Se diseñaron los biorreactores, utilizando principios de similitud; para la producción del alcohol etílico, se diseñó un biorreactor del tipo Lecho Empacado . Inmovilización de levaduras en residuos lignocelulósicos para la producción de etanol en biorreactor de lecho empacado. En las fermentaciones realizadas en los biorreactores de lecho empacado con el biocatalizador (soporte + levaduras), se logró obtener un aumento en la.

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Yeast immobilization in lignocellulosic wastes for ethanol production in packed bed bioreactor. Bloque 5 laboratorio Recibido el 5 de abril de Aceptado el 24 de febrero de This study is focused on the development of an immobilization process of yeast cells in waste lignocellulosic materials and their evaluation in the ethanol production by using packed bed bioreactors.

We evaluated four different waste lignocellulosic materials: The characterization made it possible to establish the microscopic structural differences between the four materials, as well as the differences in composition of lignin, cellulose, hemicelluloses and ash. A protocol for packaging biorreacttor and a quantification methodology of immobilized biomass was developed. An experimental design was conducted to determine the effects of the size of lignocellulosic materials on cell biorreactot and to establish the effects of the flow rate in the immobilized cells when the fermentation is performed in packed bed bioreactors.

Under the established experimental conditions we determined the size and flow rate that provided the better operational stability for the fermentation in packed bed bioreactor.

The result of the study showed that the material in which the biggest amount of cells was immobilized was the sugar cane bagasse, and we obtained a value of 0. This amount of immobilized biomass is significant compared to the values reported by other authors.

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As a result of the experimental design of the influence of flow and size of the carrier on the immobilization, it was established that there is no significant statistical difference in the range of the values used in the experiment size of 3. The size of the carrier selected was 3. Emmpacado results have established the implementation potential of ethanol production by continuous fermentation with immobilized cells in waste lignocellulosic materials.

Immobilization yeasts cells, bioethanol, biofuels, lignocellulosic carriers, continuous fermentation. Se evaluaron cuatro diferentes residuos de materiales lignocelulosicos: Esta cantidad de biomasa inmovilizada es significativa comparada con los valores reportados llecho otros autores. High oil prices and decrease of world stock, in addition to the concerns about the continuous deterioration of the environment, have given a strong push to the search for alternative energy sources in recent years, particularly the development of liquid biofuels to replace oil and its derivatives, and reduce the negative impact on the atmosphere caused by burning fossil fuels.

In the last two decades there has been an increase of research focused on developing new technologies for ethanol production from renewable sources, and the efforts have focused towards the development of much more efficient production processes [1- 3]. Most industrial processes for ethanol production use batch systems, which are being implemented in Colombia in new production plants [4]. Some of the disadvantages of these processes are: As an alternative, continuous processes are brought up, which can reduce production costs, improve process efficiency and increase ethanol yield.

Continuous processes that use immobilized cells show great advantages over those using free cells, since they facilitate the separation of the product, allow re-use of the biocatalyst, they have a high volumetric productivity, and facilitate the control of process variables as well as reducing the risk of contamination [5].

However, fermentations with immobilized cells also have some disadvantages, such as the difficulty to predict changes in cell growth, physiology and metabolic activity, and the presence of mass transfer limitation by diffusion. Other authors have bioorreactor improvements [6, epacado. There are also demanding requirements for materials used as carriers, since chemical, physical and biological stability has to be ensured, and the carriers and the technique must be low cost and easy to implement on an industrial scale.

Among the most widely used immobilization matrices are hydrophilic polymer gels such as alginate, carrageenate, agarose, etc.

However, this method of immobilization is impractical on an industrial scale because of the cost of raw materials and the relative complexity in the preparation of biocatalysts with respect to the operational lifetime in the process [8- 10]. Other methods of immobilization in relation to the union of the cells to the carrier are: In recent years, raw materials lecno from agro-industrial waste and their application as promising carriers for the immobilization of cells have been evaluated.


For instance, the sugar cane bagasse [12], delignified cellulosic materials [13], orange peel [14], spent grains [15, 16], wood chips [17], among lexho. On these carriers the cells are immobilized by adsorption, a simple and economically favorable technique.

The cellulosic materials are regenerable, reusable, sterilizable on e,pacado, biologically and chemically stable under different fermentation conditions and with adequate mechanical resistance [18].

The main mechanisms that occur during immobilization by adsorption are: Most of these mechanisms are spontaneously generated during contact between cells and carrier, so adsorption is one of the simplest mechanisms to perform, both in a laboratory and industrial level. The biorreator of this study is to establish a process of cell immobilization on lignocellulosic waste and their potential use in the production of ethanol. Leccho proposed hypothesis is that these materials are appropriate matrices for the immobilization of yeast cells, which allow high cellular activity, lasting stability and they enable continuous operation for the production of ethanol.

The lignocellulosic wastes used in this research are obtained from agro-industries and they are abundant in our country. The medium for inoculum and fermentation is composed of: The pH was adjusted to 5. Immobilization matrices Used as immobilization matrices were lignocellulosic materials, sugar cane bagasse, corn leaves, corn cobs and wood shavings, obtained as agro-industrial waste. These materials were subject to a conditioning process that involved the implementation of empaxado protocol for cleaning, drying and size reduction.

The average sizes of the pieces obtained were 3.

Biomass quantification Protocol dry weight modified technique After completion ofthe 12 hours of immobilization baseline, the biomass quantification process developed was as follows: Drying time elapsed, biocatalysts were left idle until they reached room temperature, then 9 samples of each one were taken, with an approximate weight of 0.

