Research Article

Received: 7 June 2018 Revised: 25 September 2018 Accepted article published: 1 November 2018 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.5837

Treatment of real aquaculture wastewater from

a fishery utilizing phytoremediation
with microalgae
Yang Liu, Junping Lv,* Jia Feng, Qi Liu, Fangru Nan and Shulian Xie*
Abstract
BACKGROUND: In this study, five microalgal species (Chlorella vulgaris, Chlorococcum sp. GD, Parachlorella kessleri TY,
Scenedesmus quadricauda, and Scenedesmus obliquus) were cultivated in batch mode to evaluate their respective potential for
the treatment of real aquaculture wastewater from a fishery. Subsequently, the microalga with the best performance was culti-
vated with different initial inoculation concentrations to evaluate the effect of initial inoculation on pollutant removal efficiency.

RESULTS: When real aquaculture wastewater was inoculated with exogenous microalgae, the growth of both indigenous
microalgae and bacteria was significantly inhibited. Pollutant removal was closely related to exogenous inoculation of microal-
gae. Parachlorella kessleri TY had high growth potential and pollutant removal capability in aquaculture wastewater, com-
pared with the other four microalgae. When the wastewater was inoculated with low biomass concentrations of P. kessleri TY
(50–100 mg L−1 ), it grew well and degraded most of the encountered pollutant. In particular, P. kessleri TY with 100 mg L−1 of
inoculation concentration removed 94.4% of COD, 96.2% of ammonium, 99% of nitrite, 94.3% of nitrate, and 95.6% of phospho-
rus after 3 days of cultivation.

CONCLUSIONS: Both the screening for microalgal species and the regulation of initial inoculation concentrations are promising
approaches to enhance pollutant removal efficiency from real aquaculture wastewater.
© 2018 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: inoculation concentrations; microalgal species; Parachlorella kessleri TY; pollutant removal; real aquaculture wastewater

INTRODUCTION from the wastewater to produce biomass, which is different from

Over recent decades, the land-based aquaculture industry has the conventional process of precipitation of phosphorus with
been intensely developed to meet the increasing demand metal salts and conversion of nitrogenous compounds to N2
of aquatic product consumption. The fast development of the gas.5–7 In this process, no toxic substances are produced and
aquaculture industry has caused severe problems to the envi- released, and microalgal biomass can be simultaneously harvested
ronment, especially the problem of wastewater treatment.1,2 and further processed to produce high value-added products.10
The effluent from the aquaculture industry is commonly rich At present, several studies have demonstrated that microal-
in dissolved organic carbon, nitrogenous compounds (ammo- gae can grow in aquaculture wastewater.3,11,12 In addition to
nia, nitrite, and nitrate), and phosphorus.1,3 This effluent will the removal of nitrogen, phosphorus, and other pollutants
potentially induce harmful algal blooms and deteriorate the from aquaculture wastewater, microalgae can also accumu-
natural aquatic ecosystem if wastewater is not treated properly.4 late protein and/or lipid, which can subsequently be used to
To date, several biological and chemical techniques have been produce aquaculture feed and biofuel.13 However, aquaculture
successfully used to treat aquaculture wastewater, such as biolog- wastewater is typically pretreated via filtration, sterilization, boil-
ical nitrification/denitrification processes to remove nitrogenous ing, and ultraviolet irradiation before it is used for microalgal
compounds5,6 and chemical precipitation to remove phosphorus.7 culture.3,12,14–16 Due to the existence of indigenous bacteria
Although these techniques can remove pollutants from aquacul- and microalgae in real aquaculture wastewater, it is not clear
ture wastewater, they are not environmentally friendly and also whether exogenous microalgae can adapt to the conditions of real
increase the cost of aquaculture.
Although phytoremediation of wastewater utilizing microalgae
is not a new idea and was first reported in the 1950s,8 it has ∗ Correspondence to: J Lv or S Xie, School of Life Science, Shanxi Uni-
received much attention during recent years.9 Microalgae can versity, Taiyuan, 030006, China. E-mail: [email protected] (Lv);
produce oxygen through photosynthesis, which is beneficial as it [email protected] (Xie)
reduces the electricity costs of energy-intensive electromechanical Yang Liu and Junping Lv contributed equally to this work
aerators in microalgae-based wastewater treatment processes.
Moreover, microalgae can assimilate nitrogen and phosphorus School of Life Science, Shanxi University, Taiyuan, China

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 www.soci.org Y Liu et al.

