Renewable Energy 130 (2019) 439e445

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Continuous cultivation of Chlorella minutissima 26a in a tube-cylinder

internal-loop airlift photobioreactor to support 3G biorefineries
Geronimo Virginio Tagliaferro a, *, He
lcio Jose

Iza
rio Filho b, Anuj Kumar Chandel a,

rio da Silva a, Messias Borges Silva b, c, Júlio Ce
Silvio Silve
sar dos Santos a
a ~o Paulo, CEP 12602-810, Lorena, SP, Brazil
Department of Biotechnology, Engineering School of Lorena, University of Sa
b ~o Paulo, CEP 12602-810, Lorena, SP, Brazil
Department of Chemical Engineering, Engineering School of Lorena, University of Sa
c
, Sa
Department of Production, Engineering School of Guaratingueta ~o Paulo State University, CEP 12516-410 - Guaratingueta
, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Microalgae Chlorella minutissima 26a was cultivated in a tube-cylinder internal-loop airlift photo-
Received 4 August 2017 bioreactor under continuous cultivation conditions. The goal was to investigate the influence of different
Received in revised form nitrate levels on the growth and composition of microalgae. Three nitrate concentrations (75, 150 and
11 April 2018
225 mg L
1) were assessed under a fixed flow rate and the outlet flow was analyzed for concentration of
Accepted 12 June 2018
Available online 14 June 2018
biomass, lipid, carbohydrate and protein. Nitrate concentration at higher level (225 mg L
1) in the me-
dium promoted biomass growth (188.6 mg L
1 d
1) and lipid production (92.8 mg L
1 d
1), and
decreased carbohydrate amount (29.1 mg L
1 d
1) without any change in protein content
Keywords:
Chlorella minutissima
(37.7 mg L
1 d
1). Use of tube-cylinder internal-loop airlift photobioreactor in continuous mode could be
Continuous microalgae cultivation a promising approach in algal biorefineries so called 3G biorefineries, resulting in high biomass pro-
Tube-cylinder internal-loop airlift ductivity in a simple cultivation system.
photobioreactor © 2018 Published by Elsevier Ltd.
3G biorefineries
Nitrogen repletion effect

1. Introduction [4e6]. In addition, microalgae can also be harnessed for the pro-
duction of large number of bioactive compounds for different ap-
Emission of carbon dioxide and other gases into the atmosphere plications in food supplements, cosmetics, pharmaceuticals, among
by excessive burning of fossil fuels has considerably impacted on others [7]. Thus, a flexible multiproduct industry can be envisioned
the greenhouse effect, thus promoting global warming and unde- through integrated installations of 3G biorefinery by harnessing the
sirable climate changes [1]. Strategic actions to reduce the green- potential of microalgae [8e10].
house gas emissions and their harmful effect on the environment However, there are several obstacles that must be overcome in
are urgent and mandatory. Gas emissions caused by burning of order to take maximum advantage from microalgae in large-scale
renewable fuels can be sequestered through photosynthesis during processes such as production cost reduction, biomass yield and
the biomass growth [2,3]. In this context, microalgae have been productivity maximization, increase in the productivity of lipids,
considered as one of the most promising alternative sources for carbohydrates and other interesting compounds [11]. In order to
biofuel production because of their fast growth rate and high increase biomass production and to modify its composition,
biomass productivity thriving on cheap and renewable carbon appropriate strategies for example - evaluation of the effect of ni-
sources. Microalgae biomass can be employed as a raw material for trogen source concentration in the medium [12,13], promotion of
fuels production as it comprised of lipids and carbohydrates which intense interaction of microalgae with the cultivation medium,
are considered as promising feedstock for biodiesel, bioethanol and supply of optimized light and carbon source during cultivation, and
biochemicals production. Cultivation of microalgae require small the choice of an alternative photobioreactor and operation mode
physical spaces as compared to the terrestrial crops, and signifi- are necessary to adopt [14,15].
cantly contribute to the greenhouse gases reduction by CO2 capture For the appropriate yields from bench-scale microalgal cultiva-
tion, more studies are requires that allow to measure, evaluate and
control environmental and nutritional factors affecting growth and
* Corresponding author. composition of biomass production [16]. These benchmark studies
E-mail address: [email protected] (G.V. Tagliaferro).

