Bioresource Technology 130 (2013) 75–8

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortec

The effects of wavelength and wavelength mixing ratios on microalgae growth and
nitrogen, phosphorus removal using Scenedesmus sp. for wastewater treatment
Tae-Hyeong Kim, Yunhee Lee, Su-Hyun Han, Sun-Jin Hwang ⇑
Department of Environmental Science and Engineering, Center for Environmental Studies, Kyung Hee University, Seochon-dong, Giheung-gu, Yongin-si, Gyeonggi-do
446-701, Republic of Korea

highlights

" Effects of multiple wavelengths on wastewater treatment by microalgae were evaluated.

" Mixed red and blue light increased algae production rate rather than white light.
" N removal rate could be increased using wavelength mixing with red and blue light. " Blue light was
effective on phosphorus removal.
" Mixing light was effective in terms of electric consumption compared to white light.

article info abstract

Article history: In wastewater treatment using microalgae, the effects of wavelength and wavelength mixing ratio on microalgae growth and
Received 14 August 2012 removal of nitrogen and phosphorus were evaluated using LEDs (white light, 670 nm, 450 nm, and 525 nm). Microalgae
Received in revised form 26 November 2012 production rates were enhanced by a maximum of 45% with 400–700 nm white light compared to that of a single wavelength.
Accepted 28 November 2012 Available The phosphorus removal rate was as high as 90% with blue light. When red light and blue light were mixed and supplied, the
online 8 December 2012 microalgae production rate was about 50% higher than the rate of the culture with white light. Nitrogen and phosphorus
removal rates were as high as approximately 15 mg/L/day at a wavelength mixing ratio of 7 (red light):3 (blue light) and 2.1
Keywords: mg/L/day at a wavelength mixing ratio of 5 (red light):5 (blue light).
Microalgae 2012 Elsevier Ltd. All rights reserved.
Wastewater treatment
Nitrogen
Phosphorus
Wavelength mixing ratio

