Algal Research 41 (2019) 101549

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Continuous cultivation of Chlorella minutissima 26a in landfill leachate-based T

medium using concentric tube airlift photobioreactor
⁎
Geronimo Virginio Tagliaferroa,b, , Hélcio José Izário Filhob, Anuj Kumar Chandela,
Silvio Silvério da Silvaa, Messias Borges Silvab,c, Júlio César dos Santosa
a
Department of Biotechnology, Engineering School of Lorena, University of São Paulo, CEP 12602-810, Brazil
b
Department of Chemical Engineering, Engineering School of Lorena, University of São Paulo, CEP 12602-810, Brazil
c
Department of Production, Engineering School of Guaratinguetá, São Paulo State University, CEP 12.516-410 Guaratinguetá, SP, Brazil

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

Keywords: The use of landfill leachate was studied as nutrient source for Chlorella minutissima 26a microalgae in mixo-
Chlorella minutissima trophic cultivation in a continuous concentric tube airlift photobioreactor, concomitantly promoting the leachate
Lipids production bioremediation. Medium was composed of leachate, water and sodium chloride and the cultivation process was
Carbohydrates production performed in continuous mode under fluorescent light with a photon flux of 80–90 μmol m−2 s−1. The effects of
Landfill leachate
different leachate concentrations were evaluated for the microalgal growth, biomass composition (lipid, car-
Bioremediation
bohydrate, protein and ash) and for the removal of organic compounds, nitrate and metals from the medium.
Increase in leachate concentration from 5 to 10% in the medium, caused the reduction in the lipid amount but
increased the carbohydrate amount with no marked effect on the protein content. In the steady state, the
maximum productivity of biomass, lipids, carbohydrates and proteins were 232.0 ± 7.6 (7.5% of leachate
concentration), 96.6 ± 7.8 (5% of leachate concentration), 33.4 ± 2.1 (10% of leachate concentration) and
47.1 ± 3.0 mg L−1 d−1(7.5% of leachate concentration), respectively. Maximum values obtained for the re-
duction in chemical oxygen demand, removal of total organic compounds and inorganic nitrate from the landfill
leachate during cultivation in steady state were 92.8% (7.5% of leachate concentration), 90.5% (7.5% of lea-
chate concentration) and 100% (5% of leachate concentration), respectively. Maximum values of removal of
metals from the medium were 63%, 72%, 100%, and 67% for Cr, Fe, Al and Ba, respectively. These results
indicated that the use of the landfill leachate could be a possible source for the sustainable production of mi-
croalgal biomass, also resulting in concomitant removal of undesirable components, i.e. organics and metals.

1. Introduction cultivation conditions, resulting in uniformity in biomass production

Microalgae based biotechnology platform provides a number of bio-
based products such as pigments, polysaccharides, bioplastics, organic
compounds including biofuels (bioethanol and biodiesel) [1]. Typically,
microalgal biomass have been used as raw material for biorefineries to
obtain different products of industrial interest [2]. Microalgae cultiva-
tion has been carried out in different photobioreactors, such as tubular,
flat plate, bubble column and airlift with different configurations [3–5].
These reactors have several advantages compared to the open ponds
bioreactors, such as low risk of contamination, easy control and high
gas-liquid mass transfer rate, reduction of water loss, high productivity
and low cost of harvesting [6,7]. In continuous process, these bior-
eactors can lead to high biomass productivity due to the reduction of
non-productive time, simultaneously allowing a better control of
[8].
Microalgal metabolism directly depends on the availability of the
nutrients (e.g. nitrate, phosphate, carbon, metals, vitamins and others)
in the medium and culture conditions (e.g. autotrophic: light and CO2,
heterotrophic: organic carbon source, and mixotrophic: light, CO2, or-
ganic carbon). Mixotrophic cultivation synergistically employing on
carbon and organic compounds has shown rapid growth and high
biomass yield, besides the possibility of producing higher amount of
valuable components such as lipids to obtain biodiesel and other pro-
ducts of commercial significance [9,10].
Microalgae are able to utilize the nutrients present in wastewater for
their growth, generating biomass with high yields in lipids, carbohy-
drates and proteins [1,11–15]. In addition, microalgae are known as
promoting of bioaccumulation, up-taking of metals and thus removal of

⁎
Corresponding author at: Department of Chemical Engineering, Engineering School of Lorena, University of São Paulo, CEP 12602-810, Brazil.
E-mail address: tagliaferro@dequi.eel.usp.br (G.V. Tagliaferro).

