Ecological Engineering 51 (2013) 24– 32

Contents lists available at SciVers ScienceDirec

Ecological Engineering
j o ur nal homep age : www.elsevier.com/locate/ecolen

EffectsofvariousLEDlightwavelengthsandintensities ontheperformanceof purifyingsynthetic

domesticsewagebymicroalgae atdifferent influentC/N ratios
Cheng Yan, Li Zhang, Xingzhang Luo∗,1, Zheng Zheng∗,1

Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, PR China

a rt ic le i nf o ab st ra ct

Article history: In Chinese rural areas, domestic sewage is one of the main sources of water pollution that may cause water eutrophication if directly
Received 13 July 2012 discharged into natural waters without appropriate treatment. This study demonstrates that the microalgae Chlorella vulgaris can
Received in revised form effectively remove nutrients from domestic sewage. In this process, red is the optimal light wavelength, with an intensity strategy as
18 September 2012 follows: Phase
Accepted 3 December 2012 Available
1, 0–48h with 1000molm−2 s−1; Phase 2, 48–96h with 1500molm−2 s−1; Phase 3, 96–120h with
online 3 January 2013

Keywords:
COD removal 2000molm−2 s−1; and Phase 4, 120–144h with 2500molm−2 s−1. The optimal cultivation time was
Nitrogen removal
Phosphorus removal
Dry weight
Chlorella vulgaris 144 h. Nutrient removal efficiency was significantly affected by the light wavelength, light intensity, the effect of the interaction
between light wavelength and intensity, as well as the effect of the interaction among light wavelength, light intensity, and influent C/N
ratio. Moreover, the total nitrogen and total phosphorus removal efficiencies were significantly affected bythe influent C/N ratio. In this
study, the optimal influent C/N ratio is 5:1.
© 2012 Elsevier B.V. All rights reserved.

