Evaluation of bioethanol production from five different varieties of sweet and forage sorghums (Sorghum bicolor (L) Moench)

Industrial Crops and Products 33 (2011) 611–616

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Evaluation of bioethanol production from five different varieties of sweet and forage sorghums (Sorghum bicolor (L) Moench) F.J. Davila-Gomez a , C. Chuck-Hernandez a , E. Perez-Carrillo a , W.L. Rooney b , S.O. Serna-Saldivar a,∗ a b

Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología, Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, CP 64849, Monterrey, N.L., Mexico Department of Soil and Crop Science, Texas A&M University, College Station, TX 78843,USA

a r t i c l e

i n f o

Article history: Received 1 October 2010 Received in revised form 16 December 2010 Accepted 17 December 2010 Available online 15 January 2011 Keywords: Sweet sorghum Sugars Alpha amino nitrogen Bioethanol

a b s t r a c t Cultivars of sweet (Rio, M81E and Della) and forage sorghums (Tato and Thor) were planted in Northeast Mexico in order to estimate optimum harvesting time, sugar production, biomass composition and ethanol yields. The juices were characterized in terms of sugar composition, free amino nitrogen (FAN) and phenolics and then yeast (Saccharomyces cerevisiae)-fermented into ethanol. The cultivars yielded different volumes of sweet juice and total sugars. They also had different optimum harvesting times. Glucose was the most abundant sugar in raw juices, followed by fructose and sucrose. FAN concentration ranged from 19 to 36 mg L−1 therefore, nitrogen supplementation was required for adequate fermentation. After 18 h fermentation, there were no differences in efficiencies among cultivars but the sweet sorghums yielded more ethanol Ha−1 compared to the two forage sorghums (approximately 1000 L Ha−1 versus 770 L Ha−1 ). Della was the cultivar with the highest productivity with 1051 L Ha−1 ethanol produced after the first cut. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The growing interest in energy alternatives for fossil fuels has boosted ethanol production worldwide from 12 to 17 billion gallons in the period of 2005–2007, with the United States of America and Brazil being the two largest producers representing 52 and 37% of total production, respectively (RFA, 2010). In the U.S. alone, production has increased at least six times in the current century. At present, almost all of the world’s ethanol production is obtained from two major crops: maize and sugarcane (Berg, 2004; RFA, 2010). The main advantages of sugarcane in relation to starchy feedstocks are: fewer process stages as well as less energy requirements, which allow savings between 20 and 60% (Berg, 2004; Chuck-Hernandez, 2009). Nevertheless, sugarcane is a crop with relatively high agronomic requirements due to the excessive water requirements, temperature and length of growing season and consequently this limits production areas (Prasad et al., 2007b). Sweet sorghum (Sorghum bicolor (L) Moench) represents an analogous crop to sugarcane with similar accumulation of sucrose but with a higher agronomic stability to temperature fluctuations, less water requirement and better tolerance to salinity, alkalinity and drought (Almodares and Hadi, 2009; Prasad et al., 2007a). In addition, the crop is annual with a typical growing season of 3–5 months

∗ Corresponding author. Tel.: +52 81 83284322; fax: +52 81 83284262. E-mail address: [email protected] (S.O. Serna-Saldivar). 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.12.022

instead of 9–12 months required by sugarcane. Additionally, the sweet sorghum bagasse has a comparatively higher nutritional value for ruminants because of its more favorable fiber composition (Almodares and Hadi, 2009) and is a better alternative to further hydrolysis and fermentation of the fiber portion. Because of its agronomic flexibility and productivity, sweet sorghum is viewed as a viable feedstock option for ethanol production in some regions of the world. Sweet sorghums have potential for specific tropical, subtropical and arid regions of the U.S., Mexico, China, India, Southern Africa and other developing countries where the use of maize and other crops for ethanol production is not feasible due to either economic, agronomic or social considerations (Chuck-Hernandez et al., 2009; Reddy et al., 2005; Wu et al., 2010; Zhang et al., 2010). Almodares and Hadi (2009) reported that sweet sorghum has a ratio of energy output to fossil energy input comparatively higher compared to sugarcane, sugar beet, maize and wheat and its fermentation efficiency has been reported higher than 90% (Wu et al., 2010). Lastly, sorghum is known as one of the most variable in terms of genetic resources and germplasm that allows the breeding and development of new cultivars adapted to different regions around the globe (Zhang et al., 2010). Despite these advantages, some challenges must be resolved before sweet sorghum will be widely planted to provide feedstocks for ethanol biorefineries. These include a high rate of sugar degradation at ambient temperature and the need of nitrogen supplementation for yeast growth (Mei et al., 2009; Wu et al., 2010). Fermentation performance of sweet sorghum can be affected by the