https://doi.org/10.1016/j.algal.2019.101549
Received 4 November 2018; Received in revised form 7 May 2019; Accepted 14 May 2019
Available online 25 May 2019
2211-9264/ © 2019 Elsevier B.V. All rights reserved.
G.V. Tagliaferro, et al.
metals from wastewater [16].
The landfill leachate is a black liquid wastewater formed by the
decomposition process associated to the percolation of the rainwater
and the moisture of the solid waste. It is considered a source of metals,
nitrogen and phosphates, and can be used as an interesting alternative
for the cultivation of microalgae. The landfill leachate composition is
dependent on the constituents of the landfills, type of microbial flora,
soil characteristics, rainfall pattern and landfill age [17]. However,
most of the leachates have the same basic composition characterized by
four major components: dissolved organic matter, CH4, volatile fatty
acids and some refractory compounds such as fulvic and humic acids,
macroinorganic components (Ca2+, Mg2+, Na+, K+, NH4+, Cl−,
SO42−, HCO3−), heavy metals, and xenobiotic organic compounds such
as aromatic hydrocarbons, phenols and chlorinated aliphatic com-
pounds [18].
The use of landfill leachate as culture medium for microalgae can
also be a method for wastewater treatment as well as producing valu-
able products [16,19,20]. The use of landfill leachate as viable substrate
for cultivation of microalgae has not been explored much except few
reports, wherein the microalgae C. pyrenoidosa or a microalgae-bacteria
consortium have been evaluated [20,21].
Use of adequate culture conditions and bioreactor configurations
can result in high biomass productivity, thus allowing the production of
high amount of lipids, carbohydrates and other components. The airlift-
type photobioreactor offers some unique advantages, such as favoring
mixing and gas-liquid mass transfer, as well as representing a suitable
configuration for light contact with microalgal cells, favoring the high
amount of algal biomass production with greater productivities [22].
Following this approach, we studied in the present work the mixo-
trophic cultivation of the marine microalgae C. minutissima 26a in
medium containing landfill leachate, using a concentric tube airlift
photobioreactor under continuous mode. The main focus of the present
study was to produce a valuable biomass that can be a source of in-
teresting compounds in biorefineries, concomitantly promoting the
bioremediation of the landfill leachate.
To the best of our knowledge, this manuscript reports first time, the
use of landfill leachate for continuous mixotrophic cultivation of C.
minutissima, besides the evaluation of the potential of the system as a
treatment method and an option for reuse of leachate from landfills.
2. Methods
2.1. Microalga, inoculum preparation, and cultivation conditions
The microalga Chlorella minutissima 26a was provided by the Marine
Cultures Collection of the Oceanographic Institute at the University of
São Paulo (São Paulo, SP, Brazil). It was kept in artificial marine f/2
medium [23] containing 33.3 g L−1 NaCl, 75.0 mg L−1 NaNO3,
5.0 mg L−1 NaH2PO4·H2O, 3.15 mg L−1 FeCl3·6H2O, 22.2 μg L−1
ZnSO4·7H2O, 180 μg L−1 MnCl2, 6.3 μg L−1 Na2MoO4·2H2O, 10 μg L−1
CoCl2·6H2O, 9.8 μg L−1 CuSO2·5H2O, 100 μg L−1 Thiamine (B1),
0.5 μg L−1 Cyanocobalamin (B12) and 0.5 μg L−1 Biotin (B7). The stock
cultures were sub-cultured every six weeks in 250 mL Erlenmeyer flasks
containing 100 mL of medium (inoculated with 10% v/v of an aliquot
of the previous stock culture), kept at 20 °C under fluorescent light
conditions (photon flux of 80 to 90 μmol m−2 s−1). The seed cultures
were prepared by mixing 100 mL of stock culture with 900 mL of f/2
medium containing 20.0 g L−1 of NaCl [24]. In this case, the auto-
trophic culture was performed for 10 days using a bubble tubular
photobioreactor made up of transparent polyethylene terephthalate
(PET) of 120 mm in diameter and 280 mm in height, in a batch process
with aeration of 0.24 vvm.
2.2. Source and storage of landfill leachate
The landfill leachate was collected from the leach tank at Cachoeira
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Algal Research 41 (2019) 101549
Paulista/SP, Brazil, which is under the management of Vale Soluções
Ambientais (VSA, Cachoeira Paulista, SP, Brazil). Sample of leachate
was taken in 200 L plastic drum and stored at temperature of 9 °C. The
leachate was filtered with a quantitative filter paper (150 mm diameter,
0.2 mm thickness, 4–7 μm pores and 80 g m−2 density) before using it
in the culture medium preparation.
2.3. Batch and continuous culture of microalga in concentric tubes airlift
photobioreactor
The culture medium was prepared with 5%, 7.5% or 10% (v/v)
leachate diluted in water and added with 20 g L−1 NaCl. The cultivation
process was first performed in batch mode to obtain the maximum
specific growth rate (μmax) followed by the continuous mode operation.
A concentric tubes airlift photobioreactor sized 550 mm in length
and 120 mm in diameter of the outer tube and 340 mm in length by
60 mm in diameter of the central tube, as described earlier in our
previous study of Tagliaferro el al. [25]. The cultivation was carried out
with 3.8 L working volume and inoculated with 10% (v/v) microalgae
suspension as described in Section 2.1.
Batch process was performed with medium containing 5% of landfill
leachate to determine the maximum specific growth rate (μmax) and
feed rate during continuous cultivation. The maximum specific growth
rate (μmax) was estimated by the slope regression in the linear ex-
ponential phase obtained by the natural log of the dry cell weight as a
function of time.
For continuous cultivation, the hydraulic residence time (HRT) was
calculated by HRT = 1/D, where D (h−1) is the dilution rate and its
