Bioresource Technology 292 (2019) 121955

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

Microalgal biorefineries: Integrated use of liquid and gaseous effluents from T

bioethanol industry for efficient biomass production
Eduardo Bittencourt Sydneya, Carlos José Dalmas Netob, Júlio Cesar de Carvalhob,
Luciana Porto de Souza Vandenbergheb, Alessandra Cristine Novak Sydneya,
Luiz Alberto Junior Lettib, Susan Grace Karpb, Vanete Thomaz Soccolb,
⁎
Adenise Lorenci Woiciechowskib, Adriane Bianchi Pedroni Medeirosb, Carlos Ricardo Soccolb,
a
Federal University of Technology of Paraná, Department of Bioprocess Engineering and Biotechnology, 84016-210 Ponta Grossa, Paraná, Brazil
b
Federal University of Paraná, Department of Bioprocess Engineering and Biotechnology, Centro Politécnico, 81531-990 Curitiba, Paraná, Brazil

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

Keywords: A new method for CO2 recovery was proposed for cultivation of different microalgae. First, a chemical fixation,
Carbon dioxide fixation where CO2 was injected in alkalinized vinasse to form (bi)carbonate salts, was performed. In addition, biological
Vinasse fixation with CO2-enriched air injection was also accomplished for evaluation of the most promising results. Two
Microalgae bioreactor systems, a stirred-tank reactor and a bubble column reactor, were employed. A higher carbon transfer
Bioproducts
rate (43.35 g.L−1.h−1) was achieved in the bubble column reactor using NaOH-alkalinized vinasse, along with
Carbon transfer rate
reductions of the chemical oxygen demand (COD), biological oxygen demand (BOD) and turbidity (TD). This
allowed the cultivation of microalgae and cyanobacteria at vinasse concentrations between 70 and 100%,
reaching a biomass production of 2.25 g.L−1 in 15 days of culture. The viability of chemical CO2 fixation to-
gether with the use of 100% treated vinasse from a bioethanol production unit for microalgae cultivation has
been demonstrated in a successfully integrated biorefinery approach.

1. Introduction
Ethanol production from sugarcane is one of the largest biopro-
cesses in the world in terms of processed volume. The process generates
large amounts of solid, liquid, and gaseous wastes that are increasingly
seen as potential substrates for different processes. Among these re-
sidues, vinasse and carbon dioxide can potentially be used to grow
microalgae for biomass and biochemical production.
An average Brazilian ethanol industrial unit (1500 m3.day−1
ethanol) produces 1132 tons of CO2 daily (stoichiometrically, 1 m3 of
ethanol produces approx. 754.7 kg of CO2 through fermentation). With
98% purity, this CO2 is not reused in the industry and is just released to
the atmosphere. This is the same carbon dioxide fixed during photo-
synthesis of sugarcane and, thus, does not contribute to increasing its
concentration in the atmosphere, consequently, not increasing global
warming. However, reusing it is a strategy to add value to the pro-
duction chain (new products, carbon emission market) and make it
more environmentally friendly.
Different CO2 capture strategies have been investigated and can
generally be classified into two categories: (1) chemical reaction-based
and (2) biological. Microalgae and cyanobacteria are interesting alter-
natives for promoting the development of CO2 mitigation processes
because they naturally consume CO2 through photosynthesis. The
growth of microalgal and cyanobacterial biomass in liquid media is
usually limited by the low solubility of CO2, which depends on factors
such as temperature, pressure, and partial pressure of other gases.
Microalgae and cyanobacteria biomass productivity and, consequently,
the CO2 fixation rate depend directly on its concentration and are af-
fected by chemical and biochemical factors that are linked to photo-
synthesis.
Vinasse is a liquid waste generated in ethanol industries, where it is
the bottom product of distillation columns. This waste consists of
ethanol-free fermented broth and contains organic solids in suspension,
as well as minerals, residual sugars, and some volatile compounds.
Vinasse is generated in an average proportion of 15 L for each liter of
alcohol produced (Sydney et al., 2014). In 2018, approximately 1.6
billion m3 of vinasse were generated worldwide for a corresponding
ethanol production of approx. 108.6 million m3 (Renewable Fuels
Association (RFA), 2019). Sugarcane vinasse is characterized by its high
chemical and biochemical oxygen demands (COD and BOD, 30,000 and