https://doi.org/10.1016/j.renene.2018.06.041
0960-1481/© 2018 Published by Elsevier Ltd.
440 G.V. Tagliaferro et al. / Renewable Energy 130 (2019) 439e445

will pave the outcome for acquisition of necessary information for light penetration. The cylindrical tube was located concentrically
large-scale microalgae propagation. In such a case, photo- towards the outer column with 30 mm space between them, aim-
bioreactors enable cellular photosynthetic metabolism and offer ing to promote an upstream flow towards the central tube and
several unique advantages, such as low possibility of contamina- downstream flow towards the outer tube. Compressed air was
tion, better control of gas-liquid mass transfer rate, higher biomass pumped through a sterile filter and supplied to the reactor via
density, greater exposure to light, high productivity per area and porous stones placed centrally at the base of the draft tube at the
low cost of biomass harvesting. However, the main disadvantage of flow rate of 0.24 vvm. For all experiments, temperature was kept at
photobioreactors is the low penetration of light due to the 30 ± 2  C and continuous light supply was provided via fluorescent
increased cell density in the medium during the cultivation time lamps with photon flux density of 125e130 mmol m
2 s
1. A peri-
[15]. The later needs careful evaluation of reactor designing, inter- staltic pump was used to feed the photobioreactor during the
nal geometry of reactor, appropriate air flow rate and other oper- continuous mode operation.
ating conditions, enabling ideal amount of oxygen transfer and
removal, mixing, recirculation time and light exposure frequency 2.3. Batch cultivation in airlift photobioreactor
for adequate microalgae growth [15,17e19]. In continuous culti-
vation of microalgae, high biomass productivity can be achieved by Batch experiments were carried out to determine the maximum
reducing non-productive time, employing a uniform and optimized specific growth rate (mmax), with the estimated feed flow rate dur-
culture medium which facilitates the design of downstream steps ing the continuous cultivation. Culture medium was consisted of
[20]. synthetic seawater f/2 with 150 mg L
1 NaNO3, and the seed culture
Despite their advantages, airlift photobioreactors have been (as described in section 2.1) was added at a volumetric fraction of
scarcely reported in literature for microalgae cultivation under 10%. Samples were taken periodically taken for dry cell weight
continuous mode [16,21,22], with no previous report on Chlorella (DCW) measurements. Specific growth rate was estimated by the
minutissima cultivation in a tube-cylinder internal-loop airlift slope of regression in the exponential phase (linear region) ob-
photobioreactor. Chlorella sp. have been reported as a promising tained from the curve of natural logarithm of dry cell weight as a
candidate, as they have a fast and easy growth [23,24], with lipid function of time. The hydraulic residence time (HRT) was calculated
content ranging from 10.0 to 48.0% (wt) [25,26], carbohydrate as a function of the dilution ratio (“D”) the by following equation:
content from 12.0 to 17.0% (wt) and protein content from 40.0 to HRT ¼ 1/D where D ¼ 0.8  mmax.
60.0% (wt) [27,28] depending on cultivation conditions.
This study reports the effect of different nitrogen concentration 2.4. Continuous cultivation in the airlift photobioreactor
on growth and composition of C. minutissima 26a in an autotrophic
culture by using a tube-cylinder internal-loop airlift photo- In all experiments, seed culture was prepared as described in
bioreactor. The cultivation was operated in a continuous mode, and section 2.1, was added into the medium at a volumetric fraction of
biomass production and chemical composition was assessed in 10%. For continuous cultivation, firstly, the airlift photobioreactor
terms of lipids, carbohydrates, protein and ash content. was operated in batch mode until the microalgae growth reached
to the exponential phase. Thereafter, the airlift photobioreactor was
2. Methods fed with the culture medium at a flow rate of 0.4 mL min
1, cor-
responding to a hydraulic residence time (HRT) of 155 h. The har-
2.1. Microalgae strain, storage and seed culture vesting was performed daily (approximately after 24 h), and the
culture solution was centrifuged at 1800  g and dried in an oven at
The microalgae C. minutissima 26a was provided by the Seaweed 60  C for 24 h.
Culture Collection of the Oceanographic Institute at the University
of S~
ao Paulo (S~ao Paulo, SP, Brazil). Microalgae were stored and kept
in artificial seawater f/2 medium [29], composed of: 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 mg L
1 ZnSO4$7H2O, 180 mg L
1 MnCl2, 6.3 mg L
1
Na2MoO4$2H2O, 10 mg L
1 CoCl2$6H2O, 9.8 mg L
1 CuSO2$5H2O,
100 mg L
1 Thiamine (B1), 0.5 mg L
1 Cyanocobalamin (B12) and
0.5 mg L
1 Biotin (B7), in 250 mL Erlenmeyer flasks kept at 20  C
under illuminating conditions (photon flux of
80e90 mmol m
2 s
1), and sub-cultured every six weeks. Seed
cultures were prepared in the same f/2 medium (900 mL) with the
addition of a stock culture (100 mL). Thus, autotrophic cultivation
was carried out during 10 days by using a batch tube photo-
bioreactor which was consisted of a transparent plastic bottle cyl-
inder of 12.0 mm of diameter and 28.0 mm of height, and aerated at
0.24 vvm. Temperature was kept at 30 ± 2  C with continuous light
supply at photon flux of 130 ± 2 mmol m
2 s
1.

