Beruflich Dokumente
Kultur Dokumente
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
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 and (2) biological. Microalgae and cyanobacteria are interesting alter-
natives for promoting the development of CO2 mitigation processes
Ethanol production from sugarcane is one of the largest biopro- because they naturally consume CO2 through photosynthesis. The
cesses in the world in terms of processed volume. The process generates growth of microalgal and cyanobacterial biomass in liquid media is
large amounts of solid, liquid, and gaseous wastes that are increasingly usually limited by the low solubility of CO2, which depends on factors
seen as potential substrates for different processes. Among these re- such as temperature, pressure, and partial pressure of other gases.
sidues, vinasse and carbon dioxide can potentially be used to grow Microalgae and cyanobacteria biomass productivity and, consequently,
microalgae for biomass and biochemical production. the CO2 fixation rate depend directly on its concentration and are af-
An average Brazilian ethanol industrial unit (1500 m3.day−1 fected by chemical and biochemical factors that are linked to photo-
ethanol) produces 1132 tons of CO2 daily (stoichiometrically, 1 m3 of synthesis.
ethanol produces approx. 754.7 kg of CO2 through fermentation). With Vinasse is a liquid waste generated in ethanol industries, where it is
98% purity, this CO2 is not reused in the industry and is just released to the bottom product of distillation columns. This waste consists of
the atmosphere. This is the same carbon dioxide fixed during photo- ethanol-free fermented broth and contains organic solids in suspension,
synthesis of sugarcane and, thus, does not contribute to increasing its as well as minerals, residual sugars, and some volatile compounds.
concentration in the atmosphere, consequently, not increasing global Vinasse is generated in an average proportion of 15 L for each liter of
warming. However, reusing it is a strategy to add value to the pro- alcohol produced (Sydney et al., 2014). In 2018, approximately 1.6
duction chain (new products, carbon emission market) and make it billion m3 of vinasse were generated worldwide for a corresponding
more environmentally friendly. ethanol production of approx. 108.6 million m3 (Renewable Fuels
Different CO2 capture strategies have been investigated and can Association (RFA), 2019). Sugarcane vinasse is characterized by its high
generally be classified into two categories: (1) chemical reaction-based 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. Bioresource Technology 292 (2019) 121955
15,000 mg.L−1 O2, respectively); high potassium content temperature of 250 °C, with H2 as a carrier gas and velocity in the
(3600 mg.L−1); low pH (approx. 4.0); and considerable amounts of column controlled at 35 cm.s−1.
nitrogen, phosphorus, and micronutrients (Fuess et al., 2018). Because Raw and alkalinized vinasse supplementations were done by the
its simple disposal in nature can cause underground water contamina- additions of nitrogen (NaNO3) and phosphorus (K2HPO4) to reach C:N:P
tion, lixiviation, and soil desertification (Sydney et al., 2010), the de- 100:15:1, Redfield ratio (Loladze and Elser, 2011). The carbon con-
velopment of technologies to promote a more rational destination for centration in vinasse, as bicarbonate/carbonate, was quantified by ti-
vinasse is of great importance. It has been shown elsewhere that vinasse tration with phenolphthalein (carbonates) and methyl orange (bi-
is a feasible culture medium for microalgae (Barrocal et al., 2010; carbonates).
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 2.3. Biological CO2 fixation
of nutrients and end up limiting biomass production.
In this work, the biological carbon fixation capacity of different Microalgae and cyanobacteria were grown in a raw vinasse-based
microalgae and cyanobacteria species and a method to recycle vinasse medium with the automatic addition of 99.9% CO2 at 100 mL.min−1
and CO2 from the ethanol industry are described in a biorefinery ap- for 30 min during the light phase (at night CO2 injection was inter-
proach. CO2 is chemically fixed in the vinasse, resulting in bicarbonate rupted). Carbon dioxide fixation in microalgal and cyanobacterial cul-
salt production that leads to a significant reduction in turbidity and tures was calculated considering that 1.83 kg of CO2 was captured by
COD with improvement of microalgal biomass production. each kg of biomass produced (Chisti, 2007).
2. Material and methods 2.4. Effect of the addition of different alkalis on vinasse composition
2.1. Vinasse Five alkaline substances were evaluated to determine their effect on
vinasse composition. The experiments were performed by adding one of
Vinasse was kindly supplied by an ethanol and sugar refinery lo- the following alkalis to vinasse with a final concentration of 1 M: so-
cated in Cravinhos/SP, Brazil. Experiments were performed using the dium carbonate, sodium hydroxide, calcium hydroxide, calcium car-
same lot of vinasse, which was kept frozen at −20 °C until use. bonate, and potassium carbonate. After homogenization, the alkali-
nized vinasse was allowed to rest for 24 h and was then centrifuged. A
2.2. Microalgae and cyanobacteria cultivation raw vinasse (without alkaline substances but centrifuged to remove
solids) was employed for comparison.
