Xylitol Production from Rice Straw Hemicellulose Hydrolyzate by Polyacrylic Hydrogel Thin Films with Immobilized Candida subtropicalis WF79

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1 JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 105, No. 2, DOI: /jbb , The Society for Biotechnology, Japan Xylitol Production from Rice Straw Hemicellulose Hydrolyzate by Polyacrylic Hydrogel Thin Films with Immobilized Candida subtropicalis WF79 Wen-Chang Liaw, 1 * Chee-Shan Chen, 2 Wen-Shion Chang, 3 and Kuan-Pin Chen 1 Department of Chemical Engineering, National Yunlin University of Science and Technology, 123, Section 3, University Road, Touliu, Yunlin 640, Taiwan, R.O.C., 1 Department of Applied Chemistry, Chaoyang University of Technology, 168 Jifeng E. Rd., Wufeng, Taichung County 413, Taiwan, R.O.C., 2 and Jinwen University of Science and Technology, 99 An-Chung Rd., Shin-Tien, Taipei County 231, Taiwan, R.O.C. 3 Received 21 June 2007/Accepted 2 November 2007 Xylose from rice straw hemicellulose hydrolysate was fermented for xylitol production using Candida subtropicalis WF79 cells immobilized in polyacrylic hydrogel thin films of 200 µm thickness. Cell immobilization was conducted by first suspending the yeast cells in a mixture of 2-hydroxyethyl methacrylate (HEMA, hydrophilic monomer), polyethylene glycol diacrylate (PEG-DA, crosslinking agent), and benzoin isopropyl ether (photoinitiator). The mixture was then allowed to form polyacrylic hydrogel thin films, between two pieces of glass sheets, by UV-initiated photopolymerization. The hemicellulose of rice straw was hydrolyzed using dilute sulfuric acid at 126 C. The hydrolysate was neutralized with calcium hydroxide. After separating the solid residues and calcium sulfate precipitates by filtration, the hydrolysate was treated with charcoal to partially remove potential inhibitory substances, followed by vacuum concentration to obtain solutions of desired xylose concentrations for yeast fermentation. The thin films with immobilized yeast cells were submerged in the xylose solution from rice straw hydrolysate for fermentation in an Erlenmeyer flask. The maximum yield was 0.73 g of xylitol per gram of xylose consumed. In the 52.5-day long durability test, after 40 d of repeated batchwise operation, the fermentation activities of the cell immobilized in thin films began to decline to a yield of 0.57 g/g at the end. [Key words: xylitol, rice straw, hydrolysis, polyacrylic hydrogel, thin film immobilization] * Corresponding author. liawwc@yuntech.edu.tw phone: fax: In Asia, rice straw is produced in large quantities as an agricultural by-product. It is a fibrous lignocellulosic material that contains about 25% hemicellulose, which can be further utilized through bioconversions to produce other useful compounds. It is not economically favored as an energy source because of its low heat of combustion (1). The hemicellulose fraction of rice straw can easily be hydrolyzed using acids to yield a xylose solution. Many studies have demonstrated that the obtained xylose solution can be used to produce xylitol by fermentation (2 6). Xylitol is a five-carbon sugar alcohol with beneficial health properties which has been successfully marketed. Because of the absence of carbonyl groups, it does not participate in Maillard-type reaction (7). Being able to inhibit the growth of bacteria living around the teeth, xylitol can be utilized for the prevention of cavities (8, 9). The applications of xylitol in the food and pharmaceutical industries have been increasing in the last decade. Xylitol is present naturally in fruits. However, its quantity is minor, which makes its extraction difficult and not economically feasible (10). A major industrial method of xylitol production involves the chemical reduction of xylose obtained by acid hydrolysis of xylan present in the hemicellulose of plant structural tissues (11). Owing to the use of high pressure and temperature, as well as the need for expensive separation and purification steps in the chemical reduction process, xylitol production through bioconversion has been proposed as an alternative process utilizing microorganisms such as yeasts, bacteria and fungi (12, 13). Among these, yeasts have been shown to possess some desirable properties as a potential xylitol producer. From the viewpoint of engineering, immobilized cell fermentation systems have generally been considered as efficient in terms of overall productivity and production costs, as compared to free cell fermentation systems (14). The main reason is that immobilized cell systems allow repeated use of the same cells and direct most of the energies consumed to the desired bioconversions. Moreover, immobilized cell systems are advantageous because they are suitable for continuous operation. Many research efforts have been devoted to developing durable, self-maintaining cell immobilization systems for efficient and continuous fermentation operation. In the literature, different immobilization strategies for 97

