Evaluation of The Production of Second Generation Ethanol by Co-Culture of Saccharomyces Cerevisiae and Pachysolen Tannophilus Immobilized in Sodium Alginate.

10 The production of an alternative form of fuel that replaces fossil fuels has been increasingly studied due to the 11 environmental impacts generated by its excessive use, as well as the depletion of these fossil energy sources. 12 Ethanol obtained from the crushing of sugar cane has been used as a substitute for these fuels, mainly in the 13 automotive area. However, alternative sources are being studied to produce the so called second generation 14 bioethanol. This would avoid competition for food producing agricultural areas and agroindustrial waste is a 15 great source for obtaining it. In general, these residues are not always completely reused and are disposed of 16 inappropriately in the environment, becoming contaminants. Therefore, the use of agroindustrial waste can 17 become a renewable source of energy, in addition to reducing environmental impacts. The objective of this work 18 is to produce second generation bioethanol as an alternative to the one currently used, using the rice husk 19 hydrolyzate by the consortium formed by Saccharomyces cerevisiae and Pachysolen tannophilus . For this, an 20 acid hydrolysis was performed with 2% sulfuric acid during 10 minutes of heating in an autoclave, after which 21 the hydrolyzate was detoxified with the use of activated carbon. The crude and detoxified hydrolysates were 22 used as a substrate for the fermentation medium with an initial concentration of 50 mg/mL of reducing sugars. 23 The fermentation process with the use of both yeasts in the crude hydrolyzate medium, in the detoxified medium 24 and in a synthetic medium composed of glucose, was carried out for 24 h, 30º C, 0 rpm and pH 6.5.The best 25 results for the ethanol production of Saccharomyces cerevisiae was the synthetic medium with 20.6 mg/mL. For 26 the yeast Pachysolen tannophilus , its highest production was in a synthetic medium with 11.67 mg/mL. The 27 intercropping of the two yeasts proved to be efficient with a greater ethanol production reaching 21.5 mg/mL, the 28 hydrolyzed and detoxified media showed great potential for ethanol production both in intercropping and in 29 monoculture. 30


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The concern to reduce the emission of gases related to the greenhouse effect, mainly derived from The inoculum was prepared by the addition of microorganisms previously cultivated in YM broth in 119 Erlenmeyer bottles of 250ml containing 100ml of YM broth (pH 5.0). The inoculum was standardized by 120 spectrophotometry with an absorbance of 0.6 and wave length of 600nm.

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The production of ethanol was performed in culture media (pH 7) composed by yeast extract (5 g.L -1 );

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The fermentation was evaluated using the monoculture of S. cererevisiae and P. tannophilus and by 141 the co-culture of both yeasts, immobilized in sodium alginate spheres.

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The fermentation occurred during 24 h, where, samples were taken every 4 h at the phase-lag and at 143 every 2 h at the phase-log for the purpose of evaluating the ethanol production, cellular biomass values, pH 144 change, sugar values (reducing) and productivity calculation.

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For analysis by the yeast consortium, it was performed with the inoculation of the yeast S. cerevisiae 146 in the first 12 h of the fermentation, after, P. tannophilus was removed and inoculated.

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The values of total ethanol productivity, expressed in mass of the product formed per unit of time and 160 per unit of volume (g.L-1.h-1), will be obtained through Equation 1. The initial pH of the culture media for fermentation containing, synthetic medium (MS), rice husk 178 hydrolyzate (MH) and detoxified hydrolyzate (MHD) was 6.5. Figure 1 shows the performance in relation to pH 179 during the alcoholic fermentation process during the 24 hour period. It becomes possible to see the reduction that 180 occurred sharply during the first 12 hours, followed by a less mild reduction during the following hours. The

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final pH values after fermentation were 4.93 for hydrolyzed medium, 5.1 for detoxified and 2.93 for synthetic 182 medium. It is possible to observe a small difference in relation to hydrolysates, however, considerable with the 183 synthetic medium.

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The performance of pH during the fermentation process acts directly on its yield, considering that 190 these changes can influence the metabolism of the micro-organism as well as yield in the process. PH can play 191 an important role in the face of fermentative inhibition, and consequently lead to changes in cellular metabolism.

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During ethanol production, the pH of the medium tends to decrease due to the consumption of substrates and by-193 product formations, as it gradually decreases during this process (16). The inoculum of the yeast S. cerevisiae in the fermentation media was used to evaluate cell growth.

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The best results obtained for ethanol production were 20.6 mg / mL for synthetic medium in 24 hours,

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As cell growth increased, ethanol production also increased, especially after the 12-hour fermentation 229 period. As noted earlier in the 4 to 10 hour fermentation period, cell growth is still low, which corroborates the 230 low ethanol production.

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The authors (18)  which showed a higher production until the 14-hour fermentation period, followed by a stability in production.

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The behavior of the synthetic medium, in turn, showed less ethanol production in the first 16 hours of 248 fermentation, however, after this period it was more efficient for production, following a constant increase until 249 the end of the analysis in 24 hours.

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With the best parameters determined, they were carried out for synthetic, hydrolyzed and detoxified hydrolyzed 287 medium, during the 24-hour period.

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The sugars that were used by the yeast Pachysontannophilus as a substrate for fermentation and 376 ethanol production to occur. Where the initial concentration was 50 mg / mL for all media. Figure 10 shows

Cell Growth (Biomass)
426 The yeasts S. cerevisiae and P. tannophilus were used in fermentation media to evaluate cell growth,

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with the first 12 hours of S. cerevisiae followed by P. tannophilus by intercropping until the end.
428 Figure 12 shows the results obtained in the fermentations during the time of 24 hours. This figure   429 shows that over time during the fermentation process, biomass increased for the 3 media used during this process

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The yeasts Saccharomyces cerevisiae and Pachysolen tannophilus showed potential for ethanol 515 production, highlighting the efficiency of S. cerevisiae in monoculture for productivity.

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The intercropping between the two yeasts proved to be efficient and promising, due to a higher 517 production of ethanol and consumption of fermentable sugars in lignocellulosic material compared to 518 monoculture. Cellular immobilization in sodium alginate was effective for all parameters of the study,

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emphasizing the importance of this technology to improve performance and facilitate the process.

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All autors are consent to participate.

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All autors are consent to the publish.

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Availability of data and materials

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Not applicable.  Kinetics of cell biomass growth of Saccharomyces cerevisiae during the 24-hour period in the 2-hour interval.

Figure 3
Kinetics of ethanol production in fermentation by Saccharomyces cerevisiae during the period of 24 hours in the interval of 2 hours.

Figure 4
Productivity of ethanol in Saccharomyces cerevisiae fermentation during the 24-hour period in the 2-hour interval.

Figure 5
Sugar consumption present in the fermentation media used by Saccharomyces cerevisiae during the 24hour period in the 2-hour interval.  Kinetics of ethanol production in fermentation by Pachysolen tannophilus during the 24-hour period in the 2-hour interval Productivity of ethanol in fermentation by Pachysolen tannophilus during the period of 24 hours in the interval of 2 hours.

Figure 10
Sugar consumption present in the fermentation media used by Pachysolen tannophilus during the 24hour period in the 2-hour interval.

Figure 11
Fermentation pH kinetics by the consortium of Saccharomyces cerevisiae and Pachysolen tannophilus during the 24-hour period determined in the 2-hour interval in the MS, MH and MHD media.      Sugar consumption present in the fermentation media used by the Saccharomyces cerevisiae and Pachysolen tannophilus intercropping during the 24-hour period in the 2-hour interval.