Bioresource Technology 244 (2017) 400–406

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

Assessment of Chlorella vulgaris and indigenous microalgae biomass with MARK

treated wastewater as growth culture medium
⁎
Luis C. Fernández-Linares , Claudia Guerrero Barajas, Enrique Durán Páramo,
Jesús A. Badillo Corona
Departamento de Bioprocesos, Unidad Profesional Interdisciplinaria de Biotecnología – Instituto Politécnico Nacional (UPIBI – IPN), Av. Acueducto s/n Col. Barrio la
Laguna Ticomán, 07340 Ciudad de México, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of the present work was to evaluate the feasibility of microalgae cultivation using secondary treated
Chlorella vulgaris domestic wastewater. Two Chlorella vulgaris strains (CICESE and UTEX) and an indigenous consortium, were
Treated wastewater cultivated on treated wastewater enriched with and without the fertilizer Bayfolan®. Biomass production for C.
Fertilizers vulgaris UTEX, CICESE and the indigenous consortium grown in treated wastewater was 1.167 ± 0.057,
Lipids
1.575 ± 0.434 and 1.125 ± 0.250 g/L, with a total lipid content of 25.70 ± 1.24, 23.35 ± 3.01and
FAME
20.54 ± 1.23% dw, respectively. The fatty acids profiles were mainly composed of C16 and C18. Regardless of
the media used, in all three strains unsaturated fatty acids were the main FAME (fatty acids methyl esters)
accumulated in a range of 45–62%. An enrichment of treated wastewater with Bayfolan® significantly increased
the production of biomass along with an increase in pigments and proteins of ten and threefold, respectively.

1. Introduction efficiency of foliar fertilizers in microalgal culture has been demon-

Biofuels are an interesting alternative to cover part of the energy
demand of fuels derived from petroleum. Microalgae have been taken
into consideration as a feedstock for renewable biofuel such as ethanol,
butanol, biodiesel, hydrogen and methane (Ramanna et al., 2014;
Aguirre et al., 2013; Hadj-Romdhane et al., 2012; Chisti, 2007). The
most positive impact of the use of biofuels is the reduction on the
emissions of greenhouse gases during both, the production and con-
sumption of such fuels. Atmospheric CO2 is consumed for the biomass
which in turn can be a renewable resource of energy (Singh et al.,
2011a; b).
The use of microalgae for the production of biofuels has emerged as
a promising alternative to the utilization of fossil fuels (Amaro et al.,
2011; Chisti, 2007). Not only microalgae have greater photosynthetic
efficiency and are more efficient in the assimilation of CO2 (fixing 183
tons per 100 tons of biomass) and other nutrients, they do not require
arable land, and demand less water with respect to plants.
In the production of microalgae at large scale, the culture media
represents 30–40% of the operating costs (Jad-Allah, 2012). However,
commercial agricultural fertilizers, which contain macronutrients or a
combination of micronutrients and vitamins that are essential for the
rapid growth of microalgae and are a cheaper option than conventional
media, could then reduce the operating costs (Richmond, 1999). The
strated by Jad-Allah (2012); with a yield greater or equivalent to bio-
mass productivities compared to conventional media such as BG11 and
f/2. Besides, the use of chemical fertilizers for microalgal cultivation is
unsustainable. Instead, organic fertilizers containing high value nu-
trients that can support microalgae growth can be used. Lam and Lee
(2012) reported the feasibility to use Baja Serbajadi Humus 27 (organic
fertilizer or compost) to cultivate C. vulgaris for biodiesel production in
outdoor conditions. A better option are piggery industry and domestic
wastewaters that can be used for microalgal growth (Olguín, 2012; Park
et al., 2011). Microalgae nutrients removal from these wastewaters has
been widely reported (Viruela et al., 2016; Alcántara et al., 2015; Wang
et al., 2015; Cai et al., 2013). In addition, Acién et al. (2012) suggested
the use of fuel gases as carbon source and wastewater as growth
medium to significantly reduce the costs of producing biofuels from
microalgae with current technologies. On the other hand, microalgae
cultivation in wastewaters or treated sewage promotes an effective
treatment or polishing for these waters. This culturing approach con-
tributes to the production of microalgae biomass at reduced costs with
lower environmental impact, since nutrients supply and freshwater are
not required (Olguín, 2012; Silva-Benavides and Torzillo (2012); Boelee
et al., 2011).
Recent studies have demonstrated the ability of C. vulgaris to grow,
produce lipids and treat different types of wastewater (Ww). Some