2 98 LIAW ET AL. J. BIOSCI. BIOENG., fermentation have been described. For xylitol production, various solid support matrices for cell adsorption immobilization have been reported (15 21) such as ceramics, nonwoven fabrics, microporous glass beads and zeolite. Entrapments of cells in the gels of calcium alginate or carrageenan have also been reported by other research groups (22 34). For years, the hydrophilic crosslinked polyacrylic hydrogels have been widely used on soft contact lenses, suggesting that the success of such hydrogels is attributable to their high water content, good biocompatibility, satisfactory mechanical strength and mass transfer rate, and these attributes make them a good candidate for the type of immobilization under consideration (35, 36). In our separate report (submitted), we have tried various formulations of polyacrylic resin to prepare hydrogel films (of 200 µm thickness) with entrapped viable yeast cells for the bioconversion of pure xylose to xylitol. The optimized polyacrylic hydrogel for fermentation using immobilized cells has been demonstrated to be capable of converting pure xylose-to-xylitol at a yield of 0.81 g/g in a fermentation medium containing yeast extract. However, when it comes to rice straw hydrolystate, the fermentation medium tends to be more complex owing to the presence of harmful substances derived during acid hydrolysis (37 41). This study is divided into two parts. The first is to evaluate the effects of rice straw hydrolysis and the pretreatment of the hydrolysate on xylitol yield. The second part focuses on the effects of several fermentation parameters, such as initial xylose concentration in the hydrolysate of rice straw and cell loading, on the bio-reduction of xylose. As pointed out by Yahashi et al. (15, 16), when xylose was used as the only carbon source available for the yeast s cell maintenance as well as xylitol production, the diverted energy utilization would decrease the xylitol yield. Glucose upplementation in xylitol fermentation has been proposed and tested by other researchers (15, 16, 42, 43) and this research will also cover this aspect. The durability of the prepared immobilization thin films for the production of xylitol from rice straw hydrolysate is another important aspect that needs to be looked into. MATERIALS AND METHODS Microorganism and inoculum The yeast strain used in this research was isolated using selective medium from waste sugarcane bagasse recovered from central Taiwan. The obtained xylose metabolizing strains were further screened for xylitol producers on the basis of high xylitol yield and fast fermentation rate. Cells of the yeast C. subtropicalis WF79 were isolated and maintained at 4 C on malt extract agar slants. The inoculum was prepared by cultivation of the yeast in 125 ml Erlenmeyer flasks with liquid medium consisting of 3 g/l yeast extract, 3 g/l malt extract, 5 g/l peptone and 20 g/l xylose. Yeast cells were cultivated at 100 rpm and 30 C for 24 h and were recovered by centrifugation (2000 g, 20 min, at 10 C). Shaker flasks were used to grow the required amount of cells for immobilization under the conditions described above (xylose concentration was increased to 100 g/l). Yeast cells collected by centrifugation were weighed (wet yeast cake) and suspended by stirring in the solution described below. One gram of wet yeast cake sample was oven-dried (60 C) for 48 h, and the dry/wet weight ratio was used to obtain the cell loading (dry weight) in the films. Preparation of polyacrylic hydrogel as carriers for cell immobilization The following polymer components were first mixed with viable yeast cells: 2-hydroxyethyl methacrylate (HEMA), polyethylene glycol diacrylate (PEG-DA, MW=1000 g mol 1 ) (HEMA: PEG-DA=9:1 by weight), and 1 wt% of benzoin isopropyl ether was added to the above solution. The polyacrylic hydrogel film was prepared by injecting the solution into the space between two pieces of glass ( mm) separated by a 200-µm-thick spacer, followed by free-radical reaction initiated by UV light. Length