considered value was 80% of μmax. The biomass productivity (P,
mg L−1 d−1) was calculated as Eq. (1). For calculation of lipids, car-
bohydrates and proteins productivities, “P” was multiplied by their
correspondent mass fraction in the cells.
P b = C × D × 24 × 1000 (1)
−1
where, C is the dry cell weight (g L ).
The airlift photobioreactor was run in batch mode, at the beginning
of the continuous culture, with medium containing 5% of landfill lea-
chate until the microalgae growth reached the exponential phase.
Subsequently, the feeding was started in the reactor at a flowrate ob-
tained by multiplying the dilution rate (D) by the reactor working vo-
lume (3.8 L). Continuous cultivation was carried out in three phases,
each one with 3 HRT (totalizing 309.6 h) and with concentrations of
leachate in the medium of 5%, 7.5% and 10% (v/v) for first, second and
third phase, respectively. The total continuous cultivation time was
1082 h.
Triplicate samples were taken during the process and analyzed with
relation to biomass, number of cells, absorbance, cells composition and
inorganic nitrate concentrations.
2.4. Analytical methods
For the measurement of dry biomass (g L−1), 10 mL aliquots sample
of the medium were centrifuged at 1800 ×g for 10 min, kept in oven at
60 °C for 24 h and weighed on analytical balance. Absorbance of bath
culture medium was measured by UV–visible spectrophotometry
(Jenway spectrophotometer, model 7305, Stone, Staffs, UK) at 680 nm.
The cell counting was performed in a microscope (BA400, Motic
(Xiamen) Electric Group Co., China) aided by a Neubauer chamber
hemocytometer of 0.1 mm depth. Average cells diameter were esti-
mated by ‘Image J 1.8’ software [26].
Total lipid of the microalgal biomass was extracted following the
protocol of Bligh and Dyer [27]. The lipid content (%) was calculated as
the dry biomass of the sample before and after the extraction. The
carbohydrate (%) content in microalgal biomass was determined fol-
lowing the method of Moxley and Zhang [28] and total sugars de-
termination using the phenol‑sulfuric method [29]. High performance
G.V. Tagliaferro, et al.
Fig. 1. Chlorella minutissima 26a growth profile during batch cultivation in airlift photobioreactor in the first (a), second (b), third (c) and fourth (d) day of culture in
medium containing 5% (v/v) leachate.
liquid chromatography (HPLC) was used for the measurement of the
monomeric sugars present in the microalgae biomass hydrolysate. For
this, an Agilent 1200 HPLC series chromatograph (Agilent Technolo-
gies, Inc., USA) was fitted with an HPX-87H (300 × 7.8 mm) column
(Bio-Rad, USA) was used and the analysis conditions were as following:
column temperature 45 °C, H2SO4 (0.01 N) as mobile phase with flow
rate of 0.6 mL min−1 and 20 μL of injection volume with RID-6A re-
fractive index detector. The crude protein content was estimated ac-
cording to Lourenço et al. [30], considering “protein con-
tent = nitrogen concentration × 4.25”, with the total nitrogen
concentration measured by the Kjeldahl method [31]. The ash content
(%) was calculated according to the analytical method of Wychen and
Laurens [32]. All of the analytical assays were performed in triplicate.
Inorganic nitrate concentrations (NO3–N) in the culture medium
were determined by spectrophotometry according to the APHA 4500-
NO3− standard method [33]. For this, triplicate samples were taken
periodically from the bioreactor during cultivations of microalgae.
TOC (Total Organic Carbon) and COD (Chemical Oxygen Demand)
analyzes of the medium containing landfill leachate before and after the
microalgal cultivation were carried out using methods 5310-A and
5520-B, respectively, as described in the Standard Methods for the
Examination of Water and Wastewater [34]. Because a saline medium
was used, the COD analysis was done after dilution in water and ad-
dition of mercury sulfate, according to Kayaalp et al. [35]. The TOC
value was determined by Shimadzu TOC-V CPN analyzer (Shimadzu,
Japan) and a blank test was performed with a 20 g L−1 sodium chloride
solution, so that the influence of the salt in the analysis could be taken
into consideration. Metal analysis of the samples was performed using
the Optical Emission Spectrometry (ICP-OES) technique (model Optima
8000, PerkinElmer, USA). In all cases, coefficients of variation for re-
sults of TOC and COD was 0.5% and metal were lower than 3%.
3. Results and discussion
3.1. Batch cultivation of C. minutissima 26a in a concentric tubes airlift
photobioreactor: determination of the maximum specific growth rate
The microalgae were cultivated in mixotrophic form in airlift pho-
tobioreactor of concentric tubes containing landfill leachate and sodium
chloride. For the cultivation, the concentration of sodium chloride used
(20 g L−1), as reported by Cao et al. [24].
Based on the growth profile of microalgae in the batch process, the
maximum specific growth rate (μmax) was 0.01247 h−1 (0.30 d−1), a
value similar to that reported by Zhao et al. [21] (0.28 d −1) for the
3
Algal Research 41 (2019) 101549
microalgae C. pyrenoidosa cultivated in 500 mL flasks containing 10%
(v/v) of landfill leachate.
A control experiment with autotrophic cultivation was not per-
formed. Landfill leachate is a very complex material and its exact and
detailed composition is not known and, thus, the elaboration of a de-
fined autotrophic medium to simulate landfill leachate and proceed
with a control experiment is very difficult. However, in our previous
work (Tagliaferro et al. [25]), the autotrophic cultivation of C. minu-
tissima showed the μmax (0.19 d−1) in seawater f/2-based medium,
lower than the observed in the present work for mixotrophic cultiva-
tion.
About heterotrophic cultivation, previous work performed in our
