ARTIGO

ORIGINAL

VINASSE FROM THE BRAZILIAN LIGNOCELLULOSIC ETHANOL

PROCESS: CHEMICAL COMPOSITION AND POTENTIAL FOR
BIOPROCESSES
VINHAÇA DO PROCESSO DE ETANOL LIGNOCELULÓSICO BRASILEIRO:
COMPOSIÇÃO QUÍMICA E POTENCIAL PARA BIOPROCESSOS

VINAZA DEL PROCESO DE ETANOL LIGNOCELULÓSICO BRASILEÑO:

COMPOSICIÓN QUÍMICA Y POTENCIAL PARA BIOPROCESOS

Recebido em: 19/08/2020 - Aprovado em: 06/01/2021 - Publicado em: 20/04/2021

http://dx.doi.org/10.18011/bioeng2021v15n1p42-68

Manuella Souza Silverio1 ([email protected])

Rubens Perez Calegari2 ([email protected])
Gabriela Maria Ferreira Lima Leite1 ([email protected])
Laysa Maciel Lewandowski Meira Prado1 ([email protected])
Bianca Chaves Martins1 ([email protected])
Eric Alberto da Silva1,3 ([email protected])
José Piotrovski Neto4 ([email protected])
André Gomig4 ([email protected])
Antonio Sampaio Baptista1 ([email protected])

1 University of São Paulo. College of Agriculture, Agroindustry, Food and Nutrition, Piracicaba, SP, Brazil.
2 University of São Paulo. Center of Nuclear Energy in Agriculture, Piracicaba, SP, Brazil.
3 Nuclear and Energy Research Institute. Technology Center of Radiation, São Paulo, SP, Brazil.
4 Engie Brasil. Florianópolis, SC – Brazil.

ABSTRACT

Brazil is the second-largest producer of ethanol and the alcoholic fermentation wastes have become a
concern for both environmental and economic reasons. Recently, the Brazilian industry has implemented
the second generation (2G) process to attend the growing for biofuel. In this study, we aimed to investigate
whether the 2G vinasse faces the same environmental challenges that first generation (1G) vinasses do,
meaning vinasses from ethanol processes using sugarcane juice and/or molasses. Thus, vinasse was
obtained from one of the recently-started 2G ethanol facilities in São Paulo State and then chemically
characterized. Considering glycerol, mannitol, residual sugars, and organic acids concentrations altogether,
it was determined that 2G vinasse had a total carbon source of 23,050 mg L-1 (compared to 4,800 mg L-1
in 1G vinasse). Magnesium, calcium, potassium, and others salts were determined as well. Based on its
chemical composition, vinasses could be considered as nutrient sources for other bioprocesses. Finally, we
brought some perspectives into bioprocesses with nutritional requirements that might be fully or partially
provided by vinasses, leading to the production of bioenergy or bioproducts.
Keywords: Sucroenergetic sector. Chromatographic analyses. Waste valorization. Biorefinery. 2G vinasse.

Artigo publicado sob a licença Creative Commons - Atribuição 4.0 Internacional (CC BY 4.0).

BIOENG, v. 15, n. 1, p. 42-68, 2021. DOI: http://dx.doi.org/10.18011/bioeng2021v15n1p42-68

 Manuella Souza Silverio et al.

1 INTRODUCTION

The sucroenergetic sector plays a very important role in the Brazilian economy,
accounting for about 2% of the Brazilian Gross Domestic Product (UNICA, 2019a).
Internationally, Brazil is an important player in ethanol production as the second-largest
producer in the world. During the 2018/19 crop, Brazil produced 33 billion L of ethanol, and
the largest ethanol producer, the United States, produced about 60 billion L in 2017 (UNICA,
2019b; U.S. ENERGY INFORMATION ADMINISTRATION, 2019). In this scenario, the
Brazilian industry aims to more efficient processes so the growing demand for biofuels may
be met.
According to recent studies on technical-economic evaluation, second generation
(2G) ethanol processes have great potential in systems integrated with first-generation (1G)
processes. Because 2G ethanol production requires obtaining fermentable sugars from
lignocellulosic feedstocks, different methods and operations are required, such as physical-
chemical pretreatments and enzymatic hydrolysis, so production costs are higher when
compared to 1G ethanol process (STEPHEN et al., 2012). Thus, a stand-alone 2G ethanol
process might be more expensive than 1G processes. However, once they are integrated,
2G process provides a higher ratio of volumetric production per sugarcane ton. In addition
to the optimization of facilities use, the integrated process becomes more attractive than
specialized 1G or 2G production facilities (MACRELLI et al., 2014; DIAS et al., 2012).
Co-fermentation of hexoses and pentoses is still challenging for industrial scales.
Therefore, integrated 1G and 2G processes might be configured as separate operations for
1G and 2G fermentations and a single distillation process, with a mixed (1G + 2G) fermented
broth as input (MACRELLI et al., 2014).
Feedstocks, operations and process conditions have significant effects on industrial
wastes composition. As for the 2G ethanol process, it requires different sugar sources and
different operations by employing severe physical-chemical treatments and enzymatic
hydrolysis on lignocellulosic feedstock to obtain fermentable sugars. Regarding ethanol from
sugarcane, 2G ethanol process means using bagasse to obtain a sugar hydrolysate, rich in
hexoses and pentoses. Though some undesired byproducts are commonly generated as
well, such as organic acids, phenolic compounds, and furfuraldehydes (furfural and 5-
hydroxymethylfurfural). During alcoholic fermentation, these byproducts are not significantly
consumed, therefore they might be found in vinasse (JARDINE et al., 2009).
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BIOENG, v. 15, n. 1, p. 42-68, 2021. DOI: http://dx.doi.org/10.18011/bioeng2021v15n1p42-68

 Vinasse from the brazilian lignocellulosic ethanol process: chemical composition and …

