Improved sugar co-utilisation by encapsulation of a recombinant Saccharomyces cerevisiae strain in alginate-chitosan capsules
© Westman et al.; licensee BioMed Central Ltd. 2014
Received: 28 January 2014
Accepted: 19 June 2014
Published: 3 July 2014
Two major hurdles for successful production of second-generation bioethanol are the presence of inhibitory compounds in lignocellulosic media, and the fact that Saccharomyces cerevisiae cannot naturally utilise pentoses. There are recombinant yeast strains that address both of these issues, but co-utilisation of glucose and xylose is still an issue that needs to be resolved. A non-recombinant way to increase yeast tolerance to hydrolysates is by encapsulation of the yeast. This can be explained by concentration gradients occuring in the cell pellet inside the capsule. In the current study, we hypothesised that encapsulation might also lead to improved simultaneous utilisation of hexoses and pentoses because of such sugar concentration gradients.
In silico simulations of encapsulated yeast showed that the presence of concentration gradients of inhibitors can explain the improved inhibitor tolerance of encapsulated yeast. Simulations also showed pronounced concentration gradients of sugars, which resulted in simultaneous xylose and glucose consumption and a steady state xylose consumption rate up to 220-fold higher than that found in suspension culture. To validate the results experimentally, a xylose-utilising S. cerevisiae strain, CEN.PK XXX, was constructed and encapsulated in semi-permeable alginate-chitosan liquid core gel capsules. In defined media, encapsulation not only increased the tolerance of the yeast to inhibitors, but also promoted simultaneous utilisation of glucose and xylose. Encapsulation of the yeast resulted in consumption of at least 50% more xylose compared with suspended cells over 96-hour fermentations in medium containing both sugars. The higher consumption of xylose led to final ethanol titres that were approximately 15% higher. In an inhibitory dilute acid spruce hydrolysate, freely suspended yeast cells consumed the sugars in a sequential manner after a long lag phase, whereas no lag phase was observed for the encapsulated yeast, and glucose, mannose, galactose and xylose were utilised in parallel from the beginning of the cultivation.
Encapsulation of xylose-fermenting S. cerevisiae leads to improved simultaneous and efficient utilisation of several sugars, which are utilised sequentially by suspended cells. The greatest improvement is obtained in inhibitory media. These findings show that encapsulation is a promising option for production of second-generation bioethanol.
Second-generation bioethanol has long been suggested as a contender to become the main type of renewable liquid fuel . Nevertheless, there are issues with its production that still limit its commercialisation. One of the main problems is the issue of inhibitors produced during the pretreatment and hydrolysis of the raw material into fermentable sugars. Another problem is the fact that pentoses are not fermentable by wild-type Saccharomyces cerevisiae, although there are a few exceptional cases of slow consumption . The most popular approach to this problem has been to create recombinant strains of S. cerevisiae utilising xylose, arabinose or a mixture of the two . However, these strains still have the problem of poor simultaneous co-utilisation of the pentoses together with hexoses . Xylose will not be consumed in considerable amounts until the concentration of glucose is low [6, 7]. The reason for this is that there are no specific pentose transporters in S. cerevisiae, and pentoses are instead transported by the native hexose transporters [8, 9]. However, these transporters have higher affinity for glucose, and therefore there is a strong preference for glucose uptake as long as it is present.
Cell retention through immobilization of the cells can be used as a means to increase the volumetric productivity of biological processes . Numerous examples of immobilization of various microbial cells in alginate beads can be found in the literature, for example, the immobilization of Debaryomyces hansenii for xylitol production [11, 12] or of S. cerevisiae for ethanol production . Immobilization produces several benefits to the process, such as easier cell reuse at high biomass concentration. Cell encapsulation in a semi-permeable membrane differs from bead immobilization in that the cells grow inside a liquid core, forming a dense cell pellet inside the capsule rather than being dispersed in the pores of the alginate matrix . Encapsulation appears to be a good solution to the first of the aforementioned problems, the inhibitor tolerance. It has been shown that encapsulation of the yeast increases its tolerance towards convertible inhibitors such as the furan aldehydes. This effect is believed to be a result of concentration gradients, which are formed when cells close to the membrane convert inhibitors; the cells closer to the core of the pellet are then surrounded by sub-inhibitory levels of the inhibitors, and can still ferment the medium efficiently . It has also been shown on a proteomic level that there are cells in the capsule that are starved despite the presence of high ‘extracapsular’ levels of glucose . This is most likely an effect of concentration gradients of glucose occurring throughout the cell pellet inside the capsules, owing to consumption of glucose by the yeast cells and limitations in mass transfer. Such concentration gradients could hypothetically also promote glucose and xylose co-utilisation. Inside a capsule, some cells will experience a low glucose concentration at the same time that other cells experience a high glucose concentration; however, because glucose inhibits xylose consumption all cells may experience relatively high xylose concentrations. It is plausible to assume that the co-utilisation of the sugars will be improved in such a system, because the cells that experience low glucose levels will take up more xylose.