laboratory in a medium with 5% of landfill leachate showed that cell
growth was faster at the beginning of the process, reaching a μmax value
of 0.395 d−1 (Fig. S1). However, the biomass production in this case
was 0.46 g L−1 in 48 h of cultivation and declined thereafter. Thus,
heterotrophic cultivation was limited by carbon sources available in the
medium. The mixotrophic batch cultivation, although resulting in lower
μmax value compared to heterotrophic cultivation, but resulted in higher
biomass production, reaching 1.42 g L−1 in 95 h of the cultivation
process. This behavior is related to the availability of CO2 as an addi-
tional carbon source in mixotrophic cultivation.
The calculated μmax in the mixotrophic batch cultivation was used as
a base to determine the dilution rate (D) to be used in the continuous
runs. In order to avoid washout in bioreactor and to favor high biomass
productivity, the D value (0.009 h−1) was 20% lower than that of μmax
was used. This value corresponded to a hydraulic residence time (HRT)
of 103.2 h. Based on this information, the airlift photobioreactor was
fed, during the continuous process, with a flow rate of 0.62 ml min−1 of
culture medium with a working volume of 3.8 L.
During batch cultivation the characteristic green color of the
“chlorophyll-a” pigmentation was progressively intensified (Fig. 1),
presented higher intensity on the fourth day of cultivation (absorbance
of 0.519, dry biomass of 0.52 g L−1).
3.2. Effect of the leachate concentration on cell growth using the concentric
tubes airlift photobioreactor in continuous process
Process was started as batch cultivation and the continuous process
started after 103.2 h by feeding of medium. Bioreactor was fed with a
flowrate of 0.62 mL min−1 of medium containing different concentra-
tions of landfill leachate. The continuous operation was carried out
until 1082 h of cultivation and the results obtained for dry biomass and
concentration of nitrate ions remaining in the culture medium are
G.V. Tagliaferro, et al.
Fig. 2. Biomass production (a) and nitrate consumption profile in the medium (b) corresponding to cultivation carried out with different concentrations of leachate
(5, 7.5 and 10% (v/v)). Data were shown as mean ± standard deviation, n = 3.
shown in Fig. 2. The nitrate concentration was measured in the stream
at the exit of the reactor and was a representative value of inorganic
nitrogen sources available in the medium.
Cultivation of the microalga C. minutissima 26a using leachate
based-medium as a growth medium was feasible under the studied
conditions. The profile of the biomass concentration in the continuous
process presented small variation of values as a function of the increase
of the leachate concentration in the medium (Fig. 2a). The low varia-
tion in the concentration of biomass as a function of the different
concentrations of leachate in the medium corresponded to a behavior
similar to that observed by Markou et al. [36]. Those authors reported
that the initial concentration of leachate coming from poultry waste-
water varying from 10% to 25% (v/v) resulted only in small changes in
the biomass production of C. vulgaris, showing the values between 1.76
and 1.86 g L−1 in batch culture.
As shown in Fig. 2a, after starting feed flow rate with medium
containing 5% of leachate, a transient phase of about 100 h was ob-
served and then system reached an average biomass concentration at
steady state of 0.96 ± 0.04 g L−1. A transient phase also occurred
between 480 h and 600 h of culture, when the leachate concentration in
feed flow rate was raised from 5% to 7.5% (v/v). Then, a new steady
state was reached, with an average biomass concentration of
1.09 ± 0.02 g L−1. When leachate concentration in feed flow rate was
increased to 10%, there was an initial decrease in the biomass con-
centration, indicating that the cells needed a period of adaptation to
this new condition. The biomass decrease is due to the response of cells
4
Algal Research 41 (2019) 101549
to the increase in toxic components in the medium when 10% of lea-
chate concentration was used. Indeed, landfill leachate has compounds
that are toxic to microorganisms [37], such as phenol, benzene, to-
luene, acetone, ammonical‑nitrogen, phosphate, heavy metals (Pb, Cd,
Cu, Zn, Cr, Ni and others), sulfide, acids, alkalis and inorganic salts
[18,38,39].
After this phase of cells adaptation, system reached a new steady
state with a biomass concentration (average value of
1.01 ± 0.04 g L−1) similar to the observed when 7.5% of leachate was
used. This behavior was also evidenced in Fig. 2b, which showed in-
crease in leachate concentration in feed flowrate to 7.5% and to 10%
resulted in an initial increase of the remaining nitrate concentration in
the medium, followed by reduction in this value. Biomass production
values were lower than those obtained by Zhao et al. [21], which re-
ported the batch culture of other microalgae species - C. pyrenoidosa
(FACHB-9) - in medium containing diluted landfill leachate, varying its
initial concentration from 0 to 20% (v/v). The highest concentration of
biomass obtained in that work was 1.58 g L−1 in the medium con-
taining 10% (v/v) diluted leachate.
Fig. 2b shows that the remaining nitrate concentration varied with
the concentration of leachate in the culture medium. The landfill lea-
chate initially showed about 90 mg L−1 of nitrate. In the batch phase
(until 103.2 h) with 5% (v/v) initial leachate concentration, the re-
maining nitrate concentration in the medium decreased with the bio-
mass increase, demonstrating that the inorganic nitrogen source in the
leachate was consumed by microalgae for the biomass growth. After the
G.V. Tagliaferro, et al.
Table 1