The 2G ethanol process is an emerging technology in the Brazilian industry and

extensive research is needed so that effective treatment and management may be
established for 2G organic wastes, based on their specific characteristics and composition.
Vinasse is the most important waste from alcoholic fermentation since it is generated
at very large amounts: in the sugarcane 1G process, every 1 L of ethanol generates an
average ratio of 10-15 L of vinasse (CORTEZ, 2010; ESPAÑA-GAMBOA et al., 2012;
MORAES et al., 2015). For the 2G ethanol process, there is no information for such a
production ratio yet, although the undesired byproducts mentioned above might have
inhibitory effects on fermentative microorganisms and the process might require diluted
fermentation broths.
Vinasse fertirrigation became a common practice in Brazil during the 80s, when
ethanol production had a fast increase because of government incentives. In the 1980/81
crop, Brazil produced 3.7 billion L of ethanol and about 37 billion L of vinasse. By the 1989/90
crop, Brazil produced 11.9 billion L of ethanol and about 119 billion L of vinasse, meaning
an increase of over three times in less than ten years (UNICA, 2020).
Before the 80s, vinasses with no previous treatment were disposed of in rivers or
other water bodies (RIBEIRO et al., 1983; SANTOS et al., 1981). Given the significant
increase in vinasse generation during the 80s, fertirrigation came up as an immediate and
satisfactory alternative for an increasing volume of vinasse in such a short time.
Over the last forty years, the Brazilian industry has continuously increased the annual
ethanol production, which also led to an increase of almost ten times in vinasse volume from
the 1980/81 crop until 2018/19 crop. In the meantime, the Brazilian industry has kept the
same practices regarding vinasse management from the 80s until nowadays.
So, considering a process that employs severe physical-chemical treatments on
lignocellulosic material, such as 2G ethanol process, would vinasse from such process bring
even more risks of soil contamination once applied in fertirrigation? Would legislation based
on potassium content be enough to regulate the safe amounts of 2G vinasse for
fertirrigation? Would there be any other applications for vinasses that could be safer for the
environment, human health, and bring economic benefits as well?
In this study, we characterized the vinasse generated in one of the recently-started
2G ethanol facilities in Brazil, which employs the integrated 1G and 2G process. By
analyzing vinasse composition, we aimed to propose biotechnological applications that
might lead to the production of important bioproducts and bioenergy.
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 Manuella Souza Silverio et al.

2 MATERIALS AND METHODS

2.1 MATERIALS AND SAMPLES PREPARATION

In this study, two types of vinasse were analyzed. A sample of vinasse from an
ethanol process using sugarcane molasse (1G) was used as control for analyses. The 2G
vinasse was obtained from an integrated production unit, 1G + 2G ethanol process. Thus,
in this study, we name 2G vinasse the one composed of a mixture of 1G and 2G vinasses.
Further details on the integrated process, such as 1G and 2G ratios, which operations are
common to 1G and 2G processes or characteristics of fermentative microorganisms were
not provided by the industry due to corporate and legal reasons related to patent deposit.
Both vinasses were obtained in a concentrated form from distilleries in São Paulo
State, Brazil. Before analyses, they were both diluted to in natura concentrations: 1G
vinasse, 3,2 ºBx; 2G vinasse, 3,8 ºBx. Diluted vinasses were not submitted to any other pre-
treatments, except those required by the analytical methods described below. Samples were
stored in 4ºC and kept in room temperature before analyses.

2.2 ANALYTICAL PROCEDURES

2.2.1 Chemical Oxygen Demand (COD)

Vinasses were characterized using the colorimetric method (APHA, 2012). The
following solutions were prepared: (i) 2.04% K2Cr2O7 (m v-1), 3.33% HgSO4 (m v-1) and
0.0167% H2SO4 (v v-1) in distilled water; (ii) 1.012% Ag2SO4 (m v-1) in concentrated
H2SO4. Reactions were prepared using 2.0 mL of diluted sample (1:50), 1.2 mL solution (i)
and 2.8 mL solution (ii).

2.2.2 Total Phenolic Compounds (TPC)

We employed the procedure described by JULKUNEN-TIITO (1985). Samples were
diluted 50 times to fit into the calibration curve.

2.2.3 Organic acids and furfuraldehydes

Organic acids, furfural, and 5-hydroxymethylfufural (HMF) were analyzed in a UFLC
Prominence high-performance liquid chromatography system (SHIMADZU).

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 Vinasse from the brazilian lignocellulosic ethanol process: chemical composition and …

For organic acids characterization, acetic acid, propionic acid, butyric acid, iso-butyric
acid, and lactic acid were analyzed. The system consisted of Aminex HPX-87H (300 mm x
7.8 mm; Bio-Rad) column, at 64 ºC, eluted with 0.005 M H2SO4 at a flow rate of 0.4 mL
min-1, and a UV-Vis/DAD detector (208 nm). Samples were diluted 100 times, acidified with
concentrated H2SO4, until pH ≤ 2.0, as described elsewhere (PENTEADO et al., 2013), and
filtered in 0.45 μm cellulosic membranes. The volume sample was 100 μL.
The furfural and HMF analyses consisted of a system with a Shim-pack VP-ODS (5
μm) of 250 x 4.6 mm column, at 25 ºC, eluted with acetonitrile and acetone (1:8 v v-1) in
acetic acid (1% v v-1), at a flow rate of 0.8 mL min-1, and DAD detector (SHIMADZU SPD-
M20A) (275 nm). Samples were diluted 50 times, filtered in 0.45 μm cellulosic membranes
and analyzed in a volume of 20 μL.