F.J. Davila-Gomez et al. / Industrial Crops and Products 33 (2011) 611–616

microorganism used, bioreactor configuration, free amino nitrogen and sugar content and composition of juices. For this reason, the evaluation of different sweet sorghum cultivars is critically important (Zhang et al., 2010). The aim of this research was to evaluate the agronomic performance and bioethanol yield of three improved sweet sorghum cultivars (with higher biomass, juice and sugar yield), and two commercial forage sorghums planted in Northeast Mexico.

18.00 16.00 14.00 12.00

°Brix

612

10.00

Río

8.00

M81E Della

6.00

2. Materials and methods 2.1. Experimental design

2.2. Physical and chemical characterization of sweet sorghum juices

Thor

2.00 0.00

The three varieties of sweet sorghums (Río, M81E and Della) were provided by the Texas Agrilife Research Sorghum Breeding Program at College Station and the two local forage sorghums (Tato and Thor) were purchased from a local seed company. These cultivars were grown at the ITESM’s (Instituto Tecnológico y de Estudios Superiores de Monterrey) Experiment Station located at Hualahuises, Nuevo León, Mexico (24◦ 53 09 N 99◦ 40 22 W, 400 m above sea level). Sorghum was sown during the first week of March of 2006 at a rate of 15 seeds m−1 . The seeds were planted at a depth of 5 cm in 80 cm interspaced rows. After planting, all plots were irrigated with 15 cm surface water. Further irrigation was not needed because the experiment station received 320 mm rain during the three and a half subsequent months. Two mechanical weed controls were performed 30 and 60 days after sowing. Each experimental plot consists of four rows of 10 m each. Each sorghum type was planted in four plots that were randomly assigned in a Complete Block Design. Some of the plants in the two edge rows were harvested every week after anthesis in order to determine optimum harvesting time. Then, the two central rows of each plot containing plants at optimum maturity were cut in preparation for crushing and milling. The plants located within 1 m of the perimeter of the plots were not sampled in order to reduce border effects and experimental error. Juice was extracted from stalks for five consecutive weeks after anthesis (approximately 90 days after sowing), with a sugarcane three roller mill. The juice was filtered (0.25 mm sieve) to remove plant residues, transported to the university laboratory and frozen.

Tato

4.00

0

1

2

3

4

5

6

Post anthesis time (week) Fig. 1. Post-anthesis weekly evolution of ◦ Brix for five juices extracted from cultivars of sweet (Rio, M81E, Della) and forage sorghums (Tato and Thor) immediately after harvest.

sweet sorghum juice was sealed and placed in a shaker at 30 ◦ C with 125 rpm for 48 h (Lab-Line Model 3526) according to recommendations reported by Perez-Carrillo et al. (2008). At least three replicas were made for each treatment. 2.4. Sugar and ethanol during fermentation Sugar concentrations throughout 48 h fermentation were quantified by HPLC-IR, using the same parameters described in Section 2.2. Ethanol content was determined by gas chromatography (GCFID, Agilent 6850) with a flame ionization detector operating at 280 ◦ C. The GC was equipped with an HP Innowax column (30 m × 0.53 mm × 1.0 ␮m) and helium at 1.5 ml min−1 was used as carrier gas. Injection volume was 1 ␮L at a split rate of 1:10 (Davila-Gomez, 2009). 2.5. Calculation of kinetic parameters Ethanol fermentation rate was calculated using the methodology proposed by El-Mansi and Ward (2007) using data from exponential stage of fermentation. Fermentation velocity constant (k) was obtained with the following equation: k = ln(C/C0 )/t, where C was ethanol concentration in t, C0 was initial concentration of ethanol and t was time of fermentation (in hours). 2.6. Statistical analysis