Count and mean size of microalgae cells as a function of leachate concentration in the medium in the airlift photobioreactor feed stream during continuous culture at
steady state (mean ± standard deviation, n = 3).
Leachate concentration in the medium (% v/v) Average population of microalgae (×10−6) (cell/mL)
5 7.16 ± 0.61a
7.5
10
Results corresponds to average triplicates ± standard derivation. Same letter in the column indicates no difference according Tukey's range test (95% of confidence
level).
starting of the continuous feed in the photobioreactor, the nitrate
concentration decreased slowly until undetectable values, observed
from 288.7 h. When the reactor was fed with 7.5% (v/v) of leachate, the
nitrate concentration increased in a transient phase until reaching
45.5 mg L−1, decreasing until reaching values close to zero in 650 h.
The increase in leachate concentration in the feed to 10% (v/v) resulted
in an increase in the remaining nitrate concentration until reaching the
value of 74.8 mg L−1, then decreasing to the an average value of
24.3 mg L −1, thus not fully consumed in the process during this phase.
Fig. 2 clearly showed that, when the leachate concentration in-
creased from 5% to 7.5%, with the consequent increase in the con-
centration of nitrate and other nitrogen sources present in the medium
concomitantly showing slightly higher concentration of biomass.
However, the increase of leachate concentration from 7.5% to 10% did
not have the same effect, resulting in partial nitrate consumption and
not increasing biomass concentration. This behavior can be attributed
to the stress caused by the increased concentration of toxic components
present in the leachate, which inhibited microalgal growth [20,40].
Table 1 shows the average number of cells produced and average
size of microalgae cells during continuous cultivation at different lea-
chate concentration in feed stream. As can be observed, the average
number of cells decreased when concentration of leachate in the feed
stream was increased from 7.5% to 10%. This pattern could be related
to the higher concentration of toxic compounds present in the medium.
Population reduction, however, was accompanied by an increase in the
average size of microalgal cells (Table 1 and Fig. 3), which resulted in
biomass concentration similar for 7.5% and 10% (Fig. 2). The increase
in the average cell size at higher concentrations of leachate in the
medium is possibly due to the stress conditions imposed to microalgae.
In this case, in response to the increased presence of toxic compounds,
but with greater abundance of carbon sources, cells can accumulate
reserve compounds present in the leachate, such as starch or other
polysaccharides [41–43].
3.3. Effect of leachate concentration on biomass composition and
productivity in airlift photobioreactor of concentric tubes in continuous
process
The growth profile of C. minutissima 26a grown in medium con-
taining 5%, 7.5% and 10% (v/v) of leachate was regularly observed.
Samples of biomass cultivated at steady state were analyzed for the
amount of lipids, carbohydrates, protein and ashes and average values
are shown in Fig. 4.
The variation in leachate concentration in the medium showed
marginal influence on biomass composition in terms of proteins, which
was approximately 22% in all cases. On the other hand, increase in the
concentration of leachate from 5% to 10% (v/v) resulted in the re-
duction in the content of lipids from 47.3% to 37.0%. The increase in
leachate concentration also resulted in a higher ash content in micro-
algal cells. In addition, the carbohydrate content of about 9% was ob-
tained at concentrations of leachate of 5% and 7.5% (v/v), this value
increased to 15% when the concentration of leachate in the feed stream
was 10%.
The variation in lipid content in the biochemical composition of the
Algal Research 41 (2019) 101549
Average size of cells (×104) (mm)
1.01 ± 0.03c
7.02 ± 0.29a 1.10 ± 0.02c
4.41 ± 0.33b 1.53 ± 0.23d
microalgal biomass is dependent of different factors, including the
availability of nitrogen source [44]. The limitation of nitrogen sources
or even the submission of the cells to a period during the absence of
nitrogen (“starvation”) can lead to the accumulation of lipids in mi-
croalgae in several types of culture media [45,46]. However, this be-
havior may differ depending on the species, the range of nitrogen
source concentrations evaluated, the composition of the medium and
type of cultivation, with the possibility of reducing the lipid content
with increased availability of the nitrogen source [40]. In the present
work, although concentration of inorganic nitrogen sources was re-
duced to low values in the medium when leachate concentration in feed
stream was 5% or 7.5%, possibly there were other sources of nitrogen to
the microorganisms, such as ammoniacal nitrogen, which were not
measured. Besides, when effluents were used in the composition of the
medium for mixotrophic culture, cellular metabolism is also affected by
other factors, such as temperature, light intensity, etc. [13]. For ex-
ample, in the work of Ansari et al. [47], increased supplementation of
NaNO3 from 200 to 1500 mg L−1 in aquaculture wastewater in batch
process decreased the lipid content of the microalgae C. sorokiniana and
Ankistrodesmus falcatus, a behavior similar to that observed in the
present study. However, in the work of Zhao et al. [21], increase in
landfill leachate concentration in the culture medium of microalgae C.
pyrenoidosa resulted in higher lipid content in the cells for culture
media with up to 10% of leachate diluted in municipal effluent. Higher
concentration of leachate (> 10%) caused lower lipid production.
Fig. 4 also showed the metabolism of microalgae which was di-
rected to the accumulation of carbohydrates when the highest con-