2.2.4 Carbohydrates, anions, and cations

Glycerol, mannitol, sugars, cations, and anions were analyzed using ionic
chromatographic systems (930 IC Compact, Metrohm). All samples were diluted 100 times,
filtered in 0.45 μm cellulosic membranes and analyzed with a sample volume of 20 μL.
Glycerol mannitol, sucrose, glucose, fructose, xylose, and arabinose were
determined in ionic chromatographic system, using Metrosep Carb 1 150/4.0 column, at
30 ºC, eluted with 100 mM NaOH and 10 mM Sodium acetate at a flow rate of 1.0 mL min-
1 and amperometric detector (METROHM, 2016).
Sodium, potassium, ammonium, iron, magnesium, and calcium were determined
using the cation system in ionic chromatograph: Metrosep C4 250/4.0 column, at 30 ºC,
eluted with 7.5 mM tartaric acid, 0.135 mM dipicolinic acid and 3.0 mM ascorbic acid, at a
flow rate of 0.9 mL min-1 and a conductivity detector (METROHM, 2015a).
Chloride, nitrate, nitrite, phosphate, and sulfate were determined in ionic
chromatograph using the anion system: Metrosep A Supp 5 250/4.0, at 25 ºC, eluted with
3.2 mM Sodium carbonate and 1.0 mM Sodium bicarbonate, at a flow rate of 0.7 mL min-1
and a conductivity detector (METROHM, 2015b).

2.2.5 Statistical analysis

All analyses were carried out in triplicates. Calibration curves, descriptive statistical
analyses of means and standard deviation were performed using Microsoft Excel 2010Ⓡ
software.
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 Manuella Souza Silverio et al.

3 RESULTS AND DISCUSSION

3.1 VINASSES COMPOSITION: FEEDSTOCKS AND OPERATIONS INFLUENCE

Table 1 details the chemical characterization for 1G and 2G vinasses. It also provides
reference values, which were obtained from previous studies with 1G vinasse
characterization.

Table 1 – Physical-chemical characterization of 1G vinasse and 2G vinasses.

(mg L-1) 1G Vinasse 2G Vinasse Reference

CORTEZ, 2010;
COD (mgO2 L-1) 26,715.19 ± 161.49 30,969.25 ± 28.18 21,000 - 33,600 MORAES et al.,
2014

Sucrose 285.50 ± 16.02 67.95 ± 7.14

Glucose 88.78 ± 8.91 66.51 ± 1.98

Total residual FERREIRA et
Fructose 130.71 ± 13.05 324.70 ± 11.29
sugars: 962 al., 2011
Xylose ND 105.00 ± 3.29

Arabinose ND 12.16 ± 0.78

Furfural ND ND

HMF ND ND

ORTIZ-MUNIZ
Glycerol 1,970.12 ± 3.43 701.28 ± 2.10 1,400
et al., 2010
DOWD et al.,
Lactic Acid 571.27 ± 14.21 6,869.19 ± 739.71 7,740
1994

Propionic Acid ND ND

Iso-butyric Acid ND ND

PRADO et al.,
Butyric Acid ND 1,021.18 ± 1,444.17 325.61
2016
ESPAÑA-
GAMBOA et al.,
Acetic Acid 1,641.90 ± 700.45 13,844.38 ± 4,189.51 2,200
2012; DOWD et
al., 1994
DOWD et al.,
Mannitol 130.41 ± 1.50 112.98 ± 1.03 89,00
1994
FERREIRA et
TPC 510.28 ± 8.26 2,407.21 ± 21.17 1,100
al., 2011

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Vinasse from the brazilian lignocellulosic ethanol process: chemical composition and …

ESPAÑA-
GAMBOA et al.,
Sulfate 3,440.85 ± 237.40 2,467.17 ± 29.54 23.93 - 5,336
2012; PRADO
et al., 2016
ESPAÑA-
GAMBOA et al.,
2012; MORAES
Potassium 8,746.78 ± 526.10 38,966.43 ± 764.26 600 - 13,000 et al., 2015;
PATHAK et al.,
1999; BISWAS
et al., 2009
FERREIRA et
al., 2011;
PEDRO-
Sodium 4,959.23 ± 236.05 22,264.89 ± 259.31 50 - 31,300
ESCHER et al.,
2014; COELHO
et al., 2018
ESPAÑA-
GAMBOA et al.,
Calcium 960.59 ± 87.77 6,850.61 ± 347.12 450 - 5,180
2011; SOUZA
et al., 2015
ESPAÑA-
GAMBOA et al.,
Magnesium 1,739.63 ± 420.63 8,015.92 ± 231.21 420 - 520
2011; COELHO
et al., 2018
PEDRO-
ESCHER et al.,
Phosphate 109.52 ± 0.61 ND 1.3 - 3,796
2014; REIS et
al., 2015
PEDRO-
ESCHER et al.,
2014;
Nitrate 513.74 ± 0.54 631.69 ± 0.55 1.3 - 17.6
CASSMAN et
al., 2018;
SYDNEY, 2013
NASPOLINI et
Chloride 1,546.98 ± 5.08 978.21 ± 10.22 3,500
al., 2017

Ammonium ND ND

ND: Not Detectable

Source: Original results.