It has been shown that the most efficient transport of xylose is achieved by strains overexpressing single hexose transporter genes in the order HXT7 > HXT5 > GAL2 > HXT1 > HXT4. HXT7 was also reported to be the best xylose transporter in another study . Encapsulation of yeast has been shown to lead to higher expression of Hxt6/7p, at glucose levels outside the capsules of more than 10 g/l . This is a strong indication that cells in different parts of the capsule have differences in physiology, in that some cells can sense low while others sense high concentrations of glucose. It also indicates that xylose uptake may be stimulated by encapsulation.
Based on this hypothesis, we simulated the sugar consumption of cells in a capsule half-filled with yeast, using finite element modelling, and a kinetic model for glucose and xylose uptake and conversion of furfural and 5-hydroxymethylfurfural (HMF). The simulations showed that the simultaneous utilisation of glucose and xylose would indeed benefit from encapsulation, especially in an inhibitory medium. To validate these results, a xylose-utilising strain of yeast was constructed and encapsulated. The fermentation performance was compared with that of the same yeast in suspended culture in defined inhibitory media and dilute acid spruce hydrolysate.
Results and discussion
Finite element modelling shows increased co-consumption due to encapsulation
Efficiency factors ( β j ), rate response coefficients ( ) and efficiency response coefficients ( ) for glucose and xylose for the base case and various simulated system changes
Modified parameters ( P)
c x = 40 g/l
High furfural and HMF
c f = 4 g/l; c h = 4 g/l
No furfural and HMF
c f = 0 g/l; c h = 0 g/l
Strong inhibition by furfural and HMF
K if =5 mM; K ih = 10 mM
C cells = 270 g/l
C cells = 330 g/l
Maximum glucose uptake rates
vgmax, L = 5.4 mmol/g/h; vgmax, H = 1.8 mmol/g/h
vgmax, L = 6.6 mmol/g/h; vgmax, H = 2.2 mmol/g/h
Maximum xylose uptake rate
v xmax = 5.4 mmol/g/h
v xmax = 6.6 mmol/g/h
Inhibition of glucose uptake by xylose
K ix = 32.4 mM
K ix = 39.6 mM
Inhibition of glucose and xylose uptake by furfural and HMF
K if =36 mM; K ih = 72 mM
K if =44 mM; K ih =88 mM
Inhibition of xylose uptake by glucose
K ig =0.36 mM
K ig =0.44 mM
Effective diffusivity in the cell pellet
D c,glucose = 1.52 × 10-10 m2/s; D c,xylose = 1.73 × 10-10 m2/s; Dc,furfura l = 2.52 × 10-10 m2/s; D c,HMF = 2.39 × 10-10 m2/s
D c,glucose = 1.86 × 10-10 m2/s; D c,xylose = 2.11 × 10-10 m2/s; Dc,furfura l = 3.08 × 10-10 m2/s; D c,HMF = 2.92 × 10-10 m2/s
By contrast, the efficiency factors for glucose uptake were generally less than 1, indicating that mass transport becomes limiting for the glucose consumption (Table 1). However, when we increased the inhibition of glucose metabolism by furfural and HMF by decreasing the inhibition constants, glucose consumption also benefitted from the encapsulation. When the inhibition constants were set to K if = 5 mM and K ih = 10 mM, encapsulation increased the glucose and xylose consumption rates by 1.69 and 222 times, respectively, compared with well-mixed conditions.