2.2. Tube-cylinder internal-loop airlift photobioreactor

The tube-cylinder airlift photobioreactor used in this study was

having following dimensions: 550 mm in height and 120 mm in
diameter with the concentric tube (one tube is placed inside
another) of 340 mm by 60 mm, with a working volume of 3.8 L. The
bioreactor lay out is shown in Fig. 1. The tubes were made of Fig. 1. Schematic diagram of continuous cultivation system used for the cultivation of
transparent acrylic polymer for the visual observation of effective microalga C. minutissima.
 G.V. Tagliaferro et al. / Renewable Energy 130 (2019) 439e445 441

2.5. Analytical methods As Fig. 2 showed, microalgae biomass increased concomitantly

by increasing nitrogen source concentration in the medium. DCW
Cell growth was monitored by measuring the dry cell weight values (Fig. 2a) in the batch phase was 0.25 g L
1, 0.40 g L
1 and
(DCW). Aliquots of 10 mL were taken from the medium, centrifuged 0.6 g L
1 for nitrate initial concentrations of 75 mg L
1, 150 mg L
1
at 1800  g for 10 min, dried in an oven at 60  C for 24 h, and and 225 mg L
1, respectively, during 155 h of culture time. Increase
weighed on an analytical balance. Humidity was determined by in biomass concentration was also observed using higher nitrogen
using a semi-analytical infrared balance (model MB25, OHAUS, Pine source concentrations after starting the continuous feed into the
Brook, USA) with a heating program to get the final temperature of reactor at 155 h. Fig. 2a shows that, during the first hydraulic resi-
135  C. Nitrate concentrations in the medium were determined dence time of cultivation, HRT1 (155e310 h), the growth profile was
according to the standard method of APHA 4500-NO-3 [30]. unstable, probably due to the transition between batch and
Total intracellular lipids were extracted by following the pro- continuous operation modes. This was expected, as HRT value
cedure proposed by Bligh and Dyer [31], and oil yield of microalgae represents the time required to exchange the entire reactor volume
(%) was calculated taking into consideration the dry mass of the for the feed flow.
sample. Carbohydrates in the algal biomass was determined by the Fig. 2 shows that, after first HRT, continuous cultivation reached
method of Moxley and Zang [32], using phenol-sulfuric method for into steady state with the DCW values (average ± standard devia-
total sugar determination [33] and high performance liquid chro- tion) of 0.70 ± 0.04 g L
1, 0.96 ± 0.09 g L
1 and 1.31 ± 0.08 g L
1 for
matography (HPLC) for measurement of different monomeric experiments where nitrate solution was fed with the feeding rate of
sugars present in biomass hydrolysate. HPLC Agilent 1200 series 75 mg L
1, 150 mg L
1 and 225 mg L
1, respectively, at the begin-
(Agilent Technologies, Inc., USA) was equipped with a HPX-87H ning of HRT2 phase.
(300  7.8 mm) column (Bio-Rad, USA) and the analysis condi- Fig. 2b shows the remaining nitrate concentration in the reactor
tions were as following: 45  C column temperature, 0.01 N H2SO4 as during the cultivation process. Depletion in nitrate concentration in
the mobile phase at 0.6 mL min
1 flow rate, 20 mL injection volume the reactor was evident during the batch phase, which decreased
and refractive index detector RID-6A. Crude protein content was up to about 10 mg L
1 from the beginning of feeding flow. During
estimated according to Becker [34] “protein concentration ¼ ni- the continuous phase, only small differences could be observed for
trogen content  6.25”, with the total nitrogen concentration experiments with feeding of different nitrate concentrations. These
measured by Kjeldahl method [35]. Ash content was determined differences were more evident at the beginning of region HRT3,
according to the analytical method of Wychen and Laurens [36]. All with nitrate concentrations of about 14 mg L
1, 25 mg L
1 and
analyses were performed in triplicate. 37 mg L
1 inside the reactor for the tests with high nitrate con-
centration at feeding flow of 75 mg L
1, 150 mg L
1 and 225 mg L
1,