of exposure to UV light was kept shorter than 30 s to minimize UV damage to the microorganism. Hemicellulose hydrolysis and treatment conditions Rice straw collected from the crop fields of central Taiwan was dried in the sun and milled to give particle sizes of approximately 1 cm length and 1 mm thickness. Acid hydrolysis was conducted in a stainless steel pressure cooker under the following conditions: liquid: solid ratio 10:1 (v/w); at temperatures of 100 C, 110 C, 120 C, and 126 C for 30, 60, 90, and 120 min; sulfuric acid concentrations used were 1%, 2%, 3% and 4%. The solid residue in the hydrolysate was removed by vacuum filtration. The ph of the hydrolysate was first raised to 10.0 with calcium hydroxide (Ca(OH) 2 ), then reduced to 5 6 with sulfuric acid (H 2 SO 4 ). After these treatments, the hydrolysate was vacuum-concentrated at 70 C to obtain solutions of desired xylose concentrations for subsequent experimentations. The concentrated hydrolysate was treated with activated charcoal for 1 h to remove potentially inhibitory compounds generated during acid hydrolysis. Charcoal treatment For comparison, two types of activated charcoal were tested. One (charcoal A) was made from peat bog and acidified, and the particle size was 200 mesh (purchased from Sigma-Aldrich, St. Louis, MO, USA; product no. C4386). The other (charcoal B) was purchased from Benson Corporation (Taipei, Taiwan), made from coconut shell with a particle size of 40 mesh. The furfural removal was quantified by measuring the changes in color intensity of the solution at 460 nm which reflects the pigment concentrations in the hydrolysate. An optical absorbance of one at 460 nm was defined as one unit of pigment per ml. Pigment in the hydrolysate was measured by serial dilution until an absorbance smaller than 0.5 (to ensure a linear relationship between absorbance and pigment concentration) was obtained; the pigment concentration in unit in the original sample was obtained by multiplying the absorbance by dilution ratio. For example, if 1 ml of sample was diluted to 12 ml, and the absorbance of the diluted sample at 460 nm was 0.33, then, the pigment concentration in the original sample was =3.6 (unit/ml). The efficiencies of furfural removal for various strategies were studied by comparing the percentages of furfural removal after charcoal treatment. Fermentation conditions Rice straw hydrolysate was vacuum-concentrated to give solutions with xylose concentrations of 80, 100, 125, 150, 200 g per liter, and yeast extract 3 g/l, malt extract 3 g/l, and peptone 5 g/l were added to the medium. Fermentations were carried out in 250 ml Erlenmeyer flasks of 100 ml working volume. The flasks were shaken at 30 C in an orbital shaker set at 75 rpm. To investigate different strategies for improved bioconversions, glucose supplementation at various initial concentrations was carried out to observe their effects on xylitol yield. Different pieces of hydrogel with immobilized cells were put into the fermentation medium. The fermentation runs that lasted 180 h were monitored through periodic sampling and analyzing for sugar and xylitol concentrations. Analytical procedures Glucose, xylose, xylitol and arabinose concentrations were determined using a high-performance liquid chromatography (HPLC) system equipped with a refractive index (RI) detector (Jasco, Tokyo). The chromatographic column used

3 VOL. 105, 2008 XYLITOL USING THIN FILMS WITH IMMOBILIZED CELLS 99 was a Carbosep CHO-620CA carbohydrate column (Transgenomic/Cetac, Ohama, NE, USA) operated at 30 C. The mobile phase was N sulfuric acid at a flow rate of 0.6 ml/min, and the injected sample volume was 20 µl. RESULTS AND DISCUSSIONS FIG. 1. Chemical structures of hydrophilic acrylic monomer (HEMA) (a) and crosslinking agent (PEG-DA) (b). Cell Immobilization For easy processing as well as for better mechanical properties of the gels (e.g., reduced number of steps for synthesis, durable gels), a spherical geometry of the beads is commonly adopted. Yahashi et al. (15) compared various strategies of cell immobilization, and they found that the nonwoven fabric immobilization method outperformed other methods. To some extent, similarity between a thin-film and a fabric structure seems to be favored in such system. Comparatively, a thin film structure, if properly designed, could offer a more exposed surface area and a shorter diffusion path than spheres. In a packed bed bioreactor, the stacking situation of the beads inherently blocks some of the bead surface