⁎
Corresponding author.
E-mail address: soccol@ufpr.br (C.R. Soccol).

https://doi.org/10.1016/j.biortech.2019.121955
Received 6 June 2019; Received in revised form 1 August 2019; Accepted 2 August 2019
Available online 05 August 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
E.B. Sydney, et al.
15,000 mg.L−1 O2, respectively); high potassium content
(3600 mg.L−1); low pH (approx. 4.0); and considerable amounts of
nitrogen, phosphorus, and micronutrients (Fuess et al., 2018). Because
its simple disposal in nature can cause underground water contamina-
tion, lixiviation, and soil desertification (Sydney et al., 2010), the de-
velopment of technologies to promote a more rational destination for
vinasse is of great importance. It has been shown elsewhere that vinasse
is a feasible culture medium for microalgae (Barrocal et al., 2010;
Montalvo et al., 2019; Santana et al., 2017; Sydney et al., 2016), but its
high turbidity and acidity are the main growth-limiting factors. Tur-
bidity can be decreased by dilution but that can reduce the availability
of nutrients and end up limiting biomass production.
In this work, the biological carbon fixation capacity of different
microalgae and cyanobacteria species and a method to recycle vinasse
and CO2 from the ethanol industry are described in a biorefinery ap-
proach. CO2 is chemically fixed in the vinasse, resulting in bicarbonate
salt production that leads to a significant reduction in turbidity and
COD with improvement of microalgal biomass production.
2. Material and methods
2.1. Vinasse
Vinasse was kindly supplied by an ethanol and sugar refinery lo-
cated in Cravinhos/SP, Brazil. Experiments were performed using the
same lot of vinasse, which was kept frozen at −20 °C until use.
2.2. Microalgae and cyanobacteria cultivation
Six species of microalgae (Botryococcus braunii SAG 801-7,
Synechococcus nidulans LEB 25, Chlorella kessleri LEB15, Chlorella vul-
garis LEB 104, Neochloris oleoabundans UTEX 1185, and Scenedesmus
obliquus LEB 22) and three cyanobacteria (Arthrospira platensis SAG
257.80, Arthrospira laxissima SAG 256.80, and Arthrospira maxima SAG
84.79) were grown in pure or diluted vinasse-based media as follows.
Biomass production was carried out with sterilized (121 °C, 15 min)
raw and alkalinized vinasse (with the addition of different alkalis – see
Section 2.4), starting from a concentration of approx. 0.25 g.L−1. The
cultures were incubated in an orbital shaker at 28 °C and 100 rpm with
a 12 h/12 h photoperiod; illumination was provided at 50 µmole pho-
tons.m−2.s−1 with cool white 32 W fluorescent lamps. Dilutions of vi-
nasse were made with distilled water. Strain adaptations to increasing
vinasse content were made starting from a culture in synthetic medium
that was inoculated in 10% (v/v) vinasse and cultivated for 15 days.
This culture was then used as inoculum for the medium containing 20%
(v/v) vinasse and so on. With this strategy, a maximum growth in
natural vinasse at a 30% (v/v) concentration was achieved. Strains
were maintained for several months at this condition until the experi-
ments described in the present work were conducted. This adapted
culture in 30% natural vinasse was used as inoculum for the treated
vinasse experiments.
Biomass quantification was performed by gravimetric analysis.
Samples were withdrawn and centrifuged in a Sorvall Legend Mach 1.6
R centrifuge (Sorvall, Germany) at 3000 g for 15 min. The cells were
washed once with bicarbonate buffer (0.1 M) and dried at 80 °C until
reaching a constant weight.
Lipids were determined from the dried biomass by extraction with
methanol:chloroform 1:1 (v/v) followed by a liquid–liquid extraction
with hexane (Sydney et al., 2010). The apolar extracts were pooled, and
the fatty acid methyl esters (FAME) were prepared by the method
proposed by Hartman and Lago (1973). The fatty acid composition of
the samples was determined using a Shimadzu GC-2010 gas chroma-
tograph (GC) equipped with a hydrogen flame ionization detector. The
GC conditions were as follows: column RTX-WAX (fused silica,
0.25 mm × 30 m), injector temperature of 230 °C, a split ratio of 1:100,
an oven temperature program of 130–240 °C (4 °C.min−1), a detector
2
Bioresource Technology 292 (2019) 121955
temperature of 250 °C, with H2 as a carrier gas and velocity in the
column controlled at 35 cm.s−1.
Raw and alkalinized vinasse supplementations were done by the
additions of nitrogen (NaNO3) and phosphorus (K2HPO4) to reach C:N:P