COD in vinasses may vary between 21,000 and 33,600 mgO2 L-1 and it is influenced
by many factors, such as type of feedstocks and their quality (concerning microbial
contamination), unit operations, process conditions and fermentative microorganisms’
metabolism (CORTEZ, 2010; MORAES et al., 2015). COD content in both 1G and 2G

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 Manuella Souza Silverio et al.

vinasses were in accordance with previous studies, however, detailed chemical analysis
revealed very different compositions.
Because lignin is the main source of phenolic compounds, detectable concentrations
are not expected to be found in 1G vinasses, which are obtained from ethanol processes
using sugarcane juice and/or molasses. In our study, TPC concentration in 2G vinasse was
over four times higher than the concentration determined for 1G vinasse. Such a difference
is clear evidence of how feedstock and operations from the ethanol processes might have
an influence on vinasse composition.
The higher concentration we found in 2G vinasse was expected because of the
physical-chemical treatment used in sugarcane bagasse for the 2G ethanol process. Lignin
is a very complex heterogeneous vegetable polymer, formed by several aromatic
compounds. Once they are submitted to severe conditions, such as high temperature and
pressure in physical-chemical treatments, several phenolic compounds may be released
and eventually found in the hydrolysate. Since they are not significantly consumed during
alcoholic fermentation, they are eventually found in vinasse (JARDINE et al., 2009).
As a less usual case for 1G vinasses, FERREIRA et al. (2011) reported 1,100 mg L- 1
of total phenolic compounds in 1G vinasse, almost twice higher than the concentration we
determined for 1G vinasse in this study. That might be explained by the fact that some
lignocellulosic parts, such as leaves and straw, are possibly processed during sugarcane
milling for syrup production. During sugar production, syrup is submitted to very high
temperatures and then molasse is obtained as a byproduct, which is further used as carbon
source in alcoholic fermentation (SAHU, 2018). Such residual compounds are not supposed
to be consumed during the fermentation process, so, as a result, they will be found in 1G
vinasse.
Lactic acid concentration in 2G vinasse was considerably higher than that determined
in 1G vinasse. However, our results were consistent with the literature (DOWD et al., 1994),
which reports that elevated lactic acid concentrations are common in industrial alcoholic
fermentation since they are a bacterial contamination product.
Other organic acids may be indicators of bacterial contamination as well, such as
propionic, iso-butyric, butyric, and acetic acids. Neither propionic acid nor iso-butyric acid
was found in detectable concentration in any of vinasses. Butyric acid was determined only
in 2G vinasse, in higher concentrations than those previously reported in the literature for
1G vinasses (PRADO et al., 2016).
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In comparison to the 1G vinasse we analyzed, and also previous reports in the

literature, acetic acid concentration in 2G vinasse was notably higher (ESPAÑA-GAMBOA
et al., 2012; DOWD et al., 1994).
In the 1G ethanol process, the acetic acid in fermented broths and vinasse results
mostly from yeast metabolism and, possibly, from acetic bacteria metabolism as well,
whenever there is contamination (LOPES et al., 2016).
In the 2G ethanol process, however, there might be acetic acid production from both
yeasts and bacteria metabolisms, but the enzymatic treatment on sugarcane bagasse may
be the main source. Enzymes are used to extract fermentable sugars from hemicellulose,
which is a heterogeneous polymer composed by units of pentoses, hexoses, and acetyl
groups. After acetyl groups are released into the fermentation broth, they are converted into
acetic acid and they are not significantly consumed during alcoholic fermentation. As a
result, high acetic acid concentrations might be found in 2G vinasses (JARDINE et al.,
2009). For these reasons, acetic acid concentration was indeed expected to be found in high
concentration in the 2G vinasse.
Furfural and HMF were expected to be found in 2G vinasse, since the 2G ethanol
process may lead to sugars degradation due to high temperature and elevated pressure
conditions in physical-chemical treatments. Furfural is formed from pentoses degradation
and HMF results from hexoses degradation (JARDINE et al., 2009). However, our analyses
found no detectable concentration of such compounds.
Residual sugars were detected in both vinasses. In 2G vinasse, xylose and arabinose
were detected, as well as other residual sugars from alcoholic fermentation, such as
sucrose, glucose, and fructose. Residual pentoses were expected to be found in 2G vinasse
since 2G ethanol exploits both hexoses and pentoses from sugarcane bagasse.
Similarly, 1G vinasse was also characterized in residual hexoses. Total residual
sugars concentrations were similar between vinasses in our study: 504.99 mg L-1 in 1G
vinasse and 576.32 mg L-1 in 2G vinasse. Because sugars are exhausted in fermentation,
very low concentrations of residual sugars are commonly detected in vinasses (FERREIRA
et al., 2011).
Glycerol was determined in both vinasses and concentrations were consistent with
previous studies (ORTIZ-MUNIZ et al., 2010). Glycerol is one of the main yeast metabolites
in alcoholic fermentation since its metabolic pathway is related to redox balance
maintenance and osmotic stress response (NEVOIGT & STAHL, 1997).
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 Manuella Souza Silverio et al.

Mannitol, along with organic acids, is an indicator of bacterial contamination and the
concentrations we determined for both 1G and 2G vinasses in our study were higher than
those reported elsewhere (LOPES et al., 2016; EGGLESTON et al., 2007).
Vinasses usually have very high concentrations of sulfate due to many operations
along the global process. During the sugar production process, sulphitation is applied for
crystal sugar production. Thus, some residual forms of sulphur are generated in molasses
and converted into sulphate, which will remain in the broth during alcoholic fermentation,
and finally in vinasse (SAHU, 2018).
As for the ethanol production process, after alcoholic fermentation, it is common to
apply H2SO4 on cream yeast, which is known as the acid treatment. The operation is useful
to decrease bacterial contamination during inoculum recycling for the next fermentation
batch (OLIVA-NETO & YOKOYA, 2001). Acid treatment may also generate residual forms
of sulphur, such as sulphate, which are eventually found in vinasse. Still, some authors
suggest H2SO4 to be used in physical-chemical pre-treatments on sugarcane bagasse for
2G ethanol process (JARDINE et al., 2009), which could be yet another possible source of
sulfates in 2G vinasses. However, this type of information was not confirmed by the industry
that provided the 2G vinasse for this study. Sulfate concentrations from our analyses were
consistent with those in literature for 1G vinasses (ESPAÑA-GAMBOA et al., 2012; PRADO
et al., 2016).
In our study, nitrate was determined in higher concentrations than those found in
literature, for both 1G and 2G vinasses (PRADO et al., 2016; PEDRO-ESCHER et al., 2014;
CASSMAN et al., 2018; SYDNEY, 2013). Ammonium was investigated and no detectable
concentration was found for neither vinasses.
Other components, such as salts of sodium, calcium, magnesium, chloride, and
potassium were determined in important concentrations. These salts have been previously
reported by other authors with highly variable concentrations in 1G vinasses. In our
analyses, 2G vinasse had higher concentrations for calcium and magnesium salts than
those reported elsewhere, for 1G vinasses. Sodium and chloride concentrations in 2G
vinasse were following other authors’ results with 1G vinasse samples. As for our analysis
with 1G vinasse, concentrations of sodium, calcium, chloride, and phosphate salts were
consistent with those reported elsewhere. No detectable concentration for phosphate was
determined in 2G vinasse (COELHO et al., 2018; PEDRO-ESCHER et al., 2014; FERREIRA