1.Introduction 0925-8574/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ecoleng.2012.12.051
blanket reactors entail expensive construction costs for collection and
One of the main water pollution sources in the rural areas of China is domestic
treatment facilities, and are therefore not suitable for use in rural areas (Ye and
sewage, which contains abundant carbon, phosphorus, and nitrogen nutrient
Li, 2009). Furthermore,suchreactors require a specific land area,whichis not
substances. In 2004, over 90% of the domestic sewage generated in rural areas
available in most rural areas because of farmland protection policies in China
was directly discharged into natural waters without appropriate treatment,
(Fang et al., 2011). Stabilization ponds (i.e., microalgae biological
eventually causing water eutrophication (Wang et al., 2004). The amount of
wastewater treatment systems) have been attracting an increasing interest as
nutrients present in domestic sewage is conducive for the implementation of a
economical and highly efficient alternatives for the purification of domestic
biological wastewater treatment method (Wang et al., 2011). Therefore, the
sewage because of their low construction and maintenance costs, minimal
identification of an optimum biosystem is urgently needed to effectively treat
energy consumption during operation, as well as high removal efficiency (Li
domestic sewage.
et al., 2010; Ferrero et al., 2012). Moreover, culturing certain species of
Existing methods for domestic sewage treatment include packed-bed
microalgae with wastewater leads to decontamination and the possible
biofilm reactors, upflow anaerobic sludge blanket reactors, and stabilization
harvest of potentially valuable microalgae biomass or metabolic products
ponds (Renman et al., 2009; Wang et al., 2010). However, centralized
(Chinnasamyet al.,2010; Pilon etal., 2011; Sandefur et al.,2011).
wastewater treatment reactors based on packed-bed biofilm reactors or upflow
However, the current microalgae biological wastewater treatment
anaerobic sludge
system suffers from inconsistent light intensity or wavelength (Pilon et al.,
2011; Wang et al., 2007). Under natural conditions, varying light intensities
likely inhibit microalgae growth because of insufficient light energy during
rainy days or excessive irradiance at noontime during summer that causes
∗ Corresponding authors at: Department of Environmental Science and Engineering, Fudan photoinhibition. Microalgae require optimal lighting conditions to achieve
University, 220 Handan Road, Shanghai 200433, PR China. maximum nutrient removal efficiency (Termini et al., 2011). Thus, the
Tel.: +86 21 65642948; fax: +86 21 65642948. indoors culturing of microalgae by using artificial light sources is an
E-mail addresses: yancheng622@126.com (C. Yan), LXZ@fudan.edu.cn (X. Luo), alternative to the microalgae biological wastewater treatment system. The
zzhenghj@fudan.edu.cn (Z. Zheng).
1
intensity and wavelength of artificial light sources are essential parameters
These authors are equal contributors to this work.
for microalgae growth (Chojnacka and Noworyta, 2004; Hirata et al., 1998;
 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32
Rochet et al., 1986). In using light for microalgae metabolism, ordinary light
bulbs or fluorescent lamps are less economical and less efficient than lights
with specific narrow bands. This result is attributed to the theory that ordinary
light sources are a combination of efficient and inefficient light spectra for
microalgae growth or that their wavelengths do not cover the absorption
bands of the chlorophyll pigments of microalgae (Zhao et al., 2011). Thus, in
this study, light-emitting diode (LED) was used as the optimal light source
for microalgae culture because of its low power consumption and narrow
band wavelength. However, the performance of domestic sewage
purification by microalgae under various LED light wavelengths and
intensities remains largely unknown. Furthermore, influent C/N ratio is a
main factor in the biological wastewater treatment process (Yan et al., 2012;
Zhao et al., 2011). Thus far, only a few studies have been reported on the
effects of different influent C/N ratios on domestic sewage treatment by
microalgae.
This research focusedon the responses of microalgae to various LED light
wavelengths and intensities, as well as to influent C/N ratios, relative to the
dry weight (DW) and nutrient removal efficiency of domestic sewage. The
optimal light wavelength and intensity were determined. Moreover, the
treatment effect in response to different influent C/N ratios was analyzed to
predict andexplainthe performance of the microalgae biological wastewater
treatment system.
2. Materials and methods
2.1. Materials
For convenience in comparison of parallel experiments and for the health
and safety of the researchers, this study used a synthetic domestic sewage that
was a modification of the OECD standard sewage (OECD, 1996) and a
mixture of several chemical substances. The concentrations of chemical
oxygen demand (COD) and total nitrogen (TN) concentrations were
adjusted whereas that of total phosphorus (TP) was kept constant. Two
experimental categories were created (Table 1). Category 1, i.e., C variation
treatment, included fixed TN/TP levels at medium strength and various
levels of COD (low, medium, and high). Three levels of C variation were
designated as C1NP (C:N = 2.5:1), C2NP (C:N = 5:1), and C4NP (C:N =
10:1). Category 1 was prepared by mixing the following components: 100,
200, and 400gm−3 glucose; 80gm−3
carbamide; 15 g m−3 NaH PO ; 1.5g m−3 KH PO ;4 gm−3 CaCl ;
2 4 2 4
3
and 2g m− MgSO . Category 2, i.e., N variation treatment, included
4 efficiency was calculated as follows:
fixed COD/TP levels at medium strength and various levels of TN (low,
medium, and high). Three experimental groups for N variation were
designated as CN1P (C:N = 10:1), CN2P (C:N = 5:1), and CN4P (C:N =
2.5:1). Category 2 was prepared by mixing the following components:
200gm−3 glucose; 40, 80, and 160gm−3 carbamide;
15g m−3 NaH PO ; 1.5g m−3 KH PO ;4 gm−3 CaCl ; and 2gm−3
2 4 2 4 2
MgSO4. All experimental groups were performed in quadruplicates. Table 1
indicatesthe characteristics of the synthetic domestic sewage.
The LEDs (CN-PT10-XX15) used in this study were purchased from the
Canal Optoelectronic Technology Co., Ltd., P.R. China. The LEDs are 26
mm wide and 600 mm long, and their characteristics are indicated in Table 2.
The Chlorella vulgaris (FACHB-31) microalgae strain was purchased from
the FACHB-Collection, Institute of Hydrobiology, Chinese Academy of
Sciences.
25
2.2. Experimentalprocedure
A jar-test used a 1000 mL Erlenmeyer flask containing 400 mL synthetic
domestic sewage and 200 mL C. vulgaris solution, which had a DW of
104.56±2.73mgL−1. All treatments were preserved
in an illuminating incubator (SPX-400I-G) purchased from the Boxun Industry
& Commerce Co., Ltd., P.R. China. The LEDs were installed in the illuminating
incubator as light sources. During the 10 d/240 h experimental period, the
temperature was maintained at 25.0 ± 0.5◦C and the light–dark cycle was
maintained at 12 h:12 h (light was from 8:00 a.m. to 8:00 p.m., and the rest was
dark). Artificial shaking was performed prior to daily sampling. The optimal
light wavelengths for efficient nutrient removal of synthetic domestic sewage
under different influent C/N ratios and microalgae reproduction were
determined by exposing the jar-test flask to various light wavelengths (i.e., red,
yellow, purple, blue, green, and white) at constant light intensity of
2000molm−2 s−1.
The jar-tests were then illuminated using optimal light wavelengths at various
intensities (i.e., 500, 1000, 1500, 2000, 2500, and 3000molm−2 s−1) to
determine the optimal light intensity strat-
egy. The required light intensities were attained by increasing the number of
LEDs. All treatments were performed in quadruplicates. The cultures were
sampled and analyzed daily at 8:00 a.m., and then the mean values were
calculated.
2.3. Sampling andanalyses
The DW of C. vulgaris was measured as follows. Culture suspensions of 15
mL were filtered using a glass microfiber filter (GF/C, Whatman, USA). Then
the C. vulgaris cell was attached to the filter, which was afterwards dried at 100◦C
for 24 h and then cooled to room temperature in a desiccator. Finally, DW was
calculated from the difference between the filter weights before and after
filtration (Kebede and Ahlgren, 1996). The culture filtrates were analyzed for
COD, TN, and TP concentrations by using standard methods (APHA-AWWA-
WPCF, 1995). The pH value and oxidation–reduction potential (ORP) were
measured using a pH meter (Orion 250 Aplus ORP Field Kit, USA). Light
2 intensity was measured using a light meter (CEM, DT-1308) purchased from the
Shenzhen Everbest Machinery Industry Co., Ltd., P.R. China. Nutrient removal
R (1)
where R is the nutrient removal efficiency (%) and C0 and Ci are the nutrient
concentrations in the initial synthetic domestic sewage and in the filtrates of
the cultures (mgL−1), respectively.
The growth rate of the DW of C. vulgaris represents its ability for microalgae
reproduction. Growth rate wascalculated asfollows (Wang etal., 2007):
G (2)
where G is the growth rate of the DW of C. vulgaris (%), D0 is the DW of C.
vulgaris at the beginning of the experiment, and Di is the highest DW of C.
vulgaris during the experimental period (mgL−1).
26 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32

2.4.Statistical analyses Table 2 Characteristics

of LED.

All statistics were analyzed using the SPSS software (SPSS, 2003). One-
way ANOVA was used to test differences in the related parameters of light
wavelengths at similar light intensities. Twoway ANOVA was used to test
differences among the effects of light wavelength, light intensity, influent C/N
ratio, and the interaction between any two of these factors on treatment
performance.
Table 1
Mean concentration ± SD of influent synthetic sewage.
treatments (Table 3) because the reproduction capacity of microalgae C.
vulgaris was highly affected by the characteristics of the light wavelengths.
The chlorophyll pigments in C. vulgaris are not good at absorbing purple,
blue, and green light wavelengths (Kebede and Ahlgren, 1996). The photons
of short wavelengths, i.e., purple (400–410 nm), blue (460–470 nm), and
green (525–550 nm), have high probabilities of striking the light-harvesting

Categories C or N level C/N ratios COD

Influent concentration TNL − 1 )
(mg TP

4pc Low C1NP (C:N = 2.5:1) 104.56 ± 5.78 7.83 42.87 ± 2.64 3.29 5.17 ± 0.96
COD Medium C2NP (C:N = 5:1) 206.71 ± 9.24 42.85 ± 1.82 5.23 ± 0.75
Category 1: C variation treatment
level High C4NP (C:N = 10:1) 407.54 ± 43.51 ± 5.19 ± 0.68

Low CN1P (C:N = 10:1) 206.34 ± 5.81 7.46 21.38 ± 2.65 2.36 5.24 ± 0.73
Category 2: N variation treatment TN level Medium CN2P (C:N = 5:1) 208.52 ± 6.83 42.72 ± 3.49 5.26 ± 0.84
High CN4P (C:N = 2.5:1) 203.79 ± 82.14 ± 5.28 ± 0.67

Three-way ANOVA was used to test differences among the effects of the
interactions among light wavelength, light intensity, and influent C/N ratio.
Finally, Duncan’s multiple range tests were used to further assess the
differencesamong the factors thatwere deemed significant in ANOVA.
3. Results anddiscussion
complex at the peak electrical energy. Therefore, purple, blue, and green
wavelengths are highly energetic and efficient for photosynthesis, resulting
in photoinhibition (Das et al., 2011). On the other hand, the red, yellow, and
white wavelengths prevented photoinhibition; red and yellow wavelengths
possess relatively longer wavelengths, whereas the white wavelength is a
combination of red and other growth-inefficient light wavelengths (Matthijs
et al.,1996).