Many of the parameter values used were rather uncertain. For the effective diffusivities, conservative estimates of the mass transfer resistances were used. For the cell pellet, the effective diffusivities were assumed to be 25% of the diffusivities in the surrounding liquid. In comparison, the effective diffusivity of glucose in flocs (that is, unencapsulated cell pellets) of S. cerevisiae NRRL Y265 has been shown to be 7 to 17% of that in the surrounding water . Furthermore, the external mass transfer resistance was ignored, and the diffusivities in the gel membrane and liquid core were assumed to be close to or equal to the diffusivity in water [17–20]. All these assumptions should lead to underestimated concentration gradients (that is, overestimated concentrations), inside the capsules, and therefore, the predicted effects of encapsulation should be conservative estimates. Furthermore, it would be very difficult to identify and validate, for example, K M values and maximum glucose uptake rates, as these inevitably vary along the radius of the cell pellet, owing to the varying glucose concentration and the consequential differential expression of the hexose transporters.
Rate response coefficients and efficiency response coefficients were calculated in order to investigate the sensitivity of the simulation results to changes in different parameters (Table 1). The glucose consumption rate was clearly increased at higher diffusivities, with rate response coefficient . It was also clearly increased by higher biomass concentration in the pellet and by higher maximum glucose uptake rates (, ). However, although increased diffusivity also led to an increased efficiency factor for glucose (), increased biomass concentration and maximum rates actually decreased the efficiency factor (, ). This clearly shows that the glucose consumption rate is diffusion-limited. The glucose uptake efficiency factor increased in response to inhibition by furfural and HMF, and to a smaller extent in response to increased xylose inhibition. At sufficiently strong inhibition, even glucose uptake benefits from encapsulation, as already mentioned above.
The xylose consumption rate was even more sensitive to increased biomass concentration and equally sensitive to v gmax , but for xylose the efficiency response coefficient increased in response to v gmax . Moreover, the efficiency factor for the xylose consumption increased with stronger inhibition (that is, lower K i ) by glucose, furfural and HMF (, ). In summary, encapsulation becomes very favourable for xylose consumption if the inhibition effects are strong and the glucose concentration in the capsule can be kept low.
Construction of the xylose-fermenting S. cerevisiae CEN.PK XXX
A xylose-fermenting strain, which we named CEN.PK XXX, was constructed by overexpression of the native RPE1, TAL1, RKI1 and XKS1 genes, and insertion of codon-optimised XYL1 and XYL2 genes from Scheffersomyces stipitis (formerly Pichia stipitis) into the genome of S. cerevisiae CEN.PK 122 MDS. The cells were shown to assimilate xylose in repeated rounds of dilutions to OD600 = 0.1 and semi-aerobic growth up to OD600 = 12 to 13 in 3 days.
Encapsulation of a xylose-fermenting S. cerevisiae to validate simulation results
To test the hypothetical results of the simulations, S. cerevisiae CEN.PK XXX was encapsulated and utilised for anaerobic batch cultivations in various media. The fermentations were operated for 96 hours, except for those with suspended yeast in medium without xylose, which were stopped after 30 hours, because all the glucose was consumed by that time.
Improved simultaneous co-utilisation of glucose and xylose by encapsulation
The xylose consumption was rather similar between the encapsulated and freely suspended yeast cells when xylose was the only carbon source (Figure 2C,D). Hence, the diffusion limitations observed for glucose, which led to slower utilisation, was not a limitation for xylose utilisation, confirming the results of the simulations.
The encapsulated CEN.PK XXX fermented xylose from the beginning of the batches, regardless of the initial furfural concentration. A slight reduction in the uptake rate of xylose was observed as the glucose was depleted in the media with mixed carbohydrates (Figure 2D). However, the final uptake of xylose after 96 hours was significantly better for the encapsulated cells than for the freely suspended cells in media containing glucose. The final residual xylose concentrations were 21.4 to 23.4 g/l. Hence, encapsulated S. cerevisiae CEN.PK XXX consumed at least 50% more xylose than the freely suspended cells, under the same conditions. This significant difference was also clearly visible in the ethanol production, and resulted in approximately 15% higher final concentrations (Figure 3).