3. Results and discussion respectively. A complete consumption of the nitrogen source was
observed by Milla
n-Oropeza et al. [38] in the batch cultivation of
3.1. Batch growth profile of C. minutissima 26a in a tube-cylinder the microalga Nannochloropsis oculata after a period of 6, 8 and 8
internal-loop airlift photobioreactor days in cultures with initial nitrate concentrations of 150, 200 and
250 mg L
1, respectively. In the present study, nitrate was added in
Considering the microalgae growth profile in batch run, specific reactor continuously before the complete depletion of nitrogen.
growth rate (mmax) was found as 0.008 h
1 (0.19 day
1), which is However, initial nitrogen source concentration had an effect on
close to the value reported by Cabello et al. [37] for the microalgae total produced biomass, and on microalgae composition.
Scenedesmus obtusiusculus when cultivated in a 20.0 L tubular airlift
photobioreactor (mmax, 0.005 h
1). However, mmax values can vary 3.3. Effect of nitrate concentration on biomass composition in airlift
widely, as shown in the work of Olivieri et al. [19] who reported photobioreactor continuous cultivation
different values from 0.1 to 0.8 day
1 for different species of
Chlorella. This variation in mmax is due to the differences in culti- Besides biomass production, nitrogen source concentration has
vation systems, growth conditions and culture medium used to been regarded as a decisive factor in microalgae biomass compo-
cultivate Chlorella spp. sition [13,38]. Fig. 3 shows biomass composition in terms of its
Although different nitrogen source concentrations were used in main components as a function of NaNO3 concentration in airlift
continuous cultivation could result in variation of mmax values, the photobioreactor continuous cultivation by considering three hy-
value calculated during the batch growth experiment was selected draulic residence times from starting the feeding flow.
to have an understanding on the dilution rate to be used in During first hydraulic residence time (155e310 h), initial nitrate
continuous experiments. Thus, a dilution rate (D) was 20% lower concentration in the feeding medium (in the feeding flowrate),
than mmax value (D ¼ 0.006 h
1) was considered with the purpose of showed a slight difference in the biomass chemical composition as
avoiding washout in reactor. This dilution corresponded to the HRT compared with the biomass composition obtained from the sta-
of 155 h. A reactor of 3.8 L working volume was used and the feed tionary state (hydraulics residences times HRT2 and HRT3,
was supplemented with the flow rate of 0.40 mL min
1. 310e620 h). However, for both of them, transition time or sta-
tionary state, there was a similar behavior regarding compositional
3.2. Effect of nitrate concentration on biomass growth in airlift changes at different nitrate concentrations.
photobioreactor continuous cultivation Protein content was not influenced by changes in initial nitrate
concentration in the medium within the evaluated range. Protein
Continuous cultivation was started as a batch mode and after content remained of about 20% in all cases. Although there are
155 h of cultivation, the feed was initiated in a continuous mode. some reports in literature which indicate change in protein content
The continuous operation was operated for 720 h. The results ob- due to variation in initial nitrogen source concentration [23,39].
tained for biomass growth and remaining nitrate concentration in However, protein content depends on the evaluated range of nitrate
the medium are shown in Fig. 2. Two main cultivation phases i.e. concentration. Kim et al. [13], found higher protein content when
initial batch and continuous operation were presented. Continuous nitrate concentration was increased from 75 mg L
1 to 375 mg L
1.
operation was presented by considering hydraulics residences Pancha et al. [40] did not observe any decrease in crude protein
times: HRT1, HRT2 and HRT3. content in microalgae Scenedesmus sp. CCNM 1077, when nitrate
442 G.V. Tagliaferro et al. / Renewable Energy 130 (2019) 439e445