Six species of microalgae (Botryococcus braunii SAG 801-7, The total precipitated mass was quantified by gravimetry (dried to a
Synechococcus nidulans LEB 25, Chlorella kessleri LEB15, Chlorella vul- constant weight at 80 °C) and analyzed for ash and organic matter ac-
garis LEB 104, Neochloris oleoabundans UTEX 1185, and Scenedesmus cording to the AOAC official method 942.05 (Thiex et al., n.d.) and the
obliquus LEB 22) and three cyanobacteria (Arthrospira platensis SAG AOAC method 967.05 (AOAC INTERNATIONAL, 2000), respectively.
257.80, Arthrospira laxissima SAG 256.80, and Arthrospira maxima SAG Ion chromatography was also performed to evaluate the ionic
84.79) were grown in pure or diluted vinasse-based media as follows. composition of raw and alkalinized vinasse. The determination of ca-
Biomass production was carried out with sterilized (121 °C, 15 min) tions (sodium, calcium, potassium, and magnesium) was done using a
raw and alkalinized vinasse (with the addition of different alkalis – see 761 Compact IC 817 Bioscan chromatograph. The column used was a
Section 2.4), starting from a concentration of approx. 0.25 g.L−1. The METROSEP C3 250/4.0 (Metrohm), with 250 mm × 4.0 mm of inner
cultures were incubated in an orbital shaker at 28 °C and 100 rpm with diameter. The analytical conditions were defined as follows: 3.5 mM
a 12 h/12 h photoperiod; illumination was provided at 50 µmole pho- HNO3, 1.0 mL.min−1, 40 °C, 20 µL injection volume, and pressure
tons.m−2.s−1 with cool white 32 W fluorescent lamps. Dilutions of vi- controlled at 11.2 MPa. A standard chromatogram was prepared with
nasse were made with distilled water. Strain adaptations to increasing the following salts: CaCl2·2H2O, MgCl2·6H2O, KCl, Na2SO4,
vinasse content were made starting from a culture in synthetic medium ZnSO4·7H2O, NH4Cl, and FeSO4·7H2O. All used reagents were of ana-
that was inoculated in 10% (v/v) vinasse and cultivated for 15 days. lytical grade (Sigma–Aldrich, EUA).
This culture was then used as inoculum for the medium containing 20% Nitrate quantification was performed using the nitration of salicylic
(v/v) vinasse and so on. With this strategy, a maximum growth in acid under highly acidic conditions (Cataldo et al., 1975), and phos-
natural vinasse at a 30% (v/v) concentration was achieved. Strains phorus was assessed using the phosphomolybdate method (Bhargava
were maintained for several months at this condition until the experi- and Raghupathi, 1993).
ments described in the present work were conducted. This adapted
culture in 30% natural vinasse was used as inoculum for the treated
vinasse experiments. 2.5. Chemical CO2 fixation in a stirred-tank reactor (STR)
Biomass quantification was performed by gravimetric analysis.
Samples were withdrawn and centrifuged in a Sorvall Legend Mach 1.6 Two experiments were conducted to determine the carbon-fixing
R centrifuge (Sorvall, Germany) at 3000 g for 15 min. The cells were potential of alkalinized vinasse with sodium hydroxide (NaOH) at
washed once with bicarbonate buffer (0.1 M) and dried at 80 °C until concentrations of 1 and 2 M. A conventional stirred-tank reactor (STR)
reaching a constant weight. with a total capacity of 5 L (working volume of 4 L, height/diameter
Lipids were determined from the dried biomass by extraction with ratio of 0.61 m/m) was employed. Agitation was maintained at
methanol:chloroform 1:1 (v/v) followed by a liquid–liquid extraction 300 rpm, and pure CO2 (99.9%) was injected at 3 L.min−1 (0.75 vo-
with hexane (Sydney et al., 2010). The apolar extracts were pooled, and lumes of air per volume of medium per minute, or vvm). The tem-
the fatty acid methyl esters (FAME) were prepared by the method perature was maintained at 40 °C, and the pH was monitored. Samples
proposed by Hartman and Lago (1973). The fatty acid composition of were collected hourly and dried to quantify the production of bi-
the samples was determined using a Shimadzu GC-2010 gas chroma- carbonate salt. The end of the reaction was determined when the pH
tograph (GC) equipped with a hydrogen flame ionization detector. The approached neutrality or when the pH stability was observed after
GC conditions were as follows: column RTX-WAX (fused silica, 5 min of continuous CO2 injection. The agitation was then turned off,
0.25 mm × 30 m), injector temperature of 230 °C, a split ratio of 1:100, and the solution was left 12 h to rest. At the end of the experiment, the
an oven temperature program of 130–240 °C (4 °C.min−1), a detector treated vinasse was titrated to determine the amount of fixed CO2.