Final yields and carbon recoveries in anaerobic batch fermentations of defined media a
Freely suspended CEN.PK XXX
52 ± 1
3 ± 0
48 ± 0
438 ± 6
97 ± 1
83 ± 6
8 ± 3
187 ± 6
41 ± 1
290 ± 0
94 ± 1
99 ± 1
5 ± 1
67 ± 21
38 ± 0
402 ± 1
94 ± 1
G40 X40 F1
102 ± 2
3 ± 0
121 ± 9
37 ± 1
402 ± 1
95 ± 1
G40 X40 F2
Encapsulated CEN.PK XXX
39 ± 1
2 ± 1
47 ± 3
428 ± 2
93 ± 1
111 ± 0
3 ± 1
209 ± 4
55 ± 2
277 ± 3
92 ± 1
104 ± 1
3 ± 0
106 ± 15
32 ± 3
387 ± 1
93 ± 2
G40 X40 F1
97 ± 2
2 ± 0
114 ± 9
30 ± 1
393 ± 1
94 ± 1
G40 X40 F2
86 ± 2
1 ± 0
122 ± 5
29 ± 2
401 ± 2
94 ± 1
Encapsulation solves two issues of lignocellulosic hydrolysate fermentation
As a result of encapsulating the relatively sensitive laboratory strain S. cerevisiae CEN.PK XXX, the inhibitor tolerance of the system as a whole and its ability to simultaneously co-utilise glucose and xylose were improved. Another benefit of cell encapsulation, which has not been stressed in this work, is that the cells are easily retained in the reactor. Thus, the volumetric productivity can be easily improved by increasing the amount of capsules in the reactor.
Results from in silico simulations and in vivo experiments showed that encapsulation of a xylose-fermenting yeast strain increased the simultaneous utilisation of glucose and xylose and improved the tolerance to furfural. All the data indicate that the reason behind these results is that diffusion limitations cause concentration gradients of convertible inhibitors and glucose within the cell pellet. High local cell density, here in the form of encapsulated yeast, can thus help in overcoming two of the remaining obstacles for successful commercialisation of second-generation bioethanol production, namely, fermentation inhibitors and sequential hexose and pentose fermentation.
Materials and methods
Diffusion and consumption of glucose, xylose, furfural and HMF by encapsulated cells was simulated using finite element modelling (FEM) and simplified kinetics. The Chemical Reaction Engineering Module in Comsol Multiphysics 4.3 (Comsol AB, Stockholm, Sweden) was used for all simulations.
Physical description of capsules and meshing for FEM
The capsule was meshed in Comsol Multiphysics 4.3 (Comsol AB, Stockholm, Sweden) using the built-in free triangular mesh algorithm, with extra fine element size in the membrane and the liquid core, and extremely fine element size in the cell pellet.
Kinetic description of cell metabolism
Because the purpose of the mathematical modelling was only to visualise potential concentration gradients inside the capsules, yeast metabolism was modelled only with reactions consuming sugars and the inhibitors furfural and HMF. Thus, in the investigated time frames, growth and product formation were assumed to have negligible effects on the diffusion of sugars and inhibitors, and on the rates of consumption.
For the purpose of the model in this work, glucose uptake was modelled as a two-component system, consisting of a low and a high affinity transporter . Xylose uptake was assumed to be facilitated by a single, low-affinity system . Because they are transported by the same hexose transporter system, each sugar was assumed to competitively inhibit the uptake of the other sugar . Xylose was assumed to inhibit both glucose uptake systems with the same inhibition constant . Moreover, both uptake rates were assumed to be non-competitively inhibited by furfural, and to a lesser extent by HMF.
where v gmax and v xmax are the maximum specific rates of consumption (mmol/g dry cells/h); C g , C x , C f and C h are concentrations; K Mg and K Mx are half saturation constants; and K if and K ih are inhibition constants. Indexes g, x, f and h refer to glucose, xylose, furfural and HMF, respectively, while L and H refer to the low and high affinity glucose transport systems, respectively.