100:15:1, Redfield ratio (Loladze and Elser, 2011). The carbon con-
centration in vinasse, as bicarbonate/carbonate, was quantified by ti-
tration with phenolphthalein (carbonates) and methyl orange (bi-
carbonates).
2.3. Biological CO2 fixation
Microalgae and cyanobacteria were grown in a raw vinasse-based
medium with the automatic addition of 99.9% CO2 at 100 mL.min−1
for 30 min during the light phase (at night CO2 injection was inter-
rupted). Carbon dioxide fixation in microalgal and cyanobacterial cul-
tures was calculated considering that 1.83 kg of CO2 was captured by
each kg of biomass produced (Chisti, 2007).
2.4. Effect of the addition of different alkalis on vinasse composition
Five alkaline substances were evaluated to determine their effect on
vinasse composition. The experiments were performed by adding one of
the following alkalis to vinasse with a final concentration of 1 M: so-
dium carbonate, sodium hydroxide, calcium hydroxide, calcium car-
bonate, and potassium carbonate. After homogenization, the alkali-
nized vinasse was allowed to rest for 24 h and was then centrifuged. A
raw vinasse (without alkaline substances but centrifuged to remove
solids) was employed for comparison.
The total precipitated mass was quantified by gravimetry (dried to a
constant weight at 80 °C) and analyzed for ash and organic matter ac-
cording to the AOAC official method 942.05 (Thiex et al., n.d.) and the
AOAC method 967.05 (AOAC INTERNATIONAL, 2000), respectively.
Ion chromatography was also performed to evaluate the ionic
composition of raw and alkalinized vinasse. The determination of ca-
tions (sodium, calcium, potassium, and magnesium) was done using a
761 Compact IC 817 Bioscan chromatograph. The column used was a
METROSEP C3 250/4.0 (Metrohm), with 250 mm × 4.0 mm of inner
diameter. The analytical conditions were defined as follows: 3.5 mM
HNO3, 1.0 mL.min−1, 40 °C, 20 µL injection volume, and pressure
controlled at 11.2 MPa. A standard chromatogram was prepared with
the following salts: CaCl2·2H2O, MgCl2·6H2O, KCl, Na2SO4,
ZnSO4·7H2O, NH4Cl, and FeSO4·7H2O. All used reagents were of ana-
lytical grade (Sigma–Aldrich, EUA).
Nitrate quantification was performed using the nitration of salicylic
acid under highly acidic conditions (Cataldo et al., 1975), and phos-
phorus was assessed using the phosphomolybdate method (Bhargava
and Raghupathi, 1993).
2.5. Chemical CO2 fixation in a stirred-tank reactor (STR)
Two experiments were conducted to determine the carbon-fixing
potential of alkalinized vinasse with sodium hydroxide (NaOH) at
concentrations of 1 and 2 M. A conventional stirred-tank reactor (STR)
with a total capacity of 5 L (working volume of 4 L, height/diameter
ratio of 0.61 m/m) was employed. Agitation was maintained at
300 rpm, and pure CO2 (99.9%) was injected at 3 L.min−1 (0.75 vo-
lumes of air per volume of medium per minute, or vvm). The tem-
perature was maintained at 40 °C, and the pH was monitored. Samples
were collected hourly and dried to quantify the production of bi-
carbonate salt. The end of the reaction was determined when the pH
approached neutrality or when the pH stability was observed after
5 min of continuous CO2 injection. The agitation was then turned off,
and the solution was left 12 h to rest. At the end of the experiment, the
treated vinasse was titrated to determine the amount of fixed CO2.
E.B. Sydney, et al.
2.6. Chemical CO2 fixation in a bubble column reactor
Experiments for evaluation of vinasse carbonation were also per-
formed in bubble column reactors with a height/diameter ratio of 4.25,
which favors a longer contact time between the CO2 and the alkalinized
vinasse. The same conditions used in the STR experiments were applied.
Different injection rates were studied: 0.75, 2, and 4 vvm of CO2
(99.9%).
2.7. Analysis of the precipitated salt
The bicarbonate content of the precipitated salt was determined by
titration. A solution of 1.5 g.L−1of precipitated salts in distilled water
was titrated with HCl, using phenolphthalein (for carbonates and hy-
droxides) and methyl orange (bicarbonates) as indicators.
2.8. Physicochemical analysis of the alkalinized vinasse
The alkalinized vinasse, treated with 1 M NaOH, was analyzed for
turbidity, BOD, and COD according to the methods described in the
Standard Methods for the Examination of Water and Wastewater
(APHA, 1999).
3. Results and discussion
3.1. Effect of different raw vinasse concentrations on microalgae growth
