Bioresource Technology 301 (2020) 122698

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

Simultaneous saccharification and fermentation of Spirulina sp. and corn T

starch for the production of bioethanol and obtaining biopeptides with high
antioxidant activity
Angela Luiza Astolfia, Alan Rempelb, Vítor Augusto Farina Cavanhic, Maycon Alvesd,
⁎
Kricelle Mosquera Deamicie, Luciane Maria Collaa, Jorge Alberto Viera Costaa,e,
a
Graduate Program in Food Science and Technology, University of Passo Fundo (UPF), Passo Fundo, Rio Grande do Sul 99052-900, Brazil
b
Graduate Program in Environmental and Civil Engineering, University of Passo Fundo (UPF), Passo Fundo, Rio Grande do Sul 99052-900, Brazil
c
Chemical Engineering Course, University of Passo Fundo (UPF), Passo Fundo, Rio Grande do Sul 99052-900, Brazil
d
Chemical Course, University of Passo Fundo (UPF), Passo Fundo, Rio Grande do Sul 99052-900, Brazil
e
Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, 96203-900 Rio Grande, RS, Brazil

G R A P H I C A L A B S T R A C T

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

Keywords: The aim was to produce bioethanol by the simultaneous saccharification and fermentation (SSF) of Spirulina sp.
Biorefinery LEB 18 biomass and corn starch, increasing the process scale and obtaining biopeptides from bioethanol residue.
Bioethanol Different temperatures of SSF and biomass/starch concentrations were tested, and the best conditions were
Biopeptides chosen to scale-up the bioethanol production. The biopeptides were obtained enzymatically with a protease. The
Antioxidant capacity
antioxidant capacity, molecular structure, thermal stability and mass loss of the biopeptides were evaluated. A
Microalgae
total of 73 g L−1 bioethanol was obtained during scale-up, and the residue presented a high protein content with
a degree of hydrolysis of 86%. The biopeptides showed 32% ABTS radical inhibition with high thermal stability.
This study showed the possibility of the biorefinery concept being able to produce bioethanol by Spirulina, and
the biopeptides from the bioethanol residue presented high antioxidant capacity and can be used in many areas
of the food industry.

⁎
Corresponding author at: Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, 96203-900 Rio
Grande, RS, Brazil.
E-mail address: jorgealbertovc@gmail.com (J.A.V. Costa).

https://doi.org/10.1016/j.biortech.2019.122698
Received 31 October 2019; Received in revised form 23 December 2019; Accepted 24 December 2019
Available online 27 December 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
A. Luiza Astolfi, et al. Bioresource Technology 301 (2020) 122698

