Ecotoxicology and Environmental Safety 174 (2019) 435–444

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Wastewater-leachate treatment by microalgae: Biomass, carbohydrate and T

lipid production
Andrea Hernández-Garcíaa, Sharon B. Velásquez-Ortac, Eberto Novelob, Isaura Yáñez-Nogueza,
⁎
Ignacio Monje-Ramíreza, María T. Orta Ledesmaa,
a
Instituto de Ingeniería, Universidad Nacional Autónoma de México, Av. Universidad 3000, Delegación Coyoacán, CP 04510 México, D.F, Mexico
b
Facultad de Ciencias, Departamento de Biología Comparada, Universidad Nacional Autónoma de México, Av. Universidad 3000, Delegación Coyoacán, CP 04510
México, D.F, Mexico
c
School of Chemical Engineering and Advanced Materials, Merz Court, Newcastle University, Newcastle upon Tyne NE1 7RU, UK

A R T I C LE I N FO A B S T R A C T

Keywords: Increases in wastewater discharges and the generation of municipal solid wastes have resulted in deleterious
Microalgae effects on the environment, causing eutrophication and pollution of water bodies. It is therefore necessary to
Wastewater investigate sustainable bioremediation alternatives. Wastewater treatment using consortia of microalgae-bac-
Landfill leachate teria is an attractive alternative because it allows the removal and recycling of nutrients, with the additional
Carbohydrates
advantage of biomass production and its subsequent conversion into valuable by-products. The present study
Lipids
aims to integrate wastewater and landfill leachate treatment with the production of microalgal biomass, con-
sidering not only its valorization in terms of lipid and carbohydrate content but also the effect of nutrient
limitation on biomass formation. The effect of treating a mixture of raw wastewater with different leachate ratios
(0%, 7%, 10% and 15%) was investigated using a microalgae-bacteria consortium. Two microalgae
(Desmodesmus spp. and Scenedesmus obliquus) were used. Nutrient removal, biomass concentration, carbohy-
drate, lipid and Fatty Acid Methyl Ester (FAMEs) content and morphological changes were evaluated. Removals
of 82% of NH4+ and 43% of orthophosphate from a wastewater-leachate mixture (containing 167 mg/L NH4+
and 23 mg/L PO43-) were achieved. The highest final yield was obtained using Desmodesmus spp. (1.95 ± 0.3 g/
L). The microalgae were observed to accumulate high lipid (20%) and carbohydrate (41%) contents under
nutrient limiting conditions. The concentration of Polyunsaturated Fatty Acids (PUFAs) also increased.
Morphological changes including the disintegration of coenobia were observed. By using a mixture of waste-
water-leachate it is possible to remove nutrients, since microalgae tolerate high ammonia concentrations, and
simultaneously increase the algal biomass concentration containing precursors to allow biofuel production.

1. Introduction decomposition of stored waste and rainwater percolation (SEMARNAT,

The generation of municipal solid wastes and wastewater discharges
has increased due to urban growth, industrial development, technolo-
gical changes and changes in consumption patterns. Of the total mu-
nicipal and industrial wastewater generated in Mexico, 57% and 32%,
respectively, is treated (CONAGUA, 2016). In 2015 around 51 million
of tons of municipal solid waste was generated, an increase of 61.2%
with regard to 2003. Globally, landfill is one of the most common
methods for the final disposal of municipal solid waste, and in Mexico it
is the most frequently used. Leachate production is one of the con-
sequences of landfill use. This is a liquid derived from the
2015).
The high levels of nutrients and other compounds in leachate and
wastewater, such as heavy metals and xenobiotic organic compounds
like phenols and pesticides (Ghosh et al., 2017; Naveen et al., 2017),
represent a risk to human health and the environment. Additionally,
wastewater discharges released without appropriate treatment may
result in eutrophication of water bodies causing algal blooms, oxygen
depletion, the loss of species and an increase in turbidity and toxicity in
water (Cai et al., 2013; Gonçalves et al., 2017). For these reasons,
feasible treatment alternatives that guarantee the reduction of toxic
characteristics using carbon neutral technologies are needed. Several

Abbreviations: FAME, Fatty Acid Methyl Ester; WWTP, wastewater treatment plant; TSS, total suspended solids; COD, Chemical Oxygen Demand; FLWTP, fa-
cultative lagoon wastewater treatment plant; TN, total nitrogen; PUFA, polyunsaturated fatty acid
⁎
Corresponding author.
E-mail address: MOrtaL@iingen.unam.mx (M.T. Orta Ledesma).

https://doi.org/10.1016/j.ecoenv.2019.02.052
Received 27 August 2018; Received in revised form 12 February 2019; Accepted 15 February 2019
0147-6513/ © 2019 Elsevier Inc. All rights reserved.
A. Hernández-García, et al. Ecotoxicology and Environmental Safety 174 (2019) 435–444

