Journal of Water and Environment Technology, Vol.13, No.

3, 2015

Evaluation of Growth Characteristics of Euglena gracilis

for Microalgal Biomass Production Using Wastewater

Kenta TORIHARA1), Naoyuki KISHIMOTO2)

1) Graduate School of Science and Technology, Ryukoku University, 1-5 Yokotani, Seta Oe-cho,
Otsu-shi, Shiga 520-2194, Japan
2) Faculty of Science and Technology, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu-shi,
Shiga 520-2194, Japan

ABSTRACT
Recently, fossil fuel depletion and global warming have become serious problems in the world.
In order to solve these problems, renewable energy has attracted much attention. Here, a
microalga, Euglena gracilis (E.gracilis), was focused on as a renewable biomass energy source.
In our idea, E. gracilis biomass produced using nutrients in wastewater is mixed with sewage
sludge, and anaerobically digested for methane recovery. It is considered that microorganisms
and suspended solids in wastewater may have a negative impact against the growth of E. gracilis.
Accordingly, their effect on the growth of E. gracilis was discussed in this research. Three types
of culture media, supernatant fluid of wastewater, suspended solids-free wastewater, and
suspended solids and microorganisms-free wastewater, were used for the cultivation test. The
culturing conditions were a temperature of 25°C and photosynthetically active radiation of 98.2
μmol/(m2·s). As a result, E. gracilis was able to increase in wastewater, though both
microorganisms and suspended solids gave a negative influence. Since the negative effect of
suspended solids was stronger than that of microorganisms, an introduction of a pre-removal
process of suspended solids would be favored for the microalgal biomass production in
wastewater.

Keywords: biomass production, euglenid, growth rate, phytoplankton, wastewater

INTRODUCTION
Recently, people live a very convenient life owing to the remarkable development of
science and technologies. After the Industrial Revolution, on the other hand, natural
ecosystem has collapsed due to global warming, air pollution by emission of acidic
substances like sulfur oxides and nitrogen oxides, and other phenomena in the world. In
addition, depletion of fossil fuels is a serious problem (Bentley, 2002). Currently,
renewable energies being characterized as carbon neutral, attract much attention in order
to solve these problems. For example, sustainable energy like solar power, wind power,
geothermal power, and biomass energy have been developed to substitute for fossil fuel
in Vietnam since the early 2000 (Nguyen et al., 2013). Japanese government is also
carrying out Biomass Nippon Strategy since 2002 (Kuzuhara, 2005). However, the
production cost of biomass energy in Canada was about 50 US$/MWh, whereas that of
fossil energy was only about 30 US$/MWh (Kumar et al., 2003). In Belgium it was
reported that biomass production cost 78% higher than fossil fuel production (Brammer
et al., 2006). Therefore, it is necessary to improve a cost-performance ratio of biomass
energy for its penetration into our society.

In this study, we paid attention to microalgae as a biomass energy source. They can
grow rapidly without soil (Widjaja et al., 2009) and incept carbon dioxide during their
growth. Therefore, they can contribute to solve the global warming problem (Avagyan,

Address correspondence to Kenta Torihara, Graduate School of Science and Technology, Ryukoku
University, Email: t14m100@mail.ryukoku.ac.jp
Received May 23, 2014, Accepted October 26, 2014.
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 Journal of Water and Environment Technology, Vol.13, No.3, 2015

2008). Although a usage of food crops like corn as a biomass energy source may arouse
a food crisis, microalgal biomass is unlikely to cause such a problem. Furthermore,
microalgae can be harvested in shorter cultivation period than other crops (Schenk et al.,
2008). Thus, the microalgal biomass is a potential substrate for sustainable biomass
energy production (Harun et al., 2010).

Everyday people are producing a huge amount of municipal wastewater, which

abundantly contains nitrogen and phosphorus. Benemann and Oswald (1996) reported
that the usage of wastewater as a nitrogen and phosphorus source was estimated to cut
down the microalgal biomass production cost by about 10 – 20%. Thus, microalgal
biomass production in wastewater is a promising process for economical biomass
production. However, there are few reports on microalgal biomass production in
wastewater. Bhatnagar et al. (2010) successfully cultivated Chlorella minutissima (C.
minutissima) using a mixture of wastewater and synthetic medium, but the cultivation in
100% wastewater deteriorated the growth of C. minutissima. Accordingly, cultivation of
microalgae in wastewater is a challenging but important subject for the economical
microalgal biomass production. In this research, we focused on Euglena sp. as a
microalga for biomass production, because Mahapatra et al. (2013) reported that
Euglena sp. was useful to wastewater treatment and might be produced in municipal
wastewater.