1. Introduction Bacillus licheniformis and amyloglucosidase (AMG® 300L) from a se-

lected strain of Aspergillus niger donated by Novozymes (Brazil) were
Significant research efforts in the development of energy sources are used in this study. To obtain the biopeptides, the commercial enzyme
currently being considered since there has been a depletion of fossil fuel Protemax 580L (Prozyn, Brazil) was used, which is a serine en-
reserves as well as a need to reduce dependence on oil and reduce dopeptidase of bacterial origin (Bacillus licheniformis).
greenhouse gas emissions (Kumar and Singh, 2019). The enzymatic activities were defined as the release of 1 μmol of
The demand for renewable energy has grown considerably world- reducing sugar per ml of broth per minute and are expressed as μmol
wide, and bioethanol production from different sources is being stu- mL−1 min−1 (Alva et al., 2007). The protease activity was defined as
died. Microalgae have been investigated as a raw material for third- the amount of enzyme that releases 1 μg of tyrosine per minute, ex-
generation bioethanol production since their biomass presents a high pressed as U mL−1 (Ma et al., 2007).
carbohydrate content in addition to the advantage of capturing atmo-
spheric CO2 and advantages in food production (Zhu and Ketola, 2012). 2.3. Fermentation conditions
Spirulina sp. biomass may act as a source of nutrients during the fer-
mentation process by providing nutrients to yeast. In addition, carbo- A total of 0.15 mL of each enzyme extract per gram of carbohydrate
hydrates present in microalgae biomass in their starch and cellulose was used. The assays were performed on a shaker table at 130 rpm in
forms facilitate the conversion to monosaccharides compared with Erlenmeyer flasks (300 mL working volume) in duplicate. Cells were
lignocellulosic materials (Ho et al., 2013). disrupted by a 24/24 freezing/defrosting cycle (Rempel et al., 2018) to
One of the most important advances in the bioethanol production release the intracellular material, after which different corn starch
process is the development of simultaneous saccharification and fer- concentrations were added after each test and the pH was adjusted to
mentation (SSF). In the enzymatic degradation of starch, it usually 5.0. The culture media was autoclaved at 121 °C for 20 min and after
combines with the fermentation of glucose obtained from the hydrolysis prehydrolysis was accomplished by α-amylase followed by amyloglu-
of polysaccharides (Mojović et al., 2006). An advantage of the SSF cosidase enzymes and fermentation using Saccharomyces cerevisiae CAT-
process is that with microalgae biomass, it is possible to obtain a large 1 yeast added at a 10% inoculation ratio.
amount of high protein residue, and this residue can be used to obtain To determine the reducing sugar (RS) and ethanol concentrations,
biopeptides. Thus, microalgal biomass can be used in a biorefinery the samples were collected at time zero, 2 h and 12 h. The tests were
context since for bioethanol production, a biomass from a renewable finished after stabilization of the bioethanol and RS concentrations.
source can be used, and its residue integrated and diversified for the The RS concentration was quantified by the 3.5 DNS method
production of many coproducts, with a minimum generation of waste (Miller, 1959). The ethanol concentration was determined by distilla-
and harmful emissions. In this context, the aim of this study was to tion of the sample in a Tecnal TE-012 microdistiller, followed by the
produce bioethanol by SSF from Spirulina sp. LEB 18 biomass and corn ethanol reaction procedure with potassium dichromate. The absorbance
starch, increasing the process scale and obtaining biopeptides from was determined by spectrophotometry at 600 nm against a blank re-
bioethanol residue production. active (Salik and Povh, 1993).

2. Materials and methods 2.4. Strategies for bioethanol production by SSF

2.1. Microorganism and biomass characterization A concentration of 20% (m v−1) biomass was used for the enzy-
matic SSF. Corn starch was added as a source of nutrients and carbo-
Spirulina sp. LEB 18 biomass was studied, and the microalga was hydrates during the fermentation of Spirulina sp. The substrates were
cultivated on a pilot scale in Santa Vitória do Palmar (Brazil, 33° 30′ 13″ prehydrolyzed with the α-amylase enzyme for 2 h at 50 °C, after which
S and 53° 08′ 59″ W) in raceway open PVC tanks located in a green- the amyloglucosidase enzyme was added at different temperatures, as
house with daytime sunlight of ~300 µmolphotons m−2s−1, average shown in Table 1.
temperature of 25 °C and using Zarrouk medium (Zarrouk, 1966). The
dry biomass of Spirulina sp. was ground in a ball mill (Model Q298, 2.4.1. Scale-up for bioethanol production
QUIMIS) to obtain the granulometry 40 mesh. The scale-up for bioethanol production was carried out in a Tec Bio
The protein, carbohydrate and lipid contents in the biomass were Flex bioreactor (3 L) with the best conditions obtained from previous
determined. The protein content was obtained according to Lowry et al. tests. The parameters of pH, dissolved oxygen (DO), temperature and
(1951); the carbohydrate content was determined by the phenol-sul- agitation were controlled during the SSF process. A total of 0.25 (v v−1)
furic method (Dubois et al., 1956), and the lipid content was de- of each amylolytic enzyme was added to make the process viable on
termined according to the method of Folch (Folch and Lees, 1957) with larger scales.
chloroform/methanol (2:1 v v−1). Samples were collected at time zero and after 2 h and 12 h of pre-
hydrolysis to determine the RS and ethanol concentrations, and the
2.2. Enzymes assays were complete after 60 h of SSF. The residue was separated by
centrifugation at 3640×g for 15 min.
The α-amylase (Liquozyme® Supra 2.2X) enzyme obtained from The bioethanol production by SSF was evaluated by the

Table 1
Variation in the corn starch, Spirulina sp. LEB 18 biomass and temperatures tested, hydrolysis efficiency (PHE) after 2 h and SSF ethanol efficiency (η).
Assay Corn starch (%) Spirulina biomass (%) Temperature (°C) Carbohydrate (g Cho L−1) PHE (%) η (%)