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 Vinasse from the brazilian lignocellulosic ethanol process: chemical composition and …

et al., 2011; ESPAÑA-GAMBOA et al., 2012; SOUZA et al., 2015; REIS et al., 2015;
NASPOLINI et al., 2017).
Potassium content in vinasses is commonly very high, ranging from 600 to 13,000
mg L-1 (ESPAÑA-GAMBOA et al., 2012; MORAES et al., 2015; PATHAK et al., 1999;
BISWAS et al., 2009). In our analyses, potassium content in 1G vinasse was consistent with
those found by other authors, whereas potassium content in 2G vinasse was highly above
that range.
São Paulo State accounts for the most important share of ethanol production in Brazil
and is the only region in the country where vinasse fertirrigation must follow a governmental
technical regulation. Since potassium salts are usually the most abundant in vinasses, its
concentration should be considered for vinasse application in soil in order to avoid nutrients
imbalance and salinization (CETESB, 2013).
In this regard, 2G vinasse management could be an even greater challenge if
fertirrigation is to be considered. According to the São Paulo regulation, lower volumes of
2G vinasse would be allowed per area, which means transporting vinasse to further fields.
Costs with fuel for trucks, labor and specialized material for vinasse storage and
transportation (by trucks, channels or lagoons) are implicated.
Considering fertirrigation, 2G vinasse might be economically very challenging.
Moreover, calcium, sodium, magnesium salts, and organic acids were found in very high
concentrations as well. So, not only from the economic point of view but also for
environmental concerns, 2G vinasse might arise the urgent need for alternative and more
innovative technologies in its management strategies.

3.2 VINASSE MANAGEMENT CHALLENGES: ADVANTAGES, DISADVANTAGES OF

FERTIRRIGATION AND OTHER TECHNOLOGICAL OPPORTUNITIES

Vinasses might have a variable composition, but in general, they are interesting
sources of salts, carbon (organic acids and glycerol), other nutrients, and even water. In
Brazil vinasse fertirrigation is an important supply of these nutrients for sugarcane fields.
Moreover, previous studies have showed that vinasse fertirrigation might indeed promote
higher sugar production by increasing the sugarcane growth and rooting (MEDINA et al.,
2002; PAULINO et al., 2002).

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 Manuella Souza Silverio et al.

An important factor about vinasse fertirrigation is that it means a low-cost investment,

with low-cost maintenance, as well. The use of vinasse in sugarcane fields demands
facilities such as piping, pumps, channels, trucks, and storage lagoons. The nutrients
recycling by fertirrigation also means purchasing less fertilizers for sugarcane crops
(CHRISTOFOLETTI et al., 2013).
In the last years many studies have been focusing on the impacts that fertirrigation
might have on the quality of soil and groundwaters.
According to literature, the continuous vinasse fertirrigation in a certain area means
the continuous addition of specific nutrients, such as salts and organic acids, which might
lead to an unbalanced composition of nutrients in the soil (SILVA et al., 2007; OLIVEIRA et
al., 2015; PEDRO-ESCHER et al., 2014).
Researchers reported that soil physical structure might be altered as a consequence
of such chemical imbalance. The result is that fertirrigated areas will eventually be salinized
and potassium, sulfate, nitrate, and metals might be leached through soil inner layers and
contaminate superficial and groundwaters (SILVA et al., 2007; CASSMAN et al., 2018;
CHRISTOFOLETTI et al., 2013).
SOTO et al. (2015) suggested that, depending on environmental conditions, soil, and
vinasse characteristics, vinasse percolation might occur between one to three years after
fertirrigation.
Salts have usually been the greatest concern in vinasse composition because of their
potentially negative effects on soil salinization. However, recent studies have quantified
greenhouse gases (GHG) emissions from vinasse fertirrigated areas. The results indicated
that emissions are significant and environmental concern with vinasse should be wider than
soil degradation (OLIVEIRA et al., 2015; CASSMAN et al., 2018).
Considering all these aspects, 2G vinasse might bring the same environmental
concerns. As determined in our study, 2G vinasse presented as many salts and organic
acids as 1G vinasse, or more. In our analyses, 2G vinasse had higher concentrations of
potassium, sodium, nitrate, lactic acid, acetic acid, butyric acid, and TPC than those
determined in 1G vinasse. Further studies with 2G are definitely needed, considering that
more vinasses from different locations and processes should be analyzed. However, our
results have already indicated that 2G vinasse might bring the same environmental
problems known to be related to 1G vinasse.