3.1. Microalgae growth atvarious light wavelengths

Table 3 indicates the growth rate of the microalgae DW under various light
wavelengths at a constant intensity of 2000molm−2 s−1. The microalgae C.
vulgaris reproduced under
3.2. Nutrient removalefficiencies with various light wavelengths
Table 3 indicates the highest nutrient removal efficiencies at various light
wavelengths with a constant intensity of 2000molm−2 s−1. The nutrient
removal efficiency of COD with

monochromatic light quality (i.e., red, yellow, purple, blue, and green) and
polychromatic light quality (i.e., white) at various influent C/N ratios.
Furthermore, the growth rate of the microalgae DW demonstrated similar
variation tendencies for each influent C/N ratio treatment exposed to different
light wavelengths. The red wavelength achieved significantly higher (P < 0.05)
microalgae DW, whereas the green wavelength achieved significantly lower (P
< 0.05) microalgae DW compared with the rest of the light wavelengths. No
significant differences were observed between the results of the purple and blue
light wavelengths (P > 0.05). In general, the order of microalgae C. vulgaris
reproduction at various influent C/N ratios is red > white > yellow > purple > blue
> green, indicating that the red light wavelength is the optimal light wavelength
for microalgae C. vulgaris reproduction.
This result agrees with that of Wang et al. (2007), who reported that the red
wavelength is the optimal light wavelength for Spirulina platensis reproduction.
Matthijs et al. (1996) reported a similar optimality of the red light wavelength for
Chlorella pyrenoidosa reproduction. These phenomena can be explained by the
theory that microalgae are good at absorbing red light through their green
pigment, chlorophyll (Matthijs et al., 1996). However, Das et al. (2011)
suggested a different result. Blue light wavelength was reported as optimal for
the growth of Nannochloropsis sp. and Botryococcus braunii Bot-144 (race B)
because different species of microalgae might require different light
wavelengths for their reproduction (Pilon et al., 2011). In this study, the DW
growth rates in the red, white, and yellow light treatments were much higher
compared with those in the purple, blue,andgreenlight
red wavelength was significantly higher (P < 0.05) compared with those with
the other wavelengths. No significant difference was observed between the
results of the blue and purple light wavelengths (P > 0.05). The yellow light
wavelength was significantly higher (P < 0.05) than the white wavelength.
The green wavelength was significantly lower (P < 0.05) than all other
wavelengths. COD removal efficiency at various influent C/N ratios was in
the order of red > yellow > white > purple > blue > green (Table 3). Therefore,
the red light wavelength between 76.02 ± 4.32% and 76.46 ± 1.52% is the
optimal light wavelength for COD removal. This result is higher than that of
Yang et al. (2008), who reported a maximum COD removal efficiency of
71.2% by cultivating C. pyrenoidosa with cassava ethanol fermentation
under continuous polychromatic wavelengths. The results demonstrate that
the optimal and not the ordinary light wavelength, can achieve significant
COD removal efficiency.
The removal efficiencies of TN and TP indicated similar variation
tendencies at all influent C/N ratios. The nutrient removal efficiency of the red
light wavelength was significantly higher (P < 0.05) compared with those of
the other light wavelengths. Furthermore, no significant difference was
observed between the results of the

Table 3

Light source Wavelength (nm ) Power consumption (W )

1000 (mol m−2 s−1) 1500 (mol m−2 s−1) 2000 (mol m−2 s−1) 2500 (mol m−2 s−1)

Red 620–630 3.58 4.53 5.37 7.94

Yellow 590–600 8.34 11.42 12.61 15.58
Purple 400–410 6.17 10.46 14.25 18.43
Blue 460–470 6.26 10.58 14.27 18.52
Green 525–550 11.14 15.79 17.42 21.81
White 380–760 3.61 5.36 6.87 9.62
 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32 27
Mean value±SD of the growth rate of the DW of microalgae and nutrient removal efficiency at various light wavelengths with a constant light intensity of 2000molm −2 s−1.

