Yucca (manihot esculenta crantz) starch polysaccharide dextrination through biological procedures.

Pedro Villalba, Antonio Bula, Homero San Juan and Adrián Ávila

Pedro Villalba. Mechanical Engineer and M.Sc. student, Universidad del Norte (UN), Baranquilla, Colombia. Junior Researcher, Grupo de Investigación en Uso Racional de la Energía y Preservación del Medio Ambiente (UREMA), Colombia.

Antonio Bula. Mechanical Engineer, UN, Colombia. M.Sc. and Ph.D. in Mechanical Engineering, University of South Florida, USF, USA. Coordinator and Researcher, UREMA, Colombia. Address: Mechanical Engineering Department, Universidad del Norte. Km 5 Antigua Vía Puerto Colombia. Barranquilla, Colombia. e-mail: abula@uninorte.edu.co

Homero San Juan. M.D., UN, Colombia. Ph.D., USF, USA. Researcher, Grupo de Investigación en Biotecnología, UN, Colombia.

Adrián Ávila. Mechanical Engineer and M.Sc. in Mechanical Engineering, UN, Colombia. Researcher, UREMA, Colombia. Mechanical Engineering Coordinator at Universidad Pontificia Bolivariana, Montería, Colombia.

SUMMARY

The yucca (Manihot esculenta) starch dextrination process using Aspergillus niger has been evaluated in order to obtain fermentable sugars from the original polysaccharide. The maximum glucose concentration (2466mg·l^-1) was obtained at 30°C for a period of three days. The statistical analysis (P=0.05) of the experimental results revealed a second order behavior for the process. The interaction between the variables time (days) and temperature significantly affected the glucose production due to the enzymatic action of the A. niger on the substrate. Response surface analysis showed an optimal point for the process at 37ºC and 2.75 days, and a significant interaction between temperature and time. It is concluded that the catalytic action of A. niger strongly depends on the environmental conditions in which the process is carried out.

El proceso de dextrinación del almidón de la yuca (Manihot esculenta) utilizando Aspergillus niger fue evaluado a fin de obtener azúcares fermentables a partir del polisacárido original. La concentración máxima de glucosa (2466mg·l^-1) se obtuvo a 30ºC en un período de tres días. El análisis estadístico (P=0,05) de los resultados experimentales reveló un comportamiento de segundo orden para el proceso. La interacción entre las variables tiempo y temperatura afectaron significativamente la producción de glucosa debida a la acción enzimática de A. niger sobre el sustrato. El análisis de la superficie de respuesta mostró un punto óptimo para el proceso en 37ºC y 2,75 días, y una interacción significativa entre tiempo y temperatura. Se concluye que la acción catalítica de A. niger depende fuertemente de las condiciones ambientales en las que se desarrolla el proceso.

RESUMO

O processo de dextrinado do amido da mandioca (Manihot esculenta) utilizando Aspergillus niger foi avaliado a fim de obter açúcares fermentáveis a partir do polissacarídeo original. A concentração máxima de glicose (2466mg·l^-1) se obteve a 30ºC em um período de três dias. A análise estatística (P=0,05) dos resultados experimentais revelou um comportamento de segunda ordem para o processo. A interação entre as variáveis, tempo e temperatura, afetou significativamente a produção de glicose devido à ação enzimática de A. niger sobre o substrato. A análise da superfície de resposta mostrou um ponto ótimo para o processo em 37ºC e 2,75 dias, e uma interação significativa entre tempo e temperatura. Conclui-se que a ação catalítica de A. niger depende fortemente das condições ambientais nas quais se desenvolve o processo.

KEYWORDS / Aspergillus niger/ Dextrination/ Manihot esculenta/ Starch/ Yucca/

Different processes are known to produce ethanol from biomass and, generally, they depend on the source used. The different reaction stages for starchy materials such as yucca, require a step previous to fermentation, in which the fermentable sugars are obtained. This brings a time delay in the process.

Ohta and Hamada (1993) and León and Chalela (1997) presented evidences of a process where the hydrolysis is not necessary as one of the stages required to obtain glucose. In order to attain this, the polysaccharides are degraded through a biological process that avoids the acid hydrolysis and allows the emergence of co-cultures, which are able to carry out a combined action of hydrolysis and fermentation. Other authors (Inloes and Taylor, 1983; Laluce and Mattoon, 1984; Ma and Lin, 2000; Rendlema, 2000) have considered the use of other systems to go directly from starch to fermentable sugars, considering combinations of different Saccharomyces sp. (cerevisiae, diastaticus). However, these studies did not include an analytical correlation between the glucose concentration obtained and the process variables. The purpose of the present study was to carry out a biological hydrolysis process and determine the experimental correlation between temperature, time and concentration of the reaction products. Thus, eliminating the problems produced by the acid hydrolysis such as pH stabilization prior to the fermentation stage. The experimental setup considered Aspergillus niger acting over a yucca (Manihot esculenta Crantz) starch substrate as the bio-system.

An experimental design was developed in order to analyze the process variables. The design included two variables (temperature and time) and two levels for each variable (low and high). Temperatures were 23-37°C and times were 1-5 days. In addition, the central point (3days and 30°C) was replicated three times to reach the required degrees of freedom for the experimental error to be determined. All results were studied by means of ANOVA.

Three batches, each of 500g of yucca were washed and cut in pieces in order to liquefy them. Then the fiber was separated using a mesh and the liquid containing the starch was left to sediment for 24h in a pan. Once the liquid and the solid separated, the starch was removed from the bottom of the pan. Aspergillus niger was obtained from an original cell from the biology laboratory at Universidad del Norte, Colombia, and kept in solid Agar Sabouraud (MERCK®). The spores were counted using a Neubauer chamber, using the central measurement area (mm).