⁎
Corresponding author.
E-mail address: lfernand36@gmail.com (L.C. Fernández-Linares).

http://dx.doi.org/10.1016/j.biortech.2017.07.141
Received 31 May 2017; Received in revised form 21 July 2017; Accepted 23 July 2017
Available online 27 July 2017
0960-8524/ © 2017 Elsevier Ltd. All rights reserved.
L.C. Fernández-Linares et al.
examples are: anaerobically pretreated Ww (Marjakangas et al., 2015),
municipal Ww (Gonçalves et al., 2016; He et al., 2013a,b), domestic
Ww (Lam et al., 2017), piggery Ww (Hossein et al., 2016; Wang et al.,
2015; Abou-Shanab et al., 2013), anaerobically digested dairy manure
(Wang et al., 2010), monosodium glutamate Ww (Jiang et al., 2016),
synthetic Ww (Miao et al., 2016; Feng et al., 2011), food Ww (Hou
et al., 2016), and saline Ww (Shen et al., 2015).
Scarce work on the use of treated wastewater has been reported.
Biomass and lipid productivities of C. vulgaris grown in a mixture of
primary (25%) and secondary wastewater (75 %) were 138.76 mg/L·d
and 45.49 mg/L·d, respectively according to Ebrahimian et al. (2014).
C. vulgaris was grown in 16-L volume laboratory ponds while operating
at dilution rates (D) in a range of 0.250–0.071 d−1 and using a mixture
of pre-treated swine and sewage as substrate. Viruela et al. (2016)
cultivated sewage’s indigenous microalgae (Scenedesmus sp. and/or
Chlorella sp.) in outdoor 0.55 m3 flat-plate photobioreactors using the
effluent from an anaerobic membrane bioreactor fed with pre-treated
sewage. The maximum biomass productivity reached was
52.3 mg VSS L/d. Furthermore, Botryococcus braunii was grown in
wastewater from the secondary treatment; the lipids and biomass yields
were considerably high (Orpez et al., 2009).
In order to overcome the economic constraints of microalgal bio-
fuels production at large scale, it must be integrated into the concept of
biorefinery (Milledge, 2010; Singh and Gu, 2010; Chisti, 2007). This
would link the biofuel production to high value compounds such as
cosmetics, nutraceuticals and compounds for human consumption.
Other low-value products as animal and human food, bio-fertilizers, as
well as wastewater treatment, nitrogen fixing and CO2 mitigation
would be obtained (Hadj-Romdhane et al., 2012). The main valuable
components of microalgae are lipids, proteins, carbohydrates, pig-
ments, vitamins, polyunsaturated fatty acids (PUFAS) and also com-
pounds that may exhibit some biological activity.
In this study, the aim was to assess the use of treated wastewater as
substrate for the growth of two C. vulgaris strains (UTEX and CICESE)
and an indigenous microalgae consortium (obtained from treated Ww)
and its effect on the production of lipids, proteins, pigments and car-
bohydrates.
2. Material and method
2.1. Microalgae strains, media and algal cultivation
Chlorella vulgaris (UTEX 26) strain was acquired from the Culture
Collection of Algae, Texas University (Austin, Texas, EE.UU.); the
second C. vulgaris strain used was obtained from Centro de
Investigación Científica y de Educación Superior de Ensenada, Baja
California, Mexico (C. vulgaris CICESE). Likewise, the indigenous strains
(consortium) was obtained from treated wastewater (TWw) and was
cultivated in 1 L flasks under white fluorescent light illumination at an
intensity of 180 μmol photons m2/s at 24 ± 1 °C, to allow the growth
of the native microflora. Prior to the growth experiments, both C. vul-
garis strains were pre-cultured individually in 1 L flasks containing
800 mL of BBM medium. The indigenous consortium was pre-cultured
in 1 L flasks containing 800 mL of treated wastewater.
2.2. Treated wastewater
The feedstock used was a secondary (after the biological reactor)
treated urban wastewater (TWw) from the wastewater treatment plant
“San Juan Ixhuatepec” located in Mexico City (15° 31′ 15″ N, 99° 07′
26″ W), Mexico. For C. vulgaris growth, TWw was filtered by 0.7 μm
pore glass fiber membrane while for indigenous strains TWw was not