and keeps the cells from being in contact with the fermentation medium. In a continuous operation, a spherical bead would experience both normal and shear stresses resulting from the fluid flow. For the thin-film structure, the bioreactor can also be operated such that the fluid flows only tangentially to the thin film with immobilized cells while providing the above-mentioned advantages. However, such thin-film design in a large scale fermentor requires a more complex frame-type support and spacer to separate and hold the films in shape. Although making the frame-type support is not technically difficult to achieve, the extra effort may be worth the while. What is worth mentioning here is that the polyacrylic hydrogel with immobilized cells, in this study, used the highly hydrophilic 2-hydroxyethyl methacrylate (HEMA, Fig. 1a) as the main acrylic monomer (90 wt%). In our previous studies (data not shown), we have tried formulations other than HEMA, such as methacrylic acid (MAA) and N,N-dimethylacrylamide (DMA), but their mechanical strengths were not satisfactory; particularly when the DMA dosage was higher than 10% of HEMA, the hydrogel could not be used to form films. The crosslinking agent used, polyethylene glycol diacrylate (PEG-DA, Fig. 1b), is also hydrophilic, and a hydrophilic environment is friendly to the microorganism concerned. One virtue of this type of formulation is that the molecular weight and the content of the crossklinking agent can be adjusted to manipulate the mechanical strength of the film for a wide spectrum of applications involving cell immobilization. A high crosslink density would lead to better mechanical properties, but the microstructure of the film would be too dense to provide sufficient diffusion passages and water holding capacity for the microorganisms. A good formulation would have to be a compromise between mechanical properties and the requirements of the microorganisms. In our previous research, we experimented with various formulations and found that the films that used 10% PEG-DA with a molecular weight of 1000 g mol 1 gave a film with acceptable mechanical strength and good diffusion properties. These materials are readily available and inexpensive which makes the idea practical and feasible. The water content of the thin films with immobilized cells was 40%, which is comparable to those of alginate, carrageenan, and contact lens as well. The tensile strength of 2.53 Kgf/cm 2 was measured (comparable to that of contact lenses, 1.23 to 5.87 Kgf/cm 2 ; 35, 36). Different cell loadings were tested and compared. One major tradeoff for cell immobilization in such system would be a decreased xylitol yield. Santos et al. (18, 19) studied the xylitol production from sugarcane bagasse hydrolysate using Candida guilliermondii immobilized on porous glass and zeolite, and found that the xylose-to-xylitol yield decreased from 0.72 (free cells) to 0.53 (porous glass) and 0.52 g/g (zeolite). Silva et al. (17) operated a semicontinuous fluidized bed reactor using C. guilliermondii immobilized on porous glass. They attained xylitol yields of 0.57 to 0.79 g/g (17). Carvalho et al. studied the Ca-alginate immobilization system (28 34), run in different modes (batch, repeated batch, stirred tank reactor). By optimizing the stirred tank reactor operation (using the response surface method and by adjusting the air flow rate, agitation speed, initial cell concentration, and initial ph), the xylitol yield was improved from 0.47 to 0.81 g/g. Yahashi et al. (15, 16) used the nonwoven fabric immobilization method, and attained a high yield of 0.69 g/g with glucose feeding of 20 g/d/ 200 ml. The intention of citing various yields obtained under different conditions here is merely to point out the range of xylitol yield for relative comparison. In this study, a xylitol yield ranging from 0.57 to 0.73 g/g, depending on operational conditions, was obtained. Hydrolysis of rice straw Low-pH hydrolysate should be neutralized before it is suitable for microbial fermentation. After neutralization, the formation of a significant amount of salt is unavoidable, which tends to increase the osmotic pressure to a level unacceptable to yeast cells. If sodium hydroxide is used to neutralize the acid, owing to the high solubility of sodium sulfate, the hydrolysate would have a high ionic strength of approximately 0.6 (calculation based on neutralizing 2.0 wt% sulfuric acid with sodium hydroxide). Therefore, calcium hydroxide was chosen because of the low solubility of calcium sulfate (Ksp= at room temperature), and most of the salt formed during neutralization will precipitate and can be collected by filtration