The six microalgae and three cyanobacteria strains used in this
study were previously screened for their capacity to produce biomass in
a vinasse-based medium (data not shown). It was observed that the
medium composed of more than 30% vinasse did not favor microalgal
growth. This could be explained by the excess of potassium in vinasse
composition or by its turbidity. Biomass production of different species
varied between 1.00 and 2.19 g.L−1 in 15 days of culture, with higher
values found for the cultures in media with 20% vinasse (Fig. 1).
Few works in the scientific literature investigated microalgal pro-
duction in vinasse media, and these reported difficulties in achieving
consistent biomass production. In most cases, the feasible concentration
of vinasse (diluted with synthetic medium) was lower than 10% (dos
Santos et al., 2016; Marques et al., 2013), except in the study performed
by Ramirez et al. (2014) where 50% vinasse diluted in synthetic
medium was used but a low biomass concentration (0.25 g.L−1) was
obtained. The higher biomass concentration achieved (Fig. 1) in com-
parison to these studies may be an effect of inoculum adaptation (see
Section 2.2).
The growth of the microalgal/cyanobacterial biomass in liquid
media can be stimulated by the addition of CO2. When CO2 dissolves in
water, it forms carbonic acid. At a slightly acidic pH (6.3), carbon
Fig. 1. Biomass production (bars) and CO2 biofixation (lines) of different strains of microalgae and cyanobacteria cultivated in raw vinasse-based media.
3
Bioresource Technology 292 (2019) 121955
dioxide dissociates into bicarbonate ions (HCO3−); while at a basic pH,
it dissociates further into carbonate ions (CO3−2). In very high pH so-
lutions, carbon dioxide reacts readily with bases, generating bicarbo-
nate and carbonate ions. These ions can be used by microalgae and
cyanobacteria as carbon sources for growth (Lohman et al., 2015;
Mokashi et al., 2016). Depending on the concentration and type of base,
bicarbonate and carbonate salts precipitate during the carbonation
process and can be removed by simple sedimentation or centrifugation
to be used as a fertilizer, in feed composition, for soil pH correction, or
even sold in purified form.
In the process developed in this work, vinasse was used instead of
water to absorb CO2 from the fermentation vats. During the tests, when
the pH of the vinasse increased above 10, the precipitation of solids
occurred. This precipitation affected the original composition of the
medium and directly influenced the algal growth process.
3.2. Effect of the addition of different alkalis on the composition of the
vinasse
Different vinasse treatments were carried out by adding different
alkalis to the vinasse media. After each treatment, the ash and organic
matter contents (Table 1) were determined.
Among the different studied alkalis, the addition of Na2CO3 caused
the removal of nearly 100% of the ashes from vinasse, which would
possibly hinder the subsequent cultivation of microalgae and/or cya-
nobacteria because of the lack of micronutrients. The lowest ash loss
(33.6%) and highest organic matter reduction (89%) were observed in
vinasse, which was treated with NaOH. BOD, COD, and turbidity ana-
lysis of this NaOH–treated vinasse showed reductions of 39.6%, 25.7%,
and 60.1%, respectively, in comparison to raw vinasse. These results
indicate that the vinasse obtained from the treatment with NaOH pre-
sents favorable conditions for the growth of microalgae.
The physicochemical treatment of sugarcane vinasse was also in-
vestigated by Prazeres et al. (2019), who compared H2SO4, Ca(OH)2,
and NaOH precipitations; FeCl3 coagulation-flocculation; and atmo-
spheric CO2 carbonation. Among these treatments, H2SO4 had a low
capacity to reduce organic matter content, while above 50% reduction
was achieved with Ca(OH)2 precipitation (at pH 12.5). Thermocalcic
precipitation (Ca(OH)2 at 95 °C) resulted in a similar COD reduction,
while NaOH treatment presented lower efficiency with 15.4% COD
reduction at pH 12.5. As for the minerals, Ca(OH)2 and thermocalcic
precipitation had the highest depletion percentages, followed by NaOH
precipitation that showed removal percentages of around 80%, 64%,
24%, 19%, and 74% for Ca, Mg, N, K, and P, respectively. Variations in
mineral depletions as a function of pH were not as significant in the
NaOH treatment as they were in the Ca(OH)2 treatment.
E.B. Sydney, et al. Bioresource Technology 292 (2019) 121955