The methane production from sewage sludge is one of the biomass energy processes.
The addition of microalgal biomass produced in wastewater into a methane fermenter
using sewage sludge is one of the potential alternatives to increase the methane
production yield. It is considered as an alternative energy to fossil fuel (Chisti, 2007).
Microalgal cultures in wastewater have also been discussed for wastewater treatment
(Noüe et al., 1992). However, wastewater contains various microorganisms and
suspended solids (SS), which may affect the growth of microalgae. Accordingly, we
discussed the effect of microorganisms and SS on the growth of microalgae in
wastewater to elucidate the feasibility of microalgal production using wastewater in this
study.

MATERIALS AND METHODS

Microalga
An axenic and clonal strain of Euglena gracilis (E. gracilis) stocked in the National
Institute for Environmental Studies, Japan (NIES-49, Kawachi et al., 2013) was used in
this research. Although euglenid is classified as both animal (Häder and Liu, 1990) and
plant (Chang et al., 1981), the classification of Euglenophyceae in Euglenozoa was
adopted in this research according to the classification by the National Institute for
Environmental Studies. This species is an oval-shaped single cell flagellate (Ascoli et
al., 1978) with a high growth rate (Mahapatra et al., 2013) and can grow under both
autotrophic and heterotrophic conditions because it is a mixotrophic alga (Takeyama et
al., 1997). It is considered to be potentially useful for biomass energy, because of the
production ability of wax esters combined with fatty alcohols and fatty acids (Inui et al.,
1982).

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Cultivation of E. gracilis in synthetic media

The HUT medium based synthetic media were used in this research. The normal HUT
medium (Kawachi et al., 2013) was used for the evaluation of light and temperature
effect. The compositions of the modified HUT media for the evaluation of nitrogen
limitation and of phosphorus limitation are summarized in Table 1 and 2, respectively.
Although vitamin B12 and thiamine HCl contain nitrogen and phosphorus, the final
concentrations derived from these chemicals were only 67 µgN/L and 0.012 µgP/L.
Accordingly, the effects of nutrients from these chemicals were negligible. In the
cultivation test, 100 mL of each medium was poured into a 200 mL Erlenmeyer flask
with a loosely closed screw cap and sterilized for 20 minutes at 120°C in an autoclave
(ES-315, Tomy Seiko, Tokyo, Japan). Then, 0.1 – 0.2 mL of pre-cultured E. gracilis
was inoculated into each flask and cultured in a biotron (LH-55-RDS, Nippon Medical
& Chemical Instruments, Osaka, Japan) for 13 days. The cultivation condition for
nutrient limitation was a temperature of 25°C and a photosynthetically active radiation
(PAR) of 98.2 µmol/(m2·s). However, the temperature and PAR were changed from 10
to 35°C for the evaluation of temperature effect and from 58.4 to 178 µmol/(m2·s) for
the evaluation of light effect. The triplicated culture was used for all tests.

Table 1 - Composition of the modified HUT medium for nitrogen limitation.

Nitrogen concentration [mgN/L] Starvation
Composition
0 5 10 20 40 medium
KH2 PO4 20 mg
KCl 0 mg
NH4 Cl 0 mg 19.08 mg 38.17 mg 76.36 mg 152.7 mg 0 mg
MgSO4 • 7H2 O 25 mg
Sodium acetate 400 mg
Potassium citrate 40 mg
Polypeptone 0 mg
Yeast extract 0 mg
Vitamin B12 0.5 µg
Thiamine HCl 0.4 mg
Distilled water 1,000 mL

Table 2 - Composition of the modified HUT medium for phosphorus limitation.

Phosphorus concentration [mgP/L] Starvation
Composition
0 1 3 5 10 medium
KH2 PO4 0 mg 4.394 mg 13.18 mg 21.97 mg 43.94 mg 0 mg
KCl 24.07 mg 21.66 mg 16.85 mg 12.04 mg 0 mg 24.07 mg
NH4 Cl 288.8 mg
MgSO4 • 7H2 O 25 mg
Sodium acetate 400 mg
Potassium citrate 40 mg
Polypeptone 0 mg
Yeast extract 0 mg
Vitamin B12 0.5 µg
Thiamine HCl 0.4 mg
Distilled water 1,000 mL

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Cultivation of E. gracilis in wastewater