1 5 15 30 66.8 34.03 ± 1.16a 46.35 ± 2.79a

2 10 10 30 111.2 53.67 ± 1.85b 64.10 ± 2.01bc
3 15 5 30 155.6 56.28 ± 2.66c 73.64 ± 0.72c
4 5 15 40 66.8 36.88 ± 2.17a 51.08 ± 3.57ab
5 10 10 40 111.2 52.52 ± 2.47b 53.02 ± 0.67ab
6 15 5 40 155.6 58.43 ± 3.62c 57.80 ± 3.12ab

Means ± standard deviation (n = 2). Different letters in the same column show significant differences at the 95% confidence level.

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A. Luiza Astolfi, et al. Bioresource Technology 301 (2020) 122698

prehydrolysis efficiency (PHE) and ethanol formation efficiency (ƞ). 2.6.3. Protein profile (SDS-PAGE)
AR2h
The PHE was obtained by the equation PHE (%) = m C . (1.1)
,where Electrophoresis (SDS-PAGE) was performed according to the
⎛ biomass ⎞
⎝ v ⎠ method of Laemmli (1970). Electrophoresis in sodium dodecyl sulfate
AR2h corresponds to the RS concentration obtained after hydrolysis (g (SDS) polyacrylamide gel was carried out in a continuous buffer system:
L−1), m is the amount of biomass utilized (g L−1), C is the carbohydrate 1.5 mol L−1 Tris buffer and 10% SDS (w v−1). The gel was prepared
content in SSF (g L−1), 1.1 is the conversion number of carbohydrates with 15% separation and 12% concentration. After the electrophoretic
to glucose and v is the working volume. The ethanol formation effi- analysis, the gel was removed from the apparatus and placed in staining
ΔE
ciency (ƞ) was obtained according to equation η (\% ) = 0.511 . C . (1.1) , solution for 2 h under constant agitation. A mixture of proteins ranging
where ΔE is the variation in the ethanol concentration (g L−1), C is the from 5 to 250 kDa was used as a reference.
carbohydrate content in SSF (g L−1) and 1.1 is the conversion number
of carbohydrates to glucose (Hang et al., 1981). 2.7. Statistical analysis