centration of leachate in the medium was used. This higher accumu-
lation of carbohydrates is probably due to the stress caused by the
greater amounts of toxic components in the culture medium, even in the
presence of high availability of carbon sources.
Table 2 shows the effect of leachate concentration in the medium on
the productivity of lipids, carbohydrates, proteins and biomass of C.
minutissima 26a cultured in the concentric tubes airlift photobioreactor
under continuous process at steady state. The increase in leachate
concentration from 5% to 7.5% increased biomass productivity by
11.7%, reaching 232.0 ± 7.6 mg L−1 d−1 when 7.5% of leachate was
used. When the concentration of the leachate changed from 7.5% to
10% (v/v), the biomass productivity variation was not statistically
different at 95% confidence level. Values for biomass productivity ob-
tained in the present study were higher compared to other reports. For
example, the maximum value obtained in the cultivation of C. pyr-
enoidosa in medium with 10% of leachate from landfill was
131.7 mg L−1 d−1 in the study of Zhao et al. [21]. The values obtained
in our work were also higher than those obtained in autotrophic culture
in synthetic medium (maximum productivity of 188.6 mg L−1 d−1) as
reported by Tagliaferro et al. [25].
Considering the results shown in Table 2, the variation in the con-
centration of leachate in the culture medium allows to direct the cul-
tivation to produce several components of industrial interest in bio-
mass. For example, the productivity in lipids decreased with increasing
leachate concentration in the medium from 5 to 10% (v/v), with the
maximum value of 96.6 ± 7.8 mg L−1 d−1 obtained in lower con-
centration of leachate. On the other hand, the carbohydrate
5
G.V. Tagliaferro, et al.
Fig. 3. Optical micrographs of Chlorella minutissima 26a cells grown in different concentrations of leachate using airlift photobioreactor. Concentration of leachate in
the reactor feed: (a) 5%, (b) 7.5% and (c, d) 10% (v/v).
productivity showed an opposite behavior, with increasing values of the
leachate increased from 5 to 10% (v/v) in the medium. Protein pro-
ductivity slightly varied with the change in leachate concentration in
the culture medium, reaching the minimum value of
42.7 ± 3.5 mg L−1 d−1 at the concentration of 5% (v/v) of leachate in
feed stream and a maximum value of 47.1 ± 3.0 mg L−1 d−1 for the
concentration of 7.5% (v/v). Thus, the continuous process of C. minu-
tissima 26a cultivation using the concentric tubes airlift photobioreactor
and diluted leachate as culture medium indicated a great potential for
the production of biomass, lipids, carbohydrates and protein. This
system can enable the production of biofuels, such as bioethanol and
biodiesel, derived from carbohydrates and lipids respectively. Ad-
ditionally, this system could effectively contribute in the bioremedia-
tion of landfill leachate [38].
3.4. C. minutissima cultivation for the treatment of leachate in a continuous
airlift photobioreactor
The landfill leachate has a complex composition and includes sev-
eral compounds which are toxic to microorganisms [37]. In addition, it
is a cloudy dark brown liquid and therefore its dilution is necessary to
reduce toxicity and increase the transparency to allow the photo-
synthesis process performed during microalgal growth.
Table 3 shows the effect of the cultivation of the microalgae C.
minutissima 26a on the removal of organic compounds from the medium
evaluated by COD and TOC, besides the removal of NO3-N (inorganic
nitrate concentration). In all tests, a reduction in the value of these
parameters was observed, with COD removal percentage (92.8%)
higher in the medium containing 7.5% (v/v) leachate, whereas for TOC,
6
Algal Research 41 (2019) 101549
the highest removal was 90.5% under the same conditions. Regarding
to the NO3-N removal, it was almost complete in the medium con-
taining 5.0% or 7.5% (v/v) of leachate. However, higher concentration
of leachate (10%) caused less removal of NO3-N from leachate.
Removal values presented in Table 3 are comparable to those re-
ported in other studies in the literature where microalgae were grown
in various effluents. For example, Wang et al. [48] studied the culti-
vation of the microalga Chlorella sp. in four different effluents types
from the municipal effluent treatment plant of St. Paul, Minnesota,
USA. This study reported that, in all assays, there was removal of
components from the medium during cultivation. The highest removal
values were 83% for COD, 90.6% for P-PO4, 82.4% for N-NH3 and
62.5% for N-NO3.
Capacity of reduction in the concentration of some metals in the
landfill leachate by C. minutissima 26a during continuous cultivation
was also evaluated (Table 4). Concentration of Cr, Fe, K, Ca, Al and Ba
showed a reduction in all the tests. Particularly, Al was completely
removed in all assays, while Ca completely removed in the assays with
5% and 7.5% of leachate in the culture medium. The removal of the Fe
and Ba metals increased with the increase in leachate concentration in
the culture medium from 5 to 10%, showing removal 71.9% and 66.5%
respectively. However, Cr removal decreased from 63.3% to 44.5%
with increasing leachate concentration from 5% to 10%. Earlier, some
studies have shown the ability of microalgae to reduce the concentra-
tion of metals from various kind of wastewater streams [16,49,50].
According to Becker [51], removal of metals from wastewater by mi-
croalgae is an economical method for the reduction of heavy metals
concentration. Microalgae assisted removal of metals from wastewater
results in a high-quality effluent to be reused, with concomitant
G.V. Tagliaferro, et al. Algal Research 41 (2019) 101549

