20–25 μm wide, and E.
anabaena var. minima is 26–30 μm long, 9–12 μm
wide. The 16S and 18S rDNA sequences were also
determined to confirm that this E. anabaena var. minor
strain is closely related to other E. anabaena strains
(Supplemental Figure 1).
All strains except E. gracilis were maintained in the
AF6 medium at 23 °C with a 14:10 h light:dark cycle
(50 μmol photons/m2s). E. gracilis was maintained in
the CM medium (pH 3.5)12) at 26 °C with a 14:10 h
light:dark cycle (50 μmol photons/m2s).
Culture media. The culture media used for the
maintenance of the cells and in the growth rate tests
were CM,12) KH,14) AF6,16) BG-11,17) C,18) mAC,19)
and TAP20) (supplemental Table 1). The TAP medium
does not include vitamin B1 and B12, which are essen-
tial for the growth of E. gracilis, so those vitamins
were supplemented at the same concentration as that
used in the AF6 medium. The CM and KH media were
prepared at pH 5.5 unless stated otherwise.
Growth tests. We used 24-well tissue culture test
plates (TPP) for small-scale culture. The plates were
placed statically in an incubator set at 23 °C and
equipped with lighting under a 14:10 h light:dark cycle
(50 μmol photons/m2s). Larger scale cultivation tests
were performed using 100 mL capacity large test tubes
that contained 40 mL culture media. During culture,
150 μmol photons/m2s of constant light illumination
was provided, and the culture was aerated with aseptic
air that included 5% of CO2. The growth rate was
evaluated on the basis of the optical density at a wave-
length of 680 nm (OD680) using a microplate reader
(SH-1200, Corona electric) and a spectrophotometer
(UVmini-1240, Shimadzu) for the 24-well plate and
large test tube cultures, respectively.
Quantification of carbohydrates. The harvested
algal cells were dried in a freeze dryer (FDV-1200,
EYELA), and the deproteinized carbohydrate compo-
nent was extracted as previous report.21) Approximately
10 mg of dried algal cells was suspended in 10 mL of
acetone, and the cells were homogenized twice for 90
s, each time using a sonicator (UD-201, TOMY), and
the extract was collected by centrifugation (5810R,
Eppendorf, 800 g, 5 min). The extract was then boiled
for 30 min in 10 mL of 1% sodium dodecyl sulfate
aqueous solution and washed two times with 10 mL of
0.1% sodium dodecyl sulfate aqueous solution and
water, in order. The extracted carbohydrate was quanti-
fied using the phenol–sulfuric acid method.22)
Results
Screening for rapidly growing Euglena species
To evaluate the growth rate of Euglena species other
than E. gracilis, we selected six strains of Euglena spe-
cies, i.e. E. anabaena var. minor, E. clara, E. deses, E.
granulata, E. schmitzii, and E. stellata, and assessed
their growth. The AF6 medium16) was used to maintain
most of the Euglena strains in our laboratory because it
can sustain the growth of a variety of algal species.
However, it was not clear whether AF6 would be the
best medium for the growth of each strain. To deter-
mine the growth rate of each strain, we tested several
typical types of algal culture media, i.e. AF6,16)
mAC,19) BG-11,17) TAP,20) and C18) as well as culture
media optimized for E. gracilis, i.e. CM12) and KH.14)
E. gracilis is tolerant to low pH and is usually cultured
at pH 3.5, but the other Euglena species tested in this
study did not exhibit growth at this pH (data not
shown). Therefore, in our trials, the CM and KH media
were adjusted to pH 5.5. Among the media tested,
mAC and KH include glucose and various other carbon
sources so they could support heterotrophic growth,
whereas the other media contained no carbon sources
or low levels, thereby indicating that the cells prolifer-
ate mainly in autotrophic conditions.
Each strain was cultured in triplicates at 23 °C in
1 mL of medium on 24-well plates and the growth
rates of cells were evaluated by determining the OD680
of each well (Fig. 1). In contrast to E. gracilis, which
grew well in the CM and KH media (Fig. 1(A)), the
other strains did not grow well in these media
(Fig. 1(B)–(G)). The sole exception to this was E.
schmitzii, which grew well in the KH medium
(Fig. 1(F)). Although E. granulata exhibited a low
level of growth in the KH medium, all descendants
died after prolonged culture (Fig. 1(E)). E. anabaena
var. minor, E. deses, and E. stellata exhibited the most
rapid growth in the TAP medium (Fig. 1(B), (D), and
(G)), E. granulata exhibited the most rapid growth in
the C medium (Fig. 1(E)), and E. clara grew the best
in the mAC medium (Fig. 1(C)). Some of the strains
expressed medium-specific phenotypes in TAP and C
media. E. anabaena var. minor and E. deses turned fat
 A screen for industrially applicable Euglena species 3