aquaculture wastewater. Moreover, the microalgae-based aqua- required for microalgal cultivation. The physicochemical parame-
culture wastewater treatment process does not predominate over ters for collected aquaculture wastewater are depicted in Table S1
the conventional biological wastewater treatment process due to in File S1, Supporting information. The chemical oxygen demand
the slow growth rate of microalgae. This results in a longer culture (COD), nitrate (NO- 3-N), nitrite (NO- 2-N), ammonium (NH- 4-N)
period of microalgae (more than 10 days) to achieve the removal and total phosphorus concentrations of the supernatant were
of most pollutants from aquaculture wastewater.12 Clearly, it will 32.4, 0.35, 24.7, 6.25, and 1.83 mg L−1 , respectively. The pH of the
not be conducive to microalgae-based aquaculture wastewater collected wastewater was 7.86. The bacterial concentration in
treatment when a large amount of aquaculture wastewater is dis- the aquaculture wastewater was found to be 2.54 × 104 colony
charged. Recently, adjustment of the initial inoculation dosages forming unit (cfu) mL-1 . Several microalgae were also found.
of microalgae has been proposed to alter pollutant removal rate The cell densities of Cyanophyta, Chlorophyta, Bacillariophyta
and shorten cultivation time. As described by Nasir et al.12 30% and Euglenophyta were 21.7 × 106 2.5 × 106 0.68 × 106 and
(v/v) inoculation dosage of Chlorella sp. achieved a faster rate of 0.16 × 106 cells L−1 , respectively. This meant that cyanobacteria
ammonia reduction, compared with other inoculation dosages. were the dominant species in this aquaculture wastewater.
94.9% of ammonia was removed by the 3rd day. Lananan et al.17
reported that Chlorella sp. with 40% (v/v) inoculation dosage Microalgal batch cultivation
had a faster degradation rate of ammonia and orthophosphate, All of the five microalgae growing in BG11 medium were collected
most of which were degraded within 3–4 days. Nevertheless, a via centrifugation at 5000 rpm for 5 min when they were in the
related investigation on controlling the pollutant degradation logarithmic growth phase. Pellets were washed with deionized
rate via initial inoculation dosages has not been found. More water and centrifuged a second time at 5000 rpm for 5 min.
importantly, 3–4 days incubation time is still long. Consequently, Pellets were then suspended in a small volume of aquaculture
a large reactor volume will be required to treat a large volume of wastewater, and subsequently inoculated in 1000 mL flasks con-
aquaculture wastewater. This is clearly not economical. To further taining 500 mL aquaculture wastewater. The initial inoculation
improve the pollutant removal rate and shorten the cultivation concentration of these five microalgae was about 100 mg L−1
time in aquaculture wastewater, further knowledge is required (equal to 21. 9 × 106 –26.2 × 106 cells L−1 ). 70 mg L-1 of suspended
regarding microalgal performance under different initial inocula- solids (mainly composed of indigenous microalgae and bacteria)
tion concentrations. Furthermore, as mentioned in other reports were resuspended in the aquaculture wastewater, and the total
on municipal sewage and piggery wastewater treatment, the suspended solids in the wastewater after inoculation reached
efficiency of removal of pollutant depends on the microalgal 170 mg L−1 . All microalgae were cultivated at a constant tem-
species.18,19 The selection of appropriate microalgal species is perature of 25 ∘ C in a shaker at a shaking rate of 160 rpm min−1 .
central for an efficient treatment of aquaculture wastewater. The cultures were illuminated with fluorescent lamps providing
In this study, several microalgal species were cultured in batch an incident light intensity of 55.5 𝜇mol photons m−2 s−1 under
mode to evaluate their potential for the treatment of real aqua- 14:10 h light:dark cycle. All experiments were performed in trip-
culture wastewater from a fishery. Subsequently, the microalga licate. In the present study, the aquaculture wastewater was not
with the best performance for pollutant degradation was culti- sterilized prior to experiments.
vated with different initial inoculation concentrations to evaluate To investigate the effect of initial microalgal concentration on the
the effect of initial inoculations on pollutant removal efficiency performance of microalgae-based aquaculture wastewater treat-
from real aquaculture wastewater. ment, an appropriate amount of microalgal biomass was added to
the aquaculture wastewater to obtain initial microalgal concentra-
tions of 0, 25, 50, 100, 200, and 400 mg L−1 for each experiment. All
MATERIAL AND METHODS experiments were carried out in triplicate. The culture conditions
Microalgae and aquaculture wastewater were consistent with the experiments described above.
Five microalgal species belonging to Chlorophyta were used in this
study. Chlorella vulgaris (FACHB-1227), Scenedesmus obliquus
Analytical methods
(FACHB-417), and Scenedesmus quadricauda (FACHB-1468) were
Determination of microalgal growth
purchased from the Culture Collection of Algae, Institute of
The microalgal concentration was determined with the method
Hydrobiology, Chinese Academy of Sciences (FACHB-collection,
of mixed liquid suspended solids (MLSS) according to the Chinese
Wuhan, China). Chlorococcum sp. GD and Parachlorella kessleri
state standard monitoring method.23 The specific growth rate and
TY were isolated from the moss Entodon obtusatus and soil
doubling time of microalgae were calculated according to the
from Shanxi Province, China, respectively. The details have been
following equations:
described in Feng et al.20 and Gao et al.21 respectively. They were
preserved in our laboratory. These microalgae were pre-cultivated ( ) ( ) ( )
Specific growth rate 𝜇, d-1 = ln Na - ln Nb ∕ ta -tb (1)
in BG11 medium. The cultivation conditions followed Fathurrah-
man et al.22 with modifications. The cultivation was conducted
at 25 ∘ C, 55.5 𝜇mol photons m−2 s−1 of incident light intensity ( )
Doubling time Td , t = ln 2∕𝜇 (2)
under 14:10 h light:dark cycle and shaking at 160 rpm on a shaker
(HY-8, Changzhou Guohua Electric Appliance Co., Ltd, Changzhou,
where Na and Nb refer to microalgal biomass (mg L−1 ) at times ta
China).
and tb , respectively.
The aquaculture wastewater was sampled from a fishery in
Taiyuan city of Shanxi Province, China. Prior to use, aquaculture
wastewater was static for 1 h to settle and remove large sus- Determination of water quality
pended particulates. Aquaculture wastewater was analyzed for To determine the water quality, the microalgal suspension
the presence of nitrate, nitrite, ammonia, and total phosphates was centrifuged at 5000 rpm for 5 min and the supernatant