2.5. Protein hydrolysis The data were assessed by analysis of variance followed by Tukey’s
test at the 95.0% confidence level.
The hydrolysates were obtained with 15.4 g of residue in 100 mL of
bicarbonate-sodium carbonate buffer (pH 9.5) and 5 U mL−1 of the 3. Results and discussion
protease enzyme. All assays were arranged in an orbital incubator at
180 rpm and a temperature of 60 °C in duplicate. After the reaction, the 3.1. Chemical characterization of Spirulina biomass
enzyme was thermally inactivated at 85 °C for 10 min (Lisboa et al.,
2014). The Spirulina sp. LEB 18 biomass presented 74.5% protein content,
To determine the degree of hydrolysis (DH), 1 mL aliquots of hy- 11.2% carbohydrate content, 2.9% lipids and 14.1% ash. The biomass
drolysate were inactivated with 9 mL of 6.25% (w v−1) trichloroacetic used in this study had low carbohydrate contents because the main
acid (TCA) solution for 10 min (Lisboa et al., 2016). After that, the objective of the crop was not to accumulate carbohydrates in the bio-
samples were centrifuged for 5 min at 3300×g to remove the insoluble mass, which influences the bioethanol yield. In this sense, the addition
material precipitated by TCA. The soluble protein content of the su- of corn starch in our study became relevant for the production of
pernatant was determined using the Folin-Lowry method (Lowry et al., bioethanol by the SSF process.
1951), and the results are expressed as mgalbumin.
The DH was determined each hour for 8 h total, estimated according 3.2. SSF using the biomass of Spirulina sp. for bioethanol production
to the method described by Hoyle and Merritt (1994) with modifica-
tions, and expressed as the amount of soluble protein in the TCA before Fig. 1 presents the results of the bioethanol and RS concentrations
and after the addition of the enzyme related to the amount of total obtained during 72 h of the SSF process at two different temperatures
protein present in the sample according to the equation (30 °C and 40 °C). According to the results shown in Fig. 1, the higher
the amount of carbohydrates in the culture medium, the higher the RS
%DH = (
PSt − PSt0
Pt )
. 100 . Here, PSt is the amount of soluble protein at a
concentrations after 2 h of prehydrolysis due to the carbohydrate source
given time after the addition of the enzyme, PSt0 corresponds to the
being higher and the α-amylase enzyme acting at its optimum tem-
amount of protein soluble in 6.25% TCA before the enzymatic addition,
perature (50 °C).
and Pt is the amount of total protein in the sample determined by the
As the corn starch and the microalgal starch were saccharified, S.
micro-Kjeldahl method (N = 6.25) (AOAC, 2005).
cerevisiae yeast cells were in their exponential phase of growth, and at
the optimum temperature (30 °C), the yeast cells used the RS for their
2.6. Biopeptide characterization own growth and ethanol production (Fig. 1a–c). This characteristic of
SSF presents advantages compared to the separate saccharification and
2.6.1. Antioxidant activity fermentation processes since these processes have high glucose con-
The biopeptides produced were characterized according to their centrations and may inhibit bioethanol fermentation during the process
antioxidant capacity with the ABTS method. The ABTS cationic radical (Eklund and Zacchi, 1995).
was prepared from a solution of 7 mmol L−1 ABTS (2,2′-azinobis(3- Inoculation of the yeast caused a reduction in the RS concentration
ethylbenzothiazoline-6-sulfonic acid)) with a potassium persulfate so- at 12 h, reaching close to zero in the assays at 30 °C (Fig. 1a–c). Singh
lution (2.45 mmol L−1) (Re et al., 1999). The resulting absorbance of et al. (2018) observed a similar trend during the SSF of Chlorella sp., in
the solution was measured at 734 nm with a spectrophotometer which the RS concentration decreased at 12 h, and at the end of the
(Biospectrometer Eppendorf 6136). The percent ABTS radical inhibition process, 98.8% of the RS had been consumed. The tests performed with
was calculated from the following equation 5% starch + 15% Spirulina (Fig. 1a) and 10% starch + 10% Spirulina
(Fig. 1b) did not show a significant difference (p ≥ 0.05) in their in-
ABSblank − ABSsample ⎞ crease in bioethanol production since in these assays, the concentration
% inhibition = ⎛ ⎜ . 100.
⎟

⎝ ABSblank ⎠ of RS remained constant until the end of the SSF process (72 h). For the
assay with a higher corn starch concentration (Fig. 1c), a significant
difference (p ≤ 0.05) in bioethanol concentration was observed during
2.6.2. Fourier transform infrared spectroscopy (FTIR) the 72 of the SSF process, achieving a maximum of 64.4 g L−1 at 60 h.
The molecular structure, thermal stability by differential scanning The assay with the highest bioethanol production was the SSF with 15%
calorimetry (DSC) and mass loss by thermogravimetric analysis (TGA) corn starch and 5% Spirulina sp. biomass (Fig. 1c), showing 73.6%
parameters of protein hydrolysate samples, bioethanol production re- conversion efficiency, since this assay contained the highest carbohy-
sidue and Spirulina sp. biomass were analyzed by Fourier transform drate concentration and was carried out at 30 °C, benefiting yeast de-
infrared spectroscopy (FTIR) to evaluate the differences in the func- velopment and product formation.
tional groups present in the biomass of Spirulina sp. The samples were For the assays carried out at 40 °C (Fig. 1d–f), the RS concentrations
pressed onto a Zn-Se crystal, and the transmittance was recorded in the after 12 h were higher than those from the assays at 30 °C, independent
range of 4000 to 650 cm−1 (Agilent model Cary 630). The thermo- of the corn starch and Spirulina biomass concentrations used. The RS
gravimetric and calorimetric curves were obtained in a thermal analysis values obtained in these assays may be related to the temperature tested
simulator (STA 6000, Perkin Elmer). (40 °C), which is not the optimum temperature for the yeast but is

3
A. Luiza Astolfi, et al. Bioresource Technology 301 (2020) 122698

Fig. 1. Profile of bioethanol and RS concentrations during the SSF process with different concentrations of Spirulina sp. biomass and corn starch: (A) 5%
starch + 15% Spirulina, (B) 10% starch + 10% Spirulina and (B) 15% starch + 5% Spirulina at a temperature of 30 °C; and (D) 5% starch + 15% Spirulina, (E) 10%
starch + 10% Spirulina and (F) 15% starch + 5% Spirulina at 40 °C.