filtered. Three TWw samples were taken at different times and analyzed
for chemical oxygen demand (COD), ammonia nitrogen (NH4+-N), ni-
trate nitrogen (NO3−-N), and phosphorus were determined as pre-
viously described (Ramírez-López et al., 2015).
401
Bioresource Technology 244 (2017) 400–406
In addition, TWw was enriched with the fertilizer Bayfolan® Forte
(1 mL/L) (TWw + B). The purposes of enriching TWw with Bayfolan®
were to establish the effect of a more economical source of nutrients
than conventional media, therefore, TWw + Bayfolan® may be con-
sidered a type of economical medium. In this way, there is also the
advantage of reusing treated water. This may reduce production costs,
on the other hand, due to the low nutrient content of TWw that could
limit the growth of microalgae, TWw was enriched with Bayfolan® as a
control of the experimentation and also for potential utilization.
Bayfolan® Forte is a concentrated formula of nutrients that contains
vitamins and phytohormones that stimulate the metabolic processes of
plants. In addition, indolacetic acid is a growth stimulant in plant
cultures and could produce the same effect on microalgal cultures.
2.3. Experimental design
The experiments were conducted in 1 L photobioreactors (PBR) with
a working volume of 0.8 L. The photobioreactors were bubbled with air
at a constant flow rate of 0.5 vvm and illuminated 12:12 h (light: dark)
by using cool-white fluorescent light at an intensity of 180 μmol pho-
tons m2/s. Illumination was placed on the surface of the reactors on one
side with cool white fluorescent tubes placed on the side of the reactors.
The temperature was maintained at 24 ± 1 °C. The growth conditions
were the same for all the experiments.
In order to determine the growth in TWw, both of the C. vulgaris
strains that were previously grown in BBM and washed in isotonic so-
lution, were inoculated (10%v/v) separately in 1 L PBRs containing
TWw and TWw enriched with Bayfolan® (1 mL/L). A third PBR was also
inoculated with the indigenous consortium grown in TWw.
Three consecutive cultures (1st, 2nd and 3rd cultures) were carried
out. For each consecutive culture, the previous culture was taken as
inoculum for the following. The biomass concentration was assessed
every day and the rest of the analyses at the end of the kinetics.
Ammonium (NH4+) and nitrate (NO3−) concentrations as well as pH
were also measured during the experiments. All the experiments were
conducted in triplicate. In the present work, the complete character-
ization of the biomass is presented only for the third culture. After the
procedure of culturing, any possible effect due to the performance of
the initial inoculum was eliminated, as a result, the third culture was
stable, synchronized and consistent for the purposes of the present
work.
2.4. Analyses
2.4.1. Growth and biomass dry weight measurement
Optical density was determined daily at 600 nm (HACH 3000
spectrophotometer). For dry biomass (g/L), 40 mL of culture was fil-
tered through a glass microfiber membrane (Ahlstrom, 4.7 cm dia-
meter, 0.7 μm pore size) to constant weight. The membrane with the
wet sample was dried at 200 °C in a Moisture Analyzer MS-70.
2.4.2. Determination of pigments, carbohydrates, proteins and lipids
Culture samples of 1.0 mL were centrifuged for 5 min at 17 000×g,
after which the supernatant was removed. Cells were suspended in
1.0 mL of methanol and incubated at 45 °C in the dark during 30 min.
Tubes were centrifuged (17 000×g/5 min) and the methanolic extract
was transferred into plastic cuvettes for measurement at 480, 652, 665
and 750 nm. Carotenoids and chlorophyll a and b concentrations were
estimated according to Eqs. (1)–(3).
Chlorophyll-A [mg/mL] = −8.10 OD652nm + 16.57 OD665nm−OD750nm (1)
Chlorophyll-B [mg/mL] = 27.44 OD652nm−12.17 OD665nm−OD750nm (2)
Carotenoids [mg/mL] = 4 OD480nm−OD750nm (3)
Proteins were measured by the Bradford method Bradford (1976);
L.C. Fernández-Linares et al. Bioresource Technology 244 (2017) 400–406