Table 1
Characteristics of the wastewater and landfill leachate used as the growth media.
Parameter Wastewater Landfill leachate Mixture Wastewater-leachate 7%

pH 8.3 ± 0.5 8.5 ± 0.4 8.4 ± 0.2

Total suspended solids (TSS) (mg/L) 70 ± 6.7 290 ± 21.3 90 ± 21.3
Total Alkalinity as CaCO3 (mg/L) 460 ± 0.5 13,200 ± 23.4 705 ± 3.4
Orthophosphate (mg/L) 27.98 ± 1.8 53.28 ± 4.5 20 ± 1.5
Chemical oxygen demand (COD) (mg/L) 457.5 ± 15.2 2061 ± 22.4 465 ± 2.4
Ammonium (mg/L) 102.06 ± 3.2 1471.4 ± 30.8 150 ± 1.2
Total nitrogen (TN) (mg/L) 112.51 ± 2.4 1566.8 ± 41.2 160 ± 41.2
Nitrate (mg/L) 12.6 ± 2.5 102.8 ± 5.2 8 ± 0.2
N:P ratio 4.02 29.4 8
As (µg/L) 5 70 6
Cd (µg/L) 2 13 2
Cr (µg/L) 17 163 20
Hg (µg/L) 0.35 8 0.8
Zn (µg/L) 56 1800 50
Cu (µg/L) 17 190 18

Table 2
Initial conditions studied using different leachate ratios, during microalgae cultivation and nutrient limitation.
Microalgae cultivation
Leachate ratio Mixture composition (%, v/v) I / W / L Desmodesmus spp. S. obliquus
NH4+ (mg/L) Orthophosphate (mg/L) N: P ratio NH4+ (mg/L) Orthophosphate (mg/L) N: P
ratio
0% 50/50/0 48.3 ± 1.9 16.3 ± 0.9 3.4 44.1 ± 1.5 16.2 ± 0.79 4.4
7% 50/43/7 167.8 ± 0.6 20.2 ± 0.1 8.4 145.2 ± 1.9 21.1 ± 0.2 7.9
10% 50/40/10 217.2 ± 2.8 22.9 ± 0.6 10.09 225.3 ± 1.6 23.9 ± 0.3 9.9
15% 50/35/15 274.9 ± 2.5 18.8 ± 0.7 15.01 279.92 ± 1.5 19.9 ± 0.7 14.8
Nutrient limitation
Cultures selected from first Culture medium added Desmodesmus spp. S. obliquus
stage NH4+ (mg/L) Orthophosphate (mg/L) NH4+ (mg/L) Orthophosphate (mg/L)
0% 3 L of wastewater 60.9 ± 1.4 19.2 ± 0.4 62.7 ± 1.1 21.8 ± 0.7
7% 1.5 L of wastewater 1.5 of 30.1 ± 1.2 10.7 ± 0.9 30.6 ± 0.5 9.6 ± 0.8
distilled water

I: Inoculum of microalgae; W: Wastewater; L: Landfill leachate.

authors have argued that bioremediation through nutrient and metals
removal is possible through the cultivation of microalgae for different
types of wastewater such as domestic, industrial (Alcántara et al.,
2015a, 2015b), agricultural (Luo et al., 2016; Li et al., 2018; Zhou et al.,
2018) and even acid mine drainage (Bwapwa et al., 2017). The mi-
croalgae cultured in wastewater-combined leachate provide a feasible
option for bioremediation due to their ability to remove nutrients,
mainly ammoniacal nitrogen, and other pollutants such as phosphorus,
metals and organic compounds (Zhao et al., 2014; Kumari et al., 2016;
Pereira et al., 2016).
In addition, the presence of other microorganisms in wastewater,
such as bacteria, also plays an important role in promoting nutrient
removal and microalgal growth. As a result, microalgae-bacteria con-
sortia are formed (Gonçalves et al., 2017). Different studies have re-
ported successful results by using these consortia to remove effectively
nutrients such as nitrogen and phosphorus from various wastewater
streams (He et al., 2013; Alcántara et al., 2015a, 2015b; Ren et al.,
2015).
In different types of wastewater, nutrient content varies (Chokshi
et al., 2016). However, in leachate, nutrients are present at higher
concentrations, mainly in the form of ammoniacal nitrogen
(1000–5000 mg/L) and this could cause the inhibition of microalgal
growth. To reduce the toxicity of leachate, some authors have made
dilutions. The use of wastewater to achieve this offers treatment for
both wastes simultaneously. During this biological treatment process,
microalgal biomass is generated that could have value through its
conversion into biofuel (Bibi et al., 2017; Faried et al., 2017).
Several publications have focused on leachate treatment using mi-
croalgae (Martins et al., 2013; Cheah et al., 2016; Kumari et al., 2016;
Paskuliakova et al., 2016; Pereira et al., 2016). These works explored
nutrient removal and toxicological assessment, but did not use a
mixture of wastewater-leachate or simultaneously valorize the micro-
algae biomass for biofuel production. Only three publications have re-
ported on nutrient removal, biomass concentration, and lipid content in
microalgae cultivated using wastewater-leachate (Zhao et al., 2014) or
investigated leachate alone with deionized water (Sforza et al., 2015;
Paskuliakova et al., 2018). Additionally, growing microalgae in was-
tewater reduces biofuel production costs. Different cultivation condi-
tions and strategies have been implemented to increase the content of
carbohydrates and lipids (Pan et al., 2011; Ho et al., 2012;
Kaewkannetra et al., 2012; Samori et al., 2013; Pancha et al., 2014; Xia
et al., 2014; Singh et al., 2016). Of these strategies, nutrient limitation
has been found to be the condition promoting the greatest accumula-
tion of such biomolecules in microalgae (Hu et al., 2008; Pancha et al.,
2014).
Subsequently, the present study aimed to integrate wastewater and
leachate treatment with the production of microalgal biomass, and
promote an increased accumulation of lipids and carbohydrates for the
potential production of biofuels. For the first time, the treatment of a
leachate/wastewater mixture using microalgae, and the effect of nu-
trient limitation on carbohydrate and lipid accumulation was assessed.
During the process, the morphological and chemical changes of mi-
croalgae were integrally evaluated. This included the initial and final
evaluation of the FAMEs profile of microalgae, characteristics closely
related to biodiesel production.
2. Materials and methods
2.1. Landfill leachate and municipal wastewater
Landfill leachate was sampled from the Bordo Poniente landfill and
municipal wastewater was collected from the “Cerro del Agua”

