Statement of Novelty

We declare that this manuscript is new because it has shown that the use of molasses was efficient in supplementing vinasse, allowing yeast growth and the production of volatile compounds. In addition, some yeasts were efficient when grown in vinasse supplemented with molasses to increase bioaroma and biomass production, combining the reduction of fermentation cost and the toxicity level of this byproduct. This paper shows the integration process between reuse and value-added products such as 2-phenylethanol and the reduction of vinasse toxicity. Thus, reduce the cost to the industry due to the use of co-product in place of glucose and reducing the degree of contaminant released into the environment.

Introduction

High biomass production and ease of growth have made sugarcane one of the most important agricultural products in the world cultivated in more than 100 countries; Brazil and India being the largest producers [1, 2]. Sugar cane is the main raw material to produce sugar, ethanol and distilled alcoholic beverages. In Brazil, spirit beverage is much appreciated specially the artisanal cachaça make by small and media farmers. Despite the great economic importance of the sector, the sugarcane industry is responsible for the generation of different types of wastes, such as vinasse (286–678 × 106 m3—estimated), bagasse (169 × 106 tons) and filter cake (22 × 106 tons) [3]. Among them, vinasse is the major waste (10 L vinasse/1 L of cachaça) [4] and more polluting because it is liquid waste reaching into the soil and water sources. The lack of inspection and the high volume produced in each cycle, become the correct disposal a challenge.

Vinasse is an acidic liquid, dark brown color that, after the distillation of the cane must to obtain the cachaça, has a temperature of approximately 107 °C and astringent odor [5]. Vinasse causes higher soil and aquatic environmental impacts in the consequence of high concentrations of phenols, heavy metals, high biological and chemical oxygen demand (BOD/COD), but low soluble carbohydrates. Part of the vinasse is used as bio-fertilizer, incineration to energy power and other [6]. However, considerable volume is not adequately treated, and it is essential to reduce the level of toxicity of these wastes before it is disposed in the environment [7]. This way, the new alternatives should be simpler and lower-cost. One of them, is aerobic microbial process, wich ally treatment and bioproducts production. Then, the transformation of simple sugarcane mills into biorefinery has allowed the diversification of the sector, generating a wide range of other products from microorganisms, such as volatile compounds and microbial biomass, SCP [8,9,10] Thus, the waste is used to a new process, which changes the physical–chemical composition of waste decreasing of the organic load [11].

Microbial biomass and volatile compounds produced by microorganisms like yeasts, bacteria and filamentous fungi from agro industrial wastes appear as economical and environmentally favorable alternatives that allow to obtain products with a high added value [12, 13]. Reis et al. [11] showed that vinasse is appropriate substrate since have carbon source supplementation. To lower costs, we test molasses as supplementation and evaluated the performance of add-value products producing and a new alternative to reuse the spent waste. Moreover, we analyzed if the quality of biomass is appropriate to use as SCP in animal feed.

Material and Methods

Substrates

Vinasse was provided by a traditional unit of cachaça production located in the state of Minas Gerais, Brazil (21° 04′ 05.9″ S, 44° 59′29.1″ W). Molasses was obtained from a commercial market, in Lavras (21° 14′ 43″ S, 44° 59′ 59″ W), Minas Gerais, Brazil.

The vinasse and molasses were collected in sufficient volumes of 20 and 15 L respectively to perform the whole experiment. Immediately after the sampling, the vinasse and molasses were frozen and, when necessary, the desired volume was thawed, filtered and sterilized at 121 °C for 15 min.

Physicochemical Analysis

The fermentation media contained vinasse and molasses in two different proportion. First one, consisted of (%) 50:50 vinasse: molasses (medium 1) and the second one, 70:30 (medium 2). Samples of vinasse and molasses in natura, no spent and spent of the media1 and 2, were submitted the physicochemical analysis of pH, total nitrogen, carbohydrates and alcohols. The pH analyzes were determined in pH meter and analyzes for total nitrogen were determined according to the American Public Health Association [14]. Moreover, these samples were analyzed on the production of volatile compounds and sub-chronic toxicity, described lately.