Table 1
Analysis of ash and organic matter in vinasse treated with different alkalis for CO2 fixation.
Raw vinasse Na2CO3 Ca(OH)2 CaCO3 NaOH K2CO3

−1
Ashes in vinasse (g.L ) 12.5 ± 0.5 0.1 ± 0.0 5.3 ± 0.2 5.8 ± 0.4 8.3 ± 0.4 1.7 ± 0.1
Organic matter (g.L−1) 47.6 ± 3.2 12.5 ± 1.7 16.1 ± 1.1 12.8 ± 0.9 5.2 ± 0.4 16.2 ± 1.0

Table 2
Effect of vinasse carbonation with the addition of NaOH (1 M or 2 M) and the effect on salt purity and process yield.
Reaction time (h) Final pH CO2 fixation rate (g.L−1h−1) Precipitated salt (g.L−1
vinasse) Salt purity (% bicarbonate)

Vinasse + 1 M NaOH 6 7.8 ± 0.2 5.79 ± 0.2 50.10 ± 2.1 62 ± 2.0

Vinasse + 2 M NaOH 5 8.1 ± 0.2 6.25 ± 0.15 31.25 ± 1.5 70.73 ± 2.5

3.3. Carbonation of vinasse alkalinized with NaOH and quantification of Table 4

CO2 capture Influence of the reactor geometry and CO2 injection rate on the carbonation of
vinasse for microalgal culture.
The chemical carbon fixation rate in the vinasse treated with 1 M CO2 injection Reaction Final pH CO2 fixation
and 2 M NaOH (Table 2) resulted in similar values (2 M was 8% higher rate (vvm) time (h) (g.L−1.h−1)
than 1 M vinasse) and was 46–50 times higher than that achieved in
Stirred Tank 0.75 6.00 7.80 ± 0.2 5.79 ± 0.05
raceway reactors supplied with flue gas (de Godos et al., 2014). The
Reactor
present CO2 chemical fixation strategy emerges as an alternative to Bubble Column 0.75 2.75 7.80 ± 0.1 8.74 ± 0.09
circumvent the limitations on biomass productivity caused mainly by 2.00 1.17 7.87 ± 0.1 19.00 ± 0.07
low carbon dioxide transfer rates in open raceway ponds. 4.00 0.75 7.85 ± 0.2 43.35 ± 0.05
When 1 M NaOH was used, the total mass of bicarbonate salt pro-
duced in the precipitated form was higher but with lower purity. The
lower precipitated salt mass observed in the experiment beginning with consisted of the injection of atmospheric CO2 in an open reactor oper-
2 M NaOH vinasse in comparison to 1 M NaOH vinasse may be a con- ated in batch mode and containing 2.6 L (0.0162 m2 of surface area) of
sequence of the difference on the final pH of both experiments. In vinasse treated by Ca(OH)2 precipitation. The vinasse was maintained
average, a pH difference of 0.3 between experiments was achieved: 8.1 at room temperature and without agitation, and the effluent could be
(2 M NaOH) versus 7.8 (1 M NaOH). For the experiment with a higher neutralized after 12 to 22 days. The concentration of bicarbonates in-
initial NaOH concentration, to decrease 0.3 in pH it would be necessary creased, in 100 h, from 3000 to 6000 mg.L−1 of CaCO3, which were
to consume approx. 0.5 mol.L−1 [OH−]. According to the bicarbonate then converted to carbonates (2000 mg.L−1, 6–190 h). After around
production equation (CO2 + NaOH → NaHCO3), this is equivalent to 240 h, precipitation of CaCO3 was observed.
0.5molCO2.L−1 (or 22gCO2.L−1) and would result in 42 g.L−1 of bi- The use of an alkalinized vinasse for carbon dioxide absorption
carbonate. At this point of the experiment, the solution is saturated with presents three advantages: (1) the medium serves as a reserve of CO2
bicarbonate and all “new” bicarbonate would precipitate; and, then, the that can be used by the microalgal culture without requiring forced
precipitated mass in the experiment beginning with 2 M NaOH would aeration; (2) a portion of the CO2 that is absorbed by the alkalinized
be greater than 1 M NaOH, which was expected. vinasse is precipitated as a salt, which is adequate for storage and
The vinasse treated with 1 M NaOH was analyzed for turbidity, transport; and (3) the absorption of gases composed of pure or con-
BOD, and COD (Table 3). When compared to standard vinasse, BOD was centrated CO2 is more efficient than absorption from air because the
reduced in 39.6%, COD in 25.7%, and turbidity in 60.1%. The capacity high concentration favors mass transport and the reaction kinetics.
of 1 M NaOH vinasse to decrease its turbidity and promote carbon di-
oxide fixation into carbonate/bicarbonate salts is of interest for algal 3.5. Cyanobacterial and microalgal biomass production in pure and treated
cultivation. vinasse