C1NP Red 126.95a ± 12.32 76.02a ± 4.32 75.08a ± 3.65 67.06a ± 3.29
White b
b
112.53 ± 13.29 c
68.76 ± 3.91 71.36 ± 2.63 61.41b ± 1.78
Yellow
Purple 107.24c ± 11.75 72.26b ± 5.21 67.59c ± 1.45 56.82c ± 2.64
Blue
d d
Green
d
55.62 ± 6.21 50.63 ± 4.22 49.42 ± 1.78 38.18d ± 3.42
C2NP Red
50.73d ± 5.43 48.42d ± 5.81 47.37d ± 2.64 36.81d ± 1.49
White
e e
Yellow e
13.21 ± 2.32 32.71 ± 3.58 29.63 ± 1.72 20.42e ± 2.47
Purple
135.37a ± 10.32 76.28a ± 1.96 74.43a ± 1.42 73.93a ± 3.28
Blue
Green b
124.42 ± 11.43 c
66.96 ± 1.64 b
69.52 ± 3.71 67.53b ± 1.21
C4NP Red
c
White 114.56c ± 13.24 b
71.71 ± 3.61 65.64 ± 2.37 61.58c ± 2.37
Yellow
61.83d ± 8.79 48.93d ± 2.16 49.25d ± 2.38 38.29d ± 1.48
Purple
d
Blue 60.32d ± 6.96 d
47.34 ± 3.24 47.71 ± 1.73 37.36d ± 2.47
Green Red
CN1P 15.67e ± 2.23 29.58e ± 2.11 29.28e ± 2.64 23.72e ± 1.45
White
Yellow a
122.95 ± 11.46 a
76.19 ± 1.34 a
75.15 ± 2.71 61.95a ± 1.53
Purple
Blue 108.72b ± 10.55 65.49c ± 2.55 68.93b ± 1.57 57.83b ± 2.65
Green c
c
99.32 ± 11.59
b
70.14 ± 2.47 64.15 ± 4.27 52.95c ± 1.81
Red
CN2P White 42.62d ± 5.54 47.24d ± 3.81 48.23d ± 1.59 31.33d ± 1.86
Yellow d
d
38.45 ± 4.75
d
46.71 ± 2.43 45.69 ± 2.53 29.42d ± 3.85
Purple
Blue 6.83e ± 1.53 29.53e ± 1.76 76.36a ± 27.63e ± 1.69 13.91e ± 2.77
Green Red
White
a
127.51 ± 12.52 4.05 a
71.53 ± 2.82 66.25a ± 1.53
CN4P Yellow c
115.32b ± 11.72 67.22 ± 3.26 67.42b ± 2.62 61.31b ± 2.37
Purple
b
Blue c
102.43 ± 13.74 71.03 ± 1.49 c
61.59 ± 1.34 57.47c ± 1.39
Green d
38.63d ± 4.28 49.19 ± 2.43 48.69d ± 2.43 43.32d ± 1.73
d
32.74 ± 4.55 47.45d ± 1.78 d
47.45 ± 1.67 41.91d ± 1.59
e
4.36e ± 1.71 31.61 ± 3.82 29.91e ± 2.48 26.64e ± 1.94
a
130.66 ± 10.64 76.46a ± 1.52 a
74.61 ± 1.38 72.86a ± 1.82
c
122.62b ± 12.55 64.61 ± 1.45 b
70.36 ± 1.74 68.24b ± 2.45

106.71c ± 11.43 70.29b ± 2.51 65.72c ± 2.66 64.91c ± 2.92

d
44.85d ± 5.74 45.44 ± 2.13 d
50.73 ± 1.58 43.84d ± 2.67

38.57d ± 4.55 45.01d ± 3.42 48.92d ± 2.59 42.94d ± 1.14

e
5.21e ± 0.43 27.85 ± 1.67 e
26.86 ± 1.93 27.51e ± 2.76

119.22a ± 9.87 76.43a ± 1.34 78.56a ± 1.27 61.29a ± 2.24

c
111.73b ± 10.84 62.16 ± 2.73 b
72.51 ± 1.54 45.23b ± 2.41

100.35c ± 13.95 68.16b ± 2.59 67.73c ± 1.69 39.48c ± 1.92

d
46.34d ± 4.56 43.46 ± 1.96 d
49.24 ± 1.71 26.89d ± 1.49

43.31d ± 3.64 42.23d ± 2.44 48.39d ± 1.85 26.21d ± 2.96

e
2.74e ± 0.88 23.24 ± 1.79 e
29.74 ± 1.96 19.37e ± 1.51
28 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32
blue and purple wavelengths (P > 0.05). The white light wavelength was
significantly higher (P < 0.05) than the yellow light wavelength, and the green light
wavelength was significantly lower (P < 0.05) than all other light wavelengths.
Both TN and TP removal efficiencies at various influent C/N ratios were in the order
of red > white > yellow > purple > blue > green (Table 3). Therefore, red light
wavelength is the optimal light wavelength for both TN and TP removal. In
addition, the highest TP removal efficiency achieved in this study was between
61.29 ± 2.24% and 73.93 ± 3.28% (Table 3), which is much higher than those of
Bhatnagar et al. (2010), who only achieved 30% TP removal efficiency in the
treatment of municipal wastewaters by using Chlorella minutissima in an oxidation
pond. This phenomenon indicates that the optimal light wavelength is highly
efficientcomparedwith the ordinaryonesfor TP removal.
These results can be attributed to the theory that C. vulgaris is highly efficient in
absorbing red light wavelength through its chlorophyll during photosynthesis, but
only partially absorbs the rest of the light wavelengths (Matthijs et al., 1996). Two
dominant influencing factors are involved in these phenomena. On one hand, the
yellow wavelength is close to the red wavelength (Table 2), whereas the purple,
blue, and green wavelengths are much farther away. Therefore, the red and yellow
wavelength treatments achieved similar COD nutrient removal efficiencies that
were significantly higher than those of the purple, blue, and green wavelengths. On
the other hand, the white wavelength demonstrated an average nutrient removal
effect compared with those of the red and yellow wavelengths because it is a
combination of red
Values with different superscript letters in the same column for the same C/N ratio indicate significant differences at P= 0.05 according to Duncan’s multiple range tests.
C/N ratio Light wavelengths Growth rate (%)
and the other less efficient light wavelengths. Moreover, the emission spectrum
band of the white wavelength completely covered that of the red wavelength (Table
2); thus, the white wavelength produced mixed effects on the entire light spectrum
for TN and TP nutrient removal (Wang et al., 2007). These abovementioned two
dominant factors combined their influence on the nutrient removal efficiencies,
and caused the reverse order of yellow and white light efficiency regarding
microalgae growth and CODremoval (Termini etal., 2011; Ferrero etal., 2012).
3.3.Physicochemical variations
Table 4 indicates the mean value of the physicochemical properties of domestic
sewage under various light wavelengths at a constantintensityof2000molm−2
s−1.Underalltypesofinfluent
C/N ratios, no significant difference (P > 0.05) was observed among various light
wavelengths for pH values. Su et al. (2011) reported a similar result, which
suggested that the pH value only slowly increased during the experimental period
when C. pyrenoidosa is cultivated in soybean processing wastewater. No
significant differences (P > 0.05) were observed among various light wavelengths
for ORP values under all typesofinfluent C/N ratios.
3.4.Optimization oflight intensity
3.4.1.Microalgae growthat various light intensities
Fig. 1 indicates similar variation trends in microalgae DW under red
wavelengths at various intensities andat different influent C/N
Nutrient removal efficiency (%)
COD TN TP
 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32 29