closer to the optimum temperature for the action of the amylolytic 3.3. Scale-up for bioethanol production
enzymes (50 °C). In the assays with 10% starch + 10% Spirulina
(Fig. 1e) and 15% starch + 5% Spirulina (Fig. 1f), the bioethanol pro- The scale-up was conducted based on the best conditions obtained
duction increased gradually during the process; however, the bioe- in Erlenmeyer flasks from SSF, which was assay 3 with 15% of corn
thanol production was lower than that in the assays at 30 °C. starch and 5% of Spirulina biomass at 30 °C. The SSF values in the
Table 1 presents the prehydrolysis efficiency (PHE) after 2 h and bioreactor are presented in Fig. 2. Two hours of prehydrolysis produced
bioethanol efficiency (ƞ) for all conditions evaluated. According to the 55 g L−1 of ethanol and a RS concentration of 70 g L−1. The pre-
results, there was no significant difference (p ≥ 0.05) between the hydrolysis efficiency of the SSF in the bioreactor was 40.2%, and the
assays with the same corn starch and Spirulina biomass concentrations ethanol efficiency was 62.9%.
(assays 1, 2 and 3 at 30 °C and assays 4, 5 and 6 at 40 °C), and when The ethanol and prehydrolysis efficiencies obtained from the bior-
testing at different temperatures, the temperature did not affect the two eactor and in the Erlenmeyer flasks were similar, indicating that the 10-
parameters evaluated. fold increase in scale did not change the SSF parameters evaluated. It
The highest (p ≤ 0.05) PHE obtained was from the assays with 15% was observed in this study that 24 h of SSF was sufficient to obtain high
corn starch and 5% Spirulina biomass at 30 °C (56.3%) and 40 °C yields of bioethanol since the bioethanol production did not change
(58.4%). The highest bioethanol efficiency obtained in this study was significantly (p ≥ 0.05) compared to the other times evaluated during
73.6%, which was obtained from assay 3. In addition, the biomass of SSF in the bioreactor. In this sense, SSF presented as a good process for
Spirulina sp., presenting a source of carbon, provides sufficient nutrients the production of bioethanol on a large scale since the product is pro-
available for the yeast during the fermentation process, as evidenced by duced in a single bioreactor, reducing costs associated with bioreactors,
the studies by Rempel et al. (2018). time and concentration of the enzyme.