culture samples of 1.0 mL were centrifuged for 5 min at 17 000×g, and

COD

100
100

100
100

100
100
the supernatant was removed. The pellet was hydrolyzed with 1 mL of
NaOH 1 N at 100 °C for 2 h. Subsequently, 100 μL of the hydrolysate
were placed in plastic cuvettes and 1 mL of the Bradford reagent was

PO4−3
added. This mixture was incubated at room temperature for 10 min and

8.05

7.76

3.47
Maximal nutrient removal (%)

ND

ND

ND
then read at 595 nm using a UV-VIS spectrophotometer.
Total sugars content was determined by the Dubois’s method Dubois
et al. (1956); culture samples of 1.0 mL were centrifuged for 5 min at 17

NO3−

49.90

49.70

47.39
000×g, and the supernatant was removed. The pellet was hydrolyzed

ND

ND

ND
with 1 mL of HCl 1 N at 100 °C for 2 h. Then, 200 μL of the hydrolysate
were placed in glass tubes with 200 μL of phenol (5% v/v) and 1 mL of
concentrated sulfuric acid (H2SO4). This mixture was let to settle for

NH4+

99.92

99.20

99.72
100

100

100
10 min, and then vortexed and re-suspended for 30 min. It was read at
490 nm using a UV-VIS spectrophotometer.
Lipids were extracted and Fatty acids Methyl esters (FAME) were

COD

0.00
0.00

0.00
0.00

0.00
0.00
determined as previously described (Ramírez-López et al., 2015).

32.10 ± 0.03

32.23 ± 0.03

33.71 ± 0.04
3. Results and discussion

3.1. Obtainment of the indigenous consortium

PO4−3

ND

ND

ND
Nutrient profile and removal efficiencies of both C. vulgaris strains and the indigenous consortium cultivated in TWw and TWw enriched with Bayfolan® (1 mL/L).
To obtain an enrichment of the indigenous consortium of TWw, four
consecutive cultures were made. The first culture was started with TWw

24.49 ± 1.02

28.19 ± 2.32

22.84 ± 1.52
only. The consecutive cultures were inoculated with the previous one,
all in triplicate. In the first culture, the microalgae growth began until

Final concentration (mg/L)

NO3−
the ninth day and was maintained until day 21 with a final biomass of

0.00

0.00

0.00
0.704 ± 0.061 g/L. In the first culture, the consortium consisted
mainly of diatoms, as well as species of Chlorella and Scenedesmus. In

0.04 ± 0.00

0.38 ± 0.02

0.14 ± 0.01
the following three cultures, the growth rate increased (Fig. 1), and the
predominant species were Chlorella and Scenedesmus. The final biomass
NH4+

obtained in the three subsequent cultures was an average of

0.00

0.00

0.00
1.088 ± 0.100 g/L.
130.86 ± 16.72

130.86 ± 16.72

130.86 ± 16.72
3.2. Use of treated wastewater for microalgae growth
38.50 ± 0.07

38.50 ± 0.07

38.50 ± 0.07

The TWw used presented the characteristics listed in Table 1. The

concentrations of ammonium (NH4+), phosphate (PO4−3) and che-
COD

mical oxygen demand (COD) were extremely low compared to those

reported in different wastewaters (Gonçalves et al., 2016; Zhou et al.,
2012a,b; Li et al., 2011b) but only a little lower than the secondary
34.91 ± 0.29