Analysis of carbohydrate (sucrose, glucose and fructose) and alcohols (glycerol, methanol and ethanol) were performed in HPLC—high performance liquid chromatography, according to the methodology of Duarte et al. [15], with some modifications. The identification of compounds was performed using a Shimadzu liquid chromatography (Shimadzu Corp., Japan) equipped with a refractive index detector (RID-10Ai) and a Shim-pack SCR-101H ion exclusion column (300 mm × 7.9 mm i.d, 10 mm) made by a combination of gel filtration and binder exchange. The operating temperature used was 30 °C with a mobile phase of perchloric acid (pH 2.1) and with a constant ratio quotient of 0.6 mL min−1. All samples were evaluated in duplicate.

Inoculum Preparation

The microorganisms used in the fermentation process belong to the Culture Collection of Agricultural Microbiology (CCMA), the Department of Biology, the Federal University of Lavras, Brazil. Five yeast strains were used: (i) Saccharomyces cerevisiae CCMA 0186, isolated from the fermentation of cachaça; (ii) S. cerevisiae CCMA 0188, isolated from the ethanol production (LNF CA-11); (iii) Candida parapsilosis CCMA 0544; (iv) C. glabrata CCMA 0193 e; (v) Meyerozyma caribbica CCMA 0198, isolated from the coffee fermentation. Yeasts used in this work were previously selected based on the production of SCP in vinasse [4].

Strains were reactivated (0.1 mL) in the YEPG broth (% m/v: 1.0 peptone, 1.0 yeast extract e 2.0 glucose) and incubated at 28 °C/48 h. After that, 20 μL were plated in the YEPG medium and incubated again at 28ºC/48 h.

For the preparation of the inoculums, each yeast strain was picked up in a test tube containing 10 mL of YEPG broth and incubated at 28 °C/48 h at 150 rpm. Subsequently, each culture was transferred to Erlenmeyer containing 100 mL of YEPG broth, incubated at 28 °C/150 rpm until reaching a population of 7 log CFU mL−1. The inoculums were centrifuged (9000×g at 4ºC/10 min.) and the biomass obtained was washed three times in sterile distilled water.

Fed Batch in Discontinuous Fermentation

The fermentation process was carried out by a fed batch process, where 10% (v/v) of the centrifuged inoculums was transferred to 2 L Erlenmeyer containing an initial volume of 450 mL of culture medium (media 1 or 2). The assays were incubated at 28 °C/150 rpm for 120 h long with a continuous feed of 10% (v/v) every 12 h, a totalizing culture medium volume of 1060 mL at the end of fermentation. Before feeding, samples of 7 mL were taken every 12 h for counting cells in Neubauer chamber, pH evaluation and chemical analyzes. The five yeast strains were tested in both media.

After 120 h of fermentation for biomass determination, the assays were centrifuged (9000×g at 4 °C for 10 min) and the cell mass was quantified after oven drying at 65ºC until constant weight. All experiments were carried out in a Randomized Block Design, in triplicate for the five yeast strains, totaling at 30 assays.

Total Protein Content in Microbial Biomass (SCP)

Biomass produced in medium 1 and 2 were evaluated according to the content of nitrogen and crude protein. Nitrogen content was determined by the Kjeldahl method [14] and crude protein content was estimated using the conversion factor of 6.25 (N × 6.25).

Production of Volatile Compounds

Analysis of volatile compounds was performed by gas chromatography (GC), according to Arellano et al. [16], with modifications. The identification of compounds was performed using a Shimadzu gas chromatograph model 17A, equipped with a flame ionization detector (FID) and DB-WAX column (30 m × 0.25 mm i.d. × 0.25 µm) (J&W Scientific, Polsom, CA, USA).