Fig. 4. Biomass composition of Chlorella minutissima 26a cultured in media with different concentrations of leachate in feed stream in the concentric tubes airlift
photobioreactor in continuous process at steady state: (a) 5% v/v leachate; (b) 7.5% v/v leachate; (c) 10% v/v of leachate. Data were shown as mean ± standard
deviation, n = 3.

Table 2 medium resulted in reduction of the lipid content and increase in the
Effect of landfill leachate concentration on the productivity of lipids, carbo- carbohydrate content, without influencing the protein value in biomass.
hydrates, proteins and biomass of Chlorella minutissima 26a during continuous Regarding to the use of the process as leachate treatment, the removal
culture in concentric tubes airlift photobioreactor at steady state (mean ± of COD, TOC and inorganic nitrate values were higher than 90% in
standard deviation, n = 3) with D = 0.009 h−1. some experiments using 5–7.5% of leachate in the medium. Removal of
Leachate Productivity (mg L−1 d−1) metals was also observed. Almost complete removal of Al and Ca was
concentration observed in all cases, whereas for Fe and Ba the maximum removal
(% v/v) Biomass Lipid Carbohydrate Protein values were 71.9% and 66.5%, respectively in medium containing 10%
5 207.9 ± 11.2 a
96.6 ± 7.8 c
19.3 ± 2.1e
42.7 ± 3.5g
of leachate. For Cr, the maximum removal was 63.3% in medium with
7.5 232.0 ± 7.6b 95.3 ± 5.2c 20.7 ± 1.6e 47.1 ± 3.0h 5% of leachate. The simultaneous production of microalgal biomass and
10 223.7 ± 3.1b 83.9 ± 3.2d 33.4 ± 2.1f 44.3 ± 2.5g,h bioremediation is a particular feature of this process supporting 3G
biorefineries. This study can contribute in design of an industrial plant
Results corresponds to average triplicates ± standard derivation. Same letter growing microalgae biomass to obtain lipids, proteins and carbohy-
in the column indicates no difference according Tukey's range test (95% of
drates to produce biofuels and other valuable compounds, con-
confidence level).
comitantly promoting bioremediation of landfill leachate, in turn re-
ducing its pollutant potential.
formation of biomass.