The hydrolysis of the yucca starch was done using liquid glucose chloramphenicol (MERCK^®) diluted to 2:1 in distilled water in order to reduce the initial glucose, and the final pH was brought to 5.4 using HCl. The reaction was carried out in 1 liter of previously prepared solid Agar Sabouraud. Twenty grams of yucca starch and 10⁵ spores of A. niger were added to the media and air was supplied artificially through a pumping system. Glucose was quantified with a spectrophotometer (HANNA Instruments^®) using near-infrared light technique, described as a non-invasive blood glucose measurement (Chen and Bai, 2007; Xiao and Wang, 2007); the calibration curve was generated in a previous experiment using standards of pure glucose (MERCK^®) at different concentrations; the relative coefficient was 0.985. From the absorptance values obtained, the glucose concentration was determined (Valencia and Bula, 2005).

The amount of yucca starch obtained from the three different batches (500g each) was 96.75, 105.2 and 92.95g. These samples were hydrolyzed using the procedure described before. The temperature, number of days for the process, as well as the absorptance measured in the chromatographer at the end of the process, are presented in Table I, together with the corresponding glucose concentration obtained. The maximum values were attained at the central point for temperature and time, 30ºC for three days.

The efficiency of the process for each of the different samples, also shown in Table I, was calculated considering the 20g·l^-1 of yucca starch introduced at the beginning of the hydrolysis process. The maximum efficiency obtained was 13%.

In order to reach fermentable sugars from yucca starch through a hydrolysis procedure using a biological approach, it was necessary to determine the effect of temperature and time on glucose production. The analysis of the experiment showed that the main effect on the process was not that exerted by the original variables, temperature (A) and time (B), but was the interaction of these two factors (AB). The results are presented in Table II and the effect of the temperature - time interaction is presented in Figure 1.

As the interaction becomes significant, for the specific case of low time (B-, 1 day), the catalytic activity increases as the temperature (A) increases, and as the result the glucose concentration improves. On the other hand, for the specific case of high time (B+, 5 days), an increment in the temperature generates a falling glucose concentration for the process. This indicates that A. niger performs a better catalytic function at higher temperatures but, as the hydrolysis process improves, the microorganism suspends the task and begins to consume the glucose generated from the process.

The variance analysis presented in Table III shows a second order trend for the process (quadratic effect), which means that the average value at the central condition surpasses the average values at the edges. This effect is added to the twisting produced by the interaction and thus, the trajectory from the low level to the high level presents a curvilinear behavior.

Figure 2 shows the glucose concentration response surface, and the twisting introduced by the quadratic term is apparent. Additionally, it can be observed that the process presents a maximum. The figure also shows the tendency of the glucose concentration to increase as the temperature increases. The results presented a maximum at three days for the different temperatures considered, however, a maximum is observed at 37ºC. The response surface is represented by:

where T: temperature (ºC), and t: time (days).

The optimization process performed over the response surface proves that the optimal point for the process is presented at 37ºC and 2.75 days, with an expected final glucose concentration of 2584.33mg·l^-1. This condition confirms that the enzymatic action of A. niger is more efficient at high temperatures, limited by the survival temperature of the biological element.

The authors thank G.E. Valencia, for help in quantifying glucose and acknowledge the support received by the Young Researchers Program, Dirección de Investigaciones y Proyectos, Universidad del Norte, Colombia.

1. Chen Y, Bai G (2007) Non-invasive blood glucose measurement system based on three wavelengths in near-infrared region. In 5^th Int. Conf. Photonics and Imaging in Biology and Medicine. Proc. SPIE 6534, part 2: 653-680.        [ Links ]

2. Inloes D, Taylor D (1983) Ethanol production by Saccharomyces cerevisiae immobilized in hollow-fiber membrane bioreactors. Appl. Env. Microbiol. 46: 264-278.        [ Links ]

3. Laluce C, Mattoon J (1984) Development of rapidly fermenting strains of Saccharomyces diastaticus for direct conversion of starch and dextrins to ethanol. Appl. Env. Microbiol. 48: 17-25.        [ Links ]

4. León A, Chalela G (1997) Diseño y puesta en marcha de un sistema semicontinuo en dos etapas: hidrólisis fermentación para la producción de etanol a partir de almidón de papa usando simultáneamente Aspergillus niger y Saccharomyces cerevisiae. Rev. Col. Quím. 26: 1-10.        [ Links ]

5. Ma YJ, Lin LL (2000) Efficient utilization of starch by a recombinant strain of Saccharomyces cerevisiae producing glucoamylase and isoamylase. Biotechnol. Appl. Biochem. 31: 55-59.        [ Links ]

6. Ohta K, Hamada S (1993) Production of high concentrations of ethanol from insulin by simultaneous saccharification and fermentation using Aspergillus niger and Saccharomyces cerevisiae. Appl. Env. Microbiol. 59: 729-733.        [ Links ]

7. Rendlema J (2000) Hydrolytic action of a-amylase on high-amylose starch of low molecular mass. Biotechnol. Appl. Biochem. 31: 171-178.         [ Links ]

8. Valencia G, Bula A (2005) Determinación experimental de las azucares fermentables en el almidón de yuca horra. Universidad del Norte. Barranquilla, Colombia.        [ Links ]

9. Xiao J, Wang L (2007) A blood glucose detecting system based on three wavelengths. J. Optoelectron. Laser 18: 1135-1138.        [ Links ] ]]> 1 2007 2 1983 46 264-278 3 1984 48 17-25 4 1997 26 1-10 5 2000 31 55-59 6 1993 59 729-733 7 2000 31 171-178 8 2005 9 2007 18 1135-1138