Resumen de Producción de xilitol por tecnologías avanzadas de fermentación a partir de zuros de maíz
The general objective of this work was to develop efficient systems for the microbial production of xylitol by Debaryomyces hansenii with the future vision to scale up the process. This general objective was addressed considering two approaches:
I. Optimizing the operation in highly productive scalable culture systems based on the use of immobilized cells, and II. Searching for alternative culture media prepared from agroindustrial wastes in order to reduce the economic cost of the large scale process and to obtain a final product that could be considered ¿natural xylitol¿ from the legal point of view.
1. Operation optimization To meet the first objective, immobilized systems for xylitol production were chosen considering the operational advantages that yeast immobilization offers such as high cell concentration, which allows reaching higher productivities, the possibility of reusing the inocula, and the easier operation in continuous fermentation. Among the different immobilization techniques, entrapment in alginate beads was selected because alginate is a stable, reusable, low cost and nontoxic immobilization support considered safe for the food industry. Considering the scalability of the process, two sub-objectives were then considered:
1.1 Optimizing the basic culture parameters affecting xylitol production by Debaryomyces hansenii immobilized in alginate beads.
The first step was to study the influence of those basic culture parameters directly related with yeast immobilization in alginate beads: bead size, chitosan addition to the alginate matrix, and the ratio between the amount of immobilizing support and the volume of the liquid medium. For this purpose a simple fermentation system consisting in batch cultures performed in Erlenmeyer flasks was selected.
The interest of studying the effect of the bead size on xylitol production was related to the importance of the mass transfer processes inside the alginate particle and their possible influence on the performance of the immobilized cells, taking also into account that the particle size can be an important design variable in large scale bioreactors.
Then, the addition of chitosan was tested to improve bead strength and stability of the immobilized cells, which can be damaged or released by shear stress and alginate dissolution in long-time continuous or fed-batch fermentation systems.
Finally, bead charge and volume of liquid media were studied as the operational variables related to biomass or inoculum concentration and Erlenmeyer flask aeration degree respectively. Since they together determine the real conditions of O2 availability in the system, they were studied by means of a factorial design in order to find the combination of both variables that generates a microaerobic environment leading to the occurrence of a redox imbalance responsible for xylitol accumulation by Debaryomyces hansenii.
1.2 Study of the potential of the air-lift bioreactor for xylitol production in different fermentation modes with Debaryomyces hansenii immobilized in alginate beads.
Although the stirred tank reactor was successfully assayed for xylitol production in previous works, they were not considered adequate for future large scale operation due to the increasing shearing forces generated by the intense agitation necessary for large volumes of operation. The packed bed reactor was also dismissed due to the difficulty of this system for introducing a gas flow, which will be foreseeably necessary for providing the minimum oxygen required for the microaerobic conditions that allow the regeneration of the NADPH and cell mainteinance if glucose is exhausted. The bubble column reactor represents the opposite situation to the packed bed reactor since the gas flow is the driving fluidizing mechanism, and it has been applied for the xylitol production, but its performance will depend on whether the minimum airflow necessary for fluidization is low enough to generate the optimal microaerobic conditions that trigger to xylitol accumulation. The fluidized bed reactor seems to be a very suitable system for xylitol production and indeed it is the most studied one with immobilized yeasts, but it usually implies the need of working with medium recirculation.
Considering all of that, airlift can be considered an interesting alternative to fluidized and bubble column reactors since it respectively avoids the need of medium recirculation and allows reducing the gas flow, due to the better performance of the fluidizing stream trough the loops specially designed for this. In spite of that, airlift has hardly been studied for this bioproduction, even at lab scale. For all these reasons airlift was selected in this work for studying its performance for xylitol production by the alginate-bead immobilized yeast.
With regard to the mode of operation, the application of fed-batch and continuous culture had shown to be an adequate alternatives to the batch culture for increasing xylitol production since they it allows reaching high concentrations of productive cells by providing high substrate inputs without substrate inhibition. Anyway, there are no references describing the behavior of xylitol production by Debaryomyces hansenii in fed-batch cultures, and just some works deals with performance of the continuous mode for this yeast, although none of them is performed in an air-lift bioreactor.
In consequence, the next objective of this work was focused on the study of the performance of the xylitol production by alginate-immobilized Debaryomyces hansenii in the airlift bioreactor assaying the fed-batch and continuous modes of operation. The influence of the operational conditions on the xylitol yield and productivity in this system, as well as how they are related to growth rate and biomass concentration were the main subjects of study.