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E.B. Sydney, et al. Bioresource Technology 292 (2019) 121955
2.6. Chemical CO2 fixation in a bubble column reactor dioxide dissociates into bicarbonate ions (HCO3−); while at a basic pH,
it dissociates further into carbonate ions (CO3−2). In very high pH so-
Experiments for evaluation of vinasse carbonation were also per- lutions, carbon dioxide reacts readily with bases, generating bicarbo-
formed in bubble column reactors with a height/diameter ratio of 4.25, nate and carbonate ions. These ions can be used by microalgae and
which favors a longer contact time between the CO2 and the alkalinized cyanobacteria as carbon sources for growth (Lohman et al., 2015;
vinasse. The same conditions used in the STR experiments were applied. Mokashi et al., 2016). Depending on the concentration and type of base,
Different injection rates were studied: 0.75, 2, and 4 vvm of CO2 bicarbonate and carbonate salts precipitate during the carbonation
(99.9%). process and can be removed by simple sedimentation or centrifugation
to be used as a fertilizer, in feed composition, for soil pH correction, or
2.7. Analysis of the precipitated salt even sold in purified form.
In the process developed in this work, vinasse was used instead of
The bicarbonate content of the precipitated salt was determined by water to absorb CO2 from the fermentation vats. During the tests, when
titration. A solution of 1.5 g.L−1of precipitated salts in distilled water the pH of the vinasse increased above 10, the precipitation of solids
was titrated with HCl, using phenolphthalein (for carbonates and hy- occurred. This precipitation affected the original composition of the
droxides) and methyl orange (bicarbonates) as indicators. medium and directly influenced the algal growth process.
Fig. 1. Biomass production (bars) and CO2 biofixation (lines) of different strains of microalgae and cyanobacteria cultivated in raw vinasse-based media.
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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)
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E.B. Sydney, et al. Bioresource Technology 292 (2019) 121955
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.
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E.B. Sydney, et al. Bioresource Technology 292 (2019) 121955
observed for the two studied microalgae, C. biconvexa and Micractinium observed in the higher amount of unsaturated C16 in the biomass cul-
sp. (Santana et al., 2017). When cultivated in synthetic Chu medium in tivated in 30% raw vinasse, and on the degree of unsaturation, which
a tubular bioreactor, B. braunii UTEX 2441 biomass presented lipid was higher in the biomass cultivated in raw vinasse (62.6% versus
concentrations as high as 32 and 37%, with oleic acid (C:18:1n9) and 52.6%), as expected.
linoleic acid (C18:2n6) as the most representative fatty acids (Pérez-
Mora et al., 2016). Similar lipid content (33%) was reported by Sydney 4. Conclusions
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 A new method of chemical CO2 fixation was proposed for micro-
components in the microalgal biomass composition, which should be algae cultivation in an efficient process with the re-use of vinasse from
evaluated together with the biomass productivity to establish optimal bioethanol production. The addition of 1 M NaOH followed by the in-
process conditions. jection of pure CO2 (4 vvm) resulted in a fixation rate of 43.35
As pointed out by Ferreira et al. (2019), the addition of waste kgCO2.m-3medium.day−1 in a bubble column reactor. High biomass pro-
streams can reduce lipid accumulation by microalgae, and the fatty duction of 2.25 g.L−1 was achieved in a medium composed of 100%
acids profile can also be affected, either positively or negatively. Al- treated vinasse with previous successful reduction of turbidity, COD,
though it is not possible to establish a pattern for the fatty acids profile and BOD. The proposed technology applied in a biorefinery concept
as a function of medium composition, since it also greatly depends on will certainly allow future productive microalgae and cyanobacteria
the strain and cultivation conditions, scientific reports usually indicate cultivation in connection with sustainable bioethanol industries.
that the presence of wastewater streams results in higher contents of
saturated fatty acids in relation to unsaturated fatty acids (Ferreira
et al., 2019). Acknowledgments
The lipids profile of the biomass produced in 30% raw and 100%
carbonated vinasse (Fig. 4) indicated higher content of C16:0, C16:1, This work was supported in part by the Brazilian National Council
and C18 in both cases (54% and 63% of total fatty acid methyl esters for Scientific and Technological Development (CNPq) [grant number
(FAME), respectively). Differences on FAME composition were mainly 407543/2013-0]; the Coordination of Improvement of Higher
Education Personnel (CAPES) [PROEX program]; and the enterprise
Fig. 4. Characterization of the fatty acid methyl esters (FAME) from B. braunii cultivated in 30% raw vinasse and 100% carbonated vinasse.
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E.B. Sydney, et al. Bioresource Technology 292 (2019) 121955