Boundary conditions and parameters
Bulk concentrations, kinetic parameters and diffusivities of glucose, xylose, furfural and HMF used as base case for mathematical modelling
C bulk , g/l
v max , mmol/g/h
K M, mmol/l
K i , mmol/l
D w , m2/s
D m , m2/s
D c , m2/s
6 (L), 2 (H)
20 (L), 0.4 (H)
36 (xylose), 40 (furfural), 80 (HMF)
6.76 × 10-10a
6.08 × 10-10
3.38 × 10-10
0.4 (glucose), 40 (furfural), 80 (HMF)
7.69 × 10-10a
6.92 × 10-10
3.85 × 10-10
1.10 × 10-9b
9.90 × 10-10
5.50 × 10-10
1.06 × 10-9c
9.54 × 10-10
5.30 × 10-10
The biomass concentration inside the cell pellet compartment in the capsule was assumed to be constant at 300 g/l, as reported previously [15, 30]. The concentrations of solutes in the bulk liquid outside the capsule were set as constant boundary conditions. Parameters for glucose and xylose transport were adapted from Lee et al. . The inhibition constant for competitive inhibition of xylose uptake by glucose was assumed to be equal to the high affinity K M value for glucose. For furfural consumption, the maximum rate and saturation constant identified during steady state conditions at high cell density in a membrane bioreactor were used . HMF is generally consumed at a lower rate than furfural. It was assumed that the maximum rate of HMF conversion was one-third of the maximum furfural conversion rate, with a saturation constant of 20 mM [32–34].
to the measured specific growth rate using non-linear regression, we estimated the inhibition constant K if is approximately 40 mM. In this equation, is the estimated specific growth rate, D is the dilution rate equalling the specific growth rate at steady state prior to each pulse addition, and C f, pulse is the concentration of the furfural immediately after pulse addition. In the absence of reliable data, and as HMF is known to be less inhibitory than furfural, we set the inhibition constant K ih at 2 K if .
Diffusivities in the liquid bulk were taken from literature data (Table 3). The effective diffusivities in the liquid core, gel membrane and cell pellet were assumed to be 100%, 90% and 25% of the diffusivity in the liquid bulk (D w ), respectively. The Sherwood number was set to 100 to exclude external mass transfer resistance.
Efficiency factors and sensitivity analysis
where and are the rate and efficiency response coefficients to the parameter P.
A xylose-fermenting strain, which we named CEN.PK XXX, was engineered in two steps using targeted homologous recombination of linearised DNA fragments directly into the yeast chromosomes XVI and VIII of S. cerevisiae CEN.PK 122 MDS . In the first step, the copy number of enzymes in the pentose phosphate pathway was increased by integration of an additional copy of the RPE1, TAL1 and RKI1 genes. The promoter of the TKL1 gene was also replaced with the glycolytic promoter of ENO1. The genetic construct was integrated into the N-terminal coding region of the TKL1 gene in chromosome XVI to replace the native TKL1 promoter. The TKL1 coding sequence was left intact, and the RPE1, TAL1 and RKI1 genes were integrated upstream of the TKL1 gene. The integration event was selected for by using the resistance gene SMR1 (a mutant ILV2) and plating the transformed cells onto minimal medium agar plates with 50 μg/ml sulfometuron-methyl (Supelco, Bellefonte, PA, USA). In the second transformation, genes active in xylose metabolism were integrated: XYL1 and XYL2 from S. stipitis, and XKS1 from S. cerevisiae. The synthetic codon-optimised coding segments of XYL1, XYL2 and XKS1 were each fused upstream with new promoter segments from −500 to −1 of three glycolytic enzymes: the promoter of PGK1 with XYL1, the promoter of TDH3 with XYL2 and the promoter of TPI1 with XKS1. The DNA segment containing the ‘xylose genes’ was flanked by segments of GRE3. The cells that had been transformed with the DNA fragment were transferred to a shake flask containing 20 g/l xylose in 2 × CBS medium , and after 5 days, clear growth was seen. The strain was maintained on minimal medium agar plates with 20 g/l D-xylose (Fisher Scientific, Leicestershire, England) as the only carbon source, and was used in all experiments.
The growth medium used for the batch cultivations was a defined growth medium (DGM), as previously reported , with ergosterol and Tween 80 (both Sigma, Steinheim, Germany) added during anaerobic cultivations, and glucose, xylose or a combination of the two as carbon source.
The hydrolysate used was made from spruce chips at 18 bar pressure for 5 to 7 minutes at pH 2.0 by addition of SO2, and kept refrigerated until use. Immediately prior to use, the pH was set to 5.5 with 10 M NaOH. The medium was autoclaved and centrifuged to remove solid particles. The final concentrations in the medium used for anaerobic fermentations were: glucose 12.8 ± 0.2 g/l, mannose 16.6 ± 0.3 g/l, galactose 3.6 ± 0.1 g/l, xylose 7.6 ± 0.1 g/l, arabinose 2.4 ± 0.0 g/l, acetic acid 3.1 ± 0.0 g/l and furfural 0.28 ± 0.08 g/l, after addition of salts, vitamins and trace metals as in the defined media.