4
A. Luiza Astolfi, et al. Bioresource Technology 301 (2020) 122698

with a high DH present high amounts of low molecular weight peptides

and, consequently, greater potential for inhibition in relation to low DH
hydrolysates.
FTIR analysis was used to evaluate the differences in the functional
groups present in the biomass of Spirulina sp. in relation to the residue
and peptides obtained by protein hydrolysis.
The FTIR analysis for Spirulina sp. biomass presented peaks at 3280
and 2924 cm−1 that have been attributed to the secondary amine
stretching NH vibration (protein) and -OH (hydroxyl) functional groups
of the proteins present in Spirulina biomass, according to Suganya et al.
(2015). The bands at 1541, 1397 and 1233 cm−1 in Spirulina biomass
can be attributed to amide I resulting from the C=O stretching vibra-
tions of the peptide bonds; amide II, which results from the curvature of
NH and CN stretching vibrations and the COO- protein side chain; and
amide band III is also present, respectively (Bataller and Capareda,
Fig. 2. Profile of RS and ethanol concentrations during SSF for the tests carried 2018). The band at 1636 cm−1 remained in the peptide among the
out in the bioreactor. three parameters evaluated in both the residue and in Spirulina sp.
biomass, while the other bands disappeared after hydrolyzing the mi-
3.4. Degree of hydrolysis (DH) and characterization of biopeptides croalgal biomass, both with the amylolytic enzymes and the protease
enzyme, indicating that there was hydrolysis of the polysaccharides
The best DH value was observed after 4 h of hydrolysis, after which that released mainly glucose and other fermentable products
the DH remained constant (p ≥ 0.05) throughout 8 h of the reaction. A (1034 cm−1). Bataller and Capareda (2018) evaluated the microalgae
high DH yield was obtained in this study, which may be due to the Spirulina plantensis by the FTIR, which showed similar characteristic
accessibility of the protease to the proteins since the enzyme needs to bands from carbohydrates, CO and CC, which were observed at
bind to the surface of the molecule to access the peptide bonds and 1034 cm−1, and a characteristic lipid band observed at 1653 cm−1,
initiate hydrolysis (Pereira et al., 2018). The enzymatic hydrolysis of presenting possible overlap with amide I.
proteins involves a structural change, where the protein is gradually In addition, cleavage of the peptide in the residue into free amino
cleaved into smaller peptide units (Martins et al., 2009), and lower acids and other lower molecular weight peptides (1541, 1397 and
molecular weight peptides are desirable because of their improved 1233 cm−1) occurred through the mechanism of action of the enzyme
functional properties, such as digestibility, solubility and water reten- used (serine peptidase) in the hydrolysis process, by which it acts on the
tion capacity, in addition to antioxidant activity (Damodaran et al., carbon atom of the carbonyl of the peptide bond.
2010). The temperature of protein denaturation obtained by DSC was
The antioxidant activity of the biopeptides as detected by the ABTS 76.4 °C for Spirulina biomass, 96.7 °C for the residue and 101.5 °C for
assay over 8 h of reaction was evaluated, and 32% inhibition of the the biopeptide. When the residue proteins were hydrolyzed, the dena-
ABTS radical was obtained after 5 h of hydrolysis, which was sig- turation temperature of the peptide increased, which is an advantage
nificantly higher (p ≤ 0.05) from the other hydrolysis times. According for food applications that require high temperatures during processing.
to Elias et al. (2008), the increase in antioxidant activity is due to The enthalpy (ΔH) involved in the biopeptide process was
changes in the protein structure resulting from the enzymatic hydrolysis 2248.9 J g−1, whereas for the Spirulina biomass, this value was
process, exposing peptides with antioxidant properties that react more 151.4 J g−1, demonstrating that with the enzymatic hydrolysis process,
effectively with the radicals than in the intact protein. Furthermore, a the energy required for denaturation increased, consequently im-
correlation between the DH and antioxidant activity was observed proving the thermal stability (Trivedi et al., 2015). This increase in
during the hydrolysis reaction, as shown in Fig. 3. As the DH increases, stability may be due to the simpler structure of the protein (absence of
the antioxidant potential also increases, leading to the conclusion that tertiary and quaternary structures) and peptide bonds and the compo-
enzymatic hydrolysis occurred and that the peptides with antioxidant sition of more stable amino acids present in the lower molecular weight
properties had been released. fraction, increasing the stability of the molecules before the tempera-
According to Lisboa et al. (2016) and Costa et al. (2019), the en- ture increase.
zymatic hydrolysis of Spirulina sp. LEB 18 is important to release bio- Regarding the TGA analysis, two phases of mass loss for Spirulina sp.
peptides with high antioxidant activity that are inactive in un- were observed. The first phase had a lower loss of 13.1% between 30 °C
hydrolyzed biomass. In addition, the authors state that hydrolysates and 125 °C, and the second phase presented a higher mass loss, 41.7%
in the temperature range of 230 to 453 °C. Jesus et al. (2018) identified
two phases involving mass loss in Spirulina sp. LEB 18 biomass, the first
in the 90 °C range with dehydration and devolatilization, and the
second in the 350 °C range, associated with the decomposition of the
compounds present in the biomass. The mass loss for the residue was
76.3%, and for the biopeptides, this value was 95.4%, which occurred
in a single phase. The loss of mass obtained in this study is character-
ized by the amount of water, which is dependent on the moisture
content of the samples. According to the results obtained, it has been
suggested that the peptides can be added into processes with tem-
peratures up to 100 °C without losing their properties, demonstrating
their potential of applications in functional foods.

3.5. SDS-PAGE

Fig. 3. Correlation between the antioxidant potential and the degree of hy- The SDS-PAGE analysis of the Spirulina sp. biomass, residue and
drolysis. biopeptides are presented in Fig. 4, and a decrease in the molecular

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A. Luiza Astolfi, et al. Bioresource Technology 301 (2020) 122698

Acknowledgments

The authors are pleased to acknowledge the Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – fi-
nance code 001; Novozymes and Prozyn for supplying the enzymes
used in this work; and the University of São Paulo, campus of Ribeirão
Preto for the donation of the yeast used in this work.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.biortech.2019.122698.