Wastewater drained from a restaurant in Ryukoku University was used in this research.
As E. gracilis was reported to use ammonia nitrogen as nitrogen source rather than
nitrate nitrogen (Cramer and Myers, 1952), we assumed that a cultivation tank for E.
gracilis was installed just after a primary sedimentation tank. Therefore, two hours of
sedimentation was applied to the wastewater and supernatant fluid (called “raw
wastewater” hereafter) was used for cultivation. To omit the effect of large and abiotic
SS, raw wastewater was filtered through a glass fiber filter with 2.7 μm particle
retention (Whatman GF/D, GE Healthcare, Tokyo, Japan). This filtrate was referred to
as large SS-free wastewater hereafter. The large SS-free wastewater was further filtered
through a filter paper with particle retention of 0. 45μm (HAWP04700, Merck Millipore,
Billerica, MA, USA) to remove the microorganisms. This filtrate was referred to as
microorganism-free wastewater hereafter. The chemical oxygen demand (CODCr), total
nitrogen (TN), and total phosphorus (TP) concentrations of each wastewater analyzed in
pursuance of APHA-AWWA-WEF (2012) were respectively 485 mg/L, 72.9 mgN/L
and 3.67 mgP/L for raw wastewater; 368 mg/L, 70.8 mgN/L and 3.32 mgP/L for large
SS-free wastewater; and 281 mg/L, 62.5 mgN/L and 3.16 mgP/L for
microorganism-free wastewater. The 100 mL portion of each wastewater and 0.3 – 0.4
mL of pre-cultured E. gracilis were placed into a 200 mL Erlenmeyer flask with a
loosely closed screw cap and cultured in a biotron (LH-55-RDS, Nippon Medical &
Chemical Instruments, Osaka, Japan) for 20 days at a temperature of 25°C and a PAR of
98.2 µmol/(m2·s). The triplicated culture was used for all cultivations.

Evaluation of specific growth rate

Cultivation was continued until the cell density reached the maximum. The medium was
sampled from each Erlenmeyer flask every 2 – 3 days and the cell density of E. gracilis
was counted using a plankton counting chamber (MPC-200, Matsunami, Osaka, Japan)
under an upright microscope (CX41, Olympus, Tokyo, Japan) after fixation by the
addition of glutaraldehyde solution at a final concentration of 53.2 g/L. The cell density
increases exponentially in the logarithmic growth phase. Accordingly, the growth curve
was expressed by equation (1) (Suzuki et al., 2013),

lnXt = lnXt0+µt (1)

where µ is the specific growth rate [1/day], Xt is the cell density at day t [cells/mL], and
Xt0 is the cell density at the beginning of the logarithmic growth phase [cells/mL]. The
specific growth rate was determined by the regression analysis based on equation (1)
using the data in the logarithmic growth phase.

RESULTS AND DISCUSSION

Temperature effect
Figure 1 shows the specific growth rate and the maximum cell density observed in the
HUT medium. The specific growth rate and the maximum cell density reached the
maximum level at water temperature ranging from 25 – 30°C and 20 – 25°C,
respectively. These results mostly accorded with the suitable temperature of 20 – 35°C
reported by Buetow (1962), though 35°C was not favorable for the growth of E. gracilis
in this study. Judging from the experimental results, the optimal temperature for E.

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 Journal of Water and Environment Technology, Vol.13, No.3, 2015

×105
2.0 6.0
(a) (b)

The maximum cell density

5.0
Specific growth rate

1.5
4.0

[Cell/mL]
[1/day]

1.0 3.0

2.0
0.5
1.0

0.0 0.0
10 20 30 10 20 30
Temperature [℃] Temperature [℃]

Fig. 1 - Dependency of specific growth rate (a) and maximum cell density (b) on
temperature. The error bar represents the unbiased standard deviation of
triplicated culture.

gracilis biomass production was thought to be 25°C, at which the suitable temperature
ranges for the specific growth rate and for the maximum cell density were overlapped.
Under the optimal temperature of 25°C, the specific growth rate observed was 1.19
1/day. As the specific growth rates of E. gracilis were reported to be in the range of
0.43 – 1.08 1/day (Cramer and Myers, 1952; Maly, 1975; Ogbonna et al., 1998), the
strain used in this study had a slightly higher growth rate than the other strains
previously reported.

Nakayama et al. (2007) reported that the temperature of municipal wastewater in Tokyo
ranged from 16 to 28°C in influent and from 14 to 29°C in effluent. Accordingly, E.
gracilis has a potential to grow in the municipal wastewater, though biomass
productivity is reduced by half in winter.