436
A. Hernández-García, et al. Ecotoxicology and Environmental Safety 174 (2019) 435–444

Fig. 1. a) Growth of Desmodesmus spp. and S. obliquus with different leachate ratios. b) Orthophosphate removal in Desmodesmus spp. and S. obliquus cultures with 0%
and 7% leachate ratio. c) NH4+ removal in Desmodesmus spp. and S. obliquus cultures with 0% and 7% leachate ratio. d) Nitrate removal in Desmodesmus spp. and S.
obliquus cultures with 0% and 7% leachate ratio. e) Growth of Desmodesmus spp. during nutrient limitation.

Wastewater Treatment Plant (WWTP). Both sites are located in Mexico
City. Sample volumes of 20 L were collected separately for both was-
tewater and leachate. They were then filtered to remove solids and
stored at 4 °C. The physicochemical properties of the wastewater and
leachate were evaluated. Alkalinity, total suspended solids (TSS) and
ammonia concentrations were measured according to the APHA
standard methods (APHA-AWWA-WPCF, 2005). Orthophosphate, che-
mical oxygen demand (COD), and nitrate concentrations were de-
termined by colorimetric methods using a HACH 3900 spectro-
photometer. Total nitrogen was measured by a TOC-LCPH/CPN analyzer
(TOC-LCPH/CPN Shimadzu). All analyses were carried out in triplicate.

437
A. Hernández-García, et al. Ecotoxicology and Environmental Safety 174 (2019) 435–444

0.2
0.1
0.1
0.1
0.2
0.1
0.2
0.1
2.2. Microalgae

1
±
±
±
±
±
±
±
±
±
mg/L

3
3
3
2
3
2
3
2
93
The microalgae used in this study were Scenedesmus obliquus
(Turpin) Kützing, provided by Instituto Politecnico Nacional, Mexico

0.3
0.4
0.4

0.3
0.5
0.2
City, Mexico, and Desmodesmus spp. from ‘‘Lago Nabor Carrillo’’, an
1

6
±
±
±
±
±
±
±
±
±
artificial lake located in Texcoco, Mexico. This lake is fed with effluent
Nitrates

50
37
30
13
60
37
30
14
−1795
from the facultative lagoon wastewater treatment plant (FLWTP) also
located in Texcoco. Desmodesmus spp. were characterized considering
%

their morphology via light microscopy observations with the aid of

0.1
identification manuals (Hegewald et al., 1990). Species Desmodesmus
1
2
1
1
2
3
2
2
±
±
±
±
±
±
±
±
±
intermedius (Chodat) Hegewald, Desmodesmus magnus (Meyen) Tsar-
mg/L

2
115
107

138
48

74
52

98
58 enko, Desmodesmus communis (Hegewald) Hegewald y Desmodesmus
opoliensis (Richter) Hegewald were identified.
Previous laboratory work has demonstrated that the aforemen-
0.01
0.8
0.9

1.3

0.9
1
1

tioned strains showed better growth and adaptability in wastewater.

±
±
±
±
±
±
±
±
±
TN

2
90
74
45
25
93
77
42
20
%

2.3. Microalgae cultivation using wastewater-leachate

2
3
3
1
2
3
2
3
1

A mixture of municipal wastewater with different leachate ratios:

±
±
±
±
±
±
±
±
±
Total Alkalinity as CaCO3

mg/L

0%, 7%, 10% and 15%, was used for microalgal growth. Cultures with
87

10
178
300
318

168
315
311
141

0% leachate ratio were cultivated in wastewater alone. Microalgae in-

oculum, used for all the experiments, was previously acclimatized to
wastewater for 6 months. Each experiment was carried out in triplicate
0.02
0.3
0.1

0.3
0.2
0.1

using non-aerated bottles with a working volume of 4 L, this gave a total

1
1

1
±
±
±
±
±
±
±
±
±

of 18 experiments for each microalga, conducted in series. Biological

62
43
20

59
44
19
4

7
2
%

controls, without addition of microalgae, were also conducted in tri-

plicate to monitor if nutrient removal could be due to the assimilation
of microalgae or other microorganisms such as bacteria and fungi. The
3
3
2
2
1
2
2
1
3
±
±
±
±
±
±
±
±
±

density of the inoculum was similar among all the experiments and was
mg/L

114

137
42

92
63
46

86
54
95

measured as TSS (318.4 ± 24.7 mg/L). The initial conditions are

shown in Table 2.
Nutrient removal in percentage terms and in mg/L in Desmodesmus spp. and S. obliquus cultures over 28 days.

All cultures were maintained with manual shaking and illumination

0.3

0.5

0.6
0.2
0.4
1

1
1

at a light intensity of 53 µmol/m2/s and light cycles of 12:12.