Fig.1. Microalgae DW over time under the red wavelength at various intensities and different influent C/N ratios: (a) C1NP,(b) C2NP,(c) C4NP,(d) CN1P,(e) CN2P,and (f)
CN4P.
7.28a 55.10a ± 6.18
7.26a 55.21a ± 4.83
Table 4 7.19a 54.86a
± 6.72
Mean value ± SD of the physicochemical properties of domestic sewage at various light 7.23a 55.19a
a ± 4.33
7.74a 54.84
wavelengths with a constant light intensity of 2000molm−2 s−1. Values with
7.52a 57.46a ± 3.28
7.26a 58.29a ± 4.46

± 3.94
different superscript letters in the same column for the same C/N ratio indicate significant ± 7.58
differences at P= 0.05 according to Duncan’s multiple range tests.
± 4.18
C/N ratio Light wavelengths pH ORP (mV)
± 3.64

C1NP Red 7.34 a

± 0.25 0.31 a
56.42 ± 5.21 ± ± 4.29
White 7.26 a ± 0.28 0.29 57.89a ± 3.63 ± ± 6.93 ±
Yellow 7.40a ± 0.18 0.22 54.28a ± 7.20
Purple ± 0.33 ± 4.72 ratios. The variation trends in microalgae DW were similar under all the
7.44a 55.84a
Blue ± 0.40 a ± 3.54 influent C/N ratios. However, the DW values of C. vulgaris at intensities of
7.37a 54.79
Green Red ± 0.36 0.28
7.56a 55.74a ± 4.83 500 and 3000molm−2 s−1 were both much lower
C2NP White ± 0.13 0.18
Yellow 7.19a ± 0.37 0.62 54.35a ± 3.45
Purple 7.26a ± 0.29 0.21 53.53a
± 4.48
Blue 7.52a ± 0.17 0.32 56.73a
Green Red 7.15a ± 0.16 0.18 53.34 a ± 3.83 comparedwith all other lightintensitiesbecause500molm−2 s−1
White 7.19a ± 0.14 0.18 55.57a ± 7.59
C4NP Yellow ± 0.19 0.33
7.24a 54.73a ± 6.52
Purple ± 0.14 0.24
7.25a 55.27a was insufficient in maintaining the growth of C. vulgaris. Wang et al. (2007)
Blue ± 0.33 ± 4.75
Green Red
7.32a
± 0.14 57.49a reported a similar result, in which the treatment with a light intensity of
White 7.18a ± 0.10 55.52a ± 6.26 300molm−2 s−1 and the dark treatment achieved
Yellow 7.21a ± 0.11 0.18 56.35a ± 4.33
CN1P Purple 7.64a ± 0.24 0.21 57.83a ± 3.69
Blue 7.23a ± 0.34 0.41 58.74a
Green Red ± 0.35 ± 4.85
7.42a 55.13a similar amounts of algae biomass, whereas the light intensity of 3000molm−2 s−1
White 7.35a ± 57.35a ± 5.74 was too high to avoid photoinhibition.
Yellow ±
7.22a 55.22a ± 4.27
Purple ±
CN2P 7.11a 54.94a ± 6.84
Blue ±
Green Red 7.32a ± 55.28a Accordingto the DW variation trend (i.e., 1000,1500, 2000,and
± 4.74
White 7.38a 57.32a
±
a ± 3.27
2500molm−2 s−1), the DW curve in Fig. 1 was divided into five
Yellow 7.17a ± 58.53
Purple 7.26a ± 57.12a ± 4.85
Blue 7.23a ± 58.82a ± 3.37
CN4P Green 7.30a ± 57.19a
± ± 4.93
7.24a 55.38a
±
30 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32
phases. Phase 1 (from 24 h to 48 h) indicated a relatively lower microalgae
DW. However, the exposure of microalgae to various light intensities of
1000, 1500, and 2000molm−2 s−1 demon-
strated equal or even slightly higher DW than that exposed to 2500molm−2 s−1.
This result can be explained in that relatively
lower light intensities (1000, 1500, and 2000molm−2 s−1) are
sufficient for photosynthesis at lowmicroalgae cellconcentrations, and that the
relatively higher light intensity (2500molm−2 s−1)
may lead to photoinhibition. Phase 2 (from 48 h to 96 h) indicated that
microalgae exposure to 1500 and 2000molm−2 s−1 achieved
equal or even higher DW than that exposed to 2500molm−2 s−1.
2 1
However, the microalgae exposed to 1000molm− s− treatment
exhibited a relatively lower DW because of the higher microalgae
concentration in Phase 2 than in Phase 1; thus, relatively higher light
intensities were required to conquer the mutual microalgae cellshadings, and
the relatively lower light intensity (1000molm−2 s−1) was clearly
insufficient for photosynthesis
(Das et al., 2011; Pilon et al., 2011). Phase 3 (from 96 h to 144 h) indicated that the
microalgae exposed to 2000 and 2500molm−2 s−1
achieved similar DW values, whereas those exposed to 1000 and 1500molm−2
s−1 achieved relatively lower results. Phase
4 (from 144 h to 192 h) indicated that the microalgae exposed to
2500molm−2 s−1 achieved the highest DW values. Finally, Phase
5 (from 192 h to 240 h) indicated that DW began to quickly decrease
under various light intensities.
The maximum DW was achieved at 2500molm−2 s−1 and
192 h under various influent C/N ratios (Fig. 1). Considering only the curves
for the treatment at a light intensity of 2500molm−2 s−1,
a four-phase growth curve was obtained. The DW of C. vulgaris quickly
increased from 24 h to 144 h, gradually increased from 144 h to 192 h, and
quickly decreased from 192 h to 240 h. These phenomena can be attributed to
the four-phase bacterial growth curve theory of Zwietering et al. (1990). The
period from 24 h to 144 h served as the exponential phase, in which the C.
vulgaris cells rapidly reproduced because the nutrients in the synthetic
domestic sewage medium were sufficient and the metabolic waste has not
thickly accumulated. The period from 144 h to 192 h served as the stationary
phase, in which the reproduction process slowed due to nutrient depletion
and toxic product accumulation during the metabolic process of the C.
vulgaris cells. The characteristic of the stationary phase is that the death and
birth rates of the C. vulgaris cells were nearly equal; thus, the DW of C.
vulgaris was at maximum during the stationary phase (Fig. 1). However, the
stationary phase was short because the nutrients in the synthetic domestic
sewage medium were soon depleted and the waste products have thickly
accumulated (Das et al., 2011). Then, the death phase (from 192 h to 240 h)
ensued, in which the C. vulgaris cells exhausted all the available nutrients and
resources,andquickly wastedaway.
3.4.2.Nutrientremoval efficiencies at various light intensities
Figs. 2–4 indicate the variation tendencies of the COD, TN, and TP
removal efficiencies under the red wavelength at various intensities and
different influent C/N ratios. The variation trends of nutrient removal were
similar under all types of influent C/N ratios. Furthermore, the removal
efficiencies of various nutrients likewise indicated similar variation trends.
The nutrient removal efficiencies at 500 and 3000molm −2 s−1 were much
lower com-
pared with those under other light intensities throughout the entire
experiment. The intensity of 500molm−2 s−1 was insuf-
ficient to maintain the metabolic process of microalgae C. vulgaris, whereas
3000molm−2 s−1 was too high to avoid photoinhibition.
Moreover, the variation tendencies in nutrient removal efficiencies (Figs. 2–
4) agreed with the results of the DW variations (Fig. 1). The highest nutrient
removal efficiency was achieved at 2500molm−2 s−1 at 144h (Figs. 2–4),
which occurred at the start
of the stationary phase (144–192 h) in Fig. 1. This was the same period at
which the maximum DW of C. vulgaris was achieved. Thus, the phase
division of DW in Fig. 1 and the nutrient removal efficiency in Figs. 2–4 may
be similar.
According to the variation trends in Figs. 2–4, the COD, TN, and TP
removal efficiency curves can be divided into the previously described five
phases. In Phase 1 (from 24 h to 48 h), the microalgae exposed to 1000, 1500,
and 2000molm−2 s−1 resulted in equal
or even slightly higher nutrient removal efficiency compared with those
treated with 2500molm−2 s−1. The relatively lower light
intensities (1000, 1500, and 2000molm−2 s−1) were sufficient
for photosynthesis of low cellular concentrations of microalgae, whereas a
relatively higher light intensity (2500molm−2 s−1)
may lead to photoinhibition. In Phase 2 (from 48 h to 96 h), the microalgae
exposed to 1500 andmolm−2 s−1 achieved
 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32 31