34.91 ± 0.29

34.91 ± 0.29

municipal wastewater reported by Ebrahimian et al. (2014). However,

that one contained 224.78 mg/L of NO3−-N, while the TWw used for
PO4−3

the present work did not contain nitrates. In secondary treated waste-
ND

ND

ND

water ammoniacal nitrogen and phosphates are found in concentrations

of 20–40 mg/L and 1–10 mg/L, respectively, which can support high
64.08 ± 3.27

64.08 ± 3.27

64.08 ± 3.27

microalgae biomass productivities (McGinn et al., 2011). Nevertheless,

the wastewater composition varies within a municipal wastewater
Initial concentration (mg/L)

NO3−

treatment plant itself (Wang et al., 2010) and it also depends on the
ND

ND

ND
0.56 ± 0.024
47.90 ± 2.34

47.90 ± 2.34

47.90 ± 2.34
0.56 ± 0.02

0.56 ± 0.02
NH4+

TWw + B

TWw + B

TWw + B
Medium

TWw

TWw

TWw
C. vulgaris CICESE
C. vulgaris UTEX

consortium
Indigenous

Fig. 1. Obtainment of indigenous consortium from TWw, in four consecutive cultures. In

Strain
Table 1

the initial culture, autochthonous microflora was grown from native microflora; con-
secutive cultures were inoculated with the previous culture.

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L.C. Fernández-Linares et al. Bioresource Technology 244 (2017) 400–406

Fig. 2. Effect of TWw and TWw enriched with Bayfolan® in the

growth of the two C. vulgaris strains and the indigenous consortium
during three consecutive cultures.

period of the year. Despite the low concentration of nutrients in the

TWw used in the present study (Table 1), both strains of C. vulgaris and
the indigenous consortium showed growth in the three consecutive
cultures. In the third culture, biomass production of C. vulgaris UTEX,
CICESE and the indigenous consortium was of 1.167 ± 0.057,
1.575 ± 0.434 and 1.125 ± 0.250 g/L, respectively (Fig. 2). In C.
vulgaris cultures with non-sterilized TWw, the inoculated strains were
the ones that prevailed throughout the culture. When observing under
the microscope, only Chlorella was present. No rotifers (microalgae
grazer) were spotted.
Ebrahimian et al. (2014) obtained the highest concentration of C.
vulgaris biomass (1.13 g/L) at the end of the cultivation when 25% of
the primary wastewater was used (99.5 mg Ntotal/L) and 1.03 g/L under
optimum conditions when using secondary treated wastewater (NH4+- Fig. 3. Effect of TWw and TWw enriched with Bayfolan® (1 mL/L) on microalgae pro-
N, 0.63; NO3−-N; 224.78; PO−34 -P, 0.63; and COD, 96 mg/L) enriched duction in the third culture.
with 1 g/L of NaHCO3. Feng et al. (2011) grew C. vulgaris in artificial
wastewater with 20 mg/L nitrogen as NH4+-N (45 times higher than
ammonium, nitrate, phosphate, and COD in both, TWw and TWw + B,
the TWw used in this work), reaching a biomass concentration of
as well as the contribution of nutrients due to Bayfolan®. The COD and
1.58–1.72 g/L in 7 days. Lam et al. (2017) cultivated C. vulgaris in
ammonium were almost completely removed in both media while the
different concentrations of unsterilized wastewater (0.02 to –0.2 v/v).
nitrates supplied by the fertilizer were removed by 47–49%. Only
Chlorella vulgaris was found to have a higher biomass yield when sup-
3.47–8.05% of the supplied phosphate was consumed (Table 1). Algae
plied with the lowest concentration of wastewater studied (0.02 v/v).
could directly uptake inorganic nitrogen such as nitrate, nitrite, and
Under these conditions, the microalgae biomass productivity was
ammonia, which is the nitrogen species that is the easiest to be as-
0.0409 g/L·d, and the biomass production of 0.4908 g/L, three and two
similated by microalgae. Only the ammonium is utilized when cultures
times lower than those obtained in the present work with C. vulgaris
contain both, nitrate and ammonium.
CICESE and UTEX cultured in TWw, respectively. Li et al. (2011b) grew
C. vulgaris sp. in raw concentrated municipal wastewater (212, 116, and
2304 mg/L of P, N and COD, respectively), and obtained 1.06 g/L as the 3.4. Effect of media on lipid accumulation and fatty acid profiles
final biomass concentration.
In the treated water enriched with Bayfolan®, which provides am- The total lipid content was 8.73–20.22 and 20.54–25.70% in TWw
monium and nitrate in a concentration of 51.14 ± 3.09 and + B and TWw, respectively. The concentration of lipids is strongly
64.08 ± 3.28 g/L, respectively (53.89 mg of Ntotal/L) (Table 1), the related to the limitation of the source of nitrogen in the medium. The
biomass production increased significantly. However, there was no TWw enriched with the fertilizer contains a higher concentration of
significant difference in biomass production between strains grown in nitrogen; thus, in this medium, all strains showed a lipid content sig-
the same medium (Fig. 3). In all cultures, the pH increased two pH units nificantly inferior to those grown in TWw (Table 2). The lipid content
during the cultures and remained in values between 8 and 10 (data not was higher in the TWw because it contained sufficient nutrients for
shown). There are few reports on work done with the utilization of adequate growth but at the same time became nitrogen limiting, which
fertilizers, for example, Lam and Lee (2012) cultivated C. vulgaris with allows a greater accumulation of lipids. However, there was no
an organic fertilizer (Baja Serbajadi Humus 27) in different con-
centrations, the maximum biomass produced with enough supplement Table 2
of nutrients was 0.29–0.31 g/L. Lipids content in both, C. vulgaris strains and indigenous consortium grown in TWw and
TWw + B.