NH4+

±
±
±
±
±
±
±
±
±

Biomass concentration (measured as TSS) was used to monitor the

95
79
41
22
96
82
40
20
90
%

growth of microalgae. For nutrient removal, the liquid samples from the
cultures were filtered using a glass microfiber filter with 1.6 µm pore
7
5
4
5
6
3
3
4
3

size (Whatman, GF/A). All measurements were performed in duplicate,

±
±
±
±
±
±
±
±
±
mg/L

468
295
208
223
345
315
193
200
150

both at the beginning and at the end of each experiment. The growth
and the nutrient removal of Desmodesmus spp. and S. obliquus were
evaluated over 28 days. Morphological changes of microalgae cells
0.9

0.8

were observed by optical microscopy at 40x magnification (Axio Lab.

1

1
2
1
0
1
1
±
±
±
±
±
±
±
±
±

Al, Carl Zeiss).

COD

86
64
38
26
86
67
38
24
33
%

0.02

0.05
0.04
0.02
0.9
0.4

2.4. Microalgae cultivation: nutrient limitation

1

1
1
±
±
±
±
±
±
±
±
±
mg/L

9
5
1
9
8
5
1
3
10

From the results obtained at different leachate ratios (0%, 7%, 10%
Ortho-phosphate

and 15%), the highest concentration of microalgal biomass (7%) was

selected for further evaluation. Microalgae were cultivated for an ad-
0.9
0.5

0.9

0.6
0.5
1
1

ditional 68 days in order to observe the effect of nutrient limitation on

±
±
±
±
±
±
±
±
±

lipid and carbohydrate accumulation. First, the biomass of the cultures

60
43
20

57
41
20
5

6
9
%

was concentrated by letting them settle for 5 days. Subsequently, the

Nutrients removal in percentage and mg/L

supernatant (3 L) was removed and only the biomass was kept in the
% leachate ratio

bottles. For cultures that were cultivated at a 0% leachate ratio, was-

tewater (3 L) was added as the culture medium. For the cultures that
were cultivated at the selected leachate ratio, the same amount of
wastewater (3 L) was added but diluted with distilled water (50:50
10
15

10
15
0
7

0
7

ratio) in order to subject them to nutrient limitation. Observations were

made at the beginning and at the end of the experiment using optical
Desmodesmus spp.

microscopy.
Carbohydrates and lipids were quantified in duplicate for each ex-
Microalgae

S. obliquus

periment and twice per week during nutrient limitation trials. The total
Control

carbohydrate quantification was carried out by the phenol-sulfuric acid

Table 3

colorimetric method (Dubois et al., 1956). The lipids were quantified by

the colorimetric method of sulfophosphovanillin (Mishra et al., 2014).