slightly higher or equal efficiency for nutrient removal compared with those
treated with 2500molm−2 s−1. However, the treat-
ment of 1000molm−2 s−1 resulted in relatively lower nutrient
removal efficiency because the higher microalgae concentration in Phase 2
than in Phase 1 required higher light intensity to conquer mutual microalgae
cell shading (Das et al., 2011; Pilon et al., 2011). In Phase 3 (from 96 h to 120
h), the microalgae exposed to 2500 and 2000molm−2 s−1 resulted in equal
nutrient removal
efficiency, whereas those exposed to 1500 and 1000molm −2 s−1 yielded
relatively lower removal efficiencies. In Phase 4 (from 120 h to 144h), the
microalgae exposed to 2500molm−2 s−1 resulted
in the highest nutrient removal efficiency. In Phase 5 (from 144 h to 240 h), the
nutrient removal efficiency rapidly decreased at all light intensities. The
stationary phase (Fig. 1) began at 144 h, in which the reproduction and
metabolism processes of C. vulgaris cells slowed down. Therefore, 144 h is the
optimal cultivation time to achieve the best nutrient removal effect.
strategy
Table 5 indicates the growth rates of microalgae DW and the nutrient
removal efficiencies under the optimal light intensity strategy at various influent
C/N ratios. No significant differences (P > 0.05) were observed for the COD
removal efficiency and the growth rate of the microalgae DW in all C and N
variation treatments. Furthermore, the highest COD removal efficiencies for C
and N variation treatments were achieved by the C2NP (C:N = 5:1) and CN1P
(C:N = 2.5:1) treatments, respectively. Considering the results in Table 3 and
Fig. 2, the COD removal efficiency was significantly affected by light
wavelength and intensity, the effect of the interaction between light wavelength
andintensity, aswell as the effectofthe interaction among light wavelength, light
intensity, and influent C/N ratio (Table 6). No significant differences among the
C variation treatments (C1NP, C2NP, and C4NP) were observed in terms of TN
removal efficiency (P > 0.05). However, the CN4P treatment produced a
significantly higher (P < 0.05) TN removal efficiency than those of the CN2P and
CN1P treatments. The highest TN removal efficiency was achieved by the C2NP
(C:N = 5:1) and CN4P (C:N = 10:1) treatments for the C and N variation
treatments, respectively. Considering the results in Table 3 and Fig. 3, the TN
removal efficiency was significantly affected by influent C/N ratio, light
wavelength and intensity, the effect of the interaction between light wavelength
andintensity, aswell as the effect of the interaction among light wavelength, light
intensity, andinfluent C/N ratio(Table 6).The removal efficiencies ofTP in the C
and N variation treatments were similar. The medium C/N ratio (C2NP and
CN2P) treatments exhibited significantly higher (P < 0.05) TP removal
efficiencies than the other treatments. Considering the

3.5.Light intensity strategy

3.5.1.Optimal strategyfor light intensity

The economic benefit of the red LED light wavelength was highest
compared with the other wavelengths at the same light intensity because it
consumed the least power (Table 2). Furthermore, the light intensity used in this
study should have been relatively lower during the initial culture time. The high
light intensity for a relatively lower microalgae cell density in the culture
medium during such period may lead to photoinhibition. The relatively lower
light intensity during the initial culture time can cause similar or even a better
effect on microalgae growth and nutrient removal. In addition, low light intensity
saves power consumption. On the other hand, at the end of the culture time, the
relatively higher microalgae cell density caused mutual cell shading. Therefore,
a relatively higher light intensity was essential during this period to avoid the
effects of insufficient lighting. Consequently, considering the phase division in
microalgae DW in Fig. 1, the nutrient removal efficiencies in Figs. 2–4, and the
optimal cultivation time (determined as 144 h), the optimal light intensity
strategy is introduced as follows: Phase 1, 0–48h (0–2 d) with 1000molm −2
s−1; Phase 2,

48–96h (2–4 d) with 1500molm−2 s−1; Phase 3, 96–120h (4–5

d) withmolm−2 s−1; and Phase 4, 120–144h (5–6 d) with

2500molm−2 s−1. Cultivation was concluded at 144h.