(A) E. gracilis (B) E. anabaena var. minor

2.5 1
0.9
2.0 0.8
0.7

O.D. 680
O.D. 680

1.5 0.6
0.5
1.0 0.4
0.3
0.5 0.2
0.1
0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
days days

(C) E. clara (D) E. deses

0.18 0.35
0.16
0.30
0.14
0.25
O.D. 680

O.D. 680
0.12
0.10 0.20
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0.08 0.15
0.06
0.10
0.04
0.02 0.05
0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
days days

(E) E. granulata (F) E. schmitzii

0.09 0.30
0.08 0.25
0.07
0.20
O.D. 680
O.D. 680

0.06
0.05 0.15
0.04
0.03 0.10
0.02 0.05
0.01
0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
days days

(G) E. stellata
0.35
0.30
0.25
O.D. 680

0.20
0.15
0.10
0.05
0
0 2 4 6 8 10 12 14 16
days

Af6 BG11 C TAP

mAC CM KH

Fig. 1. Growth of Euglena strains in basic culture media.

Notes: (A–H) Growth curves of Euglena strains: E. gracilis (A), E. anabaena var. minor (B), E. clara (C), E. deses (D), E. granulata (E), E.
schmitzii (F), and E. stellata (G). The cultures were incubated at 23 °C in the AF6, BG-11, C, TAP, mAC, CM, or KH media. The starting cell
concentration was adjusted between 0.01 and 0.02 (OD680) for each condition. Culture was conducted in three wells for each condition. The error
bars indicate the SEM based on the three wells.

and dark in color in the TAP medium, while their the growth rate of E. anabaena var. minor was one-half
movement was accelerated in the C medium. To com- to one-third of that of E. gracilis in the CM medium.
pare the growth rates of E. gracilis and the other spe-
cies, we determined the growth curves for E. gracilis
in the CM medium (pH 5.5) and for the other species Optimization of the culture conditions for E.
in the C medium (Fig. 2). The growth of E. gracilis in anabaena var. minor
the CM medium was the most rapid. Among the spe- E. gracilis has been reported to exhibit rapid growth
cies other than E. gracilis, E. anabaena var. minor at 29 °C.23) Because E. clara, E. granulata, and E.
exhibited by far the fastest growth. In the C medium, schmitzii were relatively sensitive to high temperatures,
 4 K. Suzuki et al.
1.8 poor growth due to heat stress. Therefore, the succes-
1.6 sive experiments were performed at 29 °C.
In the culture media trials (Fig. 1), E. anabaena var.
1.4
minor exhibited the fastest growth in the TAP medium,
1.2 although the cultured cells were large and darkly colored
O.D. 680

1.0 in appearance. These results suggest that the TAP med-

0.8
ium may have specific effects on E. anabaena var. minor
cells. To further understand the effects of the TAP med-
0.6
ium, we compared the growth of E. anabaena var. minor
0.4 in the C, TAP, and equivalent mixtures of C and TAP
0.2 media (Fig. 4). These experiments were performed in
100 mL test tubes that contained 40 mL of each medium,
0
0 2 4 6 8 10 12 14 16 and the OD680 was recorded every day. The equivalent
days mixtures of C and TAP media supported similar rapid
growth rates to the TAP medium alone. Furthermore, the
E. anabaena var. minor
E. gracilis
appearance of the cells cultured in the mixture was the
E. clara
E. deses E. granulata same as that in the TAP medium, i.e. large and dark in
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E. schmitzii E. stellata
Fig. 2. Comparison of the growth of Euglena species.
Notes: Growth curves of E. anabaena var. minor, E. clara, E.
deses, E. granulata, E. schmitzii, and E. stellata in the C medium,
and the growth curve of E. gracilis in the CM medium (pH 3.5).
Each culture was incubated at 23 °C in 24-well plate. The starting
cell concentration was adjusted between 0.01 and 0.02 (OD680) for
each condition. Culture was conducted in three wells for each condi-
tion. The error bars indicate the SEM based on the three wells.
color, thereby suggesting that the cell phenotype in the
TAP medium was related to a specific ingredient in the
medium rather than the lack of some elements. We also
compared the growth of E. anabaena var. minor and E.
gracilis in the CM medium (pH 3.5) using the same cul-
ture conditions except for the culture media (Fig. 4). E.
anabaena var. minor could exhibit a growth rate about
half of E. gracilis in the 40 mL scale culture.