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was filtered through a 0.45 𝜇m cellulose acetate membrane. However, there were several differences in the growth of these five
The filtered supernatant was then used to determine COD, microalgae. After 5 days of cultivation, P. kessleri TY and Chlorococ-
nitrate, nitrite, ammonium, and total phosphorus. COD, nitrate, cum sp. GD achieved higher biomass concentrations of up to 260
nitrite, ammonium, and total phosphorus were analyzed via and 255 mg L−1 , respectively. C. vulgaris and S. quadricauda had
the dichromate method, ultraviolet spectrophotometric screen- moderate biomass concentrations of 235 and 240 mg L−1 , respec-
ing method, N-(1-naphthyl)-1,2-diaminoethane dihydrochloride tively. The biomass concentration of S. obliquus slowly increased
spectrophotometry, Nessler’s reagent spectrophotometry, to 225 mg L−1 . As shown in Table S2, the specific growth rate dif-
and ammonium molybdate spectrophotometric method, fered significantly among these five species (P < 0.05). P. kessleri
respectively.22 The pH, electrical conductivity, temperature, TY and Chlorococcum sp. GD achieved the highest specific growth
and dissolved oxygen were measured at the time of sample col- rate. C. vulgaris and S. quadricauda showed moderate growth rate.
lection via portable multiparameter analyzer DZB-718 (Shanghai S. obliquus had the lowest specific growth rate. Similar to the spe-
INESA Scientific Instrument Co., Ltd, China). Bacterial count was cific growth rate, there were also differences in the doubling time
determined via heterotrophic plate count.24 Microalgal count was of these five microalgae (P < 0.05). P. kessleri TY and Chlorococ-
determined by a CCD (DP72, Olympus, Tokyo, Japan) mounted on cum sp. GD had the shortest doubling time and S. obliquus had
the microscope. Microalgae were identified at the phylum level the longest doubling time. P. kessleri TY and Chlorococcum sp. GD
according to taxonomic descriptions.25–28 achieved the highest growth potential in real aquaculture wastew-
ater compared with the other three tested microalgae. The chl-a
Determination of chlorophyll content and chl-b content of P. kessleri TY also indicated that P. kessleri TY
The chlorophyll content was determined according to Mera et al.29 grew best in real aquaculture wastewater compared with the other
with some modifications. The microalgal cells were harvested via tested microalgae (Fig. 1(b) and (c)). Although indigenous bacteria
centrifugation at 4500 rpm for 15 min, and were suspended in 95% and microalgae were present in the original aquaculture wastewa-
(v/v) alcohol. The suspension was kept at 4 ∘ C in the dark for 24 h ter, a small amount of growth in biomass (15 mg L−1 of increase)
and centrifuged at 8000 rpm for 10 min to remove all cell debris. was detected after 5 days of cultivation in the control (Fig. 1(a)).
The absorbances of the supernatant were measured at 649 nm and The final biomass was significantly lower than that in wastew-
665 nm in a UV-visible spectrophotometer (TU-1810 DAPC, Beijing ater inoculated with these five microalgae (P < 0.05). This sug-
Persee General Instrument Co Ltd, China). A 95% alcohol solution gests that the increase of biomass in wastewater mainly derived
was used as blank. The concentration (mg L−1 ) of chlorophyll-a and from the exogenous inoculation of these five microalgae. In gen-
chlorophyll-b was calculated with the following equations: eral, all presented results suggested that the growth of microal-
gae in real aquaculture wastewater was species-dependent. This
Chlorophyll − a = 13.95 ∗ A665 − 6.88∗ A649 (3) was consistent with previous reports that indicated that microal-
gal growth in municipal sewage and piggery wastewater was
Chlo r ophyll-b = 24.96 ∗ A649-7.32 ∗ A665 (4) microalgae-dependent.18,19 Therefore, it is very important to select
suitable microalgal species for growth in aquaculture wastewater.
Because real aquaculture wastewater was used in this experi-
Determination of chlorophyll fluorescence
ment, a large number of indigenous microalgae were contained in
Chlorophyll fluorescence was measured according to Markou
this wastewater. The number of Cyanophyta and Chlorophyta was
et al.30 using the portable PAM fluorometer AquaPen-C AP-C 100
21.7 × 106 and 2.5 × 106 cells L−1 , respectively, in the aquaculture
(Photon Systems Instruments, Drassov, Czech Republic). After
wastewater (Fig. 2(a)). It was clear that Cyanophyta was the most
30 min of dark adaptation of the microalgal culture at room tem-
dominant group in the real aquaculture wastewater. The experi-
perature, values of Fv /Fo (potential activity of photosystem II) and
ment lasted for 5 days. On the 5th day, the count of Cyanophyta
Fv /Fm (maximum quantum yield of photosystem II photochem-
in the control had increased significantly to 25.6 × 106 cells L−1
istry) were automatically obtained via the PAM fluorometer using
(P < 0.05) (Fig. 2(a)). The number of Chlorophyta still remained
the OJIP (the typical Chl fluorescence signal rise displays four steps,
at a low level of 2.82 × 106 cell L−1 on the 5th day (Fig. 2(a)).
i.e. the O-, J-, I-, and P-steps) test.
This indicated that the species Cyanophyta had growth advan-
Statistical analysis tages in wastewater without exogenous inoculation of microal-
gae. When the real aquaculture wastewater was inoculated with
In this study, the measured values were expressed as the
exogenous microalgae, the number of green algae gradually
mean ± standard deviation. If necessary, statistical testing involv-
increased with passing time. After 5 days of cultivation, the count
ing ANOVA was implemented via SPSS software (version 19.0). A
of Cyanophyta and Chlorophyta in the experimental group of
confidence level of 95% was selected to strictly determine the sig-
S. quadricauda were 18.1 × 106 and 34.7 × 106 cells L−1 , respec-
nificance, and a statistically significant difference was defined
tively (Fig. 2(b)). In the experimental group of S. obliquus, the
for P < 0.05. Linear regression correlation analysis was conducted
cell densities of Cyanophyta and Chlorophyta were 18.1 × 106
with SPSS software, and there was a significant relationship when
and 33.2 × 106 cells L−1 , respectively (Fig. 2(c)). When the wastew-
P < 0.05.
ater was inoculated with C. vulgaris, the number of Cyanophyta
and Chlorophyta was 16.6 × 106 and 34.9 × 106 cells L−1 , respec-
RESULTS AND DISCUSSION tively (Fig. 2(d)). The number of Cyanophyta and Chlorophyta
Phytoremediation of real aquaculture wastewater from a was 16.9 × 106 and 37.1 × 106 cells L−1 in the P. kessleri TY experi-
fishery with microalgae (Chlorophyta) mental group (Fig. 2(e)). In the experimental group of Chlorococ-
The performance of microalgal growth in real aquaculture wastew- cum sp. GD, Cyanophyta and Chlorophyta reached 17.3 × 106 and
ater is shown in Fig. 1(a). No lag phase was observed in the five 35.9 × 106 cells L−1 , respectively (Fig. 2(f )). The result of the statisti-
microalgal growth curves, which indicated that all of these microal- cal data analysis indicated that the number of Cyanophyta from
gae adapted well to the conditions of real aquaculture wastewater. aquaculture wastewater inoculated with exogenous microalgae