3.3. Nutrient removal Strain Medium Lipid content DW (%)

C. vulgaris UTEX TWw 25.70 ± 1.24

The objective of the present work was to demonstrate that TWw can TWw + B 8.73 ± 1.71
support the growth of microalgae, but not the water treatment itself
C. vulgaris CICESE TWw 23.35 ± 3.01
since it already comes from an established wastewater treatment main TWw + B 20.22 ± 1.60
process. Nonetheless, it is important to consider the removal of nu-
Indigenous consortium TWw 20.54 ± 1.23
trients from both, TWw and TWw + B as a potential polish or for re- TWw + B 13.04 ± 2.10
utilization. The data in Table 1 summarized the variation of

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L.C. Fernández-Linares et al. Bioresource Technology 244 (2017) 400–406

Table 4
Fatty acids composition profiles of Chlorella strains and indigenous consortium on dif-
ferent media (% of total FAME).

Strain Culture Acids

Media
Palmitic Stearic Oleic Linoleic Linolenic
(C16:0) (C18:0) (C18:1) (C18:2) (C18:3)

C. vulgaris TWw 30.58 11.51 26.94 13.35 17.63

UTEX TWw 34.78 20.09 11.20 11.14 22.80
+B

C. vulgaris TWw 35.08 11.23 32.72 9.81 11.17

CICESE TWw 29.42 10.62 14.99 13.42 31.54
+B
Fig. 4. Effect of TWw and TWw enriched with Bayfolan® (1 mL/L) on lipid production by Indigenous TWw 31.31 9.04 38.94 5.88 14.84
the two C vulgaris strains and indigenous consortium. consor- TWw 27.80 9.82 – 14.14 48.25
tium +B