Nutrient concentration effect

The specific growth rate and the maximum cell density observed under nitrogen
limitation and phosphorus limitation are shown in Figs. 2 and 3, respectively. When E.
gracilis was incubated in the nutrient-free medium, an increase in the cell density was
observed during the first 4 days of incubation. The initial growth in E. gracilis would be
caused by the carryover of nutrients with the pre-cultured E. gracilis inoculated.
Accordingly, the specific growth rate was evaluated by the data after 4 days of
incubation. The observed specific growth rates and maximum cell densities in Figs. 2
and 3 were relatively smaller than those at 25°C shown in Fig. 1. The original HUT
medium contains 600 mg/L of polypeptone and 400 mg/L of yeast extract, which
involved nitrogen at a concentration of 56 mgN/L and 20 mgN/L, and phosphorus at a
concentration of 4.4 mgP/L and 2.9 mgP/L, respectively. Therefore, both polypeptone
and yeast extract were removed in nutrient limitation tests. However, polypeptone and
yeast extract also involve minerals and trace chemicals. Accordingly, the relatively
small growth rate and small maximum cell density shown in Figs. 2 and 3 might be
caused by the lack of some minerals and/or trace chemicals.

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×105
1.0
(a) (b)
3.0

The maximum cell density

0.8
Specific growth rate

0.6 2.0

[Cell/mL]
[1/day]

0.4
1.0
0.2

0.0 0.0
0 10 20 30 40 0 10 20 30 40
Nitrogen concentration [mgN/L] Nitrogen concentration [mgN/L]
Fig. 2 - Dependency of specific growth rate (a) and maximum cell density (b) on
nitrogen concentration. The error bar represents the unbiased standard
deviation of triplicated culture.

×105
1.0
(a) (b)
3.0
The maximum cell density

0.8
Specific growth rate

0.6 2.0
[Cell/mL]
[1/day]

0.4
1.0
0.2

0.0 0.0
0 2 4 6 8 10 0 2 4 6 8 10
Phosphorus concentration [mgP/L] Phosphorus concentration [mgP/L]

Fig. 3 - Dependency of specific growth rate (a) and maximum cell density (b) on
phosphorus concentration. The error bar represents the unbiased standard
deviation of triplicated culture.

The specific growth rate under nitrogen limitation gradually increased with the rise in
nitrogen concentration and reached the maximum at 20 mgN/L (Fig. 2(a)), but the
difference in the specific growth rate in the range of 5 – 40 mgN/L was not so large.
The maximum cell density showed similar dependency on nitrogen concentration with
the specific growth rate (Fig. 2(b)), though the significant difference between ≤ 10
mgN/L and ≥ 20 mgN/L was observed. Thus, 20 mgN/L or higher concentration was
thought to be suitable for the growth of E. gracilis.

The specific growth rate under phosphorus limitation jumped up at 1 mgP/L and
reached a plateau at a higher concentration (Fig. 3(a)). The dependency of the maximum
cell density on phosphorus concentration was unclear, but was similar to the specific
growth rate (Fig. 3(b)). Thus, 1 mgP/L or higher concentration was thought to be
suitable for the growth of E. gracilis.

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Japan Sewage Works Association (2006) reported that the total nitrogen (TN) and total
phosphorus (TP) concentrations in influent to 8 municipal sewage treatment plants in
Japan were in the range of 23 – 36 mgN/L and 2.0 – 5.2 mgP/L, respectively. The
suitable concentration range observed in this study covered the concentration range in
municipal wastewater. Consequently, E. gracilis was evaluated to be applicable to
biomass production in municipal wastewater from the viewpoint of nutrient
concentration.

Light effect
Figure 4 shows the specific growth rate and the maximum cell density observed in the
HUT medium. The specific growth rate and maximum cell density observed were
almost constant in the PAR range tested. However, solar radiation is more than 1000
µmol/(m2·s) of PAR, even though it is cloudy (Keller et al., 2001), and contains
ultraviolet (UV) radiation. Actually, inhibition of the growth of E. gracilis by UV
radiation was observed (Ekelund, 1993). Therefore, when E. gracilis is cultured under
the sunlight, its growth may be inhibited by the UV radiation. Accordingly, it may be
required to cut off the UV radiation in sunlight with an optical filter or to deepen the
depth of a culture tank for providing an evacuation site.

Growth characteristics in the wastewater

Figure 5 summarizes the specific growth rate and the maximum cell density in three
wastewater-based medium, raw wastewater, large SS-free wastewater, and
microorganism-free wastewater. The specific growth rates in large SS-free wastewater
and microorganism-free wastewater were significantly higher than that in raw
wastewater. However, the specific growth rates observed were much smaller than that in
the synthetic wastewater. The maximum cell density in microorganism-free wastewater
was the largest of the three, but the density was also much less than that in the synthetic
wastewater. Similar result of C. minutssima was reported by Bhatnagar et al. (2010),
though the reason for the growth deterioration in 100% wastewater was not identified.
Nevertheless, our results indicate that some factors except temperature, light, nitrogen,
and phosphorus affect the growth of E. gracilis in wastewater.

