Received: 23 de Junio de 2020
Accepted: 11 de Noviembre de 2020
Available: 16 de Diciembre de 2020
Yam is a starchy tuber mainly used in food preparation but with high potential applications in other fields such as pharmaceutical and bioplastic production. Colombia is among the top twelve yam producing countries worldwide and ranked first in terms of yield of tons per hectare planted. Yam production has specifically been concentrated in the Caribbean region, which is why this tuber is very little known in the inland regions. In this study, we evaluated Simultaneous Saccharification and Fermentation (SSF) for bioethanol production from yam (Dioscorea rotundata) using Saccharomyces bayanus. Ethanol production technologies involve the fermentation and hydrolysis of consumable raw materials (i.e., sugar cane and corn) which are quite mature around the world. For this reason, the process under analysis combined three phases: 60 min of gelatinization, enzymatic hydrolysis (divided into 40 min of liquefaction with α-amylase and 20 min of saccharification with glucoamylase), and 27 h of fermentation with no enzyme recovery. We used different yam concentrations (10, 12.5, 15, and 18 % w/w) in a wet basis. SSF was monitored along time, and total reducing sugars and ethanol concentration were quantified. The hydrolysis yield, was calculated based on the theoretical starch available in the tuber, was 90 % of starch mass for samples with a yam concentration of 10 and 15 % w/w. Regarding ethanol, the best result (a productivity of 0.19 g/Lh-1) was obtained with the sample with a yam concentration of 10 % w/w. Therefore, yam is a starchy material suitable to produce bioethanol via SSF.
Keywords: Yam, Starch, Enzymatic hydrolysis, Simultaneous saccharification and fermentation, Bioethanol.
El ñame es un tubérculo de almidón utilizado principalmente en alimentos, pero con un alto potencial de aplicaciones en otros campos, como la farmacéutica y la producción de bioplásticos. Colombia se encuentra entre los 12 países con la mayor producción mundial de ñame, ocupando el primer lugar en rendimiento de toneladas por hectárea plantada. La producción de ñame se ha ubicado explícitamente en la región del Caribe, y es muy poco conocida en el interior del país. Este estudio evaluó el proceso simultáneo de sacarificación y fermentación (SSF) para la producción de bioetanol a partir de ñame (Dioscorea rotundata) como materia prima utilizando la cepa de levadura Saccharomyces bayanus. Las tecnologías de producción de etanol hacen referencia a procesos de fermentación e hidrólisis de materias primas comestibles (caña de azúcar y maíz), las cuales, a nivel mundial, están bastante maduras. Por esta razón, el proceso evaluado implicó la combinación de tres pasos: 60 min de gelatinización, hidrólisis enzimática (dividida en 40 min de licuefacción con α-amilasa y 20 min de sacarificación con glucoamilasa) y 27 h de fermentación sin recuperar las enzimas añadidas. Se usaron concentraciones de 10 %, 12.5 %, 15 % y 18 % p/p de ñame en base húmeda y el monitoreo de la SSF se realizó a lo largo del tiempo de fermentación, cuantificando la concentración de azúcares reductores totales y etanol. El rendimiento de hidrólisis fue del 90 % de la masa de almidón para las concentraciones de solidos de 10 % y 15 % p/p, basado en el almidón teórico disponible en el tubérculo. Para el etanol, el mejor resultado fue de 0.19 g/Lh-1 de productividad para el ensayo de 10 % p/p de concentración de sólidos. Por lo tanto, el ñame es un material amiláceo adecuado para producir bioetanol mediante un proceso de SSF.
Palabras clave: ñame, almidón, hidrólisis enzimática, sacarificación y fermentación simultánea, bioetanol.