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A B
280 5
B io m a s s c o n c en t r a tio n (m g /L )

240 4

C h lo r o p h y ll-a (m g /L )
200
3

160 100mg/L Scenedesmus quadricauda

100mg/L Scenedesmus obliquus 2 100mg/L Scenedesmus quadricauda
100mg/L Chlorella vulgaris 100mg/L Scenedesmus obliquus
120 100mg/L Parachlorella kessleri TY 100mg/L Chlorella vulgaris
100mg/L Chlorococcum sp. GD 100mg/L Parachlorella kessleri TY
without inoculation in aquaculture wastewater 1 100mg/L Chlorococcum sp. GD
80 without inoculation in aquaculture wastewater

0 1 2 3 4 5 0 1 2 3 4 5
Culture Time (day) Culture Time (day)

C 1.6

1.4
C h lo r o p h y ll-b (m g /L )

100mg/L Scenedesmus quadricauda

100mg/L Scenedesmus obliquus
100mg/L Chlorella vulgaris
1.2 100mg/L Parachlorella kessleri TY
100mg/L Chlorococcum sp. GD
without inoculation in aquaculture
1.0 wastewater

0.8

0.6
0 1 2 3 4 5
Culture Time (day)
Figure 1. Growth and chlorophyll concentration of five microalgae in real aquaculture wastewater.

was significantly lower than that in the control (P < 0.05) after and bacterial community structure in wastewater, it became clear
5 days of culture, independent of the type of inoculated microal- that biomass accumulation was closely related to exogenous inoc-
gae. At the same time, the number of Chlorophyta significantly ulation of these five microalgae and the growth of indigenous
increased (P < 0.05) compared with the control. Based on these microalgae and bacteria was significantly inhibited.
results, it was clear that the growth of indigenous Cyanophyta Pollutant removal by the five tested microalgal species in real
was inhibited while green microalgae grew well after inocula- aquaculture wastewater is depicted in Fig. 3. The pollutant con-
tion of the five tested microalgae in real aquaculture wastewa- centration in all of the tests showed a sharp decrease during
ter, especially aquaculture wastewater that was inoculated with the first 2 days. The removal of pollutant gradually leveled off
P. kessleri TY. In addition to microalgae, real aquaculture wastew- until the end of treatment. After 5 days of treatment, the max-
ater also contained a specific number of indigenous bacteria. As imum COD, nitrate, nitrite, ammonium, and total phosphorus
listed in Table 1, the bacterial density of real aquaculture wastew- removal efficiency were 88.9–94.4%, 85.7–97.1%, 94.3–99.8%,
ater reached 2.54 × 104 cfu mL−1 . After 5 days of cultivation, the 97.9–98.9%, and 90.2–98.9%, respectively. All of these five
bacterial density significantly decreased to 2.08 × 104 cfu mL−1 in microalgae (especially P. kessleri TY) exhibited high pollutant
the control (P < 0.05). When the wastewater was inoculated with degradation capability in real aquaculture wastewater, which was
C. vulgaris, Chlorococcum sp. GD, P. kessleri TY, S. obliquus, and comparable with the performance of microalgae in aquaculture
S. quadricauda, the bacterial density significantly decreased to wastewater reported previously.12,16 The maximum COD, nitrate,
0.82 × 104 –0.98 × 104 cfu mL−1 (P < 0.05) after 5 days of cultiva- nitrite, ammonium, and total phosphorus removal efficiencies
tion, compared with the original wastewater. This suggests that from aquaculture wastewater without inoculation of these five
the significant decrease of bacterial concentration in wastewa- microalgae were 50%, 42.86%, 18.49%, 14.88% and 13.67%,
ter was caused by competition for organic carbon or nutrients. respectively. Compared with wastewater inoculated with these
Based on the integrated analysis of these results on microalgal five microalgae, pollutant removal efficiency of the control was

