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

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.


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 physicalchemical 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 5hydroxymethylfurfural). During alcoholic fermentation, these byproducts are not significantly consumed, therefore they might be found in vinasse (JARDINE et al., 2009 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.

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 pretreatments, except those required by the analytical methods described below. Samples were stored in 4ºC and kept in room temperature before analyses.

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

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.
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).

Statistical analysis
All analyses were carried out in triplicates. 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). 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 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.  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.
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.

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 PAULINO et al., 2002). 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. 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 wellestablished 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 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. 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.
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 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.

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.