The initial temperature used was 40ºC, increasing 3 °C/min to 120 °C and increasing 6 °C/min, to 200 °C. The injection and detection temperature were maintained at 240 °C and 250 °C, respectively. The carrier gas (N2) was maintained at a flow rate of 2 mL min−1. All samples were evaluated in duplicate. Volatile compounds (acetaldehyde, amylic alcohols, butanol, ethanol, ethyl acetate, ethyl butyrate, phenethyl acetate, 2-phenyl ethanol, ethyl hexanoate, isoamyl acetate, isobutanol, ethyl lactate, methanol, 1-propanol) were identified by comparing the retention times of the compounds found with the retention times of the pattern of compounds injected under the same conditions [10].

Morphological Analysis of Yeasts in SEM

The impact of media composition on yeast cellular morphology were prepared as described previously by Oliveira et al. [17] and evaluated using Scanning Electron Microscopy (SEM). If in stress condition, there are morphological changes. So, the budding and cellular shape of the yeasts grown in media 1 and 2, at the end of the fermentation time was compared to the morphology yeasts in YEPG broth (control).

The samples were observed in a scanning electron microscope (LEO EVO 40XVP) coupled to a dispersive energy system (X-ray microanalysis-EDS).

Analysis Subchronic Toxicity

Onion seed (Allium cepa) germination was used to phytotoxicity test [18], with modifications]. To carried out this, vinasse and molasses in natura, media 1 and 2 spent by yeast growth was used at five different concentrations (5, 10, 15, 20 and 25%) plus distilled water (as control). The experiment was carried out in triplicate, using a Randomized Block Design, containing 15 seeds for each concentration analyzed.

The seeds were placed on filter paper soaked with the concentrations tested. Petri dishes were incubated at 22 °C for 10 days in the absence of light. After the incubation period, the number of seeds germinated and the root length were analyzed, and the relationship between the samples and the control was used to calculate the relative germination roots (RG) (Eq. 1), root length (RL) (Eq. 2) and the Germination Index (GI) (Eq. 3).

Statistical Analysis

Biomass production data were subjected to the analysis of variance, followed by comparison of means by Scott–Knott test at 5% probability. Phytotoxicity test were subjected to analysis of variance, followed by comparison of means by Tukey’s range test at 5% probability. Both were analyzed using Sisvar® statistical software [19].

Results and Discussion

Production of Biomass and Kinetics of Growth

During the fermentation, pH and number of viable cells were monitored. The pH presented negligible variation from 4.8 to 4.2 in medium 1, and from 4.4 to 3.9 in medium 2. The small variations in pH values is because the basic pH of molasses, usually around 7.8 [20], and to the fed batch process during the fermentation. This pH behavior differs from that found by other researchers in fermentation processes carried out only with vinasse, where the final pH had a value higher than the initial pH [11, 21].

Based on the initial population (6.0 log CFU mL−1), maximum growth was observed for C. parapsilosis CCMA 0544 reaching to 8.26 log CFU mL−1 (medium 1) and 8.51 log CFU mL−1 (medium 2) and for C. glabrata CCMA 0193 that reached 8.27 log CFU mL−1 (medium 1) and 8.25 log CFU mL−1 (medium 2). Similar to S. cerevisiae CCMA 0188 in media 2 (8.23 log CFU mL−1). The others yeast strains show slight growth (maximum 7.91 log CFU mL−1). The vinasse is a media which shows many stress compounds, such as metal elements and inorganic compounds that can avoid the growth and take the strains to stationary phase early. Moreover, the yeasts in vinasse medium invested more in volatile compounds than biomass. This is true, especially to S. cerevisiae strains as observed for Reis et al. [11]. The strains S. cerevisiae used in this work do not show better growth despite being isolated from cachaça and ethanol production and used fed batch that avoids catabolic repression.