1. Introduction nitrification process oxidizing NHþ4 to NO3 , the denitrification

Microalgae are used in the medical field and in the areas of
dye and protein production. Vigorous studies are on-going to
apply microalgae for bio-fuel production and wastewater
treatment (Wang and Lan, 2011; Brennan and Owende, 2010;
Spolaore et al., 2006).
A general wastewater treatment system supplies air to
aerobic microbes via aeration to remove organic matter in the
wastewater. Approximately 1 kW h of electricity is required to
supply the air needed for 1 kg of BOD (biochemical oxygen
demand) removal (Oswald, 2003). Providing aeration is around
40% of the total electricity cost in wastewater treatment plants
(MOE, 2010), so aeration is a disadvantage due to increased
operating costs. In addition, complex processes of the
⇑ (Oilgae, 2010).
Corresponding author. Tel.: +82 31 201 2497; fax: +82 31 203 4589. E-mail
address: sjhwang@khu.ac.kr (S.-J. Hwang).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.11.134
process reducing NO3 to N2 for nitrogen removal and the
aerobic–anaerobic process for phosphorus removal are required
in general wastewater treatment plants.
In contrast, wastewater treatment technology using microalgae
has a variety of benefits compared to existing wastewater
treatment technology. Organic matter, nitrogen, and phosphorus
can be simultaneously removed in a reactor; indeed, high removal
efficiency of nutrients such as nitrogen and phosphorus is
expected (Aslan and Kapdan, 2006; Perez-Garcia et al., 2011;
Sancho et al., 1999; Shi et al., 2007). In addition, oxygen produced
from photosynthesis process by microalgae is used as a source for
aerobic microbes, thereby reducing aeration costs.
Autophototrophic microalgae cannot only remove nitrogen and
phosphorus, but can fix CO 2 by using the CO 2 as a carbon source.
Typically 1.8 tons of CO 2 are required to produce 1 ton of algae
At present, studies on the utilization of sludge produced from
general wastewater treatment systems are on-going, but sludge use
suffers from low efficiency (Liu et al., 2012). On the other hand,
76 T.-H. Kim et al. / Bioresource Technology 130 (2013) 75–80
microalgae biomass can be used in various ways such as bio-fuel
production and feedstock because the microalgae contain about
40–50% carbohydrate, lipid and protein contents in vivo (Singh
and Gu, 2010).
Photoautotrophic microalgae absorb light energy (photons) and
convert it to chemical energy such as ATP and NADP. These
reactions occur in photosystems in the microalgae and the
absorption of light energy occurs by chlorophyll pigments and
carotenoid that compose a photosystem antenna complex in the
photosystem. The absorbed light energy is sent to the reaction
center and is converted to chemical energy for photosynthesis
(Richmond, 2003).
Generally, microalgae use light of wavelengths from 400 to
700 nm for photosynthesis. The wavelengths absorbed by
microalgae differ depending on the species. For instance, green
microalgae absorb light energy for photosynthesis through
chlorophylls as a major pigment absorbing light energy in the
range of 450– 475 nm and 630–675 nm and carotenoids as an
accessory pigment absorbing light energy of 400–550 nm
(Richmond, 2003).
Several studies reported that the growth of microalgae is
different depending on wavelength. Red light (600–700 nm) and
blue light (400–500 nm) stimulate the growth of microalgae, and
growth rates and lipid content of the microalgae differ with light
intensity (Chen et al., 2011; Cheirsilp and Torpee, 2012; Wang et
al., 2007; Das et al., 2011).
Almost all previous studies focused on the growth of
microalgae and on providing nutrients for growth using
wastewater rather than on nutrient treatment in the wastewater
(Christenson and Sims, 2011). A study on nitrogen and
phosphorus removal in wastewater depending on wavelength and
mixing ratios is insufficient.
In this study, when multiple wavelengths were applied for
photoautotrophic microalgae in wastewater treatment, effects of
the wavelength and mixing ratio on microalgae growth and
removal of nitrogen and phosphorus in the wastewater were
investigated.
2. Methods
2.1. Microalgae strain and culture conditions
The strain of Scenedesmus sp. obtained from KORDI (Korea
Ocean Research & Development Institute) and cultured using
Bold’s Basal Medium (BBM) consisted of NaNO1 250 mg/L,
MgSO4 7H2O 75 mg/L, NaCl 25 mg/L, K2HPO4 75 mg/L, KH2PO4
175 mg/L, CaCl22H2O 25 mg/L, H2BO3 114 mg/L, 1 mL of a trace
1 . Results and discussion
2 .1. Effects of wavelength on microalgae production and nutrient
removal by Scenedesmus sp.
3 .1.1. Microalgae production rate and CO2 fixation
The production rate of Scenedesmus sp. and the CO2
fixation rate were evaluated at different wavelengths using
green, blue, and red light. The results were compared with
those of white light. Microalgae production was about 45%
higher with white light compared to green, and the
corresponding microalgae production rate for green light
was about 64 mg/L/day. The microalgae production rate was
highest in white light, followed in order by red, blue, and
green light. Therefore, white light providing both 450–475
nm and 630–675 nm, the ranges used for photosynthesis by
microalgae, is more appropriate for the growth of
microalgae than providing a single wavelength. The CO 2
elements solution (ZnSO47H2O 8.82 mg/L, MnCl24H2O 1.44
mg/L, MoO40.71 mg/L, CuSO45H2O 1.57 mg/L, Co(NO3)26H2O
0.49 mg/L), 1 mL of EDTA stock (EDTANa 2 50 mg/L, KOH 31
mg/L), and 1 mL of Fe solution (FeSO47H2O 4.98 mg/L and
H2SO4 1 mL) in a 250 mL cell culture flask. Temperature was
maintained at 25 ± 1 C, and light with a photosynthetic photon
flux density (PPFD) of 100 lmol/m2/s was used.
2.2. Batch experiment-1: effects of wavelength
To evaluate nitrogen and phosphorus removal efficiencies and
the growth of microalgae by wavelength, light-emitting diodes
(LEDs) were used because it is possible to control wavelengths.
Blue light (400–500 nm, with a peak at 450 nm), green light (470–
580 nm, with a peak at 525 nm, and red light (600–700 nm, with a
peak at 670 nm) were used. White light (400–700 nm) was used as
a control group.
BBM and pre-cultured Scenedesmus sp. were mixed at a 9:1
ratio in a 1 L Erlenmeyer flask, with a working volume of 600 mL.
The concentration of microalgae was 0.3 OD (optical density), and
the initial pH was adjusted to 8. Inorganic carbon for growth of
microalgae was supplied at 1 vvm (aeration volume/medium
volume/minute) using
Table 1
Mixing ratios of red LED and blue LED.
PPFD Wavelength mixing ratio
Red light (wavelength range Blue light (wavelength range
600–700 nm, peak wavelength 400–500 nm, peak wavelength
670 nm ) 450 nm )
100 lmol/ 9 1 m2/s 7 3
5 5
3 7
1 9
an air pump and air stone. Batch tests were performed for 6 days at
25 ± 1 C and 100 lmol/m2/s of PPFD for each wavelength.
2.3. Batch experiment-2: effects of wavelength mixing ratio
To evaluate nitrogen and phosphorus removal efficiencies
and the growth of microalgae by wavelength mixing ratio based
on light intensity, Blue light (400–500 nm, with a peak at 450
nm) and red light (600–700 nm, with a peak at 670 nm) were
used. The evaluated mixing ratios are presented in Table 1.
Green light was eliminated for the experiment due to large
electric power consumption and low microalgae production
rate. Batch tests were performed for 6 days, and other operating
conditions were the same as those in batch experiment-1.
2.4. Analysis
Suspended solids (SS) were measured to determine
microalgae growth(APHA-AWA-
WEF,2005),andODwasanalyzedusingaspectrophotometer (X-
ma 2000, Humas crop, Korea) at 660 nm. TC (total carbon),
TOC (total organic carbon), and TIC (total inorganic carbon)
fixation rate in all types of light was 1.8–1.9 mg CO 2/g
microalgae and was similar to literature values (Oilgae,
2010) (see Fig. 1).
4 .1.2. Nitrogen and phosphorus removal
Nitrogen and phosphorus removal efficiencies by
Scenedesmus sp. were evaluated depending on wavelength.
The removed nitro-
were measured with a total organic carbon analyzer (TOC- 3.1.3. Electric power consumption
Vcsn, Shimadzu, Japan). T-N and T-P were analyzed using a Microalgae wastewater treatment and a culture method
water analyzer (HS 3100, Humas, Korea) after pre-treatment using a photobioreactor are not affected by the surrounding
using an exclusive water analysis reactor (HS R-200, Humas, environment and have benefits in terms of 10–20% higher
Korea). The pH was measured with a pH meter (pH-200L, efficiency than an open pond, but the construction and
Istek, Korea), and electric power consumption for each LED operating costs are high
was computed by measuring the voltage and current using a (Oilgae, 2010).
digital multimeter (M-3860D, Metex Corporation, Korea). Table 2
T.-H. Kim et al./ Bioresource Technology 130 (2013) 75–80 77