Sorghum juice was thawed at refrigeration temperature (4 ◦ C) for pH determination (Potentiometer Model 8005, Orion Research, Osaka, Japan). An aliquot of 80 ␮L was taken for ◦ Brix (Atago Palette PR-32␣, Bellevue WA, USA). Free amino nitrogen (FAN) was determined using the ninhydrin official method 945.30L (AOAC, 1980) and total phenolics using the colorimetric methodology proposed by Vinson et al. (2001). Glucose, fructose and sucrose in the juices were determined by HPLC-RI (Waters 2414, Water Co, Milford, MA, USA) equipped with a Restek Ultra Amino column (Restek US, Bellefonte, PA, USA) at 30 ◦ C. A mix of acetonitrile:water (90:10) at a rate of 0.3 mL min−1 was used as the mobile phase (Davila-Gomez, 2009). 2.3. Fermentation Four hundred mL of juice of the five different varieties (harvested four weeks after anthesis) was pasteurized at 75 ◦ C for 45 min and immediately placed in an ice bath. Saccharomyces cerevisiae (ATCC 24858) previously propagated (YM broth for 12 h at 30 ◦ C and 125 rpm) was used as inoculum at a rate of 14 × 106 cells mL−1 . One liter Erlenmeyer flasks with 500 mL of

The data was analyzed using analysis of variance and means compared with Fisher test (Least Significant Difference Test), except for pH data where Tukey’s tests were utilized (alpha = 0.05). 3. Results and discussion 3.1. Agronomical characterization of juice from sweet sorghum In order to estimate the ideal harvesting period for the sweet and forage sorghum, soluble solid contents were determined every week after anthesis (Fig. 1). According to Prasad et al. (2007a) variety or genotype and environmental conditions are the principal aspects that influence the optimal maturation time and this makes necessary a continuous evaluation of the maturation progress of sorghum in the field. Almodares and Hadi (2009) indicated that nonstructural carbohydrates of sorghum are also affected by temperature, time of day, maturity, cultivar, spacing and fertilization. In the first week post-anthesis, ◦ Brix in all genotypes averaged around 8, except for M81E variety which contained <6. Prasad et al. (2007a) reported 12.5 ◦ Brix as sugar concentration in

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613

Table 1 Effect of sweet or forage sorghum cultivar harvested four weeks after anthesis on juice yield and composition. Cultivar

◦

Río M81E Della Tato Thor

13.2 10.3 11.2 10.1 10.6

Brix

pH ± ± ± ± ±

0.9a 0.7b 0.9a,b 0.8b 0.9b

4.85 4.43 4.43 4.47 4.63

± ± ± ± ±

0.27a 0.10b 0.16b 0.09b 0.09a,b

Free amino nitrogen (mg L−1 )

Total phenols (g eq. gal. acid L−1 )

35 ± 1a,b 19 ± 6c 33 ± 6a 23 ± 6b 36 ± 7a

0.227 0.204 0.184 0.180 0.239

± ± ± ± ±

0.024a 0.022a 0.021a 0.022a 0.034a

Total sugarsa (g L−1 ) 112.96 70.34 102.00 75.72 92.24

± ± ± ± ±

7.34a 4.16c 5.28a,b 4.16c 5.26b

Juice yield (L Ha−1 ) 16,400 28,300 19,900 20,900 15,000

Sugar yield (Kg Ha−1 ) 1852.5 1990.6 2029.8 1582.5 1383.6

Values with different letter(s) within columns are statistically different (P < 0.05). a Sucrose, fructose and glucose