Biomass production varied from 3.00 to 8.89 g L−1, reflecting the influence of yeast strains and culture medium (Table 1). C. parapsilosis CCMA 0544 presented the highest and similar biomass production in both media (Table 1). However, it was possible to observe that the time the strain remained in the log phase was higher in medium 1. S. cerevisiae CCMA 0186, S. cerevisiae CCMA 0188, C. parapsilosis CCMA 0544 and C. glabrata CCMA 0193 did not present significant differences in biomass production between the culture medium. However, Meyerozyma caribbica CCMA 0198 showed a significant difference, being the highest biomass production occurred in medium 2 (Table 1).

Table 1 Biomass production by yeasts strains in molasses and vinasse as nutrient source
Full size table

The highest biomass yield was achieved by C. parapsilosis CCMA 0544 at 1.77 g/day in medium 1 and 1.52 g/day in medium 2. Biomass production showed direct relation with cell counting, that is, C. parapsilosis CCMA 0544 presented the highest viable cells (8.51 log CFU mL−1) and Meyerozyma caribbica CCMA 0198 presented the lowest viable cell (7.68 log CFU mL−1) count and biomass (3.00 and 5.12 g L−1).

Microbial biomass is primary metabolite and is produced during the exponential phase, ie, the longer the microorganisms remain in this phase, the greater the biomass production. Thus, C. parapsilosis CCMA 0544 showed high adaptation to the both culture medium presented a exponential phase of 48 h and 36 h (medium 1 and 2, respectively) (Table 1). Although there is no statistically significant difference in biomass production from C. parapsilosis CCMA 0544, from an economic point of view this represents an increase of 14% in the production and therefore, should be considered in increasing production scale. These results are higher than those obtained by Reis et al. [11]. Part of this better biomass production is because here, in this work, the supplementation was molasses. Another point is the fed batch system employed that reduces the inhibitory factors.

Previous studies have evaluated the production of microbial biomass in vinasse supplemented with pure laboratory ingredients such as glucose and yeast extract [11, 21]. In the Pires et al. [21] the biomass production was higher than other literature (18.70 g L−1 to 73.20 g L−1); however, the costs of production is also high.

From statistically analyses, biomass of C. parapsilosis CCMA 0544 and Meyerozyma caribbica CCMA 0198 have analyzed to total protein content and SEM, and the medium 1 and 2 analyzed to production of volatile compounds, physicochemical characteristics and residual toxicity.

Physicochemical Analysis

The disposal of agroindustrial waste in Brazil is controlled, based on physicochemical parameters, by the government environmental agencies, COPAM and CONAMA [21] and WHO [22]. The yeasts were able to use vinasse and molasses as a substrate for their growth during the fermentation process and therefore, promoted changes in the physicochemical characteristics of these wastes.

Regarding the concentration of alcohols (Table 2) present in vinasse and molasses in natura, it was possible to observe that vinasse had glycerol (3.09 g L−1), methanol (1.47 g L−1) and ethanol (7.72 g L−1), while they were not detected in molasses. The higher ethanol concentration in vinasse is due to the distillation step during cachaça production as already noted by Reis et al. [10]. These compounds, sometimes, can have an effect on microbial growth. The opposite was observed about carbohydrates concentration. The higher sucrose (596.79 g L−1), glucose (47.42 g L−1) and fructose (41.68 g L−1) (Table 2) were in the molasses. Concentrations of carbohydrates and alcohols found in vinasse are due to the fermentation processes of sugarcane juice, where the fermentable sugars are consumed by yeasts and alcohols are produced as the final product of fermentation [23]. In fact, these results were expected owing to the origin of vinasse and molasses. Vinasse is the waste formed after the distillation of sugarcane juice fermented for ethanol and cachaça production, while molasses is formed after the concentration and cooking of sugarcane juice for production of sugar [24,25,26].