Because vinasse carbonation and the reduction of its turbidity were

3.4. Effect of the reactor type on vinasse carbonation successfully achieved (Table 3), experiments were performed to eval-
uate cyanobacterial and microalgal growth in the treated vinasse. To
To compare the effect of CO2 contact time with 1 M NaOH alkali- the best of our knowledge, this is the first description of a carbon di-
nized vinasse and to improve the process, experiments were also per- oxide fixation technology where liquid (vinasse) and gaseous (CO2 from
formed in 2 L bubble column reactors (BCRs) with a height/diameter fermentation) effluents from the ethanol industry are used for cyano-
ratio of 4.25. Different CO2 injection rates were evaluated (0.75, 2, and bacterial or microalgal culture in an integrated process. Three Ar-
4 vvm), and the results are presented in Table 4. The change in reactor throspira species and Botryococcus braunii were cultivated in different
geometry significantly influenced the CO2 fixation rate, resulting in an concentrations of supplemented raw vinasse and 1 M NaOH treated
increase of carbonation of approximately 7.5-fold. vinasse (100:15:1 C:N:P), and biomass was quantified by gravimetric
The carbonation strategy evaluated by Prazeres et al. (2019) analysis of the dry mass (Table 5). Supplementation of vinasse was
carried out because phosphorus and nitrogen concentrations are gen-
Table 3 erally growth-limiting in raw vinasse, and their concentrations de-
Evaluation of BOD, COD and turbidity of vinasse treated with 1 M NaOH.
creased after alkalinization (Table 2).
Raw vinasse Vinasse treated with 1 M NaOH The treatments of the vinasse with NaOH resulted in a significant
increase in biomass concentrations in media with vinasse contents
BOD (mg.L−1) 10,151.0 6,126.0
higher than 30% in comparison to raw vinasse, especially for vinasse
COD (mg.L−1) 15,371.0 11,415.0
Turbidity (NTU) 1,148.1 458.0 contents of 50 and 70%. Among the tested Arthrospira strains, A.
maxima exhibited the best adaptation to treated vinasse. This

