Eciency and Fermentative Yield of Sweet Sorghum Using Cat-1 And Pedra-2

Background: The increase in demand for fuels has intensied the search for renewable energy sources, mainly biomass, with sugarcane being the most important factor in the production of bioethanol. In addition, sorghum cultivation presents high potential mainly for using the same industrial production complex and being processed in the off-season of sugarcane. However, information on the chemical and technological characteristics of sweet sorghum in the national literature is scarce. These are of extreme importance considering the preparation and adaptation stages of musts and yeasts, preceding the process of ethanolic fermentation. The objective of this research was to evaluate the fermentation process of the CVSW80007 genotypes; CVWS80147 and BRS610 in laboratory scale, evaluating the performance of CAT-1 and PEDRA-2 yeasts by determining yields and fermentative eciencies and alcohol production (L.ha -1 and L. t-1 ). Results: The experimental design was the randomized blocks in a 3x2x6 Factorial model in the fermentation process (for each of the yeasts studied). The results indicated that CAT-1 and PEDRA-2 yeasts demonstrate similar behavior, being ecient in the fermentation process. The fermentation of must was prepared from stalks with leaves and without leaves (135 days after sowing) of the three genotypes, results in 47.0 and 39.8 L.t -1 ethanol, respectively, using sweet sorghum. Conclusions: The conditions studied on a laboratory scale demonstrate that the different sweet sorghum genotypes have potential for ethanol production, with the stalks with leaves and panicles increasing the alcohol content and fermentative eciency.


Background
The growing global concerns about environmental pollution from the use of non-renewable energy sources coupled with future dependence of energy on petroleum and the release of greenhouse gases have stimulated the global community to nd alternative fuels, for the increase and e ciency of energy [1].
Moreover, it is growing the demand for reducing costs and sustainability about the environment, this results in the importance of the use of biofuels to supply the global energy demand. In addition to being produced from biomass, they emit lower amounts of carbon dioxide and other pollutants particulates and have a great advantage as they are renewable fuels [2].
In this context, sugarcane has highlighted as raw material, utilized for ethanol production in Brazil. Similarly, other countries have adopted this method, based on sugarcane, which has excellent results and bringing in the paradigm changes in this new time.
Evaluating the quantities of fuels produced and consumed, both with the future statistical forecasts, it is assumed that the use of a single source of raw material may not be enough for all expected demand. Among the most economically promising alternatives, sweet sorghum highlight as a raw material for the ethanol production, both from the agronomic and industrial process. Sweet sorghum has stalks rich in fermentable sugars, similar to sugarcane. [2] For obtaining good industrial yields it is essential that the raw material to be processed presents high levels of fermentable sugars. However, there is not a lot of information about the chemical and technological characteristics of sweet sorghum in the literature. These parameters are extreme importance considering the preparation and adequation stages of broth and for the fermentation process by the yeasts.
In this context, the goals of this research were to evaluate the fermentation process of the genotypes CVSW80007, CVWS80147 and BRS610 in laboratory scale, evaluating the performance of the industrials yeasts CAT-1 and PEDRA-2 (PE-2) determining the yields, fermentative e ciencies, and ethanol production (L.ha − 1 and L.t − 1 ).

Sweet Sorghum
Sweet sorghum was grown in the experimental area of the Vegetable Production Department of FCAV/UNESP Jaboticabal -SP, Brazil. The sorghum seeds of the CVSW80007 and CVWS80147 genotypes were donated by the company Canavialis (Monsanto do Brasil) and the BRS610 genotype donated by the company Embrapa. The Planting was done with a combined spacing of 90x70 cm between the lines. Excess seeds were used at sowing and at 15 days after sowing, thinning was done leaving 10 plants per meter, to obtain a nal stand of 100.000 plants per hectare.

Experiment design
The experimental design was in random blocks at a factorial of 3x2x6 model. Each plot consisted of 10 lines of 10 meters in length, with a combined spacing of 90 and 70 centimeters between the lines. The treatments used were: Factor A: three genotypes (CVSW80007, CVWS80147, and BRS610); Factor B: two harvests (whole stalks and stalks without leaves and panicle), and Factor C: six different days for the process (100, 105, 110, 118, 135 and 160 days after the planting).
The theoretical yield was calculated considering that 1g of Total Reducing Sugar (TRS) produces 0.6475 mL of ethanol (stoichiometric reaction). The practical yield (considering the ethanol content of the broth after fermentation), Fermentation E ciency and Ethanol Production (L.ha − 1 and L.t − 1 ) were calculated according to Fernandes (2011).
The results were submitted to variance analysis (F test), comparison of averages test (Tukey 5%) and polynomial regression analysis using the System for Statistical Analyzes of Agronomic Tests -AgroEstat [4].