sweet sorghum immediately post-anthesis. All cultivars accumulated approximately 2 ◦ Brix per week, with the highest increase from week two to three before arriving to a steady state after the fourth and fifth weeks (Fig. 1). Subsequent fermentation trials were made with juices harvested from the fourth week after anthesis for all cultivars (Fig. 1). According to Prasad et al. (2007a), optimal harvesting stage is when the juice contains 15.5–16.5 ◦ Brix and this parameter is one of the most important in order to obtain a juice of high fermentable quality and thus maximize ethanol yield per hectare. At week four none of the varieties evaluated were in the range recommended by these authors but they were used in fermentation trials with the aim of standardization. 3.2. Physical and chemical characterization of juice from sweet sorghum Table 1 depicts physical and chemical properties of juices as well as agronomical yield of sweet sorghum four weeks post anthesis. The range of ◦ Brix before fermentation was between 10.1 for Tato and 13.2 for the Rio sweet cultivar. These quantities are in low range reported by Almodares and Hadi (2009) and other authors who reported roughly 18 ◦ Brix as average (Gnansounou et al., 2005; Zhang et al., 2010). It is important to point out that ◦ Brix before fermentation (Table 1) were 11–24% lower than those of the values reported immediately after harvesting, presumably due the delay in stabilizing the sample. It took at least 3 h to place samples in Monterrey after harvesting and milling and the average temperature was higher than 32 ◦ C. During this time the juice probably lost

fermentable sugars due to natural occurring microorganisms. Thus, it is critically important to stabilize samples to prevent microbiological growth that decreases ethanol yields. ◦ Brix is a term that represents an approximation of total solids content, as expected it had a positive correlation with total sugar concentration (Table 1). This positive correlation has been reported in sweet sorghum by several authors like Tsuchihashi and Goto (2004) and Davila-Gomez (2009). The range of sugar content obtained was from 7 to 11%, near 8–10% reported by Prasad et al. (2007a), but below 10–25% and 14.3–22.9% from Wang et al. (2009) and Almodares and Hadi (2009), respectively. In all five varieties evaluated, the principal sugar detected was glucose followed by fructose and sucrose (Fig. 2). Della cultivar was the highest in glucose, followed by Rio and Thor. The same trend was observed for fructose content. In the case of sucrose, the highest concentrations were obtained for Tato followed by Rio (Fig. 2). These results differ from those reported by Phowchinda et al. (1997), Wang et al. (2009) and Krishnaveni et al. (1984), where sucrose was the most abundant sugar. Gnansounou et al. (2005) reported a sweet sorghum sugar composition of: 85% (wt) sucrose, 9% glucose and 6% fructose and Prasad et al. (2007a) indicate a percentage of 60% sucrose, 33% glucose and 7% fructose. Results reported by Almodares and Hadi (2009) for 36 sweet sorghum cultivars indicate sucrose concentrations between 7.26 and 16.06%. The highest amount of glucose and fructose can be related to several factors namely: inversion of sucrose catalyzed by a slightly acidic pH characteristic of sweet sorghum juices (Whistler and BeMiller, 1999) and high level of endogenous invertases in the juice that rend sucrose in monomers fructose and glucose (Coleman,

Fig. 2. Fructose, glucose and sucrose concentration (g L−1 ) in juices from five different sweet and forage sorghum cultivars.

80.0

70.0

70.0

60.0

60.0

50.0

50.0 40.0 40.0 30.0 30.0 20.0

20.0

Ethanol Concentration (mL/L)

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Glucose Concentration (g/L)

614

10.0

10.0 0.0

0.0 0

6

12

18

24

30

36

42

48

Fermentarion Time (h) Río Glucose Río Ethanol

Tato Glucose Tato Ethanol

Della Glucose Della Ethanol

M81E Glucose M81E Ethanol

Thor Glucose Thor Ethanol

Fig. 3. Glucose and ethanol profiles through fermentation of juices extracted from five sweet and forage sorghum cultivars.