2. Alternative culture media made from agro-industrial wastes In order to extend the xylitol production to an industrial process scale is important to consider and reduce the high costs due to the culture medium. For this purpose, the use of some agro-industrial wastes as culture media ingredients has been yet explored successfully for different microbial productions. As a result, some wastes have been transformed in new highly value raw materials for the pharmaceutical industries among others, as in the case of sugarcane molasses or corn steep liquor. But the search of new uses for wastes not only provides to industry with new cheap raw materials, but also contributes to the sustainability of the productive systems. Additionally, the use of natural resources not subjected to chemical transformations as substrates for microbial productions is a good strategy to obtain food additives that can be considered ¿natural¿, thus increasing their attractive for consumers and value. Taking it into account, two sub-objectives were addressed:
2.1 Evaluation of residual lees from the wine, beer and cider industries as nitrogen and micronutrients sources for xylitol production.
Winery, beer and cider lees are the by-products resulting from the fermentation step in the corresponding industries. They are liquid wastes consisting in the exhausted medium (wine, beer and cider), but they also contain a large solid fraction composed by precipitated compounds (tartrates in the case of wine lees, proteins, polyphenols¿) and yeast cells. In consequence, they are organic matter rich materials and, consequently, they have a high DBO that implies high depuration costs. Different valorization procedures have been proposed for winey lees including animal feeding based on their protein content, soil amendment after lees composting, biocontrol of fungal phytopatogens, and nutritional supplements for microbial culture media as sources for vitamin, growing factors and yeast extract after cells disruption. Nevertheless, considering this last valorization destination, there are hardly works referred to beer and cider lees and, in the case of winery lees, there are no studies about the different value of the solid and liquid fractions as culture media ingredients.
Taking into account the importance of the beer industry all over the world and the traditional establishment of the cider industries in the North of Spain, this objective was focused on the evaluation of these lees in comparison to winery lees for the formulation of culture media suitable for xylitol production by free Debaryomyces hansenii, considering both the whole wastes and the separated contribution of the solid and liquid fractions. Additionally, a culture medium only composed by wastes consisting in xylose-rich hydrolyzates obtained from corn cob and the best lees assayed as nitrogen and mineral salts sources was optimized and tested as a cheap alternative to synthetic media for the immobilized cultures studied in the sub-objective 1.1.
2.2 Study of different enzymatic treatments for xylose generation from residual corn cob to be finally used as substrate for the microbial production of ¿natural¿ xylitol by Debaryomyces hansenii. Preliminary assays for future work.
The hydrolyzates obtained from acid hydrolysis of lignocellulosic wastes as corn cob contain some fermentation inhibitors and toxic compounds for human consumption such as furfural, 5-hydroxymethyl furfural, acetic acid, phenolic compounds, aromatic acids and aldehydes. Enzymatic hydrolysis does not generate these toxic compounds due to the high specificity of the enzymatic processes and the milder conditions of pressure and temperature of the process. For this reason, enzymatic hydrolysis is an interesting strategy for generating xylose solutions as substrates for xylitol microbial production since it avoids the need of detoxification steps of the hydrolyzates prior to fermentation, and allows designing as ¿natural¿ the xylitol thus obtained since enzymatic treatments are accepted for the production of ¿natural¿ food additives.
Nevertheless, the enzymatic hydrolysis of hemicelluloses is usually hindered by the close and complex structure of the lignocellulosic matrix, but also by the xylan cross-linking between chains through diferulic bridges, which structurally impede the enzymatic access to the glycosidic bonds, and by the high chemical heterogeneity of the polymer depending on the source. In consequence, low xylose yields are often obtained.
Considering all that, the objective of the last part of this thesis was to evaluate, trough preliminary assays, the suitability of different strategies for a future work aimed to the efficient enzymatic hydrolysis of the xylan present in corn cob for obtaining xylose-rich hydrolyzates free of toxics of chemical origin.
Thus, two approaches were considered for this purpose:
a. Assay of different enzymatic extracts for sequential or simultaneous corn cob hydrolysis and fermentation with Debaryomyces hansenii for xylitol production.
Although the central enzymes involved in hemicellulose hydrolysis are xylanases, their performance depends on the xylanase specificity for each xylan according to its origin, and on the cooperative action of feruloyl esterases, which helps to eliminate the diferulic bridges between xylan chains, and, additionally, of other enzymatic activities contributing to the opening of the lignocellulosic matrix as cellulases or pectinases.