Broth preparation, fermentation process and broth after fermentation
In each day of the process, there were collected 25 whole stalks (stem + leaves) and 25 clean stalks (without leaves). These were sent to the Technology of Sugar and Alcohol and Microbiology of Fermentations Laboratory at the FCAV/UNESP, being weighed and submitted to the extraction of the juice using a laboratory scale mill. The obtained juice was used for the accomplishment of the chemical and technological analyzes. The extracted juice was subjected to a clari cation process to remove impurities.
The broth for fermentation was prepared with the clari ed juice. It was adjusted the soluble solids content (Brix) (16° ± 0,1), the pH (corrected with sulfuric acid to 4.5 ± 0.3), and the temperature (32°C). That were determined the Total Reducing Sugars (TRS) and Total Acidity [5].
At the fermentation process were evaluated the industrial yeasts CAT-1 and PE-2, which have been shown to be persistent in the fermentation processes carried out in the State of São Paulo and are presented as excellent fermenters. The broths were inoculated at a concentration recommended by Amorim (2005) and kept at 32° ± 1°C throughout the process.
During the fermentation process, after 50 minutes of the inoculation of the yeast and at the end of the processes, there were evaluated viability of yeast cells, bud rate and viability [7].
The fermentation process was carried out in batch with the recovery of yeast by centrifugation. The process was monitored by densimetry, and the end of the fermentation was established when the Brix was less than or equal to 1, or when the stabilized within 30 minutes. At the end of each fermentation, the broth after fermentation was centrifuged at 1650g, 25 o C for 5 minutes (HIMAC CR 21G centrifuge) to separate yeast and from the broth. In the broth after fermentation was determined the pH, Sulfuric Acidity, Glycerol [8], Total Residual Reducing Sugars (TRRS) [9].

Results
The results observed for the Total Residual Reducing Sugar (TRRS) content of the broth after fermention process are at the Table 01 indicate it was no signi cant difference between the genotypes when the yeast CAT-1 was used in the fermentation process. Table 1 Results of the analysis of variance (F test) and Tukey test comparison of means (5% probability) for the chemical-technological characteristics of the broth after fermentation process. Jaboticabal-SP.