Fig. 1. Microalgae production rate and CO 2 fixation rate of Scenedesmus sp. under different wavelengths of light.

Fig. 2. Nitrogen and phosphorus removal amount of Scenedesmus sp. under different wavelength of light.

gen amount was about 20–22 mg and the nitrogen removal rate Spectral electric power consumption.
LED light PPFD (lmol/m2/s) Watt
was about 8.5 mg/L/day with no significant difference at white
light, red light, and blue light. The lowest nitrogen removal rate White 100 34
presented about 6.8 mg/L/day with green light. Phosphorus Red 29
removal amount was about 6.5 mg with blue light at 6 days
Blue 34
operated, and the corresponding removal rate was about 1.8 mg/L/
day. With white light, the phosphorus removal amount and
removal rate were 3.4 mg and 1.0 mg/L/day, respectively, about
50% lower in phosphorus removal amount and about 45% lower
in phosphorus removal rate than the results with blue light.
Therefore, with blue light (400–500 nm, with a peak at 450 nm), it
is appropriate for increased phosphorus removal amount and
removal rate in wastewater treatment (see Fig. 2).
78
Green
In this study, the electric power consumed at the same light
intensity (PPFD 100 mol/m2/s) with each LED (white light, red
light, blue light, green light) was measured to evaluate the
efficiency of the light supply (Table 2). Green light consumed
significant electric power at 95 W. Red and blue light consumed
T.-H. Kim et al. / Bioresource Technology 130 (2013) 75–80
29–34 W, 65% lower than with the green light and similar to the
white light. The green light was unsuitable for wastewater
treatment using microalgae and for microalgae culture due to low
biomass production and larger electric power consumption than
red or blue light. Therefore, based on these results, the effects of
red and blue light mixing ratios on microalgae growth and N and P
removal were evaluated in batch experiment-2.

Fig. 3. Effects of wavelength mixing ratios on microalgae production rate and CO 2 fixation rate.
Fig. 4. Optimization of wavelength mixing ratio for nitrogen removal (a) rate and (b) amount.
T.-H. Kim et al./ Bioresource Technology 130 (2013) 75–80 79

Fig. 5. Optimization of wavelength mixing ratios for phosphorus removal.