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A B
70 70
Cyanophyta Bacillariophyta Cyanophyta Bacillariophyta
Chlorophyta Euglenophyta Chlorophyta Euglenophyta
60 60
wastewater without exogenous inoculation of microalgae wastewater inoculated with Scenedesmus quadricauda
cell density (x106cells/L)

cell density (x106cells/L)

50 50

40 40

30 30

20 20

10 10

0 0
0 1 2 3 5 0 1 2 3 5
Culture Time (day) Culture Time (day)

C 70
D 70
Cyanophyta Bacillariophyta Cyanophyta Bacillariophyta
Chlorophyta Euglenophyta Chlorophyta Euglenophyta
60 60
cell density (x106cells/L)

wastewater inoculated with Scenedesmus obliquus

cell density (x106cells/L)

wastewater inoculated with Chlorella vulgaris

50 50

40 40

30 30

20 20

10 10

0 0
0 1 2 3 5 0 1 2 3 5
Culture Time (day) Culture Time (day)

D E
70 70
Cyanophyta Bacillariophyta Cyanophyta Bacillariophyta
Chlorophyta Euglenophyta Chlorophyta Euglenophyta
60 wastewater inoculated with Parachlorella kessleri TY
60
wastewater inoculated with Chlorococcum sp. GD
cell density (x106cells/L)

cell density (x106cells/L)

50 50

40 40

30 30

20 20

10 10

0 0
0 1 2 3 5 0 1 2 3 5
Culture Time (day) Culture Time (day)
Figure 2. Microalgal community composition at phylum level in aquaculture wastewater inoculated with exogenous microalgae.

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Table 1. Bacterial density in the course of wastewater treatment

Bacterial density (×104 cfu mL−1 ) in the course ofwastewater treatment

Microalgae species 0 day 1 day 2 days 3 days 5 days

Chlorella vulgaris 2.58 ± 0.01a 1.83 ± 0.01a 1.65 ± 0.01a 1.02 ± 0.01a 0.98 ± 0.03a
Chlorococcum sp. GD 2.62 ± 0.01b 1.53 ± 0.02b 1.44 ± 0.01b 1.04 ± 0.01a 0.85 ± 0.03b
Parachlorella kessleri TY 2.55 ± 0.01c 1.37 ± 0.01c 1.34 ± 0.02c 0.96 ± 0.02b 0.82 ± 0.02c
Scenedesmus obliquus 2.49 ± 0.02d 1.75 ± 0.02d 1.53 ± 0.01d 1.15 ± 0.02c 0.95 ± 0.01d
Scenedesmus quadricauda 2.61 ± 0.01b 1.44 ± 0.02e 1.32 ± 0.01c 1.07 ± 0.01d 0.89 ± 0.02e
Without inoculation in aquaculture wastewater 2.54 ± 0.02c 2.47 ± 0.01f 2.32 ± 0.01e 2.29 ± 0.03e 2.08 ± 0.02f

Values within the same column with different letters represent significant difference (P < 0.05).