3.5.2.Effect ofinfluent C/N ratio onoptimal light intensity

32 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32

Time (h) Time (h) Time (h)

Fig. 2.COD removal efficiency over time under the red wavelength at various intensities and different influent C/N ratios: (a) C1NP, (b) C2NP, (c) C4NP, (d) CN1P, (e) CN2P,
and (f) CN4P.

results in Table 3 and Fig. 4, the TP removal efficiency was significantly affected
by influent C/N ratio, light wavelength and intensity, the effect of the interaction
between light wavelength and light intensity, as well as the effect of the
interaction among light wavelength, light intensity, and influent C/N ratio
(Table 6).
Nutrient removal was relatively lower when the carbon source was low
(C:N =2.5:1) or when the nitrogensource was insufficient (C:N =10:1). Thus,
supplementation of carbon or nitrogen sources and the controlofinfluent C/N
ratios are important factors for nutrient removal. This result is in agreement
with those of Zhao etal.
 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32 33

Fig.3. TN removal efficiency over time under the red wavelength at various intensities and different influent C/N ratios: (a) C1NP, (b) C2NP, (c) C4NP, (d) CN1P, (e) CN2P, and (f) CN4P.

Fig. 4.TP removal efficiency over time under the red wavelength at various intensities and different influent C/N ratios: (a) C1NP, (b) C2NP, (c) C4NP, (d) CN1P, (e) CN2P, and (f) CN4P.

Table 5
Mean concentration ± SD of the growth rate of the microalgae DW and nutrient removal efficiency under optimal light intensity strategy. Values with different superscript letters in the same column for

each category indicate significant differences at P= 0.05 according to Duncan’s multiple range tests.

Categories C/N ratio Growth rate (%) Nutrient removal efficiency (%)

COD TN TP
34 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32
C1NPC2NP 191.43a ± 13.75 14.21 92.45a ± 2.76 2.43 93.47a ± 2.38 87.47b ± 2.46
C4NP 195.96a ± 12.53 94.29a ± 3.58 95.87a ± 2.84 93.56a ± 1.74 ±
C variation treatment
185.39a ± 93.08a ± 95.26a ± 1.05 82.14c 3.07

CN1PCN2P 195.64a ± 14.28 11.34 93.95a ± 2.18 3.66 88.25c ± 1.49 2.74 87.94b ± 2.42
CN4P 194.71a ± 12.59 95.74a ± 2.49 92.83b ± 1.59 92.51a ± 1.57
N variation treatment
179.54a ± 93.32a ± 96.43a ± 82.46c
± 3.95
Table 6
P-values of factors and combined effects of factors for each parameter based on analysis of variance. Influent C/N ratios: C variation treatments (C1NP, C2NP, and C4NP) and N variation treatments
(CN1P, CN2P, and CN4P); Light wavelengths: red, yellow, purple, blue,green, and white; Light intensities: 500,1000,1500,2000,2500,and 3000molm−2 s−1.

Factor COD removal (%) TN removal (%) TP removal (%)

*
Influent C/N ratios 0.062 0.035 0.026*

Light wavelengths <0.0001** <0.0001**

Light intensities <0.0001** <0.0001**

Influent C/N ratios × Light wavelengths 0.014 0.013

Influent C/N ratios × Light intensities 0.017 0.019

<0.0001** <0.0001**
Light wavelengths ×Light intensities
0.012* 0.013*
Influent C/N ratios × Light wavelengths × Light intensities
*
P< under CN2P. The effluent under both treatments could meet China’s
0.05.
**
Wastewater Discharge Standard (GB 18918-2002). Thus, the effluent can be
P<
0.01. directly dischargedinto the water bodies.

Acknowledgments
(2011) and Yan et al. (2012), who both suggested that influent C/N ratio
This study was sponsored by the Beijing Green Future Environment
significantly affects the nutrient removal efficiency. In this study, the
Foundation, the National Key Special Project for Water Pollution Control and
balance of the influent carbon and nitrogen sources affected the growth of
Treatment (Grant no. 2012ZX07102004), and the National Science Funds of
microalgae, consequently affecting nutrient adsorption during the
China (Grant no. 51102136/E021301). We thank our colleagues and students
metabolic process (Yan et al., 2012; Zhao et al., 2011). Therefore,
from FudanUniversity for helping with the measurements.
considering all types of nutrient removal, the optimal influent C/N ratio was
5:1.
4.Conclusion References