4
E.B. Sydney, et al. Bioresource Technology 292 (2019) 121955

Table 5 Santana et al. (2017) achieved a maximum microalgal biomass

Arthrospira spp. and B. braunii biomass produced in treated and raw vinasse productivity of 0.22 g.L−1day−1 in 3 days, using a strain of Chlamydo-
after 15 days of culture. B. braunii was not tested in different vinasse con- monas biconvexa cultivated in 100% clarified vinasse and an air lift flat
centrations. plate photobioreactor, thus producing around 0.66 g.L−1 of biomass.
Strain Vinasse (%)a Biomass production (g.L−1) The clarification procedure consisted of a treatment with Ca(OH)2,
followed by centrifugation to remove the insoluble solids. There is no
Supplemented raw Supplemented treated information about longer cultivation periods in this bioreactor model in
vinasse vinasse
this study.
A. platensis 10 0.90 ± 0.10 0.93 ± 0.04 The possibility of using very high concentrations of vinasse (up to
30 0.83 ± 0.07 0.90 ± 0.05 100%) in microalgal cultures, achieving high biomass concentrations
50 0.45 ± 0.07 0.94 ± 0.03 and productivities as highlighted in the present study, is un-
70 0.33 ± 0.09 1.02 ± 0.07
precedented. The possibility to use higher volumes of vinasse, asso-
100 0.21 ± 0.03 0.12 ± 0.04
ciated with chemical and biological treatment and carbon dioxide
A. maxima 10 1.09 ± 0.05 1.06 ± 0.02
fixation, significantly improves sustainability and the possibilities to
30 0.94 ± 0.05 1.08 ± 0.06
50 0.56 ± 0.07 1.37 ± 0.08 cultivate microalgae in large scales at low costs.
70 0.17 ± 0.06 2.22 ± 0.05 Considering the application of this technology in the ethanol in-
100 0.10 ± 0.02 1.57 ± 0.04 dustry that produces 1500 m3ethanol/day, where 10% of the produced
A. laxissima 10 0.51 ± 0.08 0.55 ± 0.06 vinasse is directed towards CO2 chemical fixation, it will be possible to
30 0.38 ± 0.02 0.45 ± 0.03 capture 99 tons of CO2 and produce 6.48 tons of bicarbonate in pre-
50 0.89 ± 0.03 1.39 ± 0.08 cipitated form. The use of the carbonated vinasse for cyanobacterial and
70 0.17 ± 0.02 1.21 ± 0.08
microalgal cultivation would produce approximately 7.42 tons of A.
100 0.14 ± 0.02 0.15 ± 0.04
maxima biomass cultivated in 70% treated carbonated vinasse or 5.27
B. braunii 100 0.08 ± 0.02 2.25 ± 0.11
tons of B. braunii biomass cultivated in 100% carbonated vinasse in a
a
Dilution with distilled water.
culture with 15 days of hydraulic retention. The mass balance for one
average fermentation vat of 200 m3 (working volume) is presented in
cyanobacterium was capable of growing at higher concentrations, Figs. 2 and 3.
reaching 2.2 g.L−1 in a 70% vinasse medium, which is 20–fold higher
than the biomass achieved in supplemented raw vinasse after 15 days of 3.6. Lipids from B. braunii cultivated in raw and carbonated vinasse
culture. B. braunii also showed great adaptation to treated vinasse, re-
sulting in the production of 2.25 g.L−1 of biomass in treated vinasse The biomass of B. braunii cultivated in raw and carbonated vinasse
concentrations of 100%. An average increase of cyanobacterial biomass was analyzed for its lipid content. Although the lipid content of the
production of 54% was observed using treated vinasse, in comparison biomass from the medium composed of 100% carbonated vinasse was
to raw vinasse. lower than that cultivated in 30% raw vinasse (13.3% versus 19.5%,
Santos et al. (2016) reported Spirulina maxima biomass concentra- respectively), a two–fold higher biomass concentration was achieved.
tion values of around 0.5 and 0.6 g.L−1 after 7 days of cultivation in the Thus, the total lipid production would reach 300 and
presence of sugarcane vinasse. In this study, vinasse was applied at very 215 g.m−3medium.day
−1
from 100% carbonated and 30% raw vinasse,
low concentrations, at 0.1 and 1.0% (v/v) in synthetic medium. respectively. It must be considered, though, that 3x more vinasse can be
Montalvo et al. (2019) cultivated A. maxima in sugarcane vinasse for used if it is carbonated.
15 days, and a biomass productivity of 0.15 g.L−1day−1 was reached, When comparing synthetic BBM medium with two vinasse-based
while BOD and COD were reduced by 89 and 81%, respectively. This media, diluted raw vinasse (50% in water) and Ca(OH)2-treated vinasse
biomass productivity value is similar to that of the present study, (not diluted), Santana et al. (2017) observed a reduction in carbohy-
however, A. maxima was cultivated in diluted sugarcane vinasse (30% drate, carotenoid, and fatty acid contents, and an increase in protein
in water). and ash contents when the vinasse-based media were utilized. This was