Declaration of Competing Interest

4. Conclusion
Cultivation of microalgae C. minutissima 26a in airlift photo-
bioreactor using landfill leachate-based medium can be a sustainable
process for biomass production and bioremediation simultaneously.
The variation in concentration of leachate in the medium modified the
microalgae composition, allowing to obtain biomass with varied com-
position for different purposes. Higher concentration of leachate in the
The authors wish to confirm that there are no known conflicts of
interest associated with this publication.
Acknowledgements
The authors would like to acknowledge Jaime Alves Capucho (EEL/
USP) for the technical design of airlift photobioreactor and The

7
G.V. Tagliaferro, et al. Algal Research 41 (2019) 101549

Table 3
COD, TOC and NO3-N analysis of the medium containing different concentrations of leachate before and after the continuous cultivation of Chlorella minutissima 26a
in concentric tubes airlift photobioreactor (values obtained from output of the reactor and steady state). NO3-N values: mean ± standard deviation, n = 3;
Coefficients of variation for results of TOC and COD were lower than 0.5%.
Leached concentration (%) COD (mg O2 L−1) TOC (mg C L−1) NO3-N (mg L−1)

Before After Removal (%) Before After Removal (%) Before After Removal
(%)

5.0 158.7 40.7 74.4 77.4 30.1 61.1 88.9 ± 2.5 n.d.a 100.0 ± 1.3
7.5 238.1 17.2 92.8 116.6 11.7 90.5 131.1 ± 3.2 0.2 ± 0.1 99.8 ± 3.2
10.0 317.4 63.7 79.9 154.8 25.6 83.5 174.9 ± 3.5 23.5 ± 1.6 86.6 ± 3.8

a
n.d. = not detected.

Table 4
Metals concentration in the medium and their removal during continuous cultivation of Chlorella minutissima 26a using different concentrations of leachate (values
obtained from steady state reactor in the input and output streams). Coefficients of variation for results were lower than 3%.
Leachate concentration (% v/v) Stream Metals (mg L−1)

Cr Fe K Ca Al Ba

5.0 Input 0.270 23.270 116.000 6.862 1.194 0.744

Output 0.099 14.481 115.453 n.d.a n.d.a 0.499
Removal (%) 63.3 37.8 0.50 100.0 100.0 32.9
7.5 Input 0.405 34.905 174.000 10.293 1.791 1.116
Output 0.198 17.751 121.182 n.d.a n.d.a 0.397
Removal (%) 51.1 49.1 30.4 100.0 100.0 64.4
10.0 Input 0.539 46.539 232.001 13.723 2.387 1.488
Output 0.299 13.092 197.985 0.470 n.d.a 0.499
Removal (%) 44.5 71.9 14.7 96.6 100.0 66.5

a
n.d. = not detected.

Department of Biological Oceanography, University of São Paulo (USP)
for kindly donating the microalgae Chlorella minutissima 26a of the
Seaweed culture collection. We also thank CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior), Brazil, for the financial
support.
Authors' agreement to authorship and submission
All persons designed as author agree to submit the manuscript for
peer review.
Authors' contribution
G. V. Tagliaferro: collection and assembly of data. H. J. Izário Filho:
analysis of TOC, COD and metals and corresponding results inter-
pretation. G. V. Tagliaferro, A. K. Chandel, J. C. Santos and S. S. da
Silva: analysis and interpretation of results. M. B. Silva and J. C. Santos:
conception and design. G. V. Tagliaferro and J. C. Santos: draft of the
article. A. K. Chandel and S. S. da Silva: critical revision of the article
for important intellectual content.
Statement of informed consent, human/animal rights
No conflicts, informed consent, human or animal rights applicable.
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