Optimization of ethanol fermentation from discarded carrots using immobilized Saccharomyces cerevisiae

Adriana L. Clementz 0 1 Nora R. Aimaretti 0 1 Debora Manuale 0 1 Agustn Codevilla 0 1 Juan C. Yori 0 1 0 N. R. Aimaretti A. Codevilla Laboratorio de Investigaciones Aplicadas, Facultad de Qu mica, Universidad del Centro Educativo Latinoamericano , Av Pellegrini 1332, 2000 Rosario , Argentina 1 A. L. Clementz N. R. Aimaretti D. Manuale J. C. Yori (&) Facultad de Ingenier a Qu mica, Instituto de Investigaciones en Cata lisis y Petroqu mica, Universidad Nacional del Litoral, CONICET , Santiago del Estero 2654, 3000 Santa Fe, Provincia de Santa Fe , Argentina Discarded carrots are a valuable source of biomass amenable for valorization. Their use as raw material for ethanol production by fermentation, using yeasts immobilized in Calcium alginate, was proposed. The biocatalyst immobilization method, the existence of internal and external mass transfer limitations, the effect of the initial pH and the reuse of immobilized yeasts were particularly evaluated. Results indicate that beads made with a 2 % solution of Sodium alginate and a 30 % solution of Saccharomyces cerevisiae were strong enough to allow an efficient nutrient transfer into the matrix and to prevent cell leaking. A stirring rate of 200 rpm was needed to avoid external mass transfer limitations. These beads were used in three successive fermentations. An initial pH of 5.5 reached the best fermentation parameters. Non-enriched, non-sterile carrot must was fermented through immobilized yeasts; and values of ethanol concentration (29.9 g L-1), Yp/s (0.409 g g-1), and productivity (7.45 g L-1 h-1) were obtained. These values were similar to those registered when free cells were used. - Nowadays, the conversion of biomass into biofuels represents a significant economic option [1]. Ethanol is one of the most important renewable fuels contributing to the reduction of negative environmental impacts generated by the worldwide utilization of fossil fuels [2]. Bioethanol is generally produced from raw materials containing fermentable sugars, such as sugar cane, sweet sorghum, beet molasses, corn, wheat, etc. [3, 4]. However, the current focus on ethanol production is the conversion from non-edible sources or wastessuch as agricultural and forest residuesinto second generation ethanol. It is the abundance and the low-cost of these raw materials that make them attractive as such [5]. In the Santa Fe region (Argentina), 50100 tons of carrotsalmost 30 % of the total productionare discarded every day during the harvest time. Discards are generally made up of carrots that do not meet commercial standards with regard to size or shape, or whose economic value would not be enough to compensate for the harvesting costs [6]. Such a high percentage of discards is not exclusively seen in Argentina: similar values are found in other countries as well. In general, only 10 % of total discards is used for animal consumption; the rest remains in the fields generating odors and causing land deterioration, which are the main agents producing a highly adverse environmental impact. As a consequence, reutilization of this type of discard is very important. In previous papers [7, 8], it was concluded that the must extracted from discarded carrots could be used as raw material for ethanol fermentation in a stirred tank bioreactor using free cells of Saccharomyces cerevisiae as biocatalyst, with neither sterilization pretreatment nor addition of nutrients. It was also reported that the maximum productivity and ethanol yield were 6.2 g L-1 h-1 and 0.295 g g-1, respectively. Apart from the results, difficulties found in the purification of the fermented must (cell separation) were also highlighted. Some of them are the high costs of installation and microbial recycling, the high contamination risks, and the susceptibility of the microorganism to environmental variations [9]. These problems could be overcome using immobilized yeast. The most extensively studied method in cell immobilization is entrapment. This technique is based on the inclusion of cells within a rigid network to prevent cells from spreading into the reaction medium. Sodium alginate (Na-alginate) solutions form gels of Calcium alginate (Ca-alginate) in the presence of cations such as Ca2? [10]. Immobilizing cells in alginate is simple, economic, and not toxic. Therefore, Ca-alginate is frequently used for immobilization because of the simplicity of the procedure to prepare the beads and the mild conditions required. Several reports indicating the use of this compound are available [1114]. Although many works have been done on the production of ethanol immobilizing yeasts in Ca-alginate, in none of them have discarded carrots been used as raw material for fermentation. The aim of this work was to learn the feasibility of producing second generation ethanol through the use of carrot discards, particularly through the immobilization of S. cerevisiae yeast in Ca-alginate as biocatalyst. The biocatalyst immobilization method, the existence of internal and external mass transfer limitations, the effect of the initial pH and the possible beads reuse were specifically studied. Materials and methods Raw material, handling and storage Discarded carrots (Daucus carota) were collected in November and December 2012 from a packing shed in the Santa Fe area (31 250S, 60 200W), Argentina. As for handling and storage, the method described by Aimaretti and Ybalo [7] was used. Discarded carrots were selected leaving aside areas affected by microorganisms. Then, their juice was extracted by a continuous milling, compressing and filtering treatment. Particulates present in carrot juice were separated through centrifugation for 10 min at 3,500 rpm. The supernatant was named carrot must (CM) and its average sugar content was 89.8 1.2 g L-1 and its pH was 6.4 0.2. CM was not subject to sterilization. To evaluate the effect of the initial pH value, this was adjusted by adding sulfuric acid. Saccharomyces cerevisiae was provided by a local supplier (Danica, Argentina) in the form of pressed yeast and it was reactivated directly in the carrot juice. Whole yeast cells were kept in a sterile container, without addition of nutrients, at 4 C and saturation humidity, during six days [6]. Yeast immobilization method For the immobilization of yeasts, two solutions were required: one consisting of living cells of S. cerevisiae in water (solution A), and another one consisting of Naalginate (Protanal LM 120 LS) in water (solution B). Both solutions were mixed together at equal volumes (1:1 v v-1) and stirred until a homogeneous solution was visible. Then, with a micropipette, the solution was added dropwise to a 0.2 M CaCl2 solution prepared in a 0.05 M sodium acetate buffer (pH 5.6). Drops solidified in the form of beads and yeast cells were entrapped inside. Beads were kept in suspension for 30 min to harden. To remove calcium ion excess and free cells, beads were separated and washed with a 0.01 M sodiu (...truncated)