438
A. Hernández-García, et al.
Fig. 2. Carbohydrate and lipid yield per gram of biomass and percentage of Desmodesmus spp. during nutrient limitation. W: only wastewater, DW: diluted was-
tewater.
2.5. FAMEs profiling
At the beginning and end of nutrient limitation, 200 mL sample of
culture was taken from each bottle. This was then centrifuged at
1593 × g for 15 min and the biomass was recovered and dried at room
temperature. The dried biomass was preserved to carry out lipid ex-
traction in accordance with the Bligh and Dyer method (Bligh and Dyer,
1959), modified according to Garcés and Mancha (1993). FAMEs were
obtained by transesterification (Montes et al., 2011; Wahlen et al.,
2011). For FAMEs identification a gas chromatograph (Agilent Tech-
nologies 6809) coupled to a mass spectrophotometer (Agilent Tech-
nologies 5973) was used. A DB-5 ms fused silica capillary column (30 m
x 0.25 mm, 0.25 m film thickness) was used and the carrier gas was
helium with 99.9995%. A volume of 1 µL of sample was injected in
split-type mode at an injection port (250 °C). The oven programme was:
initial temperature 85 °C (5 min) with a ramp of 7 °C/min, until
reaching 265 °C (10 min). The spectrophotometer was operated in
SCAN mode at 50–550 m/z. Identification was corroborated by com-
paring retention time with a known standard FAMEs Mix, C8-C24
(Sigma-Aldrich, 18918-1AMP, USA).
2.6. Statistical analysis
The data reported in this work were analyzed using analysis of
variance (ANOVA) and Tukey tests for the mean differences using
Microsoft Office Excel 2013. A significant difference was considered at
the level of α = 0.05.
3. Results and discussion
3.1. Landfill leachate and municipal wastewater characterisation
Wastewater and leachate had similar pH values. However, other
physicochemical parameters were higher in the leachate with respect to
the wastewater (Table 1). Nitrogen concentration (ammoniacal and
total) in leachate was 14 times higher than in wastewater. It is im-
portant to take into account the N: P ratio for optimal growth of mi-
croalgae. According to Redfield et al. (1963), the composition of mi-
croalgae is C106H263O110N16P, with an optimal N: P ratio of 16. This
relationship changes according to the type of species (Rhee, 1978). For
the species used in this work, different authors have reported various
N:P ratios. According to the literature, a range of 5–30 has been es-
tablished (Rhee, 1978; Xin et al., 2010; Arbib et al., 2013; Samori et al.,
439
Ecotoxicology and Environmental Safety 174 (2019) 435–444
2013; Beuckels et al., 2015). It can be seen in Table 1 that the waste-
water had a N:P ratio below the established values, causing nitrogen
limitation during microalgal growth. However, leachate showed an
optimal N:P (29.4) ratio. In addition, in accordance with the literature,
the amounts of metals in the wastewater-leachate mixture were not
sufficient to influence microalgal growth negatively (Mustafa et al.,
2012; Pereira et al., 2016; Zeraatkar et al., 2016; Li et al., 2018). This
suggests that a wastewater-leachate mixture would be a good source of
nutrients for microalgal growth.
3.2. Microalgal cultivation using wastewater-leachate
Table 2 shows the initial conditions of the different leachate ratios.
Microalgae cultivated in wastewater alone (0% leachate ratio) had a
low N:P ratio (Table 2) compared to the range established in the lit-
erature (from 5 to 30), therefore growth was low due to nitrogen lim-
itation (Fig. 1a). It was observed that by increasing the leachate ratio,
the N: P ratio improved due to an increase in nitrogen concentration.
This shows that leachate can be used to enhance the N: P ratio for
microalgal growth (Table 2). However, the 7% leachate ratio produced
the highest biomass yield of Desmodesmus spp. (1.3 ± 0.10 g/L) and S.
obliquus (1.2 ± 0.07 g/L), respectively (Fig. 1a). The 7% leachate ratio
favored microalgal growth, doubling biomass yield compared to other
conditions.
The use of 10% and 15% leachate ratios was seen to limit growth,
due to high ammonia concentrations, ≥200 mg/L (Fig. 1a). Similar
effects on microalgal growth have been reported by other authors.
Cheung et al. (1992) worked with Scenedesmus and reported that con-
centrations greater than 143 mg/L of ammonia inhibited growth. Lin
et al. (2007) reported that at a concentration of 135 mg/L of ammonia,
growth was enhanced, whereas, at higher concentrations it was in-
hibited. On the other hand, Zhao et al. (2014) obtained increased
growth with 183 mg/L of ammoniacal nitrogen but higher concentra-
tions presented an inhibitory effect. The reported limits were close to
those used in cultures with a 7% leachate ratio (145–167 mg/L). For
cultures with no microalgae addition (controls) no growth was ob-
served, as shown in Fig. 1a.
When the leachate ratio was increased, an inhibitory effect on algal
growth was observed, likely a consequence of high ammonia con-
centrations inhibiting microalgal photosynthetic activity. Additionally,
the color of the leachate (dark brown) could have affected algal growth
at high volume ratios (10% and 15%) due to reduced light penetration.
The light intensity used in this work (53 µmol/m2/s) may not have been
A. Hernández-García, et al.
Fig. 3. Morphological changes in S. obliquus and Desmodesmus spp. a) Desmodesmus spp. during growth phase; b) Desmodesmus spp. during nutrient limitation phase;
c) S. obliquus during growth phase; d) S. obliquus during nutrient limitation phase; e) Desmodesmus spp. coenobia and their spines; f) S. obliquus coenobia; (a-d at 40×,
e-f at 100×).
sufficient for increased algal growth. Salinity is another factor that
probably resulted in an inhibitory effect (Cheung et al., 1992; Zhao
et al., 2014).
With regard to nutrient removal, the mechanisms involved in this
process may be different, for example, NH4+ removal can be achieved
either by microalgal uptake, volatilization due to an increase of pH (Fig.
S2, Supplementary material), or by oxidation to nitrate by nitrifying
bacteria (Liu et al., 2017).
According to the results obtained in this work, as shown in Table 3
and Fig. 1b-d (see also Table S1 Supplementary material), the bior-
emediation of wastewater-leachate through nutrient removal by Des-
modesmus spp. and S. obliquus grown in wastewater with 0% leachate
was between 57–60% for phosphorus, and 95–96% for NH4+. This
corresponded to 9.3–9.9 mg/L and 42–45.8 mg/L of phosphorus and
NH4+ removal, respectively. However, taking into consideration the
volatility of NH3, the biomass concentration and nitrogen content, the
actual removal due to biofixation was between 69% and 70%. The
apparent NH4+ removal efficiency obtained in this work is comparable
to the 97% removal reported by Martínez et al. (2000) using S. obliquus
and wastewater containing 27.4 mg/L of NH4+ and 11.8 mg/L of
440
Ecotoxicology and Environmental Safety 174 (2019) 435–444
phosphate.
The phosphate and NH4+ concentrations increased in the mixture of
wastewater and 7% leachate, and this consequently led to reductions in
the percentage of nutrient removal. However, considering NH4+ and
phosphate removal as mg/L, the NH4+ removal was higher in 7%
leachate when compared to microalgal cultures grown only in waste-
water. Phosphate removal remained unchanged (Table 3). Phosphorus
removal efficiencies were 41–43%, and 78–81% for NH4+. The actual
nitrogen removals due to biofixation were 52% and 48%, for Desmo-
desmus spp. and S. obliquus, respectively. Zhao et al. (2014) reported
similar removal percentages of NH4+ (52%) due to microalgae bio-
fixation while the apparent removal was 95% using 10% leachate ratio
containing 183.2 mg/L of NH4+. For cultures with no microalgae
(controls), as NH4+ decreased, nitrates increased. According to Table 3
90% of NH4+ was oxidized to nitrates, probably as a result of the
presence of nitrifying bacteria in wastewater (Khanzada and Övez,
2017; Ye et al., 2018). This nitrifying process was also observed in
cultures with microalgae but it was less evident as shown in Fig. 1c-d.
Therefore, the nitrogen removal efficiency at 0% and 7% leachate was
mainly a result of three factors: 1) microalgae uptake, since NH4+ can
A. Hernández-García, et al. Ecotoxicology and Environmental Safety 174 (2019) 435–444