nitrogen source depletion in the medium or by its excess avail-

ability. Sa
nchez-García et al. [44] observed that C. minutissima
when grown in the medium in which initial nitrate concentration
increased from 57 to 113 mg L
1, lipid content in the cells increased
from 23 to 37%, keeping this value when nitrate concentration was
increased to 225 mg L
1.
Table 1 shows the composition of carbohydrate fraction of
biomass cultured in different nitrate concentrations on steady state,
during 3rd hydraulic residence time (HRT3). Xylose, galactose and
mannose sugars were determined together because they were co-
eluted in HPLC column, the same behavior was observed by
Cheng et al. [45]. Biomass cultivated with 75 mg L
1 of nitrate
concentration in the feeding medium comprised of xylose, galac-
tose, and mannose in higher quantity (10.85 ± 0.55%), representing
50.2% of the total sugars, while glucose presented the second value
(5.94 ± 0.3%), representing 27.5% of the total sugars. However, for
the biomass cultivated using 225 mg L
1 of nitrate concentration,
glucose was the main sugar with higher mass fraction value
(8.18 ± 0.28%), representing 53.8% of the total sugars, while total
monomeric sugars (xylose þ galactose þ mannose) represented
only 33.6% of total value. In all cases, arabinose was found in lower
quantity (~4%), with a decreased mass fraction value when initial
nitrate concentration was increased. Increasing in the initial nitrate
concentration in the culture medium from 75 to 225 mg L
1 pro-
moted an increase in glucose mass fraction and decreased the total
sugars (xylose þ galactose þ mannose þ arabinose) mass fraction
in biomass.
Carbohydrates represent a group of reducing sugars and poly-
saccharides such as starch and cellulose. Among them, starch is the
Fig. 2. Profile of dry biomass (a), and nitrate concentration (b) left-over nitrate con-
centration in the airlift photobioreactor during the process carried out by using
most abundant polysaccharide in Chlorella sps. Ortiz-Tena et al. [46]
different nitrate concentrations in the medium. DCW: dry cell weight; HRT: hydraulic grown Chlorella vulgaris in BG-11 medium in autotrophic batch
residence time. cultivation and observed that 46.5% of the biomass carbohydrates
were composed of starch followed by 26.4% of monomeric sugars
and only 6% of structural polysaccharides (cell wall). This study
concentration in the growth medium was decreased from 247 to showed the rhamnose (29.1%), galactose (21.2%), glucose (16.2%),
61.75 mg L
1. In the present work, changes in nitrate concentration xylose and arabinose (11.4%), besides glucosamine (7.9%), mannose
showed main change in lipid and carbohydrate content (Fig. 3), but (7.4%) and glucuronic acid (6.8%). In the present study, sugars
only when nitrate concentration increased from 75 to 150 mg L
1. present in cell wall were not measured. However, glucose was
There were no considerable changes found when nitrate concen- present in small quantities when 75 mg L
1 of nitrate was used in
tration increased from 150 to 225 mg L
1. In the steady state, lipid the feeding medium. C. minutissima showed very low amount of
content increased from about 45% to about 49% when initial nitrate carbohydrate in form of starch when grown in the feeding medium
concentration was changed from 75 to 150 mg L
1. On the other composed of 75 mg L
1 of nitrate. When nitrate concentration was
hand, carbohydrate content decreased from about 21% to 16% for increased to 225 mg L
1, the increase in glucose content can indi-
nitrate concentration ranging from 75 to 150 mg L
1, and showed a cate changes in cell wall or starch storage. Increase in nitrate con-
slight decrease when the nitrate concentration changed to centration (>225 mg L
1) may lead the high starch accumulation in
225 mg L
1. Similar behavior was reported by Feng et al. [41] using Chlorella microalgae [47].
Isochrysis zhangjiangensis cultured in batch experiments with ni- The effect of nitrogen source concentration in the continuous
trate addition at regular intervals. In that work, experiments were process must be considered from a global viewpoint and thus
carried out by varying nitrate concentration at a large range productivity is an important parameter. Table 2 shows the effect of
(0.075e9.0 g L
1). The authors observed that increased nitrogen different nitrogen concentration in the medium on biomass, lipid,
concentration in the medium resulted in higher lipid content in the protein, and carbohydrate productivity during steady state. As
cells and lower carbohydrate concentrations with no major changes shown in Fig. 3, increase in nitrate concentration in the medium
in protein content. resulted in higher productivity values. When nitrate concentration
Lipid accumulation in microalgae was promoted by increased was increased from 75 to 225 mg L
1, biomass, lipid, protein and
nitrate concentration in the culture medium (Fig. 3). This is the carbohydrate productivities were 88%, 106%, 81% and 37% higher,
opposite of the observed behavior for some Chlorella species in respectively. Although carbohydrate content in the biomass
some studies [42,43]. However, in the present study, even at the decreased with increasing nitrate concentration in the medium
increased nitrogen concentration in the feed medium, remaining (Fig. 3), but productivity increased, reaching a maximum value of
nitrate was found low (Fig. 2b) causing nitrogen stress conditions 29.1 ± 1.7 mg L
1 d
1 for a concentration of 225 mg L
1 (Table 2),
for cells. The role of nitrogen in lipid accumulation in microalgae is due to high biomass growth.
a complex topic and must be carefully evaluated, resulting in Values in Table 2 are indicative of the potential of continuous
different lipid accumulation by different species. Type of nitrogen internal-loop airlift photobioreactor. Maximum values were
sources and feeding pattern also influences the lipid accumulation reached for biomass (188.6 ± 11.2 mg L
1 d
1) and lipid productiv-
in microalgae. As discussed by Feng et al. [41], metabolic changes ity (92.8 ± 5.5 mg L
1 d
1). These values are higher than those
that favor lipid accumulation can occur both by stress caused by observed in other continuous autotrophic cultivation processes
 G.V. Tagliaferro et al. / Renewable Energy 130 (2019) 439e445 443