significantly lower (P < 0.05). The results of pollutant removal and mixture can be directly fed back to the fish pond to provide both
the aforementioned results on microalgal and bacterial commu- clean water and aquatic feed, which is considered to be both
nity structure indicated that the partial removal of pollutant from economical and feasible for the aquaculture industry.
the wastewater without exogenous inoculation of microalgae was
attributed to the growth of indigenous species of Cyanophyta Performance of P. kessleri TY with different inoculation
in the wastewater. High removal efficiency of pollutants from concentrations in real aquaculture wastewater from a fishery
wastewater with exogenous inoculation of microalgae was mainly The growth of P. kessleri TY with various inoculation concentra-
dependent on the growth of exogenous green algae. tions is depicted in Fig. 4(a). No lag phases were observed in any
As reviewed by Camargo and Alonso,31 inorganic nitrogen com- of the five curves, which indicated that P. kessleri TY with various
pounds are typically toxic for aquatic animals, compared with inoculation concentrations could adapt well to real aquaculture
phosphate. Especially, unionized ammonia (NH3 -N) and nitrite wastewater. After 5 days of cultivation, the biomass concentra-
exceed the toxicity of nitrate.32,33 Safe levels were estimated at tion increased to 150, 190, 260, 350, and 545 mg L−1 , respec-
0.05 mg NH3 -N L-1 and 0.328 mg NO2 -N L-1 for Macrobrachium tively, achieving 1.57, 1.58, 1.53, 1.30, and 1.16-fold increases in
carcinu.32 For Catla catla, the median lethal concentration val- biomass concentration, respectively. Furthermore, the biomass
ues for 24 h of NH3 -N, NO2 -N and NO3 -N were 0.036, 117.43, and concentration of P. kessleri TY at all inoculation concentrations was
1484.08 mg L−1 in a continuous flowthrough test, respectively.33 low, compared with P. kessleri cultivated in brewery wastewater.35
As shown in Table S3, after 5 days of cultivation, the NH3 -N con- Low biomass concentration in the aquaculture wastewater was
centration in aquaculture wastewater without inoculation of these caused by the low nutrients concentration compared with brewery
five microalgae was 0.59 mg L−1 , which was clearly not benefi- wastewater. Interestingly, P. kessleri TY with low inoculation con-
cial for the growth of fishes and shrimps. In contrast, the NH3 -N centrations (25–100 mg L−1 ) grew faster and accumulated more
concentration in aquaculture wastewater inoculated with microal- biomass, compared with P. kessleri TY with high inoculation con-
gae decreased significantly to 0.0024–0.0044 mg L−1 (P < 0.05). centrations (200–400 mg L−1 ). This phenomenon was also demon-
These values of NH3 -N concentration did not exceed that of that strated by the accumulation of chl-a and chl-b as shown in
water quality standard for fisheries (0.02 mg L−1 ) in China. Similarly, Fig. 4(b) and (c), especially when P. kessleri TY was inoculated with
the NO2 -N concentration in aquaculture wastewater inoculated 100 mg L−1 .
with microalgae was significantly lower than that in aquaculture To further the insight into the effect of the inoculation concen-
wastewater without microalgae inoculation (P < 0.05). Moreover, tration on the growth of P. kessleri TY, the specific growth rate and
the NO2 -N concentration from aquaculture wastewater inoculated doubling time were calculated as shown in Table 2. There was a
with P. kessleri TY was the lowest (Table S3). It was clear that resid- significant difference in the specific growth rate and doubling time
ual unionized ammonia and nitrite would not adversely affect the among different inoculation concentrations (P < 0.05). The specific
growth of fishes and shrimps when aquaculture wastewater was growth rate decreased from 0.12 to 0.037 day−1 with increasing
treated with these five microalgae (especially for P. kessleri TY). inoculum concentrations. In contrast, the doubling time increased
In the present study, biomass accumulated as microalgae from 5.66 to 18.7 days with increasing inoculum concentrations.
removed pollutant from the wastewater. Microalgal biomass typ- As described by Guerrero-Cabrera et al.15 the specific growth rate
ically contained a large share of carbohydrate, protein, and lipid, of Chlorella sp., Scenedesmus sp., and Monoraphidium sp. in tilapia
which could be used as feedstock for different products. As shown effluent medium ranged from 0.006 to 0.018 day−1 . These values
in Table S4, a high proportion of protein was detected in these five were significantly lower than those of P. kessleri TY in real aqua-
microalgae. Especially for P. kessleri TY, the protein content reached culture wastewater found in this study. Therefore, the growth of P.
up to 51%, which was more than the sum content of carbohydrate kessleri TY in aquaculture wastewater had specific advantages. Fur-
and lipid. Protein was considered the most important nutrient as it thermore, regression analysis was used to reveal the relationship
affected the growth performance of fish. As reported by Rahimne- between the inoculum concentration and the growth of P. kessleri
jad et al.34 the highest growth was found in fish that received a diet TY. As shown in Figure S1, the inoculum concentration correlated
containing 50% protein and 14% lipid. Consequently, P. kessleri negatively with the specific growth rate (r = −0.955, P < 0.05).
TY is suitable as aquatic feed. When the wastewater was treated There was a significant positive correlation between the inoculum
by microalgae, the concentration of pollutant in the effluent was concentration and the doubling time (r = 0.993, P < 0.01) (Figure
considered to be much lower than that from the water quality S1). These results provide further indication that low inoculum
standard for fisheries in China. Therefore, the microalgae–water concentration was beneficial for the growth of P. kessleri TY in real

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A 35 B 0.4
100mg/L Scenedesmus quadricauda 100mg/L Scenedesmus quadricauda
100mg/L Scenedesmus obliquus 100mg/L Scenedesmus obliquus
30 100mg/L Chlorella vulgaris 100mg/L Chlorella vulgaris
100mg/L Parachlorella kessleri TY 100mg/L Parachlorella kessleri TY
100mg/L Chlorococcum sp. GD 0.3
25

nitrate (mg/L)
without inoculation in aquaculture
COD (mg/L)

wastewater
20
0.2
100mg/L Chlorococcum sp. GD
15 without inoculation in aquaculture
wastewater
10
0.1
5

0 0.0
0 1 2 3 4 5 0 1 2 3 4 5
Culture Time (day) Culture Time (day)
C D
28 7

24 6
ammonium (mg/L)

20 5
nitrite (mg/L)

100mg/L Scenedesmus quadricauda 100mg/L Scenedesmus quadricauda

16 100mg/L Scenedesmus obliquus 4 100mg/L Scenedesmus obliquus
100mg/L Chlorella vulgaris 100mg/L Chlorella vulgaris
100mg/L Parachlorella kessleri TY
12 100mg/L Parachlorella kessleri TY 3
100mg/L Chlorococcum sp. GD 100mg/L Chlorococcum sp. GD
without inoculation in aquaculture without inoculation in aquaculture
8 wastewater 2 wastewater

4 1

0 0
0 1 2 3 4 5 0 1 2 3 4 5
Culture Time (day) Culture Time (day)

E 2.0
total phosphorus (mg/L)

1.6
100mg/L Scenedesmus quadricauda
100mg/L Scenedesmus obliquus
1.2 100mg/L Chlorella vulgaris
100mg/L Parachlorella kessleri TY
100mg/L Chlorococcum sp. GD
without inoculation in aquaculture
0.8
wastewater

0.4

0.0
0 1 2 3 4 5
Culture Time (day)
Figure 3. Pollutant removal from real aquaculture wastewater by five microalgae.