4 100 LIAW ET AL. J. BIOSCI. BIOENG., Temperature ( C) TABLE 1. Effects of temperature, H 2 SO 4 concentration and reaction time on rice straw hydrolysis H 2 SO 4 (% w/w) Time (min) Xylose (g/l) Glucose (g/l) Arabinose (g/l) as a by-product for other applications (construction, food, medicine, etc.). During high-temperature hydrolysis under acidic condition, accompanying side reactions leading to products like furfurals and hydroxymethyl furfurals are taking place. These compounds, if present in significant amount, are inhibitory to microbial fermentation. Measures have to be taken to alleviate such inhibitory effect and will be discussed shortly. Temperature of hydrolysis Several reaction temperatures were tested for maximum sugar recovery. Table 1 compares the concentrations of xylose, arabinose and glucose in the hydrolysate of rice straw resulting from different reaction temperatures. Sulfuric acid (2 wt%) was used and the reaction time was 1 h. Table 1 shows that the xylose concentration in the hydrolysate increased with increasing hydrolytic temperature. At the reaction temperature of 126 C, the xylose concentration was 13.3 g/l. Similar trends can be observed for the concentrations of arabinose and glucose (2.34 and 2.50 g/l). Beyond 126 C, the increase in sugar recovery was less significant (data not shown). A visible change when we went from low to high reaction temperature was the appearance of a darkened hydrolysate owing to the presence of those brown pigments (mainly furfurals). Moreover, the xylose concentration of 13.3 g/l in the hydrolysate approximately accounted for 80% to 90% of the recovery of total xylose content in the rice straw. By considering the pros and cons, we adopted the reaction temperature of 126 C. Acid concentration Table 1 shows data of sugar concentrations in the hydrolysate as a result of acid hydrolysis under various acid concentrations. When sulfuric acid concentration was increased from 1 to 2 wt%, the xylose concentration increased from 10.0 to 13.3 g/l. However, the sugar concentrations (particularly that of xylose) dropped with a further increase in the acid concentration to 3 to 4 wt%. Reaction time Prolonged reaction times are not necessarily beneficial when considering the possible side reactions and energy efficiency. Table 1 compares the concentrations of monosugars in the hydrolysate for reaction times of 30 to 120 min at 126 C and 2 wt% sulfuric acid. When reaction time was extended from 30 to 60 min, the xylose concentration increased by approximately 43% (9.3 to 13.3 g/l). The level of increase declined to about 15% when the reaction time was further extended from 60 to 90 min, and then leveled off afterwards. We therefore chose 126 C, 60 min and the addition of 2 wt% sulfuric acid as the hydrolysis condition. Fermentation Thin films of 100 mm by 100 mm with immobilized yeast cells (of 200 µm thickness) per piece were placed in 100 ml of fermentation medium in a 250 ml Erlenmeyer flask, in a shaker set at 75 rpm. One piece of film with immobilized cells was used, unless otherwise specified. Carrying out fermentation in shaker flasks also provides an opportunity to observe the sturdiness of the cell immobilized films under the shear of fluid in motion. As mentioned above, rice straw hydrolysate, with high concentrations of inhibitory compounds, is not suitable for direct fermentation. Pretreatment for the removal of toxic substances is discussed below. Salt removal and activated charcoal treatment After hydrolysis, the ph of the hydrolysate was less than 1, which is too low for microbial fermentation. The hydrolysate was first neutralized with calcium hydroxide because of the ease of removal of the resulting precipitating salt (calcium sulfate). Precipitated calcium sulfate was removed by filtration, leaving the hydrolysate with a much lower ionic strength of about 0.012, as compared with the case of using sodium hydroxide as the acid neutralizer. Acidified activated charcoal is known for its capability of adsorbing nonpolar odor compounds and pigments. Table 2 compars, in terms of percentage pigment removal, two types of charcoal. Charcoal A (from Sigma-Aldrich) absorbed pigment very efficiently at the rate of 2.5 g/100 ml; 93% of the pigment was removed in 60 min. The 40-mesh charcoal B, adsorbed 63% of the pigment at 60 min at the same rate of 2.5 g/100 ml. Such difference may be attributable