TRRS (%)
Total Acidity When used PE-2 yeast as inoculum these differences were signi cant. Considering the genotypes, it was veri ed that BRS610 presented a signi cantly lower value than CVSW80007, which presented higher mean values. The presence of leaves resulted in a signi cantly lower value of total residual reducing sugar for PE-2, whereas for CAT-1. Although it hass no signi cant effect, the highest average was for stalks without leaves. About the harvesting time, it has no signi cant effect for PE-2, and at 160 days after sowing a signi cant effect was observed for CAT-1, presenting lower averages. These values can be considered high when compared to those obtained for broth fermented from sugarcane.
Considering the total acidity of the broth after fermentation process, it was veri ed that there was no signi cant effect for the genotypes and the processs with or without leaves, independently of the yeast used (Table 01).
There was a signi cant result, at the 1% level, only for the interaction AxBxC, that is, different behavior for the different genotypes, submitted to the harvest systems in the studied harvest time.
The pH of the fermented broth showed the same behavior for both yeasts (Table 01), for both genotypes and systems process. BRS610 presented a signi cantly higher mean than CVWS80147 and CVSW80007, which had the lowest pH. Considering the presence of leaves and panicles, it was veri ed a higher pH value in the broth after fermentation process, and for the last sampling seasons these were higher for the two yeasts.
In this study, it has evaluations carried out for the fermented broth at 135 days after sowing, which was considered one of the indicated seasons for the processing of the stalks for ethanol production. From the results obtained, the fermentation e ciency are presented in Figs. 01, 02, and 03.
The results indicated that the process without leaves was more adequate for all the genotypes studied, considering the fermentation e ciency. It can be veri ed that for the genotype CVSW80007, when the stalks was processed with and without leaves, using yeast CAT-1 showed higher e ciency than PE-2 ( Fig. 01), of the order of 6 to 8%.
However, CVWS80147 (Fig. 02) for without leaves and the yeast PE-2 provided the best e ciency, but similar to CAT-1. However, stalks with leaves in CVWS80147 showed no difference for both yeasts.
BRS610 genotype is considered forage, with differents characteristics, mainly the high potential of biomass production. However, when planning the trial was de ned as including this treatment and comparing it to the other sweet sorghum genotypes, it was observed that and stalks processed with and without leaves resulted in higher e ciency when PE-2 yeast was used as inoculum in the fermentation process (Fig. 03). When the yeast was CAT-1, results were lower than those found for PE-2 and did not differ from each other.
In order to compare the performance of the studied genotypes using CAT-1 and PE-2 yeast as inoculum of the fermentation process, the yields for theoretical and produced alcohol and were calculated in L.ha − 1 and L.t − 1 , are in Figs. 4 and 5. Using sorghum stalks without the presence of leaves and panicles, the average production of 1059 L.ha − 1 of theoretical ethanol, varying from 980 to 1062 L.ha − 1 , it was observed for the genotypes CVWS80147 and BRS610, respectively (Fig. 04).
When processing included leaves and panicles a variation of 1522 to 1905 L.ha − 1 of theoretical ethanol was observed, respectively for CVSW80007 and CVWS80147. That can be veri ed that the processing of leaves and stalks with resulted in an increase of 58% in the ethanol production (L.ha − 1 ) compared to the process without leaves. These results demonstrate the harvesting time of sorghum stalks, without leaves, does not impact the raw material for the preparation of the broth and fermentation process. From the analysis of the results obtained for the Alcohol Produced (L.ha − 1 ), it was veri ed that the processing of leaves less stalks resulted in an average production of 996 L.ha − 1 , where the presence of leaves and panicles was of the order of 1530 L.ha − 1 . Considering the two yeasts used in the fermentation process, they presented similar behaviors. During the fermentation process carried out using with leaves stalks, genotype CVWS80147 presented the highest yields (Fig. 04). that results in a reduction of about 15%. The yeasts used showed similar behavior for genotype CVWS80147. However, for genotypes CVSW80007 and BRS610, it was found that CAT-1 and PE-2 showed better behavior, respectively. Although processing stalks with leaves results in lower ethanol yields (Fig. 05), it should be considered that the higher yield (L.ha − 1 ) it was a consequence of the total biomass is 77.7% higher than that quanti ed for the treatment without leaves and panicles.

Discussion
After the end of the fermentation process, the broth became the raw material for studies and evaluations. Thus, the sugars that initially composed the broth now are in the form of ethanol, acids, alcohols, glycerol, among other molecules, which were transformed by yeast action [6]. In this context, the evaluation of the components that make up the broth and/or distillate is of fundamental importance when it comes to proposing a new technology.
This behavior due to the absence of the enzymatic treatment in order to favor the unfolding of starch compounds present after the extraction of the broths. It is evident the necessity of its use when the sorghum participates in the composition of the raw material for the preparation of the musts.
Glycerol is a secondary compound that forms in the auxiliary route of the ethanol forming pathway, using the same structures, and is therefore inversely proportional to its production [10]. Considering the central objective is the greater production of ethanol, it is veri ed that the ideal is that the obtained values are the smallest ones possible [6]. The analysis of Table 01, it is shown that the glycerol contents in the wine indicate that CVSW80007 was the one that presented the highest levels, and BRS610 the lowest for the two yeasts studied. By evaluating the harvesting system with leaves or removed, it was found that they resulted in signi cantly Page 8/15 higher glycerol values for CAT-1 yeast. For epochs no signi cant effect was observed for PE-2, while for CAT-1 at 100 and 135 days after sowing the glycerol contents were lower.
The results of fermentation e ency observed in this study (Fig. 1) are similiar those reported by Ribeiro Filho [11], who obtained yields of the sorghum broth, working with a clean stem of 32.91%, while in stem with leaves obtained values of 27.20%. The clean stalk showed an increase in broth yield of 5.71%, which explains the fact that many distilleries use the process called "clean cane", which in addition to a higher yield preserves the equipment that makes up the industrial unit [11].
In general, the observed e ciencies were similar to those obtained by Mancilha [12] and Ratnavathi [13], who obtained conversion of sugars present in the sorghum broth, with e ciencies higher than 90% regardless of the strains used and the genotypes considered.

Conclusions
The conditions studied on a laboratory scale demonstrate that the different sweet sorghum genotypes have potential for ethanol production, with the stalks with leaves and panicles increasing the alcohol content and fermentative e ciency.

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