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 www.soci.org Y Liu et al.

A B
600 9
Biomass concentration (mg/L)

8 0 mg/L
500 0 mg/L 100 mg/L 25 mg/L
7

Chlorophyll-a (mg/L)
25 mg/L 200 mg/L 50 mg/L
50 mg/L 400 mg/L 100 mg/L
400 6 200 mg/L
400 mg/L
5
300
4
200 3

100 2
1
0
0 1 2 3 4 5 0 1 2 3 4 5
Culture Time (day) Culture Time (day)

C
4.0
3.5 0 mg/L
25 mg/L
Chlorophyll-b (mg/L)

3.0 50 mg/L
100 mg/L
2.5 200 mg/L
400 mg/L
2.0
1.5
1.0
0.5
0.0
0 1 2 3 4 5
Culture Time (day)
Figure 4. Growth characteristics and chlorophyll content of Parachlorella kessleri TY with different initial inoculation concentrations in real aquaculture
wastewater.

aquaculture wastewater. A maximum growth of Spirulina platen-

Table 2. Specific growth rate and doubling time of Parachlorella
sis has been obtained at a low initial inoculation concentration kessleri TY with different inoculums concentrations in aquaculture
of 50 mg L−1 .36 Similarly, as reported by Nasir et al.12 Chlorella sp. wastewater
at 10%, 20%, and 30% inoculation dosages had higher growth
potential than Chlorella sp. at 40%, 50%, and 60% inoculation Initial inocula concentration Specific
dosages. Other investigations also reported that the growth rate of of Parachlorella kessleri growth rate Doubling
Phaeodactylum tricornutum and Chlorella sp. increased when they TY (mg L−1 ) (days−1 ) time (days)
were inoculated at low concentrations.37–39 As reviewed by Chen
25 0.12 ± 0.005a 5.66 ± 0.06a
et al.40 the light intensity tended to decrease rapidly due to the
50 0.11 ± 0.011a 6.03 ± 0.08ab
light shading effect from increases in microalgal concentrations.
100 0.11 ± 0.009a 6.53 ± 0.04b
High inoculation concentrations not only resulted in decreases of
200 0.064 ± 0.004b 10.7 ± 0.05c
light intensity due to the strong mutual shading among microalgal
400 0.037 ± 0.006c 18.7 ± 0.14d
cells but also led to strong competition for the available nutrients.
Therefore, it was very likely that P. kessleri TY with low inoculation Values within the same column with different letters represent signifi-
concentrations had high metabolic activities, which contributed cant difference (P < 0.05).
to effectively absorb and incorporate nutrients into cells and pro-
mote their growth.
As a potential microalga for aquaculture wastewater treatment, up to 94.4%, 96.2%, 99%, 94.3%, and 95.6% for COD, ammo-
pollutant removal of P. kessleri TY from aquaculture wastewater nium, nitrite, nitrate, and total phosphorus, respectively. In the
at various inoculation concentrations is shown in Fig. 5. Most of remaining tested inoculation concentrations (25, 50, 200, and
the pollutants were degraded within 3 days for all inoculation 400 mg L−1 ), 77.8–88.9% of COD, 54.2–86.1% of ammonium,
concentrations. P. kessleri TY, at an inoculation concentration of 64.1–91.7% of nitrite, 57.1–85.7% of nitrate, and 53–81.4% of
100 mg L−1 , achieved the highest pollutant removal efficiency, total phosphorus were removed from the aquaculture wastewater

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A 35 B
7
0 mg/L
30 25 mg/L 6
50 mg/L

ammonium (mg/L)
100 mg/L
25 200 mg/L 5
COD (mg/L)

0 mg/L
400 mg/L 25 mg/L
20 4 50 mg/L
100 mg/L
15 3 200 mg/L
400 mg/L
10 2

5 1

0 0
0 1 2 3 4 5 0 1 2 3 4 5
Culture Time (day) Culture Time (day)
C D
28 0.4
0 mg/L
25 mg/L
24 50 mg/L
100 mg/L
0.3
20 200 mg/L
nitrate (mg/L)
nitrite (mg/L)

400 mg/L
0 mg/L
16 25 mg/L
50 mg/L 0.2
100 mg/L
12 200 mg/L
400 mg/L
8
0.1
4

0 0.0
0 1 2 3 4 5 0 1 2 3 4 5
Culture Time (day) Culture Time (day)

E 2.0
total phosphorus (mg/L)

1.6
0 mg/L
25 mg/L
1.2 50 mg/L
100 mg/L
200 mg/L
0.8 400 mg/L

0.4

0.0
0 1 2 3 4 5
Culture Time (day)
Figure 5. Pollutant removal from real aquaculture wastewater by Parachlorella kessleri TY at different initial inoculation concentrations.