to both the origin and the size of the charcoal. We experimented with various levels of pigment removal and found that 63% is sufficient for providing satisfactory xylitol conversion. During pigment adsorption, we also monitored xylose loss in the process, and found that xylose loss was 15% (of the initial concentration) if we used charcoal A to remove 98% of the pigment (5 g/100 ml, 60 min). By using charcoal B, the xylose loss was reduced to below 5% (at 63% pigment removal). Because of economic and other considerations, we chose charcoal B, 2.5 g/100 ml, 60 min, as the conditions for pigment removal for the rest of the experiments. Before charcoal treatment, serious inhibition was observed such that only 20 g/l of xylose was utilized, and virtually no xylitol was produced by the end of 180 h. After charcoal

5 VOL. 105, 2008 XYLITOL USING THIN FILMS WITH IMMOBILIZED CELLS 101 Time (min) TABLE 2. Pigment percentage removal a (%) by charcoal g of charcoal per 100 ml of hydrolysate A (%) B (%) A (%) B (%) A (%) B (%) A, Acidified charcoal from Sigma-Aldrich, 200 mesh size; B, acidified charcoal from Benson Corporation, Taipei, 40 mesh size. a % pigment removal was calculated on the basis of initial pigment concentration of 6.05 unit/ml; pigment unit was defined in the Materials and Methods section. Concentration (g/l) 100 a b Time (h) Time (h) FIG. 2. Time courses of rice straw hydrolysate fermentation (a) before and (b) after charcoal treatment. Open circles, Xylose; closed circles, xylitol; open triangle, glucose; closed triangle, ethanol; open square, arabinose. Initial xylose concentration, 100 g/l; cell loading, 11.5 mg cell/g film; one piece of film. treatment, it can be seen in Fig. 2 that much improvement was achieved and xylose (initial concentration was 100 g/l) was completely consumed, while the xylitol yield was 0.65 g/g and a small amount of ethanol was produced by the end of 180 h. Arabinose was not fermented in either case. We have experimented with various levels of pigment removal. As discussed later, the maximum xylitol yield in this study was 0.73 g/g (90% of the performance of the same system using pure xylose, 0.81 g/g, data not shown) and it was concluded that 63% pigment removal is sufficient for providing satisfactory xylitol yield. Effect of initial xylose concentration In yeast fermentation, a high initial sugar concentration results in a high product yield and is more economically feasible in terms of product recovery, but with the tradeoffs of retarded cell growth and low fermentation rate. For the same hydrolysis conditions, pretreatments and fermentation conditions, we tested various initial xylose concentrations ranging from 80 to 200 g/l. Figure 3 shows that the xylitol yield increased when the initial xylose concentration was increased from 80 to 100 g/l. However, when we further increased the initial xylose concentration, the xylitol yield started to decline. An initial xylose concentration of 100 g/l seemed to be the best choice for obtaining the highest xylitol yield from xylose. Such contrasts between the different time courses in Fig. 3 may arise solely from the differences in the initial xylose concentration. The amounts of residual furfurals left in the FIG. 3. Time courses of rice straw hydrolysate fermentation after charcoal treatment, using various initial xylose concentrations (g/l): 80 (open triangles); 100 (closed circles); 125 (open circles); 150 (closed triangles); 200 (open squares). Cell loading, 11.5 mg cell/g film; one piece of film. hydrolysates, after charcoal had captured most of the furfurals, were different, owing to the different ratios of water evaporation that concentrated furfurals to different levels and imposed different loadings on charcoal adsorption. Therefore, for fermentation media with different initial xylose concentrations, different amounts of residual furfurals were present, which is likely to pose an inhibitory effect on the fermenting yeast, particularly in media with high initial xylose concen-