The importance of renewable energy sources has brought new research challenges. One of these scopes is reducing the greenhouse gases produced by the transport sector, which contributes 11.6 % and 10.7 % of global deaths due to exposure to Particulate Matter (PM) and ozone, respectively. These transport emissions mostly come from sub-Saharan Africa, Central America, parts of the Middle East and Central Asia, and Southeast Asia [
Tropical crops (e.g. sugar cane) have a restricted use worldwide due to their high-water requirements. Hence, the low-water requirement of amylaceous crops becomes an advantage in countries with a climate that changes throughout the year. For instance, Dioscorea sp. (yam) requires a lower water application (888 mm) compared to Manihot esculenta (cassava) and corn (1492 and 1017 mm, respectively) [
In this regard, Dioscorea rotundata is the Dioscorea sp. variety with the lowest moisture content (58.18 ± 1.22 % w/w) and the highest starch concentration (85.51 ± 1.21 % w/w) in dry basis [
However, ethanol production from amylaceous crops requires a starch hydrolysis process before fermentation, which increases direct costs. This has been observed in feedstocks such as corn, with 35 % higher production costs per liter of ethanol than sugarcane [
Consequently, evaluating new amylaceous feedstocks could minimize said costs, thus leading to a smaller difference between petrol fuel and flex fuel.
The biotechnological route could reduce energy requirements and fix CO. through farming growth. Saccharomyces cerevisiae is the most widely used fermenting yeast thanks to some of its strains resistant to high temperatures and ethanol. Bioethanol can be produced via fermentation using two methods: Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). Both employ starch to produce ethanol and comprise the same phases. In the first phase, enzymatic hydrolysis is applied employing α-amylase and glucoamylase enzymes for starch degradation, thus reducing polysaccharides to monosaccharides and some disaccharides used in further fermentation.
Nevertheless, SHF lasts over 72 h, while SSF only requires 36 h [
In [
Despite its low-water requirement and production costs, Dioscorea sp. has been little studied. This yam is mainly produced in tropical countries, with Nigeria, Ghana, and Cote d’Ivoire as the leading producing countries around the world [
In some studies, Dioscorea sp. has been employed to produce glucose and ethanol. In particular, Dioscorea alata, Dioscorea esculenta, and Dioscorea hispida have been analyzed to evaluate their potential for ethanol production. According to the results of these studies, 65.95 g/L, 64.17 g/L, and 59.19 g/L of TRS were obtained, respectively, after enzymatic starch digestion with 0.1 % w/w of α-amylase [
Dioscorea rotundata is the second of three main Dioscorea sp. varieties produced in Colombia [
However, a higher hydrolysis yield was achieved after 28 h of enzymatic hydrolysis (95 % of hydrolyzed starch) and 0.47 g/g of YP/S were obtained after 52 h of fermentation with Saccharomyces cerevisiae, as in [
In view of the above, the main purpose of this study is to evaluate TRS concentration and ethanol yield from Dioscorea rotundata via enzymatic hydrolysis and SSF, respectively, using Saccharomyces bayanus as a potential substitute for the well-known Saccharomyces cerevisiae.
Dioscorea rotundata peels were cut and removed, and the peeled yam was mashed. Subsequently, each sample was prepared using different yam concentrations (10 %, 12.5 %, 15 %, and 18 % w/w) in 250 mL of distilled water.
Samples were gelatinized for 60 min on a 10-place heater with a stirring speed of 250 rpm and at 68 °C. The temperature was increased to 90 °C for the liquefaction phase, while the stirring speed was maintained. After adding 0.007 mL of α-amylase (Amiltex 35 NP) per yam gram, the samples were rested for 40 min. Finally, they were subjected to saccharification by adding glucoamylase (Naturalzyme GA 300 L) in a ratio of 0.004 mL per yam gram and rested for 20 min at 60 °C. Subsequently, temperature was reduced to 30 °C for the SSF method to simultaneously continue with the saccharification process during the fermentation stage.
The Simultaneous Saccharification and Fermentation (SSF) method was used to make the most of its low-energy requirements and time savings. For this process, 2.5 g/L of Saccharomyces bayanus (SafCider-Fermentis®) was added to each experiment after its hydration with ten times its weight in a water mass at 35 °C for 15 min. The temperature was reduced to 30 °C, the pH was adjusted to 5 with HCl 1 N, and the stirring speed was set to 250 rpm for 27 h. Samples of each experiment were taken at the beginning and after 15, 18, 22, 25, and 27 h.
The 3,5-dinitrosalicylic acid (DNS) technique [
Ethanol concentration was analyzed by means of High-Performance Liquid Chromatography (HPLC) in a Dionex UltiMate 3000 (Thermo Scientific, USA) equipped with a Shodex RI-101 detector (Showa Denko K.K., Japan) and a Shodex SH1821 column operating at 75 °C with 0.5 mM H2SO4 as mobile phase (0.6 mL min-1).