The red light wavelength was the optimal light wavelength for purifying
synthetic domestic sewage with microalgae C. vulgaris. The optimal light
intensity strategy was as follows: Phase 1, 0–48 h (0–2 d) with mol m−2
1 2 1
s− ; Phase 2, 48–96 h (2–4 d) with 1500 molm− s− ; Phase 3, 96–120 h (4–5 d)
with 2000molm−2 s−1; and Phase 4, 120–144h (5–6 d) with
2500molm−2 s−1. The optimal cultivation time
Nutrient removal efficiency was significantly affected by light wavelength and
intensity, the effect of the interaction between light wavelength and intensity, as
well as the effect of the interaction among light wavelength, light intensity, and
influent C/N ratio. In addition, the TN and TP removal efficiencies were also
significantly affected by the influent C/N ratio. In this study, the optimal influent
C/N ratio was 5:1. Under the above optimal conditions, pollutants could be
removed from the synthetic domestic sewage at high efficiency rates. For
example,COD, TN, and TP concentrations in the effluent were 12.86 ±2.04, 3.19
±0.28, and 0.350.04mgL−1, respectively, under the treatment of C2NP;
± economy in algal culture (Chlorella pyrenoidosa). Biotechnol. Bioeng.50, 98–107.
and 12.45±1.74, 2.68±0.23, and 0.42±0.03mgL−1, respectively,
APHA-AWWA-WPCF, 1995. Standard Methods for the Examination of Water and Wastewater,
19th ed. American Public Health Association, Washington, DC.
Bhatnagar, A., Bhatnagar, M., Chinnasamy, S., Das, K., 2010.Chlorella minutissima – a promising
fuel alga for cultivation in municipal wastewaters. Appl. Biochem. Biotechnol. 161, 523–
536.
Chinnasamy, S., Bhatnagar, A., Hunt, R.W., Das, K.S., 2010. Microalgae cultivation in a
wastewater dominated by carpet mill effluents for biofuel applications. Bioresour. Technol.
101,3097–3105.
Chojnacka, K., Noworyta, A., 2004. Evaluation of Spirulina sp. growth in photoautotrophic,
heterotrophic and mixotrophic cultures. Enzyme Microbial Technol. 34, 461–465.
Das, P., Lei, W., Aziz, S.S., Obbard, J.P., 2011. Enhanced algae growth in both phototrophic and
was 144h. mixotrophic culture under blue light. Bioresour. Technol. 102, 3883–3887.
Fang, C.X., Guo, F.H., Luo, X.Z., Chen, J.Y., Zheng, Z., 2011. Performance of a three-stage tower
earthworm ecofilter for simultaneous removal of carbon and nutrients from domestic
wastewater. Fresen. Environ. Bull. 20, 235–243.
Ferrero, E.M., de Godos, I., Rodríguez, E.M., García-Encina, P.A., Munoz,˜ R., Bécares, E., 2012.
Molecular characterization of bacterial communities in algal–bacterial photobioreactors
treating piggery wastewaters. Ecol. Eng. 40, 121–130.
Hirata, S., Taya, M., Tone, S., 1998. Continuous culture of Spirulina platensis under
photoautotrophic conditions with change in light intensity. J. Chem.Eng. Jpn.
31,636–639.
Kebede, E., Ahlgren, G., 1996. Optimum growth conditions and light utilization efficiency of
Spirulina platensis (=Arthrospira fusiformis) (Cyanophyta) from Lake Chitu, Ethiopia.
Hydrobiologia 332, 99–109.
Li, X., Hu, H.Y., Gan, K., Yang, J., 2010. Growth and nutrient removal properties of a
freshwater microalga Scenedesmus sp. LX1 under different kinds of nitrogen sources.
Ecol. Eng.36,379–381.
Matthijs, H.C.P., Balke, H., van Hes, U.M., Kroon, B.M.A., Mur, L.R., Binot, R.A., 1996.
Application of light-emitting diodes in bioreactors: flashing light effects and energy
OECD, 1996. Guidelines for Testing of Chemicals Simulation Tests – Aerobic Sewage
Treatment. Technical Report. Organisation for Economic Co-operation and
Development (OECD), Paris.
 C. Yan et al. / Ecological Engineering 51 (2013) 24– 32 35
Pilon, L., Berberoglu, H., Kandilian, R., 2011. Radiation transfer in photobiological carbon
dioxide fixation and fuel production by microalgae. J. Quant. Spectrosc. Radiat. Transf.
112, 2639–2660.
Renman, A., Renman, G., Gustafsson, J.P., Hylander, L., 2009. Metal removal by bed filter
materials used in domestic wastewater treatment. J. Hazard. Mater. 166, 734–739.
Rochet, M., Legendre, L., Demers, S., 1986. Photosynthetic and pigment responses of sea-ice
microalgae to changes in light intensity and quality. J. Exp. Mar. Biol. Ecol. 101, 211–226.
Sandefur, H.N., Matlock, M.D., Costello, T.A., 2011. Seasonal productivity of a periphytic
algal community for biofuel feedstock generation and nutrient treatment.
Ecol. Eng.37,1476–1480.
SPSS, 2003.Analytical Software. SPSS Inc., Chicago.
Su, H.Y., Zhang, Y.L., Zhang, C.M., Zhou, X.F., Li, J.P., 2011. Cultivation of Chlorella
pyrenoidosa in soybean processing wastewater. Bioresour. Technol. 102, 9884–9890.
Termini, I.D., Prassone, A., Cattaneo, C., Rovatti, M., 2011. On the nitrogen and phosphorus
removal in algal photobioreactors. Ecol. Eng. 37, 976–980.
Wang, C.Y., Fu, C.C., Liu, Y.C., 2007. Effects of using light-emitting diodes on the cultivation
of Spirulina platensis. Biochem. Eng.J. 37,21–25.
Wang, D.B., Zhang, Z.Y., Li, X.M., Zheng, W., Yang, Q., Ding, Y., Zeng, T.J., Cao, J.B., Yue,
X., Shen, T.T., Zeng, G.M., Deng, J.H., 2010. A full-scale treatment of freeway toll-gate
domestic sewage using ecology filter integrated constructed rapid infiltration. Ecol. Eng.
36,827–831.
Wang, L.M., Zheng, Z., Luo, X.Z., Zhang, J.B., 2011. Performance and mechanisms of a
microbial-earthworm ecofilter for removing organic matter and nitrogen from synthetic
domestic wastewater. J. Hazard. Mater. 195, 245–253.
Wang, X.J., Zhang, W., Huang, Y.N., Li, S.J., 2004. Modeling and simulation of point–non-
point source effluent trading in Taihu Lake area: perspective of nonpoint sources control in
China.Sci. Total Environ. 325,39–50.
Yan, C., Zhang, H., Li, B., Wang, D., Zhao, Y.J., Zheng, Z., 2012. Effects of influent C/N ratios
on CO2 and CH4 emissions from vertical subsurface flow constructed wetlands treating
synthetic municipal wastewater. J. Hazard. Mater. 203–204, 188–194.
Yang, C.F., Ding, Z.Y., Zhang, K.C., 2008. Growth of Chlorella pyrenoidosa in wastewater
from cassava ethanol fermentation. World J. Microbiol. Biotechnol. 24, 2919–2925.
Ye, F.X., Li, Y., 2009. Enhancement of nitrogen removal in towery hybrid constructed wetland
to treat domestic wastewater for small rural communities. Ecol. Eng. 35, 1043–1050.
Zhao, Y.J., Zhang, H., Chao, X., Nie,E., Li,J.H., He,J., Zheng, Z.,2011.Efficiency of two-stage
combinations of subsurface vertical down-flow and up-flow constructed wetland systems
for treating variation in influent C/N ratios of domestic wastewater. Ecol. Eng. 37, 1546–
1554.
Zwietering, M.H., Jongenburger, I., Rombouts, F.M., vant Riet, K., 1990. Modeling of the
bacterial growth curve. Appl. Environ. Microbiol. 56, 1875–1881.