The capsules were prepared by the one-step, liquid droplet-forming method as described in . In short, cells were grown in 100 ml DGM (40 g/l glucose and 20 g/l xylose) for 24 hours. Yeast was harvested from 50 ml of medium at OD600 of approximately 4 by centrifugation at 3500 × g for 4 minutes, and resuspended in 50 ml of 1.3% w/v sterile CaCl2 (Scharlau, Sentmenat, Spain) solution containing 1.3% w/v carboxymethylcellulose (Aldrich, Steinheim, Germany) with an average molecular weight of 250 kDa and degree of substitution of 0.9. Capsules were formed by dripping this solution through syringe needles into a stirred sterile solution of 0.6% w/v sodium alginate (catalogue number 71238; Sigma) and 0.1% v/v Tween 20 (Sigma-Aldrich, Steinheim, Germany). After gelling for 10 minutes, the capsules were washed with sterile ultrapure water, and hardened in 1.3% CaCl2. The capsules were subsequently submerged for 24 hours in 0.2% w/v low molecular weight chitosan (catalogue number 448869; Aldrich) solution with 300 mM CaCl2 in 0.040 M acetate buffer, pH 4.5.
Cultivation and cell sampling
Propagation of encapsulated cells in capsules of approximately 15 ml was performed aerobically in 250 ml cotton-plugged conical flasks filled with 100 ml DGM containing 40 g/l glucose and 20 g/l xylose, which were incubated for 36 hours in a shaker bath (125 rpm) at 30°C. The capsules were rinsed with sterile 0.9% NaCl (Scharlau), and transferred to 100 ml DGM containing 5 g/l glucose and 40 g/l xylose, then incubated for another 24 hours prior to the start of the anaerobic batch fermentations. We chose these concentrations of glucose and xylose to ensure rapid cell growth by inclusion of a high concentration of glucose in the first step, and to maintain efficient xylose metabolism by using a low glucose and a high xylose concentration in the second step.
Propagation of suspended yeast was started with aerobic cultivation for 24 hours in 100 ml DGM (40 g/l glucose and 20 g/l xylose). Cells were harvested (3500 × g, 4 minutes) and resuspended to an OD600 of 3 in fresh medium of the same composition. After 36 hours, cells were again harvested and resuspended in 100 ml fresh DGM (5 g/l glucose and 40 g/l xylose) to an OD600 of 7.2, and cultured for another 24 hours of aerobic propagation. The residual sugar concentrations at the end of each propagation culture were similar. Hence, the propagation steps for the encapsulated and the suspended cells were as similar as practically possible.
Anaerobic batch cultivations were performed in conical flasks, equipped with a rubber stopper fitted with stainless steel capillaries for sample removal and a loop trap filled with sterile water to permit the produced CO2 to leave the flasks. Anaerobic encapsulated cultures were started by transferring 30 capsules to 120 ml of media containing combinations of glucose (0 or 40 g/l), xylose (0 or 40 g/l) and furfural (0, 1 or 2 g/l) (Sigma-Aldrich). This gave an initial cell concentration of 0.272 ± 0.014 g dry cells/l of liquid volume, meaning an average of 1.1 mg dry cells per capsule in the form of a visible pellet inside each capsule. Anaerobic cultivations of suspended cells were started at initial cell concentrations of 0.258 ± 0.030 g dry cells/l.
Analytical methods, statistics, yields and elemental balance calculations
Metabolite concentrations were quantified by HPLC using an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) at 60°C with 5 mM H2SO4 as eluent at a flow rate of 0.6 ml/min. A refractive index detector was used for the detection and quantification of glucose, xylose, xylitol, acetic acid, glycerol and ethanol. For the hydrolysate medium an Aminex HPX-87P (Bio-Rad), operated at 85°C with 0.6 ml/min ultrapure water as eluent, was used to quantify glucose, xylose, arabinose, galactose and mannose with a refractive index detector.
The cell dry weight was measured in predried and preweighed glass tubes. Cells were separated by centrifugation, and washed once with ultrapure water before drying for approximately 24 hours at 105°C. Cells from capsules were released by crushing the capsule, followed by extensive washing of the capsule debris with ultrapure water.