Table 2 Physicochemical characterization of vinasse and molasses in natura, and no spent and spent medium1 (50:50) and medium 2 (70:30)
Full size table

Concentrations of carbohydrates also evidenced the need of vinasse supplementation so that it can be used as a culture medium for the growth of microorganisms. Reis et al. [11] supplemented the vinasse with peptone obtain positive effect over microbial biomass production. However, the peptone is expensive to use in large-scale production. In this study, the peptone was replaced to molasses to allow both the best microbial growth and the reduction of the cost of fermentation process. In media, 1 g of molasses cost thirty-two times less than peptone. Molasses is a dense and viscous liquid, rich in sucrose and reducing sugars, besides other substances of organic origin such as amino acids, carboxylic acids, aliphatic, olefin, vitamins, proteins and phenols. In addition, molasses has a mineral composition of great importance, where more than 20 metals and non-metals are found in different concentrations [8, 27].

It is notorious that the nitrogen content in natura samples is higher than after microbial fermentation (Table 2), reinforcing the idea that vinasse and molasses were used to nitrogen source by yeasts. The nitrogen concentration in medium 1, no spent and spent media, showed highest than medium 2 because of the high molasses proportion.

Biomass Analyses

Total Protein Content in Biomass

The conventional protein sources used for animal feed can be replaced by protein present in the microbial biomass as already used to fish, for example. This represents new alternative to supplementation feed, being a more economical and environmentally friendly protein source [28]. It is important that for microbial biomass to be used as a healthy food supplement for animals, it is necessary that this biomass satisfy some nutritional and anti-nutritional characteristics, such as high nitrogen content and low nucleic acid content [4].

The vinasse as substrate to SCP production was already used by Pires et al. [21]. In this case, the SCP was qualitatively appropriated into essential amino acids content.

Microbial biomass produced of C. parapsilosis CCMA 0544 and Meyerozyma caribbica CCMA 0198 was analyzed for nitrogen and total protein content. To both yeasts, the protein percentage was similar, considering them in medium 2, that is, 26.93 and 27.30%, respectively. In medium 1 (50:50), C. parapsilosis CCMA 0544 showed higher protein content (22.03%) than Meyerozyma caribbica CCMA 0198 (17.55%). This value is inferior to those found early in yeast biomass grown only in vinasse; values equivalent to 53–57% of protein [4, 11, 21]. However, the protein values found (17.55–27.30%) agree with protein concentration required in the diet of some animal species, such as pigs, poultry, cats and rabbits [29]. In addition to the high protein content, yeast biomass also contains fats, carbohydrates, nucleic acids, minerals, vitamins and essential amino acids (lysines, tryptophan, threonine, etc.) which are satisfactory when used as food additives, otherwise they may be added in end of process low cost [30,31,32]. The spent medium (analyses below) has the high concentration of sucrose, glucose and fructose that means in the practical, that the spent medium could be used in a new biomass cycle being necessary just new yeast inoculums, keeping the cost low.

Analyses in Media Post Fermentation

Chemical Composition

Analyses by HPLC of vinasse, molasses and media 1 and 2 is in the Table 2. The alcohols are present in the cachaça, so is expected find them in the vinasse. However, in the spent media, there are increase of concentrations of glycerol, methanol and ethanol representing almost sixfold higher in the fermentation with C. parapsilosis CCMA 0544 and sevenfold in fermentation with Meyerozyma caribbica CCMA 0198 in medium 2 (Table 2). The increase in concentrations of ethanol and glycerol is a result of sugar metabolism by these microorganisms from the carbon source of vinasse and molasses [33]. In addition, the production of high concentrations of glycerol is associated with the presence of stressful growth conditions in the vinasse, such as the presence of toxic metabolites and osmotic stress [34].

Regarding the carbohydrate concentration, it was possible to observe that at the end of fermentation process, sucrose concentrations decreased, while glucose and fructose concentrations increased (Table 2). Many microorganisms have the ability to ferment disaccharides and promote the breakdown of these disaccharides in their components [33]. In this way, the yeasts used the promoted catabolism of sucrose molecules in glucose and fructose, justifying the results found after fermentation process.