In consequence, the aim of this step was to compare the ability of commercial xylanases and an enzymatic extract previously produced by solid state culture of Aspergillus niger on the same material to be further hydrolyzed, for hydrolyzing the xylan present in corn cob, thus generating xylose for further fermentation to xylitol by Debaryomyces hansenii. Two strategies were also assayed: sequential and simultaneous corn cob hydrolysis and fermentation with the yeast.
b. Preliminary evaluation of high hydrostatic pressurization (HHP) as a tool for enhancing the enzymatic hydrolysis of corn cob hemicellulose.
Several mechanical, thermal, physico-chemical and biological pre-treatments have been assayed for reducing the crystalline order of the lignocellulosic matrix. Among them, only those involving physical and/or biological technologies that avoid the generation of xenobiotic and potentially harmful by-products can be considered for the generation of ¿natural¿ food additives.
High hydrostatic pressurization (HHP) is an innovative technology considered non-degradative, with an increasing potential as a lignocellulosic pretreatment based on the favorable effect of high pressures on hydrogen bonds formation and the consequent changes induced in the cellulose crystalline structure. This way, HHP has been recently assayed successfully as a previous step to the enzymatic elimination of hemicelluloses in kraft pulp.
Based on these promising results, and taking into account that the effect of pressurization on enzymes stability and activity depends on the enzyme and the total volume changes occurring in the system during the enzymatic reaction, a preliminary assay is here proposed where HHP is applied not only as a pretreatment, but also as an assistant technology during the enzymatic hydrolysis of corn cob with xylanases.
Main conclusions.
The experiments aimed to study the influence of the main culture variables related to yeast immobilization on the performance of Debaryomyces hansenii immobilized in Ca-alginate beads for the production of xylitol have allowed to draw the next conclusions:
1. Xylitol production was not affected by particle sizes corresponding to bead diameters in the range 1.60-2.83 mm, which allows the selection of the most adequate bead size for any reactor culture only in terms of hydraulic and economic reasons of bead preparation.
2. Ca-alginate beads prepared at 4% alginate concentration have shown a high physical and chemical stability after repeated cultures even when they were performed in the air lift bioreactor, with both synthetic and waste origin culture media. Consequently, the use of chitosan as a coating agent does not seem to be necessary to provide firmness to the immobilizing particles.
3. Electron micrographs revealed a very low intraparticular cell growth but the generation of a dense biofilm at the bead surface with pseudohyphal morphology, which could be due to steric reasons and nutrient and oxygen limitations. For that reason, the use of chitosan as a coating agent seems to hinder the development of the immobilized biomass that is located at the particle surface.
4. The experiments performed with chitosan and with different charges of inoculated alginate beads point to the favorable effect of biomass immobilization on xylitol production, which could be related to the cell metabolic changes that lead to morphological changes in the already mentioned conditions of mass transfer restrictions.
5. These immobilized cultures were actually mixed systems where there was a profuse growth of the free cells, which probably were generated via budding from the immobilized cells at the bead surface. The free biomass accounted between 75-95% of the total biomass, thus contributing greatly to the total production of xylitol.
6. The aeration degree necessary to generate the microaerobic conditions that lead to xylitol accumulation must be fixed considering the charge of immobilized biomass in order to avoid suboptimal microaerobic conditions for cells maintenance.
From the study of the performance of the air lift bioreactor for the xylitol production by Debaryomyces hansenii immobilized in Ca-alginate beads in fed batch cultures, the next conclusions have been extracted:
7. Xylitol productivity and yield are subjected to product inhibition by xylitol, showing a hyperbolic dependence with the concentration of the inhibitor that allowed estimating the respective IC50 at 200 and 275 g/L xylitol in the culture medium. Growth was also affected, but the rate of xylose consumption did not seem to be influenced by high xylitol levels, at least at xylitol concentrations until to 180 g/L. In any case, 100 g/L xylitol seems to be a reasonable limit for avoiding a strong effect of product inhibition in fed batch cultures.
8. As well as the limitation for xylitol secretion due to a unfavorable gradient across the cell membrane yet described by other authors, another reason for the xylitol inhibition seems to be related to the osmotic pressure generated by the high xylitol concentrations, which causes a greater energy consumption for maintenance, this including the triggering of the synthesis of glycerol as the main osmoregulator metabolite.