1975). Also, the high level of glucose can be associated with the type of sweet sorghum evaluated. According to Anglani (1998), sweet sorghums are divided according to sugar composition into: saccharin and syrup type. The saccharin type, with high sucrose content, is mainly used for refined sugar production and the latter, with higher glucose concentration, for syrup production. Given these differences, the varieties evaluated in this research are more related to the syrup type. Other key factor in sweet sorghum characterization is juice yield per cultivated area and according to Krishnaveni et al. (1984) this parameter is inversely related to sugar content. Rio cultivar was the highest in ◦ Brix and total sugar content per liter but it had one of the lowest juice yields (Table 1). For evaluation of economic convenience, sugar yield per cultivated area is a better parameter since sugar content is directly associated to ethanol yield. In Table 1, the sugar yields in the first cut are depicted. The results ranged from 1.38 Ton Ha−1 for Thor to 2.0 Ton Ha−1 for Della cultivar. These yields are lower than that of reported by Prasad et al. (2007a); Almodares and Hadi (2009) and Liu and Wang (2008) who reported yields of 6, 7 and 15 Ton Ha−1 respectively on an annual basis. Unfortunately, these authors failed to mention if the yields are after two or three consecutive cuts. Other parameter evaluated in the sweet sorghum juices was the initial concentration of soluble nitrogenous compounds measured as free amino nitrogen (FAN). These components are required for structural and enzymatic protein synthesis in yeast metabolism (Taylor and Boyd, 1986). The level of FAN detected in the juices was between 19 and 36 mg L−1 (Table 1), which is below the 150 mg L−1 optimum for an adequate alcoholic yeast fermentation (Ingledew, 1995). Thus, a nitrogen supplementation may be necessary during fermentation in order to improve fermentation performance (Mei et al., 2009; Reddy et al., 2005). Total phenolics were also evaluated in the different juices and they contained statistically similar contents (around 0.2 g gallic acid equivalent L−1 ). In clarified sugar cane juices, 0.8 g gallic acid equivalent L−1 has been reported (Payer et al., 2006) which are four times the amount detected in the sweet and forage sorghum varieties used in this research. According to Billa et al. (1997), the main phenolic compounds detected in sweet sorghum are p-coumaric and ferulic acids, whose main purpose is to contribute to cell wall bonding of lignin and hemicellulose through ether and ester linkages (Billa and Monties, 1995). These phenolics are detrimental in fermentation, because

they tend to form complexes with proteins such as proteases and glucosidases, needed for hydrolysis (Salunke et al., 1982) as well as with structural components, reducing the bioavailability of nutrients (Mullins and NeSmith, 1986). Nevertheless, the 0.2 g gallic acid equivalent L−1 detected in the juices is below the 3 g L−1 reported by Mullins and Lee (1991) as critical concentration to allow the precipitation of more than 70% of proteins in the fermentation media. 3.3. Fermentation profiles of sweet and forage sorghum varieties The profile of glucose and ethanol through reaction was characteristic of an alcoholic fermentation (Fig. 3). The lowest concentration of glucose was reached at the hour eighteen of fermentation and the highest level of ethanol between hours eighteen to twenty. There were significant differences in terms of ethanol concentration for the different cultivars at the end of fermentation. These were consistent with the observed differences in initial substrate composition (Table 1). Glucose (Fig. 3), unlike fructose (data not shown), was not totally consumed and roughly 5 g L−1 of this sugar remained in the spent fermented juices. FAN initial content in juices (Table 1) was around 30 mg L−1 , but at the beginning of fermentation (Fig. 4) was approximately 200 mg L−1 . This difference is related to the use of YM broth for yeast propagation, medium that contained 3 g L−1 of malt and 3 g L−1 of yeast extract which undoubted increased the initial concentration of nitrogenous compounds. This observation coincides with Barredo-Moguel et al. (2001) who reported that worts inoculated with S. cerevisiae previously cultivated in malt–yeast extract media reached highest levels of FAN. Given the importance of FAN in yeast metabolism (Taylor and Boyd, 1986), the residual glucose content could then be related to the limitation of nitrogen supply (Fig. 4) which reduced the capacity of Saccharomyces to complete the consumption of the available substrate. 3.4. Ethanol yield and fermentation efficiency Rio and Della yielded significantly higher ethanol production compared to the other three varieties (Table 2). These results are congruent with the initial concentration of sugars of each juice (Table 1). Thor forage sorghum ranked third in ethanol production as well as in total sugar concentration (Tables 2 and 1, respectively).