6 102 LIAW ET AL. J. BIOSCI. BIOENG., FIG. 4. Time courses of the fermentation with initial xylose concentration of 100 g/l and initial glucose concentrations of 100 g/l (a), 70 g/l (b), 50 g/l (c), and 30 g/l (d). Open circles, Xylose; closed circles, xylitol; open triangles, glucose; closed triangles, ethanol. Cell loading, 11.5 mg cell/g film; one piece of film. tration. Although the produced xylitol (product inhibition) is another factor that may pose an inhibitory effect on the yeast, as pointed out by Oh and Kim (42), its effect awaits further investigation. However, in this study, differences in brown color intensities of fermentation media with various initial xylose concentrations were found to be minor, and the choice of 100 g/l of initial xylose concentration was justified. Glucose supplementation Many reports have demonstrated that glucose feeding is beneficial in xylitol fermentation (15, 16, 38, 42, 43). Oh and Kim (42) maximized xylitol yield (0.93 g/g) with a glucose/xylose feeding ratio of 15%. Kwon et al. (43) supplemented 20 g/l glucose to the xylose medium, and xylitol yield increased from 0.83 to 0.86 g/g. In this study, we added different amounts of glucose to observe its effect on xylitol yield. Time courses of same initial xylose concentration (100 g/l) were compared, with added glucose to adjust the initial glucose concentration to be 30, 50, 70 and 100 g/l. We observed that the osmotic pressure caused by the total sugar concentration of 200 g/l (100 g/l glucose and 100 g/l xylose) was not sufficiently high to seriously suppress the fermentation. The yeast consumed glucose while metabolizing a relatively small amount of xylose. Xylitol yield was low. Perhaps excessive glucose stimulated the production of 30 g/l ethanol. When the initial glucose concentration was adjusted to 70 g/l, a small improvement in xylitol yield (0.40 g/g) was attained. Following this trend, when the initial glucose concentration was decreased to 50 g/l, the xylitol yield increased to 0.60 g/g, and the ethanol concentration dropped to 5 g/l. In Fig. 4, when the initial glucose concentration was decreased from 50 to 30 g/l, the xylitol yield further increased to 0.62 g/g, which is still low compared with that without extra glucose supplementation. A comparison of the glucose supplementation data in the literature (15, 16, 38, 42, 43) showed that suitable range for glucose supplementation is 15 to 20 g/l (42, 43). In our system, for an initial xylose concentration of 100 g/l, there was 18.8 g/l (calculated from Table 1) of glucose present in the hydrolsate. This may explain the reason why additional glucose supplementation did not help. Figure 2 shows that for 100 g/l initial xylose concentration with 18.8 g/l initial glucose content, the xylitol yield was 0.65 g/g. Although oversupplementing glucose did not seem to significantly affect the xylitol yield, it still has a contribution. An increase in cell growth was observed in the fermentation broth as increased turbidity. However, the concentration of other metabolites such as ethanol increased accordingly together with active cell growth, and no net benefit in xylitol yield was observed. Other strategies such as minute glucose feeding at intermediate time intervals (15, 16) may change the result, but these were not studied in this research. Cell loading The amount of biocatalyst in a fermentation system is another major factor that governs the bioconversion yield. Too high a cell loading may affect the overall structure of the solid matrix and lead to a reduced cohesiveness; meanwhile, a high cell density tends to interfere with mass transfer inside the solid matrix, making nutrients less available while putting a heavier loading on the transport of metabolites. Cell immobilized thin films with various cell loadings ranging from to 23 mg of cell per g of film (mg cell/g film) were prepared by controlling the amount of cells added to the monomer mixture before polymerization. Figure 5 compares the time courses of xylose conversions over time. With low cell loadings (0.575 and 1.15 mg cell/g film, dry basis), a low xylitol yield was obtained (0.30 g/g at 180 h). With an increase in cell loading to 5.75 and 11.5 mg cell/g film, the final xylitol yield (>0.6 g/g) also increased. A com-

7 VOL. 105, 2008 XYLITOL USING THIN FILMS WITH IMMOBILIZED CELLS 103 FIG. 5. Time courses of xylitol yield for various cell loadings (mg cell/g film). Open circles, 0.575; closed circles, 1.15; closed triangles, 5.75; open squares, 11.5; open triangles, 23. Initial xylose concentration, 100 g/l; one piece of film. FIG. 6. Durability test. Initial xylose concentration, 100 g/l; one piece of film; cell loading, 115 mg/g film. parison of the time courses corresponding to cell loadings of 5.75 and 11.5 mg cell/g film showed that, although the final xylitol yields were not significantly different (0.62 and 0.65 g/g, respectively), the cell loading of 11.5 mg cell/g film resulted in a rapid xylitol production (the xylitol curve rose earlier). By further increasing cell