Table 4 consortium with microalgae. Bacteria play an important role in nutrient

Fatty acid profile of Desmodesmus spp. at the beginning and at the end of nu- removal, as a result of the microalgae-bacteria partnership (Gonçalves
trient limitation. et al., 2017; Liu et al., 2017). The use of wastewater negates mono-
FAME type Relative FAME composition % cultures or axenic cultures, so the participation of bacteria in bior-
Desmodesmus spp. emediation is evident. Among the advantages of these consortia as
discussed in the literature, is the cooperation that occurs during their
0% leachate 7% leachate
metabolic interaction in the culture medium, which increases the gen-
Initial Final Initial Final eral absorption of nutrients. This interaction also confers greater re-
sistance to change in ambient conditions (Gonçalves et al., 2017). The
C12:0 Lauric – – – – key advantages of this interaction are the various nutrient removal
C14:0 Myristic 1 ± 0.01 – 1 ± 0.02 –
pathways that result, allowing higher removal percentages (Liu et al.,
C15:0 Pentadecanoic – – –
C16:0 Palmitic 16 ± 0.08 13 ± 0.04 15 ± 0.07 16 ± 0.05 2017).
C16:1n9c Palmitoleic 7 ± 0.02 4 ± 0.01 6 ± 0.04 5 ± 0.03 In the case of biochar, microalgae can also act synergistically.
C17:0 Margaric – – – – Biochar is a product that can be used as an absorbent material. It is a
C18:0 Stearic 4 ± 0.02 2 ± 0.03 2 ± 0.01 2 ± 0.02 material that can be produced from microalgal biomass ash after pyr-
C18:1n9t Oleic – – – –
olysis. Biochar is a porous carbon material, suitable for bioremediation
C18:2n6c Linoleic 8 ± 0.02 10 ± 0.05 10 ± 0.06 9 ± 0.07
C18:3n3 Linolenic 21 ± 0.06 20 ± 0.09 22 ± 0.09 30 ± 0.1 through nutrient absorption (Magee et al., 2013). It has shown effective
C18:3n6 Gamma linolenic – 1 ± 0.01 – 0.8 ± 0.01 immobilization of heavy metals during the remediation of con-
C20:4n6 Arachidonic – 1 ± 0.02 – 1 ± 0.01 taminated soils (Wu et al., 2017). This property, as reported by Magee
C22:0 Behenic 0.8 ± 0.01 – 1 ± 0.01 –
et al. (2013), could be used to attract and immobilize algae to its sur-
C26:0 Cerotic 1 ± 0.02 2 ± 0.02 1 ± 0.01 2 ± 0.02
Saturated 38.78% 32.08% 34.48% 30.40%
face. This would serve as an effective method to overcome the chal-
Monounsaturated 11.90% 7.55% 10.34% 7.60% lenges of harvesting microalgae, making the production of biomass
Polyunsaturated 49.32% 60.38% 55.17% 62.01% from microalgae grown in wastewater-leachate mixture an even more
C16-C18 95.24% 94.34% 94.83% 95.44% attractive proposition.