Yields of metabolites and biomass, as well as the carbon balance, were calculated from the determined concentrations at the end of the fermentations. The biomass composition, CH1.76O0.56 N0.17, was used in the carbon balance calculations. Error intervals are given as ± 1 standard deviation with n = 3, unless otherwise indicated.
DGM: Defined growth medium; FEM: Finite element modelling; HMF: 5-hydroxymethylfurfural; HPLC: high performance liquid chromatography; OD: Optical density;
β: Efficiency factor (−); c: Concentration (mM); : Rate response coefficient to entity j (−); : Efficiency response coefficient to entity j (−); D: Diffusivity (m2/s); D: Dilution rate (per h); K i : Inhibition constant (mM); K M : Half saturation constant (mM); n: Number of repeated experiment; R: Total rate of reaction in cell pellet calculated by surface integration (mol/m/s); v: Specific rate (mM/g/h); YSi: Yield of compound i on consumed sugar (g/g);
Ace: Acetate; c: Cell pellet; EtOH: Ethanol; f: Furfural; g: Glucose; Gly: Glycerol; h: 5-hydroxymethylfurfural; H: High affinity; L: Low affinity; m: Capsule membrane; max: Maximum; P: Parameter; W: Water; X: Xylose.
We are very grateful to Joana Paula Da Costa Pereira and Gonçalo Carvalho Esteves for performing initial in silico simulation studies and to Aires Coelho for performing initial laboratory experiments. We thank Dr. Tomas Brandberg at SEKAB AB, Sweden, for providing the spruce hydrolysate. We gratefully acknowledge funding by the Swedish Research Council (grant no. 2009–4514), the Chalmers Energy Initiative (http://www.chalmers.se/en/areas-of-advance/energy/cei) and the University of Borås.
- Wyman CE: Ethanol from lignocellulosic biomass: technology, economics, and opportunities. Bioresour Technol 1994, 50: 3-15. 10.1016/0960-8524(94)90214-3View ArticleGoogle Scholar
- Zaldivar J, Nielsen J, Olsson L: Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 2001, 56: 17-34. 10.1007/s002530100624View ArticleGoogle Scholar
- Van Zyl C, Prior BA, Kilian SG, Kock JLF: D-xylose utilization by Saccharomyces cerevisiae . J Gen Microbiol 1989, 135: 2791-2798.Google Scholar
- Laluce C, Schenberg ACG, Gallardo JCM, Coradello LFC, Pombeiro-Sponchiado SR: Advances and developments in strategies to improve strains of Saccharomyces cerevisiae and processes to obtain the lignocellulosic ethanol-a review. Appl Biochem Biotechnol 2012, 166: 1908-1926. 10.1007/s12010-012-9619-6View ArticleGoogle Scholar
- Kötter P, Ciriacy M: Xylose fermentation by Saccharomyces cerevisiae . Appl Microbiol Biotechnol 1993, 38: 776-783. 10.1007/BF00167144View ArticleGoogle Scholar
- Bertilsson M, Andersson J, Lidén G: Modeling simultaneous glucose and xylose uptake in Saccharomyces cerevisiae from kinetics and gene expression of sugar transporters. Bioproc Biosyst Eng 2008, 31: 369-377. 10.1007/s00449-007-0169-1View ArticleGoogle Scholar
- Meinander NQ, Boels I, Hahn-Hägerdal B: Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TAL1 . Bioresour Technol 1999, 68: 79-87. 10.1016/S0960-8524(98)00085-6View ArticleGoogle Scholar
- Hamacher T, Becker J, Gárdonyi M, Hahn-Hägerdal B, Boles E: Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology 2002, 148: 2783-2788.View ArticleGoogle Scholar
- Sedlak M, Ho NWY: Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast . Yeast 2004, 21: 671-684. 10.1002/yea.1060View ArticleGoogle Scholar
- Westman JO, Ylitervo P, Franzén C, Taherzadeh MJ: Effects of encapsulation of microorganisms on product formation during microbial fermentations. Appl Microbiol Biotechnol 2012, 96: 1441-1454. 10.1007/s00253-012-4517-yView ArticleGoogle Scholar