In Meyerozyma caribbica CCMA 0198 media grown, there was the highest reduction of sucrose (of the 99%) and the highest increase of glucose and fructose when compared to C. parapsilosis CCMA 0544 (Table 2). However, the highest sucrose conversion in glucose and fructose did not correspond to biomass increase (Tables 1, 2). C. parapsilosis CCMA 0544 showed more efficient to use carbon source reinforcing the findings to Adoki [35], who stated that Candida presented a better regulation of anabolic and catabolic pathways, demonstrating a better efficiency in substrate consumption and therefore, being the most used for production of Single Cell Protein (SCP).

Production of Volatile Compounds

Qualitatively, the VOCs characterization from vinasse and molasses showed many compounds presents in crude waste (Table 3). Fifteen different compounds in vinasse and 19 in molasses. In media 1 and 2, no spent, the numbers of different compounds decrease to 16 and 13 compounds, respectively. After microbial growth, the numbers increase again (20 and 18 compounds, respectively), showed the influence of the growth in obtained VOCs to practical application besides microbial biomass. These VOCs have insecticidal and repellent, biofuels, fuel additives, fruity, candy, and perfume-like aromas, volatile biomarkers proprieties, etc.

Table 3 Volatile compounds from samples of vinasse and molassess in natura, media 1 (50:50) and media 2 (70:30) before (no spent) and after fermentation (spent media) by yeast strains
Full size table

The medium 2 (70:30) was more appropriated to volatile compounds production especially to C. parapsilosis CCMA 0544. Of 18 compounds, five of them, are major compounds identified as acetaldehyde, ethanol, ethyl acetate, methanol and 2-phenyl ethanol (Table 3). High methanol concentration in fermentation by C. parapsilosis is not common, and difficult to explain it. In this case, could be a barrier to yeast growth. This way, the removal of methanol by volatilization is important before the spent media be used in a new fermentation cycle.

Other volatile compounds quantified had their greatest expression in fermentations with C. parapsilosis CCMA 0544, especially for 2-phenyl ethanol, which had increased the 26-fold in assay containing CCMA 0544 (3.87 g L−1) (Table 3). Reis et al. [10] already identified seventeen compounds in vinasse media among them, 2-phenyl ethanol by four yeast strains in tequila and cachaça vinasse supplemented with yeast extract, glucose, peptone and potassium phosphate. The highest production of compound was 0.28 g L−1 in tequila vinasse and 0.16 g L−1 in vinasse of cachaça. The volatile compounds production from microbial fermentation becomes an economical and efficient alternative, since the natural extraction of these compounds is expensive [36]. When produced by yeast, bacteria or filamentous fungi, from agroindustrial wastes, the microbial volatile compounds still appear as an environmentally favorable product because it allows to obtain a product with a high added value from wastes [13].

Meyerozyma caribbica CCMA 0198 was better to produce acetaldehyde in medium 1. The many applications of these compounds are already cited as being used in the food, beverage, cosmetic and pharmaceutical industry [37] for the characteristic flavour of rose, apple aroma, fruity and sweet aroma, etc. [38,39,40,41].

Morphological Analysis of Yeasts in SEM

The survival of a cell depends on the ability to respond rapidly to environmental changes, which can cause rapid and transient changes in cellular genome [42] altering the cellular morphology [43, 44]. The morphological changes in growth cell in vinasse medium already observed by Silva et al. [4]. The impact of vinasse composition media in C. parapsilosis CCMA 0544 and Meyerozyma caribbica CCMA 0198 was evaluated by SEM (Fig. 1). The cellular morphology of yeast strains grown in YEPG broth (control) was compared to the cellular morphology of yeast strains after 120 h of culture in medium 1 and medium 2. C. parapsilosis and Meyerozyma caribicca have the normal physiology as ovoid to cylindrival cells.