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615

240 210

FAN Concentration (mg/L)

180 150 120 90 60 30 0 0

6

12

18

Río

24

30

36

Fermentation Time(h)

Tato

Della

M81E

42

48 Thor

Fig. 4. Free amino nitrogen (FAN) consumption during fermentation of five sweet and forage sorghum varieties.

Table 2 Ethanol yield (mL L−1 and L Ha−1 ), fermentation efficiency (%) and fermentation velocity constant (h−1 ) for five different juices extracted from cultivars of sweet and forage sorghums. Cultivar

Ethanol yield mL L

Río M81E Della Tato Thor

56.36 35.78 52.84 38.90 48.01

−1

± ± ± ± ±

3.09a 2.54c 2.24a,b 3.02c 2.59b

Fermentation efficiency, %

Fermentation velocity constant (K), h−1

−1

L Ha 924.34 1012.57 1051.53 813.21 720.26

± ± ± ± ±

50.65a,b 71.80a 44.59a 63.22b 38.84b

79.99 85.71 86.09 89.75 84.64

± ± ± ± ±

1.13a 2.33a 2.03a 2.98a 3.41a

0.25 0.24 0.26 0.29 0.25

± ± ± ± ±

0.03a 0.03a 0.03a 0.04a 0.03a

Values with different letter(s) within columns are statistically different (P < 0.05).

There were no significant differences in fermentation efficiency among juices extracted from the five cultivars where approximately 85% of the fermentable sugars were converted to ethanol (Table 2). The combination of agronomic productivity and ethanol yield (Table 2) indicated that the highest productivity was reached with the Della cultivar, followed by M81E and Rio, respectively. However, these differences were not statistically different (P > 0.05). On the other hand, Tato and Thor yielded lower amounts of sugars and ethanol per hectare compared with the three improved sweet sorghum varieties. These results are related with the sugar production per cultivated area (Table 1). The combination of high juice production and sugar yield made the Della cultivar the best ethanol yielder. The current study did not evaluate ratoon harvests, which would be possible in Mexico. Further research is needed to determine the feasibility and the potential ethanol yields from such a system. If two cuts are made and yields are similar in second harvests, the Della cultivar would yield approximately 2000 L anhydrous ethanol Ha−1 year−1 . However, this productivity is lower than the reported by Prasad et al. (2007a) and Brown (2006) for sweet sorghums. On the other hand, the average yield of maize in the United States was 9.65 Ton Ha−1 in 2008 and thus can generate approximately 3800 L ethanol Ha−1 (FAO, 2010). Zhang et al. (2010) obtained an annual ethanol yield of approximately 4700 and Prasad et al. (2007a) reported a production of 2800–4000 L Ha−1 . These yields do not consider the potential of the spent bagasse which accounts for approximately 30% of the original biomass weight. According to Mamma et al. (1996) sweet sorghum has almost the same quantity of soluble and insoluble sugars making the spent bagasse a poten-

tial and important source of bioethanol obtained via bioconversion of fiber components. 4. Conclusion Soluble solids content of cultivars of sweet and forage sorghum juices in the first week post-anthesis was around 8 ◦ Brix and increased 2 ◦ Brix in the next three subsequent weeks, reaching the optimum level after the fourth/fifth weeks. The principal sugars found in juices were glucose, fructose and sucrose. Rio and Della were the highest in total sugars with 112 and 102 g L−1 juice, respectively. The highest production of ethanol per hectare per cut (1051–924 L Ha−1 ) was for the sweet sorghums (Della, M81E and Rio) whereas the two forage sorghums (Tato and Thor) only yielded approximately 770 L Ha−1 . Acknowledgements This research was supported by grants from Fondos Mixtos N.L.-CONACyT NL-2006-C09-33363 “Creación del centro de investigación y desarrollo de biocombustibles del Estado de Nuevo León: producción de bioetanol y biodiesel a partir de recursos renovables” and Research Chair Fund CAT-005 from Tecnológico de MonterreyCampus Monterrey. References Almodares, A., Hadi, M.R., 2009. Production of bioethanol from sweet sorghum: a review. Afr. J. Agric. Res. 4, 772–780.

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