Fig. 2. Flowchart of the integrated system where the CO2 produced in fermentation is chemically fixed in alkalinized vinasse used for cyanobacterial and microalgal
production. The mass balance was based on the ethanol production of one 200 m3 reactor.

5
E.B. Sydney, et al.
Fig. 3. Calculations details of process mass balance.
observed for the two studied microalgae, C. biconvexa and Micractinium
sp. (Santana et al., 2017). When cultivated in synthetic Chu medium in
a tubular bioreactor, B. braunii UTEX 2441 biomass presented lipid
concentrations as high as 32 and 37%, with oleic acid (C:18:1n9) and
linoleic acid (C18:2n6) as the most representative fatty acids (Pérez-
Mora et al., 2016). Similar lipid content (33%) was reported by Sydney
et al. for B. braunii SAG 30.81 when cultivated in 3 N-MBM medium
(Sydney et al., 2010). This demonstrates the influence of the media
components in the microalgal biomass composition, which should be
evaluated together with the biomass productivity to establish optimal
process conditions.
As pointed out by Ferreira et al. (2019), the addition of waste
streams can reduce lipid accumulation by microalgae, and the fatty
acids profile can also be affected, either positively or negatively. Al-
though it is not possible to establish a pattern for the fatty acids profile
as a function of medium composition, since it also greatly depends on
the strain and cultivation conditions, scientific reports usually indicate
that the presence of wastewater streams results in higher contents of
saturated fatty acids in relation to unsaturated fatty acids (Ferreira
et al., 2019).
The lipids profile of the biomass produced in 30% raw and 100%
carbonated vinasse (Fig. 4) indicated higher content of C16:0, C16:1,
and C18 in both cases (54% and 63% of total fatty acid methyl esters
(FAME), respectively). Differences on FAME composition were mainly
Fig. 4. Characterization of the fatty acid methyl esters (FAME) from B. braunii cultivated in 30% raw vinasse and 100% carbonated vinasse.
6
Bioresource Technology 292 (2019) 121955
observed in the higher amount of unsaturated C16 in the biomass cul-
tivated in 30% raw vinasse, and on the degree of unsaturation, which
was higher in the biomass cultivated in raw vinasse (62.6% versus
52.6%), as expected.
4. Conclusions
A new method of chemical CO2 fixation was proposed for micro-
algae cultivation in an efficient process with the re-use of vinasse from
bioethanol production. The addition of 1 M NaOH followed by the in-
jection of pure CO2 (4 vvm) resulted in a fixation rate of 43.35
kgCO2.m-3medium.day−1 in a bubble column reactor. High biomass pro-
duction of 2.25 g.L−1 was achieved in a medium composed of 100%
treated vinasse with previous successful reduction of turbidity, COD,
and BOD. The proposed technology applied in a biorefinery concept
will certainly allow future productive microalgae and cyanobacteria
cultivation in connection with sustainable bioethanol industries.
Acknowledgments
This work was supported in part by the Brazilian National Council
for Scientific and Technological Development (CNPq) [grant number
407543/2013-0]; the Coordination of Improvement of Higher
Education Personnel (CAPES) [PROEX program]; and the enterprise
E.B. Sydney, et al. Bioresource Technology 292 (2019) 121955

Ourofino Agrociência, Brazil. 1111/j.1461-0248.2010.01577.x.

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Declarations of interest ment. Appl. Biochem. Biotechnol. 171, 1933–1943. https://doi.org/10.1007/s12010-
013-0481-y.
None. Mokashi, K., Shetty, V., George, S.A., Sibi, G., 2016. Sodium bicarbonate as inorganic
carbon source for higher biomass and lipid production integrated carbon capture in
Chlorella vulgaris. Achiev. Life Sci. 10, 111–117. https://doi.org/10.1016/j.als.2016.
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