The hydrolysis results were evaluated based on the hydrolysis yield (1). The theoretical moisture was 58 % w/w, value that was used to calculate the theoretical starch mass concentration [
Where
YH = Hydrolysis yield.
cf = Final starch concentration.
ct = Theoretical starch concentration.
The SSF results were assessed based on the product-substrate yield (2), the fermentation yield (3), and the ethanol productivity (4) from the product and substrate concentrations (denoted as P and S, respectively).
Where
Yp/s = Product-substrate yield.
YF = Fermentation yield.
Pf = Final product concentration.
Po = Initial product concentration.
Sf = Final substrate concentration.
So = Initial substrate concentration.
t = Time.
An ANOVA single factor test was conducted to evaluate the dependency of available TRS (after enzymatic hydrolysis) and remaining TRS (after SSF) on initial yam concentration.
The hydrolysis yield per each yam concentration was calculated considering the theoretical starch mass content (Figure 1). According to the results, the samples with yam concentrations of 10 % and 15 % w/w exhibited the highest yield (almost 90 % of starch hydrolyzed to reducing sugars before SSF). Nevertheless, the TRS content in the sample with a yam concentration of 15 % w/w (111.2 ± 3 g/L) was 58.9 % greater than that of the sample with a yam concentration of 10 % w/w (69.9 ± 4.5 g/L).
This hydrolysis yield is consistent with the results in [
Moreover, our results can be compared to those obtained with other Andean tubers (i.e., cassava, potato, and sweet potato). For instance, the authors in [
Compared to very high gravity systems, our results are still better than the hydrolysis yields of rye starch (25 and 28 % w/w) reported in [
The ANOVA results showed a statistical significance (p ≤ 0.05) between initial yam concentration and TRS production after hydrolysis. Consequently, initial yam concentration affects TRS concentration after hydrolysis.
TRS concentration was measured throughout the SSF process. As shown in the TRS consumption plot (Figure 2), a stabilization is observed from hour 22, with TRS concentrations close to zero for each system, as stated in [
According to the ANOVA test results, there is no statistical significance (p ≥ 0.05) between remaining TRS (at the end of SSF) and initial yam concentration. Conversely, remaining TRS depends on available TRS at the beginning of SSF (p ≤ 0.05).
The highest ethanol productivity and YP/S were observed in the system with a yam concentration of 10 % w/w (Table 1). By comparison, in the systems with a yam concentration from 12.5 % to 18 % w/w, the samples’ productivity showed a positive linear trend. However, if these results are compared to those obtained with other tubers, YP/S was 84.5% lower than the results reported in [
Dioscorea concentration (% w/w) | Volumetric ethanol concentration (% v/v) | Ethanol concentration (g/L) | Yp/s | Fermentation yield (%) | Productivity (g/L h-1) |
10 | 0.52 | 4.15 | 0.07 | 13 | 0.19 |
12.5 | 0.52 | 4.12 | 0.05 | 10 | 0.15 |
15 | 0.56 | 4.44 | 0.04 | 9 | 0.17 |
18 | 0.60 | 4.84 | 0.06 | 12 | 0.18 |
Regarding final ethanol concentration, our results are marginal if compared with the fermentation results reported in [
Despite the low fermentation performance, the resulting volumetric ethanol concentration (Table 1) was similar to that obtained in [
Nevertheless, the obtained ethanol productivity and yield confirm that the yeast strain used in this study is not feasible for fast ethanol production via SSF. Therefore, further improvements in terms of increased ethanol yields are necessary to achieve a viable economic process using this potential feedstock that has proven to have a better hydrolysis phase than other tubers.
Although the authors in [
Dioscorea rotundata has a great potential as raw material for lactic and alcoholic fermentation due to its high reducing sugar concentration after hydrolysis. However, Saccharomyces bayanus is not suitable to replace Saccharomyces cerevisiae in ethanol production via SSF due to its low ethanol productivity in the first 24 h of fermentation.
The authors would like to thank the Chemical Engineering Laboratory of the National University of Colombia for the HPLC loan. This article is not funded.
None declared.
Alfredo Enrique Villadiego del Villar: Research development and manuscript redaction.
Nicolás Sarmiento Zea: Research development.
Jeffrey León Pulido: Manuscript revision.
Lilia Carolina Rojas-Pérez: Research direction, manuscript redaction, and correction.