Series of three tubes were filled with 50 ml of water, isotonic solution and NaOH 0. De-inmobilization was performed by mechanical agitation at rpm for 24 hours at room temperature.

After drying, they were weighed again. The same process was implemented with cell-free material to be used as blank. The immobilized biomass was obtained from the dry weight difference of the carrier before and after the de-immobilization process, and the value was corrected using the blank. For the development of the protocol three solutions were evaluated: Each treatment was performed in triplicate. Study of the effect of size and flow rate for each material To determine the effect on immobilization of flow rate and the carrier size, we performed a factorial experimental design with 2 factors and 2 levels, and a central point with three replicates.

The results of this experiment were analyzed using the statistical analysis program Design Expert. Immobilization under fermentative conditions in mL bioreactor We used a glass column of ml capacity, fitted jacket for temperature control and two peristaltic pumps for feeding fresh culture medium and circulation of water through the jacket.

The baseline immobilization was performed by following the yeast immobilization protocol. Subsequently, the bioreactor was filled with fresh culture medium and allowed to operate with recirculation for 12 hours [19]. The response variables evaluated in this study were the reduced sugars consumption, and ethanol production.

Analytical methods Microbial biomass was determined by the dry weight modified protocol developed in this research. The reduced sugars in the culture medium were determined using the technique of DNS [20].

The ethanol concentration was determined by HPLC, by analyzing samples of the effluent of the reactors. We used a column Supelco gel CH with the following conditions: The method of detection was rate refraction, and the retention time was The Scanning Electron Microscopy technique SEM was used to observe the differences in the structures of materials and cell immobilization. Determination of lignin, cellulose, hemicellulose and ash contents was carried out by the detergent method of Van Soest [21].

Due to heterogeneity in nature and origin of agro-industrial waste used in the experiment, and to avoid interference in the subsequent process of cell immobilization, it was necessary to establish a cleaning treatment and adapting materials. Figure 2 shows the material upon completion of the process of conditioning. Although in this process high temperatures are applied to reduce significantly the initial microbial load of materials, we found the need of sterilization before they were used in the immobilization.


Figure 3 shows the growth of microorganisms in petri dishes containing nutrient agar and pieces of non-sterile materials; however, there is no growth in the petri dishes that were placed in sterile lignocellulosic residues.

Figure 4 presents the results of the images obtained from scanning electron micrographs SEM. Differences on the surface of the structures of the four materials can be found, these differences can lead to changes in yeast cell immobilization.

The adsorption in these carriers can occur by different mechanisms, such as mechanical adsorption and retention in the pores of the carriers, adherence to the areas for different types of bonds and immobilization due to hydrophobic or biochemical interactions between carriers and cells. These immobilization mechanisms are not only dependent of the surface characteristics of the carriers, they depend on cell surface characteristics, the carrier used and the operational conditions under which the immobilization is carried out flow, temperature, etc.

To establish which of these mechanisms were involved in immobilization on the lignocellulosic materials used, it is necessary to study each of these characteristics. Regarding materials composition, the percentages of lignin, cellulose, hemicellulose and ash that are presented in table 1there are different proportions in each material; these differences can directly affect yeast immobilization.

For example, lignin values of sugar cane bagasse, wood shavings and corncob are high, Lignin is the substance that gives stability and rigidity to lignocellulosic material, so the low value for corn leaves may cause a structural weakness of material, it could affect the continuous operation for long periods, reducing their potential for use in immobilization processes on an industrial level.

To establish immobilization capacity on each of the materials and determine the effect of size and flow rate in the process, we standardized the biomass quantification methodology on the carriers.

We studied various techniques spectrophotometry, protein quantification and dry weight ; the better results were obtained with the dry weight technique, which was modified to allow direct quantification of biomass immobilized on the carrier. The protocol developed was described in the methodology biomass quantification protocol.

By this methodology it was possible the direct determination of immobilized biomass ensuring reproducibility in the quantification. Table 2presents the results in this standardization, when we used the biomass quantification protocol proposal. In the first column the carriers studied are listed corn leaves, wood shavings, corn cob and cane bagasse.

Treatment column corresponds to the different solutions evaluated NaOH 0. Blanks were evaluated using cell-free material.

Modelado de reactores de lecho empacado en COMSOL®

As shown, the values obtained empscado the two reactors for treatment wmpacado water and isotonic solutions are not suitable for immobilized biomass determination. The results are not valid for the proposed protocol, because biiorreactor negative values represent high quantity of cells on the carrier at the end of the de-immobilization process.

In addition, the results have high deviations. However, the treatments with NaOH 0. Based on these results we chose the biomass quantification protocol modified dry weight treatmentthat uses NaOH 0. The experimental design was developed to determine the appropriate size empafado flow rate used in alcoholic fermentation in a ml bioreactor. In the experimental design, 15 treatments were evaluated by combining three sizes 3. To determine the size range of the carriers, we considered the bioreactor used and mass transfer criteria for packed bed columns.

For flow rate we considered the specifications of peristaltic pumps available in the laboratory and the residence time established previously in the bioreactor used.

Table 3 presents the consolidated experiments matrix with the results obtained. We can see that the emppacado of immobilized cells varies depending on the treatment and the material. The high variability in each of the treatments for different materials is indicated by the high deviations values and it is confirmed by the lack of statistical significance. The experimental design analysis using Design Expert software allowed us to establish that there is no significant statistical difference between treatments.