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 Vinasse from the brazilian lignocellulosic ethanol process: chemical composition and …

There is great interest in continuing with vinasse fertirrigation because nutrients from
sugarcane can be recycled to crops and have positive effects on sugarcane growth. Given
the advantages of vinasse fertirrigation, more sustainable management could start by
employing the biodigested vinasse.
MORAES et al. (2017) have compared the GHG emissions during the transportation
of in natura and biodigested vinasses for fertirrigation. Their results showed that biodigested
vinasse did not show any detectable CH4 emissions. As for N2O emissions, they observed
a decrease of about 48% to 78% in comparison to in natura vinasse.
The biodigested vinasse means the product of anaerobic digestion (AD), which is the
bioprocess that consumes dissolved carbon compounds, converting them into biogas. The
main product of AD is the biomethane, which can be further purified and used in energy
generation. Still, in an ethanol distillery scenario, the biodigested wastewater would also
have an important role as a fertilizer, since potassium, magnesium, calcium, and other salts
are not significantly removed during biogas production (BARROS et al., 2017; LÓPEZ-
LÓPEZ et al., 2015).
Fertirrigation and biogas production have important features that make them very
interesting alternatives for vinasse management in the Brazilian industry. However, vinasse
is generated in very large volumes and, despite AD being a very efficient and well-
established technology, multiple strategies are needed.
AD with concentrated vinasses has been previously investigated as a more efficient
alternative for vinasses (NACHEVA et al., 2009). And recently, many studies have been
expanding knowledge about wastewaters’ valorization. For that reason, many residues from
different processes have been studied as potential culture media components. Among
those, dairy effluents, molasses, paper mill effluents, winery wastewaters, food processing
wastes, whey thin stillage, crude glycerol, and many others have been investigated
(RATHIKA et al., 2018; KADIER et al., 2014; REVIN et al., 2018; SANTOS et al., 2016).
Table 2 provides some information from studies in which agroindustrial wastes,
including sugarcane vinasse (1G), were evaluated as a component of culture medium for
biofuel or bioproducts synthesis.
These bioprocesses employ bacteria or a consortium of microorganisms. In Table 2
there are bioprocesses with Pseudomonas spp. and Bacillus spp., for biosurfactant
production (NASPOLINI et al., 2017; MD, 2012), Xanthomonas campestris for xanthan gum
production (non-food applications) (BECKER et al., 1998), Bacillus spp. for bioplastic
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 Manuella Souza Silverio et al.

production (RATHIKA et al., 2018; DESOUKY et al., 2017), Acetobacter spp. and
Gluconacetobacter spp. for bacterial cellulose (BC) production (REVIN et al., 2018; ESA et
al., 2014) and Corynebacterium glutamicum for amino acids production (animal feed)
(BECKER et al., 2011). These species are able to consume saccharides, but glycerol and
organic acids as well, making them potentially suitable for growth and biosynthesis in
vinasse.
Some bioprocesses in Table 2 are already well-established in the industry, as AD,
xanthan gum, and amino acids production. Other bioprocesses, such as biosurfactants,
bioplastics, BC production, and microbial electrolysis cell (MCE) are not yet well-established
in large scales, so they are majorly in early stages and/or scaling up to pilot scale studies.

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Vinasse from the brazilian lignocellulosic ethanol process: chemical composition and …

Table 2. Bioprocesses, nutrient requirement, products and application.

Bioprocess Nutrient Product Product’s Current state of Does it need Complex Challenges in References
requirements for applications technology supplementation? substrates using vinasse
culture medium development previously
tested
Anaerobic COD 12,100 - CH4 Biofuel Well No Sugarcane High sulfate ESPAÑA-GAMBOA et
digestion 44,500 mgO2 L- established in vinasse, tequila concentrations al., 2012; BARROS et
1; Potassium industry vinasse, swine might lead to al., 2017; LÓPEZ-
(0.149 - 7.2 g L- wastewater, H2S production, LÓPEZ et al., 2015
1), Calcium domestic which is a
(0.005 - 0.32 g L- wastewater corrosive gas
1); Sodium (0.16

g L-1); sulfate
(1.0 - 5.336 g L-
1); phosphates
(0.141 g L-1);
Magnesium
(0.18 g L-1)
acetic acid
(2.237 g L-1);
propionic acid
(4.3 g L-1)

Biosurfactant Glycerol (30 g L- Glycolipids Mobilizing agent Laboratory Glycerol or other Sugarcane Studies on NASPOLINI et al.,
1);
production Sodium rhamnolipids in agriculture; scale carbon source vinasse, standardization 2017; BECKER et al.,
nitrate (1.2 - 4.0 detergent; supplementation; molasses, of vinasse 1998; BENINCASA et
g L-1); antimicrobial; phosphates; trace crude oils, corn application are al., 2002
phosphates (0.5 biopesticide; elements, such as steep liquor still required
- 10 g L-1); applied in crude B, Cu, Mn, Mo, Zn
Magnesium oil and
sulfate (0.2 - 0.5 hydrocarbons
g L-1); degradation
Potassium
chloride (0.1 g L-
1); ferrous
sulphate II (0.01
g L-1); Calcium
chloride (0.01 g
L-1); yeast
extract (0.01 g L-
1)

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Manuella Souza Silverio et al.

Xanthan gum Sucrose or Acidic polysaccharide Thickener and Well Carbon (sucrose or Sugarcane In industry, SANTOS et al., 2016;
production glycerol (20 g L- emulsifier for established in glycerol) and molasse; xanthan gum is BECKER et al., 1998;
1); urea (0.1 g L- food, oil, industry nitrogen (urea or glycerin; stach produced from BRANDÃO et al., 2013;
1); phosphates pharmaceutical, yeast extract); hydrolysates; sugarcane RONCEVIC et al., 2014
(1- 3 g L-1); cosmetic, paper, green coconut juices or
yeast extract (3 paint and textile shell molasses, which
g L-1); sulphate industries; hydrolysate; have a very
ammonium (1.5 gelling and straw similar
g L-1); suspending hydrolysate composition in
Magnesium agent comparison to
sulphate (0.3 g sugarcane
L-1) vinasse, except
for the carbon
source
composition.
Using vinasse
could decrease
production costs

Bioplastic Glucose, Polyhydroxyalkanoates Packaging, Mainly Carbon source Sugarcane Studies have RATHIKA et al., 2018;
production sucrose or moulded goods, laboratory and (sucrose or molasse; already DESOUKY et al., 2017;
glycerol (10 - 20 coatings, pilot scale, still glycerol); nitrogen cheese whey; evaluated KHIYAMI et al., 2011
g L-1); adhesives, films emerging in source wheat bran; sugarcane
ammonium (0.6 industry (ammonium); paper mill molasse, which
- 1.5 g L-1); phosphates effluent; dairy is a substrate
phosphates (7 g whey with similar
L-1); sulphates composition in
(0.72 g L-1); comparison to
Calcium (0.084 vinasse, except
g L-1) for the carbon
source
composition.