Fig. 1
figure 1

Analysis of impact of vinasse and molasses on cellular morphology of yeasts used in fermentation process. a CCMA 0544 cultivated in YEPG broth (control), b CCMA 0544 cultivated in medium 1, c CCMA 0544 cultivated in medium 1, d CCMA 0544 cultivated in medium 2, e CCMA 0544 cultivated in medium 2, f CCMA 0198 cultivated in YEPG broth (control), g CCMA 0198 cultivated in medium 1, h CCMA 0198 cultivated in medium 1 and i CCMA 0198 cultivated in medium 2. Arrows indicate the morphological changes observed in scanning electron microscopy

Full size image

The cellular morphology of C. parapsilosis CCMA 0544 suffer little modifications evidenced with formation of elongated buds and the non-detachment of buds from the mother cell, thus forming cells with two or more buds (Fig. 1b–e). In Meyerozyma caribbica CCMA 0198, the same behavior was observed as C. parapsilosis CCMA 0544. However, in medium 2, there was an increase of the cellular size when compared to control (Fig. 1g–i), contrary to the findings by Silva et al. [4] that observed a decrease in cell size, the presence of multiple budding per cell, the formation of pseudohyphas and clusters of cells after 24 h of incubation. However, these morphological changes are expected to both species [45]. This way, the cells could be used in the other fermentation cycle.

Subchronic Toxicity

Table 4 shows the relative germination roots (RG), root length (RL) and germination index (GI) results obtained from subchronic toxicity.

Table 4 Germination index (GI%), relative germination roots (RG%) and root length (RL%) obtained in subchronic toxicity analysis with onion seeds (Allium cepa) from samples of vinasse and molassess in natura, media 1 (50:50) and media 2 (70:30) in no spent and spent media by yeast strains
Full size table

The germination of seeds and the first days of the development of seedling correspond to a period where there is intense physiological activity. Therefore, the stages where the seed and the seedling are extremely sensitive to adverse external factors [18]. Hence, the results obtained on the effects of vinasse supplemented with molasses on onion seeds (Allium cepa) are representative of the effect of these residues on seeds and seedlings in general [18, 21].

Similar to Pires et al. [21] in a general way, the values of relative germination roots, root length and germination index showed an opposite relation to the concentration of samples. In vinasse in natura, the GI in all concentrations tested had an index that ranged from 51.40 to 81.05% (Table 4). In several countries, vinasse is commonly applied directly to the soil of sugarcane plantations, such as fertilizer because it presents high concentrations of nutrients and organic matter. However, when used in large quantities there is groundwater pollution and the salinization of soil [33, 46].

In spent media, RG, RL and GI occurred at 5 and 10%, but not at 15, 20 and 25% (Table 4). The highest RG was observed at 10% in medium 2 spent by Meyerozyma caribbica CCMA 0198. Regarding the GI, the values found in samples in spent media at a 5% concentration ranged between 13.31 and 16.74% (Table 4). There was no significant difference in the RG and in the RL between samples in concentration of 5% (Table 4).

So the 5% spent media dilution was considered the least phytotoxic concentration for discarding in soil, based on positive effects on germination. Therefore, the toxicity became low enough, but with sufficient organic load to allow root growth.

Conclusion

Molasses were efficient in the supplementation of vinasse, allowing the growth of yeasts and different volatile compounds production. C. parapsilosis CCMA 0544 is indicated to biomass and 2-phenyl ethanol production, while Meyerozyma caribbica CCMA 0198 can be indicated to produce acetaldehyde as major compound. However, the sugar concentration in spent media is high what suggest these media should be used in a new production cycle. The use as biofertilizer just is done in 10% proportion.

Appendix

Equation

$$RG(\% ) = \frac{Number\,germinated\,seeds\,samples}{{Number\,germinated\,seeds\,control}} \times 100$$
(1)
$$RL(\% ) = \frac{Average\,root\,length\,samples}{{Average\,root\,length\,control}} \times 100$$
(2)
$$GI(\% ) = \frac{RG\% \times RL\% }{{100}}$$
(3)