References

Alva, S., Anupama, J., Savla, J., Chiu, Y.Y., Vyshali, P., Shruti, M., Kumudini, B.S., 2007.
Production and characterization of fungal amylase enzyme isolated from Aspergillus
sp. JGI 12 in solid state culture. Afr. J. Biotechnol. 6, 576.
AOAC, Official Methods of Analysis of the Association Analytical Chemists, 18 ed.,
Gaithersburg, Maryland, 2005.
Bataller, B.G., Capareda, S.C., 2018. A rapid and non-destructive method for quantifying
biomolecules in Spirulina platensis via Fourier transform infrared-attenuated total
reflectance spectroscopy. Algal Res. 32, 341–352.
Costa, A.M., Bueno, K.T.L., Da Rosa, A.P.C., Costa, J.A.V., 2019. The antioxidant activity
of nanoemulsions based on lipids and peptides from Spirulina sp. LEB18. LWT-Food
Sci. Technol. 99, 173–178.
Damodaran, S., Parkin, K.L., Fennema, O.R., 2010. Química de Alimentos de Fennema.
Fig. 4. Protein profile by SDS-PAGE of Spirulina sp. biomass (S), residue (R) and São Paulo. 4. Ed. Editora Artmed, pp. 900.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric
biopeptides (B). PS: protein standards.
method for determination of sugars and related substances. Anal. Chem. 28,
350–356.
Eklund, R., Zacchi, G., 1995. Simultaneous saccharification and fermentation of steam-
mass of the proteins was demonstrated. For the Spirulina sp. Biomass, pretreated willow. Enzyme Microb. Technol. 17, 255–259.
many molecular mass bands of 100, 15, 10 and 5 kDa can be observed. Elias, R.J., Kellerby, S.S., Decker, E.A., 2008. Antioxidant activity of proteins and pep-
These alterations may be due to the enzymatic protease action during tides. Crit. Rev. Food Sci. Nutr. 48, 430–441.
Folch, J., Lees, M., 1957. A simple method for isolation and purification of total lipids
the generation of peptides of different sizes and amino acid sequences,
from animal tissues. J. Biol. Chem. 226, 497–509.
which may determine the antioxidant capacity (Lisboa et al., 2016). Hang, Y.D., Lee, C.Y., Woodams, E.E., 1981. Production of alcohol from apple pomace.
Thus, with SDS-PAGE analysis, it was possible to confirm that hydro- Appl. Environ. Microbiol. 42, 1128–1129.
Ho, S.H., Huang, S.W., Chen, C.Y., Hasunuma, T., Kondo, A., Chang, J., 2013. Bioethanol
lysis of the proteins that were present in the bioethanol production
production using carbohydrate-rich microalgae biomass as feedstock. Bioresour.
residue occurred, and that the molecular mass was higher and did not Technol. 135, 191–198.
show high antioxidant activity. Hoyle, N., Merritt, J.H., 1994. Quality of fish protein hydrolysate from herring (Clupea
harengus). J. Food Sci. 59, 76–79.
Jesus, C.S., Uebel, L., Costa, S.S., Miranda, A.L., Morais, E.G., Morais, M.G., Costa, J.A.V.,
4. Conclusion Nunes, I.L., De Souza, E., Druzian, J.I., 2018. Outdoor pilot-scale cultivation of
Spirulina sp. LEB-18 in different geographic locations for evaluating its growth and
chemical composition. Bioresour. Technol. 256, 86–94.
The bioethanol production efficiency was approximately 74% using Kumar, D., Singh, V., 2019. Bioethanol production from corn. In: Corn: Chemistry and
15% corn starch and 5% Spirulina sp. biomass. The bioethanol pro- Technology. Elsevier, pp. 615–631. https://doi.org/10.1016/B978-0-12-811971-6.
00022-X.
ductivity remained when the scale-up was 10 times greater. The peptide Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head
profile presented in the bioethanol residue was capable of inhibiting the bacteriophage T4. Nature 227, 680–685.
ABTS radical, with the potential to be added to food and biological Lisboa, C.R., Pereira, A.M., Costa, J.A.V., 2016. Biopeptides with antioxidant activity
extracted from the biomass of Spirulina sp. LEB 18. Afr. J. Microbiol. Res. 10, 79–86.
systems. This study demonstrated that it is possible to develop an in-
Lisboa, C.R., Pereira, A.M., Ferreira, S.P., Costa, J.A.V., 2014. Utilisation of Spirulina sp.
tegrated biorefinery to produce bioethanol from Spirulina sp. with and Chlorella pyrenoidosa biomass for the production of enzymatic protein hydro-
added corn starch and to obtain biopeptides with antioxidant activity, lysates. Int. J. Eng. Res. Appl. 4, 29–38.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with
which is an alternative to reduce environmental impact and supply the
Folin-phenol reagent. J. Biol. Chem. 193, 265–275.
energy demand of fuels using biomass from renewable sources. Ma, C., Ni, X., Chi, Z., Ma, L., Gao, L., 2007. Purification and characterization of an
alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide
production from different sources. Mar. Biotechnol. 9, 343–351.
CRediT authorship contribution statement Martins, V.G., Costa, J.A.V., Prentice-Hernández, C., 2009. Hidrolisado protéico de pes-
cado obtido por vias química e enzimática a partir de corvina (Micropogonias furnieri).
Quim. Nova. 32, 61–66.
Angela Luiza Astolfi: Investigation, Data curation, Formal analysis, Miller, G.L., 1959. Use of de dinitrosalicylic acid reagent for determination of reducing
Writing - original draft. Alan Rempel: Methodology. Vítor Augusto sugar. Anal. Chem. 31, 426–428.
Farina Cavanhi: Investigation, Methodology. Maycon Alves: Mojović, L., Nikolić, S., Rakin, M., Vukašinović, M., 2006. Production of bioethanol from
corn meal hydrolyzates. Fuel 85, 1750–1755.
Investigation, Methodology. Kricelle Mosquera Deamici: Writing - Pereira, A.M., Lisboa, C.R., Costa, J.A.V., 2018. High protein ingredients of microalgal
original draft, Writing - review & editing. Luciane Maria Colla: origin: obtainment and functional properties. Innov. Food Sci. Emerg. Technol. 47,
Supervision. Jorge Alberto Viera Costa: Supervision. 187–194.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999.
Antioxidant activity applying an improved ABTS radical cation decolorization assay.
Declaration of Competing Interest Free Radic. Biol. Med. 26, 1231–1237.
Rempel, A., Machado, T.P., Colla, L.M., Treichel, H., Colla, E., Margarites, A., Colla, M.L.,
2018. Saccharification of Spirulina platensis biomass using free and immobilized
The authors declare that they have no known competing financial amylolytic enzymes. Bioresour. Technol. 263, 163–171.
interests or personal relationships that could have appeared to influ- Salik, F.L.M., Povh, N.P., 1993. Método espectrofotométrico para determinação de teores
alcoólicos em misturas hidroalcoólicas. In: Congresso Nacional da STAB, Águas de
ence the work reported in this paper.