Bacterial Glycerol (2.39 - Cellulose Food packaging; Laboratory Nitrogen sources Wheat thin Studies on REVIN et al., 2018; ESA
cellulose 7,87 g L-1); lactic reinforcement scale might be required. stillage; cheese stardadization of et al., 2014;
acid (5.07 - 7.41 material for whey; waste vinasse use are RATANAPARIYANUCH
g L-1); acetic electronic and beer yeast; still required; et al., 2011
acid (0.56 - 2.72 biomedical grape skin; oil using vinasse
g L-1); succinic materials; paper mill residue could decrease
restoration

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Vinasse from the brazilian lignocellulosic ethanol process: chemical composition and …

acid (0.63 - 0.93 production

g L-1) costs.
Amino acids Sucrose (10 - 30 Lysine, threonine, Animal feed Well stablished Carbon (sucrose), Sugarcane, Amino acids BECKER et al., 2011;
g L-1); yeast tryptophan supplementation in industry nitrogen beet molasses; production with YING et al., 2014; LIU et
extract (5 g L-1); (ammonium) starch complex culture al., 2016
ammonium (10 - sources; hydrolysates media is well
36 g L-1); phosphates established,
Magnesium including
sulphate (0.2 - sugarcane
1.5 g L-1); molasses, which
phosphates have a similar
(0.25 - 3.0 g L-1) composition in
comparison to
sugarcane
vinasse, except
for the carbon
source
composition.

Microbial COD 6,500 H2 Biofuel Laboratory No Lignocellulosic MEC is a LU et al., 2009; KADIER
electrolysis cells mgO2 L-1; scale biomass recently et al., 2014
(MEC) residual wastes; developed
reducing sugars molasses; technology and
(0.2 g L-1); acetic domestic it is not yet ready
acid (0.74 g L-1); wastewater; for large scale
propionic acid fermentation implementation.
(0.35 g L-1); effluents in Most studies still
butyric acid (0.2 general; food focus on higher
g L-1) processing yields, suitable
wastewaters material for
electrodes,
costs and
substrates
optimization in
general.

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 Manuella Souza Silverio et al.

Regardless of the current state of technology development, vinasses have potential

as nutrient sources for biofuels and bioproducts generation, providing a wider view for
vinasse application.
Although many factors contribute to the exact determination of culture medium
composition for a specific bioprocess, such as the microorganism strain, bioproduct
characteristics, biosynthesis conditions, downstream operations, and others, some general
information about the main microbial nutrient requirements are presented (Table 2).
As mentioned above, the employed bacteria are capable of consuming different types
of carbon sources. Glycerol might be the chosen carbon source for biosurfactant, xanthan
gum, and BC production. Despite not being well established, amino acid production by
Corynebacterium glutamicum is also possible through glycerol consumption by engineering
strains for that purpose (RITTMANN et al., 2008). Besides, organic acids found in vinasse
might be important carbon sources in BC and MCE processes. As for AD, the bioprocess is
carried out by a consortium of microorganisms, involving many bacterial and archea species,
which consume a wide variety of carbohydrates and organic acids, especially acetic acid
(STAMS, 1994).
The vinasses we characterized in our study had an important carbon concentration
considering glycerol, mannitol, residual sugars, and organic acids altogether. For 1G
vinasse, we determined over 4,800 mg L-1 of carbon sources, and for 2G vinasse, over
23,050 mg L-1. Thus, both vinasses, but most importantly 2G vinasse, have interesting
carbon sources concentrations to be exploited, meaning energetic resources to be seized
by industrial microorganisms.
For all the bioprocesses listed in Table 2, there are possible challenges in applying
vinasse. High sulfate concentrations (AD), the lack of studies on the specific usage of
vinasse as substrate (xanthan gum, bioplastic, BC and amino acids production), replacing
the use of sugarcane molasse by sugarcane vinasse (biosurfactant, xanthan gum,
bioplastic, and amino acids) or the technological development itself (MCE). On the other
hand, all these bioprocesses have been extensively studied with complex components for
culture media, including some agro-industrial wastes.
Finally, taking into consideration the biorefinery concept, the use of vinasse in other
bioprocesses could also mean water recycling. Different from for water treatment
technologies that employ resins and membranes, with high investment and maintenance

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 Vinasse from the brazilian lignocellulosic ethanol process: chemical composition and …

costs, keeping vinasse inside a production facility and employing it in the production of
bioproducts could optimize logistics and resources use.
Expanding studies on vinasse application in bioprocesses could turn a wastewater,
with high pollutant potential, into a nutrient source for processes with economic and
environmental benefits.

4 CONCLUSIONS

Some typical compounds obtained from lignocellulosic physical-chemical and

enzymatic pretreatments were found at very high concentrations in 2G vinasse, namely
acetic acid, and total phenolic compounds.
Contents of potassium, sodium, calcium, magnesium, nitrate, sulfate, and other
organic acids were higher than those we determined for 1G vinasse. These compounds
might bring risks to the environment once vinasse is commonly employed in fertirrigation.
Therefore, 2G vinasse might bring the same risks for soil acidification and leaching.
However, these same compounds are needed in other bioprocesses. That makes
both 1G and 2G vinasses interesting materials as a potential source of nutrients for
biotechnological applications.