The comparison of results in raw wastewater and in large SS-free wastewater revealed
that large SS had a negative effect on both specific growth rate and maximum cell
density. Since aggregates of E. gracilis with SS were found under microscopic
observation in this study, the aggregation effect of SS would inhibit the growth of E.
gracilis.

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×105
1.4 6.0
(a) (b)
1.2 5.0

The maximum cell density

Specific growth rate

1.0
4.0
0.8
[1/day]

[Cell/mL]
3.0
0.6
2.0
0.4
0.2 1.0

0.0 0.0
0 50 100 150 200 0 50 100 150 200
PAR [µmol/m2 s] PAR [µmol/m2 s]

Fig. 4 - Dependency of specific growth rate (a) and maximum cell density (b) on PAR.
The error bar represents the unbiased standard deviation of triplicated culture.

0.40
(a)
0.35
Specific growth rate

0.30
0.25
[1/day]

0.20
0.15
0.10
0.05
0.00
Raw wastewater Large SS-free Microorganism-free
wastewater wastewater

×104
8.0
(b)
7.0
The maximum cell density

6.0
5.0
[Cell/mL]

4.0
3.0
2.0
1.0
0.0
Raw wastewater Large SS-free Microorganism-free
wastewater wastewater

Fig. 5 - Specific growth rate (a) and maximum cell density (b) in three types of
wastewater-based medium. The error bar represents the unbiased standard
deviation of triplicated culture.

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The microorganisms in wastewater had a negative effect on the maximum cell density,
but no significant influence on the specific growth rate. Two possible reasons for the
effect of microorganisms are as follows: an attack against E. gracilis by microorganisms
and a competition of substrate uptake. If the former occurs, a decrease in the specific
growth rate is expected. However, no difference between the specific growth rates with
and without microorganisms was observed as shown in Fig. 5(a). On the other hand, if
the latter occurs, a depletion of limiting substrates limits the final biomass, namely
maximum cell density. However, less influence on specific growth rate is expected in
comparison with the maximum cell density, because the depletion of substrate shortens
the period of logarithmic growth phase, but does not change the slope of the
semi-logarithmic plot of cell density during the logarithmic growth phase. Therefore,
Fig. 5 confirms that the latter reason was valid. Thus, it was inferred that some
competition of substrate uptake between E. gracilis and microorganisms in wastewater
limited the maximum cell density. It was unable to identify the limiting substrate at
present but it was strongly suggested that trace chemicals limited the growth of E.
gracilis as shown in the cultivation tests without polypeptone and yeast extract.
Consequently, the pre-removal of SS will be recommended for E. gracilis biomass
production in wastewater.

CONCLUSIONS
The growth characteristics of E. gracilis were evaluated by cultivation in synthetic and
wastewater-based media to elucidate the feasibility of its biomass production using
wastewater. The obtained results are summarized as follows:

(1) The suitable temperature for the growth of E. gracilis was in the range of 20 – 30°C,
and the optimal temperature was 25°C. As the temperature of municipal wastewater
in Tokyo was ranging from 16 to 28°C, E. gracilis has a potential to grow in the
municipal wastewater, though biomass productivity is reduced by half in winter.

(2) The specific growth rate and the maximum cell density of E. gracilis reached the
maximum at over 20 mgN/L as ammonia and 1 mgP/L as phosphate. As these
concentration values were within a typical range in municipal wastewater. Therefore,
E. gracilis was thought to be applicable to biomass production in municipal
wastewater from the viewpoint of nutrient concentration.

(3) The SS in the wastewater had a negative effect on the growth of E. gracilis. Since
aggregates of E. gracilis with SS were found under microscopic observation, the
aggregation effect of SS would inhibit the growth of E. gracilis.

(4) The microorganisms in the wastewater affected the maximum cell density, but did
not have any significant influence on the specific growth rate. It was inferred that a
competition of substrate uptake between E. gracilis and microorganisms in
wastewater limited the maximum cell density.

(5) The wastewater was available for the biomass production of E. gracilis if a
pretreatment of wastewater for the removal of SS was introduced. However, the
growth rate of E. gracilis in the wastewater was smaller than in the synthetic

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medium, probably due to insufficient amount of substrate like minerals and/or trace
chemicals. Accordingly, the identification of limiting substrates and the
development of culture and biomass recovery system will be required for the
application of E. gracilis to biomass production in wastewater.

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