3.2. Effects of wavelength mixing ratio on microalgae growth White light providing 400–700 nm of wavelength
and nutrient removal by Scenendesmus sp. contributed to a higher microalgae production rate than
conditions of red or blue lights; thereby, the effects of
3.2.1. Microalgae growth and CO2 fixation wavelength mixing ratio on microalgae growth were identified
80 T.-H. Kim et al. / Bioresource Technology 130 (2013) 75–80
using different mixing ratios of red light and blue light (Fig. 3).
Mixes of red light and blue light provided about 50% higher
microalgae production rates than with a single wavelength of
light, irrespective of the mixing ratio. Ruyters (1984) reported
that sufficient red and blue light should be provided for
adequate photosynthesis of microalgae and plants. For green
microalgae such as Scenedesmus sp., chlorophyll a and
chlorophyll b adsorb light at 450–475 nm and 630–675 nm,
respectively. In this study, photosynthetic efficiency increased
by selectively and simultaneously providing both red light and
blue light, which provide the wavelength ranges needed for
photosynthesis. Thereby, microalgae production rates were
higher than with only red light, blue light, or white light.
In all the mixing conditions with red and blue light, the CO2
fixation rate was about 1.5 g CO2/g microalgae, which was
slightly low compared to conditions of wavelength (red light
and blue light) or 400–700 nm (white light), while the total
CO2 removal amount was 10–25% larger. Therefore, the
microalgae production rate and CO2 removal efficiency can be
increased by supplying the selective wavelengths of 450–475
nm (blue light) and 630– 675 nm (red light) for photosynthesis
in photoautotrophic microalgae cultures.
3.2.2. Nitrogen removal
Nitrogen removal rate and removal amount by operating
time were investigated to evaluate nitrogen removal efficiency
with wavelength mixing ratio (Fig. 4).
Nitrogen removal amount was about 20–22 mg with no
significant differences under the six different conditions of
wavelengths (red and blue light), 400–700 nm (white light)
wavelength, and mixed red light and blue light ratios of 7:3,
5:5, and 3:7(Fig 4b). However, the nitrogen removal amount
was slightly lower under conditions of mixed red light and blue
light ratios of 9:1 and 1:9 than with a single wavelength (red or
blue light) or at 400– 700 nm (Fig. 4a).
At the end of the operation, no matter the wavelength or
wavelength mixing ratio, the nitrogen removal rate was about 5–7
mg/L/ day, while the nitrogen removal rate at 1–2 days of the
operation was high at about 15–13 mg/L/day under conditions of
red light and blue light at a 7:3 ratio. This rate was more than
twice that of white light. This selective supply of the proper
wavelength for the growth of microalgae stimulates both increased
photosynthesis efficiency and nitrogen consumption. Therefore,
supplying a light source of mixed red and blue light for nitrogen
removal in wastewater using microalgae can be an alternative way
to increase the nitrogen removal rate. The appropriate mixing ratio
of red light to blue light was 7:3 for nitrogen removal in
wastewater using photoautotrophic microalgae.
3.2.3. Phosphorus removal
The effects of wavelength mixing ratio on phosphorus removal
were investigated (Fig. 5). After the end of the operation, the
phosphorus removal amount and removal rate were about 9 mg
and 2.1 mg/L/day, respectively, in a reactor at a mixing ratio of 5
(red):5 (blue), and both removal amount and removal rate were
about 2.3 times higher than under white light conditions. In reactor
conditions at red: blue mixing ratios of 9:1 and 1:9, the
phosphorus removal rate was similar to that with white light
conditions, while the phosphorus removal rate was lower than that
with single red or blue light conditions. Favoring red or blue light
in the mix had a negative effect on phosphorus removal.
According to Ruyters’ (1984) study, blue light is involved in
enzyme activation, regulation of gene transcription, energy-
derivation of microalgae, and red light remediates damaged cells.
In this study, the phosphorus removal amount was significant at
red: blue mixing ratios of 5:5 and 3:7, resulting from stimulated
microalgae activity caused by the appropriate blue light.
Therefore, a mixture of blue light and red light would be
effective for phosphorus removal in wastewater using microalgae.
For nitrogen removal, the red: blue mixing ratio of 7:3 contributed
to a high efficiency of nitrogen removal, while the appropriate red:
blue mixing ratio for phosphorus removal was 5:5 or a ratio higher
in blue light (3:7).
4. Conclusion
Mixed red and blue light increased the microalgae production
rate, removal rates of nitrogen and phosphorus compared to a
single wavelength of light.
Microalgae production was not significantly different among the
mixing ratios of red and blue light, and the appropriate red:
blue mixing ratios were 7:3 for nitrogen removal, 5:5 or 3:7
for phosphorus removal, and 3:7 for simultaneous nitrogen and
phosphorus removal.
The biomass production and N, P removal efficiencies relative
to the power consumption were elevated with mixed red and
blue light compared to that with white light.
Acknowledgement
This subject is supported by Korea Ministry of Environment as
‘‘The Eco-innovation Project’’.
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