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 RESUMO
RESUMEN

RESUMO

O Brasil é o segundo maior produtor de etanol e os resíduos gerados pela fermentação

alcoólica têm levantado questões econômicas e ambientais. Recentemente a indústria
brasileira implantou o processo de segunda geração (2G) para atender às crescentes
demandas pelo biocombustível. Neste estudo, foi investigado se a vinhaça 2G tem potencial
para trazer os mesmos desafios ambientais associados à vinhaça de primeira geração (1G),
isto é, aquela proveniente dos processos de etanol a partir de caldo e/ou melaço de cana-
de-açúcar. Foi coletada vinhaça de uma das unidades de etanol 2G recentemente
instaladas no Estado de São Paulo e, então, caracterizada quimicamente. Considerando-
se glicerol, manitol, açúcares residuais e ácidos orgânicos, determinou-se que a vinhaça
2G possui um total de fonte de carbono de 23.050 mg L-1 (comparado a 4.800 mg L-1 na
vinhaça 1G). Magnésio, cálcio, potássio e outros sais também foram determinados. Com
base na composição química, vinhaças podem ser consideradas fontes de nutrientes para
bioprocessos. Finalmente, foram abordados bioprocessos cujas demandas nutricionais
podem ser completa ou parcialmente garantidas pelas vinhaças, resultando na produção
de bioenergia ou bioprodutos.
Palavras-chave: Setor sucroenergético. Análises cromatográficas. Valorização de
resíduos. Biorrefinarias. Vinhaça 2G.
RESUMEN

Brasil es el segundo más grande productor de etanol y los residuos generados por la
fermentación alcohólica han levantado cuestiones económicas y ambientales.
Recientemente la industria brasileña implanto el proceso de segunda generación (2G) para
atender a las crecientes demandas por el biocombustible. En este estudio, fue investigado
se la vinaza 2G tiene potencial para traer los mismos desafíos ambientales asociados a la
vinaza de primera generación (1G), aquella proveniente de los procesos de etanol a partir
de caldo y/o melaza de caña de azúcar. Fue colectada vinaza de una de las unidades de
etanol 2G recientemente instaladas en el Estado de São Paulo y, así, caracterizada
químicamente. Considerándose glicerol, manitol, azúcares residuales y ácidos orgánicos,
se determinó que la vinaza 2G posee un total de fuente de carbono de 23.050 mg L -1
(comparado a 4.800 mg L-1 en la vinaza 1G). Magnesio, calcio, potasio y otras sales también
se determinaron. Con base en la composición química, vinazas pueden ser consideradas
fuentes de nutrientes para bio-procesos. Finalmente se abordaron bio-procesos cuyas
demandas nutricionales pueden ser completa o parcialmente garantidas por las vinazas,
resultando en la producción de bioenergía o bio-productos.
Palabras clave: Sector sucroenergético. Análisis cromatográficas. Valorización de
residuos. Biorrefinerías. Vinaza 2G.

BIOENG, v. 15, n. 1, p. 42-68, 2021. DOI: http://dx.doi.org/10.18011/bioeng2021v15n1p42-68

 NOTAS
EDITORIAIS

LICENÇA DE USO
Este é um artigo publicado em acesso aberto (Open Access) sob a licença Creative
Commons Atribuição 4.0 Internacional (CC BY 4.0), que permite uso, distribuição e
reprodução em qualquer meio, desde que o trabalho original seja corretamente citado. Mais
informações em: http://creativecommons.org/licenses/by/4.0

CONFLITO DE INTERESSES
Os autores declaram que não há conflito de interesses neste trabalho.

CONTRIBUIÇÕES AUTORAIS
Manuella Souza Silverio: Conceptualization; Laboratorial execution; Data analysis; Writing
– original draft preparation.
Rubens Perez Calegari: Conceptualization; Data analysis; Writing – review and editing.
Gabriela Maria Ferreira Lima Leite: Laboratorial execution.
Laysa Maciel Lewandowski Meira Prado: Laboratorial execution.
Bianca Chaves Martins: Laboratorial execution.
Eric Alberto da Silva: Laboratorial execution.
José Piotrovski Neto: Resources; Funding.
André Gomig: Resources; Funding.
Antonio Sampaio Baptista: Conceptualization; Methodology; Writing – review and editing;
Resources.

FINANCIAMENTO
This study was financed by Engie Brasil Energia S.A. and by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES), Finance Code 001.

COMO REFERENCIAR
SILVERIO, Manuella Souza; CALEGARI, Rubens Perez; LEITE, Gabriela Maria Ferreira
Lima; PRADO, Laysa Maciel Lewandowski Meira; MARTINS, Bianca Chaves; da SILVA,
Eric Alberto; NETO, José Piotrovski; GOMIG, André; BAPTISTA, Antonio Sampaio.
Vinasse from the brazilian lignocellulosic ethanol process: chemical composition and
potential for bioprocesses. Revista Brasileira de Engenharia de Biossistemas (Tupã),
v. 15, n.1, p. 42-68, 2021. DOI: http://dx.doi.org/10.18011/bioeng2021v15n1p42-68.

RESPONSABILIBADE EDITORIAL
Prof. Dr. Fernando Ferrari Putti1, Prof. Dr. Paulo Sérgio Barbosa dos Santos1,
Prof. Dr. Eduardo Festozo Vicente1 e Prof. Dr. Diogo de Lucca Sartori1
1Universidade Estadual Paulista "Júlio de Mesquita Filho", FCE - Faculdade de Ciências e
Engenharia, Tupã, SP, Brasil.

BIOENG, v. 15, n. 1, p. 42-68, 2021. DOI: http://dx.doi.org/10.18011/bioeng2021v15n1p42-68