Carbohydrate content of E. anabaena var. minor

such as 29 °C, we tested the culture media (Fig. 1) in
an incubator set at 23 °C. To further improve the
growth of E. anabaena var. minor, its optimum growth
temperature was evaluated. Using 40 mL of the AF6
medium in 100 mL test tubes, the cells were cultured
at 20, 23, 26, 29, and 32 °C, and the OD680 was
recorded each day (Fig. 3). Among the temperatures
tested, the cells cultured at 29 °C exhibited the fastest
proliferation, whereas those tested at 32 °C exhibited
In the culture trials performed in large test tubes,
most E. anabaena var. minor cells settled after a speci-
fic period of time without aeration (Fig. 5(A)). In con-
trast, the E. gracilis cells did not sink within 60 min.
The rapid sinking of E. anabaena var. minor cells may
be attributable to their high carbohydrate content. Espe-
cially, paramylon is the most plausible polysaccharide
reserve in euglenoids. The particle density of paramy-
lon and other carbohydrates are approximately 1.5,24)

2.5
1
2.0
0.9
0.8
O.D. 680

1.5
0.7
0.6 1.0
O.D. 680

0.5
0.5
0.4
0.3 0
0 1 2 3 4 5 6 7 8 9
0.2
days
0.1
C (E. anabaena var. minor)
0
0 1 2 3 4 5 6 7 8 TAP (E. anabaena var. minor)
days C&TAP (E. anabaena var. minor)
CM3.5 (E. gracilis)
20°C 23°C 26°C
29°C 32°C Fig. 4. Growth curve of E. anabaena var. minor in the optimized
cell culture conditions.
Fig. 3. Optimum temperature for the growth of E. anabaena var. Notes: Cultures of E. anabaena var. minor were incubated at 29 °C
minor. in 100 mL test tubes with 40 mL of the C, TAP, or an equivalent
Notes: Growth curves of E. anabaena var. minor at 20, 23, 26, 29, mixture of C and TAP medium. For comparison, a growth curve of
and 32 °C. Each culture was incubated in 100 mL test tubes with E. gracilis in 40 mL CM medium (pH 3.5) is shown (at 29 °C). The
40 mL of the AF6 medium. The starting cell concentration was starting cell concentration was adjusted to around 0.03 (OD680) for
adjusted to around 0.03 (OD680) for each condition. Culture was each condition. Culture was conducted in triplicates. The error bars
conducted in triplicates. The error bars indicate the SEM. indicate the SEM.
 A screen for industrially applicable Euglena species 5

(A) 0 min. 10 min. 30 min. 60 min.

60 min.
gr an gr an gr an gr an upward angle

gr an

(B) C medium (C) TAP medium (D) C + TAP (E) TAP (Crushed flatly)
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(F) 50
45
40
carbohydrate (%)

35
30
25
20
15
10
5
0
CM

CM (-N)

TAP

C + TAP

E. gracilis E. anabaena var. minor

Fig. 5. Carbohydrate content of E. anabaena var. minor.

Notes: (A) Photographs showing the rapid sedimentation of E. anabaena var. minor cells. Photographs of cells cultured in 100 mL test tubes at
10, 30, and 60 min after agitation. At 60 min after agitation, a photograph captured from below is also shown where the sedimented E. anabaena
var. minor cells can be observed. “gr” and “an” indicate E. gracilis and E. anabaena var. minor, respectively. (B–D) Micrographs showing cells
cultured in the C medium (B), TAP medium (C), or an equivalent mixture of C and TAP media (D) for a week. Panels at the bottom are the
magnified images of a cell in each medium. Each cell includes paramylon-like particles. A representative particle is indicated by an arrowhead in
each cell. (E) Micrograph of cells cultured in the TAP medium, which were crushed by sandwiching between a glass slide and a cover glass. Many
paramylon-like particles can be observed. Scale bars: 10 μm. (F) Carbohydrate contents of E. anabaena var. minor and E. gracilis. One-week cul-
tures of E. gracilis in the CM medium (pH 3.5) with or without nitrogen restriction (−N), and E. anabaena var. minor in the C, TAP, or an equiva-
lent mixture of C and TAP medium were used. The carbohydrate content was calculated after quantifying the extracted paramylon particles using
the phenol–sulfuric acid method.