significant difference in lipid production between strains and media

used (Fig. 4). This could be due to the fact that the enriched medium
allowed a higher growth and biomass production, but with a lower lipid
content and the TWw exerted the opposite effect. The total lipid con-
tents achieved with TWw as the sole growth medium were similar
compared with other strains even when they were grown on a richer
Ww or with a conventional medium. Some examples of this are:
Chlorella sorokiniana (lipid content 26.3% dw) grown in municipal
wastewater (Zhou et al., 2011) and Auxenochlorella protothecoides (lipid
content 28.9% dw) also grown in municipal wastewater (Zhou et al.,
2012a,b) (see Table 3). In a richer source of nutrients such as piggery
wastewater Chlorella vulgaris CY5 presented a lipid content of 30.3% dw
(Marjakangas et al., 2015) which is not considerably higher than the
lipid contents obtained in the present work (23.4–25.7% dw, see
Table 3). However, the lipid productivities were moderate compared to
those obtained with C vulgaris grown in different wastewaters, even in a
Fig. 5. Effect of TWw and TWw enriched with Bayfolan® (1 mL/L) on protein production
typical nutrients source like the BBM and M Medium (Ramírez-López the two C vulgaris strains and the indigenous consortium. Means with different letters are
et al., 2015) (Table 3). It is worth noticing that all the wastewaters used significantly different (Tukey’s HDS, p < 0.05).
contained a higher concentration of nutrients. C. vulgaris presented the
capability of accumulating large amounts of lipids, especially under
acid (18: 1) increased significantly with respect to TWw + B, being the
limited conditions of the nitrogen source, which also generated a lipid
second FAME more produced (Table 4). In both media and strains
profile suitable for the production of biodiesel (Safi et al., 2014).
studied, unsaturated fatty acids were the main FAME in a range of 45%
The fatty acid profiles were mainly composed of C16 and C18
to 62%. The same FAME profile was obtained for C. vulgaris grown in
(Table 4). Palmitic acid (C16:0) was the main FAME in all cultures and
different domestic wastewater dilutions, but saturated fatty acids were
strains; and along with the stearic acid (C18:0), they were the single
higher than unsaturated (54–72 and 28–46%, respectively) (Miao et al.,
two unsaturated accumulated FAME. In the systems with TWw the oleic
2016). The major fatty acids obtained for C. vulgaris were palmitic and

Table 3
Comparison of lipid content, biomass and lipid productivities for microalgae cultivated in different kind of wastewaters and media.

Wastewater or Medium Type Microalgae Specie Biomass Productivity Lipid Content Lipid Productivity References
(mg/ L/ d) (%DW) (mg/ L/ d)

Artificial wastewater Chlorella vulgaris 70–296 20–42 e Feng et al. (2011)

Municipal Wastewater Chlorella sp. 231.4 33.3 77.1 Zhou et al. (2011)
Municipal Wastewater Chlorella vulgaris 120.8 17.4 21.0 Zhou et al. (2011)
Municipal Wastewater Chlorella sp. 137.5 26.8 36.9 Zhou et al. (2011)
Municipal Wastewater Chlorella sorokiniana 187.5 26.3 49.4 Zhou et al. (2011)
Municipal Wastewater Chlorella sp. 241.7 30.8 74.4 Zhou et al. (2011)
Raw concentrated municipal wastewater Chlorella sp. 65.7 11.0* 7.5* Li et al. (2011a)
Centrated wastewater Chlorella kessleri 221.8 10.9* 24.1* Li et al. (2011b)
Municipal Wastewater Auxenochlorella protothecoides 268.8 28.9 77.7 Zhou et al. (2012a)
Organic Fertilizer Chlorella vulgaris 2.6 18.1 0.5 Lam and Lee (2012)
Piggery wastewater Chlorella vulgaris CY5 233.3 30.3 70.5 Marjakangas et al. (2015)
Liquid food wastes Chlorella vulgaris 207 31.0 64.2 Hou et al. (2016)
Domestic wastewater Chlorella vulgaris 40.9 32.7 13.4 Lam et al. (2017)
Saline wastewater Chlorella vulgaris 135.3 40.1 54.3 Shen et al. (2015)
Monosodium glutamate wastewater Chlorella vulgaris ∼181.9 30.5 ∼55.5 Jiang et al. (2016)
M medium Chlorella vulgaris UTEX 36.5 38.5 14.1 Ramírez-López et al. (2015)
Treated wastewater Chlorella vulgaris UTEX 77.8 25.7 20.0 This Study
Treated wastewater Chlorella vulgaris CICESE 105.0 23.4 24.5 This Study
Treated wastewater TWw Indigenous consortium 99.9 20.6 20.5 This Study

* % as FAMES.