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 www.soci.org Y Liu et al.

A 0.75 B 2.0
0.70 1.8
0.65
1.6
0.60
Fv/Fm

Fv/Fo
1.4 0 mg/L 100 mg/L
0.55 25 mg/L 200 mg/L
50 mg/L 400 mg/L
0.50 1.2

0.45 1.0
0 mg/L 100 mg/L
0.40 25 mg/L 200 mg/L
50 mg/L 400 mg/L 0.8
0.35
0 1 2 3 4 5 0 1 2 3 4 5
Culture Time (day) Culture Time (day)
Figure 6. Characteristics of chlorophyll fluorescence of Parachlorella kessleri TY with different initial inoculation concentrations in real aquaculture
wastewater.

after 3 days of culture. The above results were compared with Similar to profiles on Fv /Fm values of P. kessleri TY, the value of
that of Chlorella sp. with 72.1–94.3% and 63.1–92.2% of ammo- Fv /Fo also increased at the end of cultivation (Fig. 6(b)). Fv /Fo rep-
nium and total phosphorus removal from Clarias gariepinus resented the change in photosynthetic quantum conversion and
wastewater,12 and were superior to that of Chlorella sp. with photochemical efficiency of PS II. The increase of this value indi-
27.34–49.73% of total phosphorus removal from Lates calcar- cated good photosynthetic activity of P. kessleri TY, especially when
ifer aquaculture wastewater.17 Furthermore, P. kessleri TY with P. kessleri TY was inoculated at a concentration of 100 mg L−1 . In
100 mg L−1 inoculation concentration had maximum COD, nitrite, contrast, both Fv /Fm and Fv /Fo values had a slight decrease in the
and total phosphorus removal efficiency of 100%, 99.8%, and control, which indicated that indigenous microalgae in aquacul-
98.9%, respectively, after 5 days of cultivation. Moreover, high ture wastewater did not grow well as shown in Fig. 4.
ammonium and nitrate removal efficiency of 98.9% and 97.1%
was achieved. To degrade most of the pollutants in the aquacul-
ture wastewater, a long HRT was needed due to the slow growth CONCLUSIONS
rate of microalgae.11,14 In the present study, the pollutant removal The growth of indigenous microalgae and bacteria was signif-
rate could be enhanced by controlling the initial inoculum concen- icantly inhibited when real aquaculture wastewater was inocu-
tration, which would be conducive to shortening the cultivation lated with exogenous microalgae. Pollutant removal was almost
period and increasing the volumetric loading in the process of totally derived from the growth of exogenous inoculated microal-
microalgae-based aquaculture wastewater treatment once a large gae. P. kessleri TY exhibited good growth potential and pollutant
amount of aquaculture wastewater was discharged. Furthermore, removal capability in real aquaculture wastewater, compared with
the inoculum concentration significantly affected residual union- C. vulgaris, Chlorococcum sp. GD, S. quadricauda and S. obliquus.
ized ammonia and nitrite concentration of aquaculture wastew- Moreover, the inoculum concentration influenced both growth
ater after 5 days of cultivation (P < 0.05). Residual unionized and the pollutant removal level achieved by P. kessleri TY. Within
ammonia and nitrite was the lowest when 100 mg L−1 of P. kessleri the tested range of inoculum concentrations (0–400 mg L−1 ), P.
TY was inoculated for the treatment of aquaculture wastewater kessleri TY with 100 mg L−1 of inoculum concentration removed
(Table S5). 94.4% of COD, 96.2% of ammonium, 99% of nitrite, 94.3% of nitrate
Photosynthesis is one of the most important metabolic activities and 95.6% of total phosphorus after 3 days of cultivation. Con-
of microalgae mixotrophically cultivated in wastewater. Therefore, sequently, controlling the inoculum concentration is conducive
the chlorophyll fluorescence of mixotrophic cells was also ana- to increasing the performance of microalgae-based aquaculture
lyzed to assess microalgal growth in response to different inocu- wastewater treatment.
lum concentrations. As shown in Fig. 6(a), Fv /Fm of P. kessleri TY
slightly increased at the end of cultivation. Fv /Fm reflected the
potential photochemical efficiency of photosystem II (PS II) in
ACKNOWLEDGEMENTS
the dark-adapted state with fully open PS II reaction centers. As This research project was financed by the National Natural Sci-
described by Samorì et al.41 the maximum Fv /Fm of Desmodesmus ence Foundation of China (No. 31700310), the Key Scientific Devel-
communis occurred on the 3rd day when the ammonium of the pri- opment Project of Shanxi Province, China (No. FT-2014-01-15),
mary wastewater effluent was almost completely depleted. Then, the Social Development Foundation of Shanxi Province, China (No.
the value of Fv /Fm gradually decreased until the end of cultivation. 201603D321008) and the Fund for Shanxi ‘1331 Project’ Key Inno-
Nitrogen starvation, therefore, caused a decrease in maximum vative Research Team.
quantum yield. In addition, previous studies reported that Fv /Fm
values decreased under light and nutrient stress.10,42 For P. kess- Supporting Information
leri TY cultivated in this study, no severe nutritional and light defi- Supporting information may be found in the online version of this
ciencies were found. Therefore, the value of Fv /Fm did not decline. article.

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