9. Despite what it is reported in the literature, there was a remarkable glycerol production by Debaryomyces hansenii in absence of glucose as substrate, which could be related mainly to the aforesaid osmoregulation function of this polyol, but also to the role of the glycerol synthesis pathway in the regeneration of NAD+ in conditions of suboptimal microaeration caused by an insufficient aeration or a high oxygen demand when high biomass concentrations were achieved.
10. The air lift bioreactor has shown to be an adequate system for this bioproduction with the yeast immobilized in Ca-alginate beads since the minimum air flow necessary for fluidization was even lower than that leading to the microaerobic conditions that trigger xylitol accumulation, while maintaining the physical and chemical integrity of the alginate beads as well as the viability of the immobilized cells until three months of reuses.
11. The fed batch operation in the air lift bioreactor allowed to extent the culture over 900 hours without loss of cell viability, as reflected by the maintenance of the substrate consumption rate during all the time of incubation without accumulation of unconsumed xylose.
12. The xylose feeding rate, the air flow and the biomass concentration at the beginning of the fed batch operation have shown to be critical parameters for achieving high productivities and yields in this system. Working at high air flows at the beginning of the incubation during the first batch step to enhance biomass growth, and increasing the xylose concentration below inhibiting concentrations (100 g/L) seems to be a good strategy for improving the performance of the fed batch xylitol production in the air lift bioreactor with Debaryomyces hansenii immobilized in Ca-alginate beads.
The assays conducted to fulfill the objective 2.1 referred to the evaluation of different lees for xylitol production by Debaryomyces hansenii have led to the following conclusions.
13. The physicochemical characteristics of the fermentation lees varied in a great extent depending on their origin.
a. Although the highest content in insoluble solids corresponded to beer lees, followed by cider and wine lees respectively, the content in soluble solids was almost the same for the three materials.
b. The most differentiating parameters for each lee considered were the high ethanol, Cu and Al content and the presence of tartaric acid in wine lees, the high concentration of Fe, Mn, phenolic compounds and lactic acid and the low N levels in cider lees, and the high Zn and N concentration, amino acids content and pH in beer lees.
14. The liquid fraction of wine and beer lees supplemented with 60 g/L xylose can be employed as a suitable culture medium for xylitol production with acceptable xylitol productivities and yields. The liquid fraction of cider lees did not allow the development of the yeast.
15. The complete lees (including the solid fraction) resulted inhibitory for Debaryomyces hansenii, but they allowed acceptable xylitol productions when they were progressively diluted, even in the case of cider lees.
16. The yeast extracts obtained by mechanical disruption of the Saccharomyces yeast cells present in the solid fraction of wine, cider and beer lees did not allow the development of Debaryomyces hansenii cultures.
17. The yeast extracts obtained by autolysis of the Saccharomyces yeast cells in the solid fraction of beer gave promising results, which must be improved optimizing the autolysis process for getting more concentrated and hydrolyzed yeast extracts.
18. The substitution of all the components of a standard synthetic medium by xylose-containing hydrolyzates originated by acid hydrolysis of corn cob and the liquid fraction of wine lees as nitrogen and mineral salts source allowed achieving a xylitol production comparable to that obtained in a synthetic medium.
Finally, the next conclusions were extracted from the preliminary experiments performed to study the enzymatic generation of xylose from corn cob:
19. The better degrees of xylan hydrolysis were obtained when an Aspergillus extract obtained from solid state cultures developed on corn cob as substrate, and Ultraflo® L (Novozymes), both containing feruloyl esterase activity, were used. Although Ultraflo showed higher ability for generating reducing sugars, the Aspergillus extract was more efficient than the commercial enzyme for producing xylose. These results point to the interest of using enzymatic extracts for the hydrolysis of corncob xylan to xylose that are specifically produced by microorganisms grown on the same material to be hydrolyzed. Future work will be done in order to increase the concentration of the Aspergillus enzymatic extract and increase the yield of the corn cob xylan hydrolysis.
20. A preliminary evaluation of the use of high hydrostatic pressurization as a pretreatment of the lignocellulosic matrix of corn cob simultaneously with the enzymatic treatment provided encouraging results, giving higher hydrolysis degrees than the thermal treatment assayed. Much more work must be done in order to increase the hydrolysis yield by optimization of the conditions of operation: pressure applied, time, temperature and enzyme concentration.