3.3. Nutrient limitation

exist at pH values less than 9.3, as shown in Fig. S2 (Supplementary
material), and pH of the wastewater and landfill leachate was 8.3 and
During nutrient limitation, the biomass concentration increased in
8.5; 2) NH3 volatilization, since as the microalgae grew, pH increased
both cultures with wastewater only and those with diluted wastewater.
until approximately 10. At pH values higher than 9.3 NH3 would be the
A maximum biomass yield of 2 ± 0.3 g/L in Desmodesmus spp. cultures
dominant form; and 3) NH4+ oxidation by nitrifying bacteria (Markou
was obtained (Fig. 1b). This is in agreement with the increases in bio-
and Georgakakis, 2011; Khanzada and Övez, 2017).
mass reported by Breuer et al. (2012) during nutrient limitation.
Phosphorus removals (43–60%) obtained from microalgae cultiva-
Desmodesmus spp. cultivations had the highest biomass yield in this
tion in wastewater containing 0% and 7% leachate were due to mi-
work, reaching 1.950 g/L. This compares with the findings of other
croalgal assimilation and phosphorus precipitation (because of pH) (Liu
authors such as Sacristan et al. (2013); Ji et al. (2014); Komolafe et al.
et al., 2017). These results are comparable to values of 55% reported for
(2014); Sforza et al. (2015) and Zhao et al. (2014). These authors re-
batch cultivations of Chlorella vulgaris and S. dimorphus in agro-in-
ported yields of between 0.412 g/L and 1.58 g/L. On the other hand,
dustrial wastewater containing 111 mg/L of phosphate (González et al.,
Samori et al. (2013) reported a value of 2.3 g/L, obtaining higher bio-
1997).
mass yield by enriching the medium with CO2 and increasing light in-
Greater nutrient removal was obtained by using a wastewater
tensity (112 µmol/m2/s). With respect to biomass productivity, Samori
mixture with 7% leachate compared to 10% and 15% leachate ratios
et al. (2013), Sacristan et al. (2013) and Zhao et al. (2014) reported
(Table 3). Similar results were obtained for both microalgae, with about
higher values (0.138, 0.073, 0.131 g/L.d) than the present work
80% of NH4+ and 40% of orthophosphates removed, respectively. The
(0.039 g/L.d). This could be due to the culture conditions and the
percentage NH4+ removal was higher compared to orthophosphates.
species they studied. These comparisons show that the culture medium
This is mainly attributable to the N:P ratio requirements of 16:1
influences both biomass yield and productivity, and that these char-
(Redfield et al., 1963). According to Table 3 the NH4+ removal was
acteristics should also be considered to establish appropriate conditions
greater than for nitrates, therefore the microalgae studied in this work
that enhance microalgal growth.
preferred NH4+ as their nitrogen source. Carbonate removal, measured
as CaCO3, was similarly observed so microalgae could have utilized
3.3.1. Lipid and carbohydrate accumulation
these carbonates to fulfill the carbon requirements for their growth, as
According to the results obtained, as shown in Table 3, there was
mentioned previously.
greater removal of carbonates and nitrogen in wastewater-leachate
Bioremediation, based on wastewater and leachate treatment using
cultures than in microalgae cultures grown only with wastewater.
microalgae, has dual benefits: biological treatment of wastewater and
Carbonate-bicarbonate equilibrium depends on the pH (Eze et al.,
leachate, as well as biomass production. Additionally, microalgae in a
2018) (Fig. S1, Supplementary material), and HCO3- is preferred by
culture can act synergistically with other biosorbent materials. In the
microalgae (Goldman et al., 1981). Inorganic carbon was assimilated
present study, as well as in the published literature, the evident ad-
via autotrophic growth as the pH increased from 8 to about 10, during
vantages of microalgae as a biosorbent material of different con-
microalgae growth.
taminants in water are shown. Both nutrients and metals can be re-
Due to the fact that carbon is used by microalgae for biosynthesis of
moved during microalgal culture through different mechanisms that
lipids and carbohydrates, its assimilation plays an important role for
occur at the cell surface level or via the metabolic processes of living
energy storage compounds (Chang et al., 2018) and this could be re-
organisms, (Alcántara et al., 2015a, 2015b). Other biological materials
flected in the increase of such biomolecules as shown in Fig. 2. Des-
reported as biosorbents and involved in the removal of these con-
modesmus spp. significantly increased the quantity of carbohydrates and
taminants are agricultural wastes, peat, algae, fungi, bacteria, yeasts,
lipids from 25% to 41% and 15% to 20%, respectively. Therefore, there
cellular products (Bulgariu and Gavrilescu, 2015), biochar (Magee
was a 64% and 33% increase, respectively.
et al., 2013; Wu et al., 2017), or duckweed (Zhou et al., 2018).
With regard to S. obliquus, the lipid content increased from 12% to
Bacteria, as described in the present study, are considered part of a
16% during nutrient limitation, an increase of 33%; the carbohydrates