Fig. 3. Biochemical composition of Chlorella minutissima 26a cultured in media with different nitrate concentrations in continuous internal-loop airlift photobioreactor. Data are
correspondent to average composition at different hydraulic residence time (HRT).

Table 1 Table 2
Carbohydrates composition after acid hydrolysis of microalgae biomass cultured at Influence of nitrate concentration on the biomass productivity, lipids, protein and
steady state (HRT3) under different nitrate concentration in the medium. carbohydrate amount in biomass during the continuous cultivation in airlift pho-
tobioreactor at steady state (average ± standard deviation) with D ¼ 0.006 h
1.
Sugars Mass fraction (%)
Nitrate concentration Productivity (mg L
1 d
1)
Nitrate concentration (mg L
1)
(mg L
1)
Biomass Lipid Protein Carbohydrate
75 150 225
75 100.2 ± 6.1 45.1 ± 2.7 20.8 ± 1.3 21.3 ± 1.3
Total carbohydrate 21.6 ± 2.10 16.1 ± 1.98 15.2 ± 1.56
150 138.8 ± 12.3 67.9 ± 6.0 27.8 ± 2.5 22.6 ± 2.0
Glucose 5.94 ± 0.31 6.07 ± 0.52 8.18 ± 0.28
225 188.6 ± 11.2 92.8 ± 5.5 37.7 ± 2.2 29.1 ± 1.7
Xyl þ Gal þ Man 10.85 ± 0.55 7.12 ± 0.43 5.11 ± 0.48
Arabinose 3.60 ± 0.62 1.51 ± 0.32 1.12 ± 0.41
Others 1.21 ± 0.42 1.40 ± 0.61 0.79 ± 0.39
using C. minutissima as reported by Tang et al. [48]. In this work,
HRT3 e 3rd hydraulic residence time after starting continuous process (from 465 h
to 620 h of cultivation).
when microalgae were cultured in a continuous stirred tank
444 G.V. Tagliaferro et al. / Renewable Energy 130 (2019) 439e445

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