so the specific gravity of the cells would be large with
high carbohydrate content. The E. anabaena var. minor
cells contained numerous paramylon-like particles,
especially the cells grown in the TAP medium
(Fig. 5(B)–(E)). To examine the carbohydrate content
of E. anabaena var. minor, carbohydrate was extracted
from the cells and then quantified after a week of cul-
ture in the C, TAP, and the mixture of C and TAP
media (Fig. 5(F)). As expected, E. anabaena var. minor
contained a high level of carbohydrate. After culture in
the TAP medium, the carbohydrate content of E. ana-
baena var. minor comprised more than 40% of its
weight. However, E. gracilis stored carbohydrate only
after nitrogen restriction, as reported previously.25) In
contrast, E. anabaena var. minor stored carbohydrate
even in an environment that was permissive to growth.
Discussion
Potential of E. anabaena var. minor for industrial
use
The nutrient requirements and preferred culture condi-
tions were relatively different according to our compar-
isons based on the culture of E. gracilis and other
 6 K. Suzuki et al.
Euglena species. Most of the Euglena species tested
could proliferate in the C medium, which contains nitrate
nitrogen, but not ammonium nitrogen, whereas
E. gracilis has been reported to exhibit negligible growth
with nitrate.12) In addition, E. clara, E. granulata, and
E. schmitzii were relatively sensitive to a higher tempera-
ture, i.e. 29 °C, and all the tested species were sensitive
to low pH (pH 3.5), except for E. gracilis.
Among the Euglena strains available, we found that
E. anabaena var. minor exhibited good proliferation.
Although this strain was not as tolerant to low pH as
E. gracilis, its tolerance to high temperature was com-
parable to that of E. gracilis. Furthermore, we found
that E. anabaena var. minor has three highly beneficial
features. First, the cell size of E. anabaena var. minor,
i.e. approximately 20 μm, is smaller than that of
E. gracilis, i.e. approximately 50 μm (Fig. 5(B)–(E)).
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The difficulty of stirring large-scale cultures increases
in proportion to cell size because Euglena is sensitive
to shear forces. Therefore, the small cell size of E. ana-
baena var. minor suggests that it would be easier to stir
cultures of this species. Second, the cells settled well
without stirring, thereby indicating that the harvest pro-
cess could be simplified. Finally, the cells stored a high
level of carbohydrate in normal culture conditions
(Fig. 5(E)). In E. gracilis, nitrogen source deprivation
is required to stimulate the storage of high levels of
carbohydrate. Therefore, the production of high carbo-
hydrate content even in an environment permissive to
growth would also be an advantage during the indus-
trial application of this species.
Nutrient requirements for E. anabaena var. minor
growth
E. anabaena var. minor proliferated well in the TAP
medium, although it was larger and darker colored than
when grown in the other media. These differences may
be attributable to ingredients that are present only in the
TAP medium because the mixture with the C medium
also obtained the same effect. Although no ingredient
was specific to the TAP medium, acetate is a component
that characterizes the TAP medium. Other than the TAP
medium, the mAC medium also includes acetate but
much less than the TAP medium20)(Supplemental
Table 2). However, the addition of acetic acid to the C
medium was detrimental, and the addition of a lower
concentration appeared to have little effect (data not
shown). Thus, acetic acid may be the main factor
responsible for specific cell appearance in the TAP med-
ium, but the combination of ingredients could also be
important for the expression of the specific phenotype.
Acetic acid was the sole carbon source in the TAP med-
ium; therefore, E. anabaena var. minor may possess an
efficient pathway for assimilating acetic acid. However,
the acetic acid content of the TAP medium was only
0.1%, and most of the acetic acid probably evaporated
during sterilization in an autoclave. Even if acetic acid
can be used as a carbon source, the main carbon source
is the fixation of carbon dioxide by photosynthesis.
In contrast to the culture of E. gracilis, the cultures of
other Euglena species have not been studied and opti-
mized well in previous investigations. Thus, in the pre-
sent study, we tested several established culture media to
determine their effects on the growth of E. anabaena
var. minor, which suggested that there is considerable
capacity for improving the culture of this species. Given
further culture improvements and basic research,
E. anabaena var. minor may have the potential for mass
culture. Although our results indicate that E. anabaena
var. minor grew faster than the other species, this does
not necessarily mean that the other species should be
excluded from industrial use because the growth rates of
these species may improve dramatically after further
screening in more suitable culture conditions. In addi-
tion, screening a wider range of Euglena species may
identify species that are more suited to mass culture.
Author contribution
K.S., S.M., O.I., and K.Y. designed experiments.
K.S., S.M., and K.Y. performed experiments. S.K. pro-
vided the strains. K.S., O.I., and K.Y. analyzed data.
K.S., O.I., T.I., S.K., and K.Y. wrote the article. T.I.,
S.K. and K.Y. supervised the project.
Supplementary material
The supplementary material for this paper is avail-
able online at http://dx.doi.10.1080/09168451.2015.
1045828.
Acknowledgment
We thank National Institute for Environmental Stud-
ies(NIES) for providing the E. gracilis strain NIES-48.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by Core Research for Evolutional
Science and Technology (CREST) Program “Creation of
Basic Technology for Improved Bioenergy Production
through Functional Analysis and Regulation of Algae and
Other Aquatic Microorganisms” of Japan Science and Tech-
nology Agency (JST).
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