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L.C. Fernández-Linares et al.
Fig. 6. Effect of TWw and TWw enriched with Bayfolan® (1 mL/L) on total pigments
production the two C vulgaris strains and the indigenous consortium. Means with different
letters are significantly different (Tukey’s HDS, p < 0.05).
oleic acids (Hou et al., 2016; Han et al., 2014). Higher saturated fatty
acids are more suitable for the production of biodiesel (Chinnasamy
et al., 2010). High ammonium concentration in the medium favors the
ratios of C16:0 and C18:2, while at low concentrations of ammonium
the composition of C18:0, C18:1 and C18:3 increases (He et al.,
2013a,b). Widjaja et al. (2009) reported that by restricting nitrogen
supplementation for several days during C. vulgaris growth, the com-
position of palmitic and stearic acids decreases and the concentration of
triglycerides increases.
3.5. Effect of media on pigments, protein and carbohydrates accumulation
The total protein content of the TWw was 14.6–27.9%, while in the
TWw + B protein content was significantly superior: 26.9–47.67%. The
protein production in TWw was 229.5–385.7 mg/L, which is sig-
nificantly lower than that produced in TWw enriched with fertilizer
(Fig. 5). Miao et al. (2016) showed that C. vulgaris produced more
protein in synthetic domestic wastewater than in BG11 medium
(40.9–50.7 vs 35.7%). This value was higher than that obtained in this
study in which the TWw had a nitrogen content 200 times lower. The
protein productivity in TWw was 12.07, 17.10, and 20.30 mg/L·d for
the Indigenous consortium, C. vulgaris UTEX, and C. vulgaris CICESE,
respectively. He et al. (2013a,b), obtained a protein productivity of
35 mg/L·d, with Chlorella vulgaris cultivated in wastewater containing
150 mg/L of ammonium, a concentration much higher than the one
used in this work with TWw.
There was no significant difference between cultures and strains
regarding productivity and concentration of carbohydrates, with values
of 1.40 to –1.80 mg/L·d, and 21.1 to –27.1 mg/L, respectively. He et al.
(2013b) reported carbohydrate productivities with C. vulgaris in was-
tewater with an ammonium concentration of 143 mg/L, 30 times
higher than the obtained in this work.
Total pigments (Chlorophyll a and b + carotenoids) obtained from
strains grown in TWw were 4.4–8.7 mg/L, which are really low; how-
ever, the addition of Bayfolan® to the medium significantly increased
the total pigments content (Fig. 6). In this case, the pigment contents
were similar to those reported in C. vulgaris by He et al. (2013b).
4. Conclusions
Treated domestic wastewater can be used for microalgae growth,
allowing a biomass production of C. vulgaris of 1.58 g/L, with a lipid
content of 24.5% and productivity of 20.3 mg/L·d. The fatty acids
proportion obtained was palmitic (35.1 %), oleic (32.7 %), stearic (11.2
%), linolenic (11.1 %), and linoleic (9.8 %) dw. The enrichment of TWw
with Bayfolan® significantly increased the production of pigments and
proteins. The indigenous consortium obtained constituted by Chlorella
and Scenedesmus, reached a biomass concentration of 1.13 g/L in TWw,
405
Bioresource Technology 244 (2017) 400–406
and productivities of lipids, proteins and pigments similar to those
obtained with C. vulgaris.
Acknowledgements
The authors are grateful for the financial support provided by the
Unidad Profesional Interdisciplinaria de Biotecnología. Instituto
Politécnico Nacional – IPN – México (Grant SIP 20161677 and SIP
20170982), and by CONACYT – México (Consejo Nacional de Ciencia y
Tecnología, Fondo Institucional Problemas Nacionales-2014 Grant
247402).
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