loading to 23 mg cell/g film, the final xylitol yield dropped to 0.50 g/g. It is possible that the reasons for this are as stated above, that is, centrifuged sticky cells added to the monomer mixtures, when presented in the matrix in too large a portion, results in the retardation of the mass transfer of nutrients and metabolites in the cells. We therefore chose 11.5 mg cell/g film as the cell loading for this research. Durability To study the durability of the cell immobilized polyacrylic thin films prepared as stated above, one and two pieces of the films were placed in 100 ml of fermentation broth in shaker flasks. To ensure that the observed results are purely attributable to the bioactivities of the films, the films were washed with buffers and the flasks were replenished with fresh medium every 180 h. Figures 6 and 7 show the performances of these films after a long-term operation of 1250 h. A maximum xylitol yield of 0.73 g/g was attained as shown in Figs. 6 and 7. In Fig. 6, one piece of film was used, and two pieces were used in Fig. 7. Comparison of the two figures shows no significant difference, although in some rounds, the maximum xylitol yield was reached slightly earlier in the two-piece batch. This suggested an important improvement that should be made in the future: the spacing between films should be finely controlled to allow for free convective fluid motion. Although small spacers were used to provide the spacing between films, the films were not rigid and most of the interior areas were stuck together and this made the two-piece film act like a one-piece system. However, durability tests confirmed that these films lasted long and the immobilized yeast cells remained active after long-term operation. Cell growth inside the hydrogel films is another phenomenon that is worth discussing here. In both Figs. 6 and 7, the final xylitol yield increased gradually from the first round of 0.65 g/g to a maximum of 0.73 g/g in the third and fourth FIG. 7. Durability test. Initial xylose concentration, 100 g/l; two pieces of films; cell loading, 11.5 mg/g film. rounds, possibly indicating some cell growth in the hydrogel matrix. After the fourth round, however, the cell growth increased to a level such that the mass transfer inside the film(s) started to decline, as was observed during the cell loading study (Fig. 5), and the final xylitol yield decreased to 0.57 g/g in the seventh round. This observation showed that continued cell growth is of serious concern, for at least two reasons: one is that part of the energy was directed to support cell growth instead of bioconversion, and the other is that a very dense population of cells tends to retard the mass transfer rate. Although continued cell growth indicates that the system is capable selfmaintenance, nutrient control that minimizes cell growth in such a system should be more efficient while considering xylitol yield. Summary This research demonstrated that the hydrophilic polyacrylic hydrogel system is a good choice for cell immobilization. The process of cell immobilization is simple and expedient given the right choice of monomers and crosslinking agent. After simple treatments of filtering precipitated calcium sulfate, water evaporation and charcoal adsorption, rice straw hydrolysate is suitable for bioconversion to produce xylitol using the hydrogel-immobilized yeast Candida sp. WF79. The maximum xylitol yield attained in this study was 0.73 (g/g), which is 90% of the performance

8 104 LIAW ET AL. J. BIOSCI. BIOENG., of the same system using pure xylose (0.81 g/g), and 80% of that of the free cell system using pure xylose (0.91 g/g). Although many other details such as carbon and nitrogen source management, as well as maximizing xylitol yield while minimizing cell growth, await further investigation, the cell immobilized films used in this research proved to be competent and durable. REFERENCES 1. Mayerhoff, Z. D. V. L., Roberto, I. C., and Silva, S. S.: Xylitol production from rice straw hemicellulose hydrolysate using different yest strans. Biotechnol. Lett., 19, (1997). 2. Kuhad, R. C. and Singh, A.: Lignocellulose biotechnology: current and future prospects. Crit. Rev. Biotechnol., 13, (1993). 3. Roberto, I. C., Felipe, M. G. A., Mancilha, I. M., Vitolo, M., Sato, S., and Silva, S. S.: Xylitol production by Candida guilliermondii as an approach for the utilization of agroindustrial residues. Bioresour. Technol., 512, (1995). 4. Roberto, I. C., Mussatto, S. 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