441
A. Hernández-García, et al.
did not show any increase, their content remained at 30%. Fig. 2 shows
the results for Desmodesmus spp. According to these results, the im-
provement in the content of energy storage compounds in microalgae
due to the strategy implemented in this study, was more evident in
these microalgae cultures.
Lipid content was similar to that reported by Zhao et al. (2014)
(14.5–20.8%), when cultivating Chlorella without a nutrient limitation
phase. This could be due to the differences between species and the
cultivation conditions. Carbohydrates accumulated faster than lipids in
both microalgae. Under nutrient limitation the storage of poly-
saccharide molecules is initially favored, and after prolonged stress
conditions the microalgae begin to degrade carbohydrates and accu-
mulate lipids (Breuer et al., 2012; Ho et al., 2012; Chen et al., 2013).
This enables them to survive under adverse conditions, since, by means
of these highly energetic reserve compounds, the cells can obtain the
necessary energy to carry out their metabolic processes and adjust their
growth to new environmental conditions (Geider and La Roche, 2002;
Markou et al., 2012). Under nutrient limitation conditions Desmodesmus
spp. reached their highest carbohydrate content on day 87 and this
started to decrease on day 90, whilst lipids started to increase on day 94
(Fig. 2). This behavior for both storage products has been previously
reported by Breuer et al. (2012), Ho et al. (2012) and Chen et al.
(2013), suggesting that both metabolic pathways for carbohydrate and
lipid synthesis are related and possibly that the products resulting from
carbohydrate catabolism could be precursors of lipid synthesis.
The results suggest that the lipid and carbohydrate contents are
considerably influenced by different factors, for instance nutrient con-
ditions such as N and C concentrations in the medium, and the growth
period. Several authors have obtained similar results (Morales-Sánchez
et al., 2013; Luo et al., 2016; Nordin et al., 2017). For example, Luo
et al. (2016) worked with microalga Coelastrella sp. and observed
changes in lipid content and fatty acid profiles at different ratios of
anaerobically and aerobically treated swine wastewater. On the other
hand, Nordin et al. (2017) used wastewater with high NO3 content to
cultivate microalgae and produced biomass with low lipid content and
significant carbohydrate and protein content. Morales-Sánchez et al.
(2013) worked with Neochloris oleoabundans at different C/N ratios and
reported that in nitrogen sufficient conditions high protein content is
obtained, while and under nitrogen limiting conditions the accumula-
tion of energy storage compounds is favored. In the present study,
nutrient limiting conditions increased lipid and carbohydrate content in
microalgal biomass (Fig. 2).
Another important factor to consider which enhances carbohydrate
and lipid content in microalgae is the duration time of the nutrient
limitation phase. This is the most studied strategy (Sajjadi et al., 2018);
however, such a condition could have negative effects on biomass
generation. Since the main component of lipids and carbohydrates is
carbon, providing a source of this element during microalgal growth,
followed by nutrient limitation could increase such compounds. Since
leachate had a greater carbonate concentration, the microalgae grown
in wastewater-leachate assimilated more carbon, thus, increasing lipid
and carbohydrate content during nutrient limitation. In this way, it is
possible to manipulate the accumulation of high-energy value biomo-
lecules and then transform them to obtain biodiesel and bioethanol.
According to these results, the microalgal biomass obtained proved to
be more viable for the potential production of bioethanol, since high
concentrations of carbohydrates were produced (41%). These results
are comparable to those reported in the literature. Different authors
have obtained various carbohydrate content (from 23% to 61%) and
biomass concentrations (from 0.26 g/L to 2.1 g/L) (Ho et al., 2012;
Chen et al., 2013; Samori et al., 2013; Pancha et al., 2014). However,
they also found that as carbohydrates increase, biomass production
decreases under nutrient limitation, and it was possible to increase
carbohydrate content without compromising microalgal biomass.
Morphological changes in both Desmodesmus spp. and S. obliquus
were observed (Fig. 3). Both microalgae showed disintegration and
442
Ecotoxicology and Environmental Safety 174 (2019) 435–444
formation of coenobia (or colonies) of 2 and 4 cells. The disappearance
of spines was observed in Desmodesmus spp., a likely consequence of the
amount of nitrogen present in the medium. This is known as phenotypic
plasticity, changes that occur in response to environmental conditions
(Cheung et al., 1992; Lürling, 2003). This has also been observed by
Pancha et al. (2014), who reported that the nitrate concentration in the
medium could cause the disintegration and formation of Scenedesmus
sp. coenobia. These morphological changes could favor the sedi-
mentation process and therefore the separation of the microalgae from
the liquid medium. It has been observed that the colonies of 4 or more
cells of both microalgae have higher settling velocities than unicellular
organisms (Lürling, 2003; Lürling and Van Donk, 2000).
Additionally, due to the presence of N. limnetica, a cell count was
carried out to determine the proportion of each species at the end of the
experiment. In S. obliquus cultures, N. limnetica was the predominant
species at 90%, whilst in Desmodesmus spp. cultures N. limnetica was
dominant only at 10%. This could possibly affect the content of lipids
and carbohydrates in S. obliquus, since greater amounts of these bio-
molecules are reported in the literature (Ho et al., 2012) than in the
current work.
3.4. FAME profiling
The fatty acid profile was obtained by GC/MS in order to evaluate if
there were significant differences at the start and end of nutrient lim-
itation conditions (Table 4). Fatty acids are more commonly synthe-
sized by green algae with chain lengths between C16 and C18 (Hu et al.,
2008; Garofalo, 2009). Of these fatty acids, palmitic acid (C16:0) and
linolenic acid (C18:3) had the greatest relative abundance (Table 4),
similar to profiles reported by Kaewkannetra et al. (2012); Samori et al.
(2013); Komolafe et al. (2014); Xia et al. (2014).
In Desmodesmus spp., polyunsaturated fatty acids (PUFA) increased
at the end of the nutrient limitation phase. These results are consistent
with those reported by Pan et al. (2011), where increased amounts of
unsaturated FAMEs were obtained after nutrient limitation.
According to Hoekman et al. (2012) an optimal composition of fatty
acids for biodiesel has low levels of saturated and polyunsaturated acids
and high levels of monounsaturated acids. The degree of poly-
unsaturation in the fatty acids obtained in this work increases under
conditions of nutrient limitation; therefore it would not be favorable for
the production of biodiesel as its oxidative stability would be affected.
To overcome this, Álvarez et al. (2015) suggested the use of a mixture
of oils, for example, the inclusion of palm oil, in which mono-
unsaturated fatty acids predominate, with jatropha and soybean oil,
where polyunsaturated fatty acids predominate. Another option could
be to vary other growing parameters to help modify the fatty acid
profile. This could include, for example, the use of nutrient limitation
with salinity, increased lighting or aeration with CO2, since it has been
reported that the combination of these parameters can help increase the
monounsaturated FAMEs (Ho et al., 2012; Xia et al., 2014, Álvarez
et al., 2015).
4. Conclusions
The use of Desmodesmus spp. and S. obliquus provides a biological
treatment for wastewater and leachate as they not only tolerate high
ammonia concentrations (≥167 mg/L) but also have the capability to
remove nitrogen and phosphorus by biotic and abiotic processes.
Therefore, the utilization of microalgae is an alternative for bior-
emediation of such wastes and results in biomass production. The
content of lipids and carbohydrates in the biomass generated increased
in response to nutrient limitation. In addition, the FAMEs profile was
modified, increasing in polyunsaturated fatty acids, when such condi-
tions were applied. Finally, this work provides a better insight into
integrating biological treatment and biomass generation and valoriza-
tion, including cultivation strategies. This may lead to the optimization
A. Hernández-García, et al.
of treatment systems and biofuel production.
Acknowledgements
This research was supported by the Fondo Sectorial CONACYT-
SENER-Sustentabilidad Energetica, project 220704. Andrea Hernández-
García was supported by Posgrado en Ciencias Biológicas, UNAM and
CONACYT (Grant no. 625020).
Conflict of interest
All the authors declare that they have no conflict of interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2019.02.052.
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