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A. Luiza Astolfi, et al. Bioresource Technology 301 (2020) 122698

São Pedro, 262–266. Trivedi, M.K., Branton, A., Trivedi, D., Nayak, G., Singh, R., Jana, S., 2015. Physical
Singh, S., Chakravartya, I., Pandey, D.K., Kundu, S., 2018. A development of a process spectroscopic and thermal characterization of biofield treated fish peptone. Eur.
model for simultaneous saccharification and fermentation (SSF) of algal starch to Biophys. J.3, 51–58.
third-generation bioethanol. Biofuels. https://doi.org/10.1080/17597269.2018. Zarrouk, C., 1966. Contribution à l’étude d’une cyanophycée. Influence de divers facteurs
1426162. physiques et chimiques sur la croissance et la photosynthèse de Spirulina maxima.
Suganya, K.U., Govindaraju, K., Kumar, V.G., Dhas, T.S., Karthick, V., Singaravelu, G., Ph.D Thesis. Université de Paris.
Elanchezhiyan, M., 2015. Blue green alga mediated synthesis of gold nanoparticles Zhu, L., Ketola, T., 2012. Microalgae as a biofuel feedstock: risks and challenges. Int. J.
and its antibacterial efficacy against Gram positive organisms. Mater. Sci. Eng. C 47, Sustain. Dev. World 19, 74–268.
351–356.