- Pérez-Bibbins B, de Souza OR, Torrado A, Aguilar-Uscanga MG, Domínguez JM: Study of the potential of the air lift bioreactor for xylitol production in fed-batch cultures by Debaryomyces hansenii immobilized in alginate beads. Appl Microbiol Biotechnol 2014, 98: 151-161. 10.1007/s00253-013-5280-4View ArticleGoogle Scholar
- Pérez-Bibbins B, Salgado JM, Torrado A, Aguilar-Uscanga MG, Domínguez JM: Culture parameters affecting xylitol production by Debaryomyces hansenii immobilized in alginate beads. Process Biochem 2013, 48: 387-397. 10.1016/j.procbio.2013.01.006View ArticleGoogle Scholar
- Galazzo JL, Bailey JE: Growing Saccharomyces cerevisiae in calcium-alginate beads induces cell alterations which accelerate glucose conversion to ethanol. Biotechnol Bioeng 1990, 36: 417-426. 10.1002/bit.260360413View ArticleGoogle Scholar
- Westman JO, Manikondu RB, Franzén CJ, Taherzadeh MJ: Encapsulation-induced stress helps Saccharomyces cerevisiae resist convertible lignocellulose derived inhibitors. Int J Mol Sci 2012, 13: 11881-11894. 10.3390/ijms130911881View ArticleGoogle Scholar
- Westman JO, Taherzadeh MJ, Franzén CJ: Proteomic analysis of the increased stress tolerance of Saccharomyces cerevisiae encapsulated in liquid core alginate-chitosan capsules. PLoS One 2012, 7: e49335. 10.1371/journal.pone.0049335View ArticleGoogle Scholar
- Vicente AA, Dluhý M, Ferreira EC, Mota M, Teixeira JA: Mass transfer properties of glucose and O2 in Saccharomyces cerevisiae flocs. Biochem Eng J 1998, 2: 35-43. 10.1016/S1369-703X(98)00015-1View ArticleGoogle Scholar
- Lebeau T, Jouenne T, Junter G-A: Diffusion of sugars and alcohols through composite membrane structures immobilizing viable yeast cells. Enzyme Microb Tech 1998, 22: 434-438. 10.1016/S0141-0229(97)00214-7View ArticleGoogle Scholar
- Hannoun BJM, Stephanopoulos G: Diffusion coefficients of glucose and ethanol in cell-free and cell-occupied calcium alginate membranes. Biotechnol Bioeng 1986, 28: 829-835. 10.1002/bit.260280609View ArticleGoogle Scholar
- Tanaka H, Matsumura M, Veliky IA: Diffusion characteristics of substrates in Ca-alginate gel beads. Biotechnol Bioeng 1984, 26: 53-58. 10.1002/bit.260260111View ArticleGoogle Scholar
- Merchant FJA, Margaritis A, Wallace JB, Vardanis A: A novel technique for measuring solute diffusivities in entrapment matrices used in immobilization. Biotechnol Bioeng 1987, 30: 936-945. 10.1002/bit.260300804View ArticleGoogle Scholar
- Talebnia F, Niklasson C, Taherzadeh MJ: Ethanol production from glucose and dilute-acid hydrolyzates by encapsulated S. cerevisiae . Biotechnol Bioeng 2005, 90: 345-353. 10.1002/bit.20432View ArticleGoogle Scholar
- Kim SR, Kwee NR, Kim H, Jin Y-S: Feasibility of xylose fermentation by engineered Saccharomyces cerevisiae overexpressing endogenous aldose reductase ( GRE3 ), xylitol dehydrogenase ( XYL2 ), and xylulokinase ( XYL3 ) from Scheffersomyces stipitis . FEMS Yeast Res 2013, 13: 312-321. 10.1111/1567-1364.12036View ArticleGoogle Scholar
- Jin Y-S, Ni H, Laplaza JM, Jeffries TW: Optimal growth and ethanol production from xylose by recombinant Saccharomyces cerevisiae require moderate d-Xylulokinase activity. Appl Environ Microbiol 2003, 69: 495-503. 10.1128/AEM.69.1.495-503.2003View ArticleGoogle Scholar
- Sárvári Horváth I, Taherzadeh MJ, Niklasson C, Lidén G: Effects of furfural on anaerobic continuous cultivation of Saccharomyces cerevisiae . Biotechnol Bioeng 2001, 75: 540-549. 10.1002/bit.10090View ArticleGoogle Scholar
- Lee WJ, Kim MD, Ryu YW, Bisson L, Seo JH: Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae . Appl Microbiol Biotechnol 2002, 60: 186-191. 10.1007/s00253-002-1085-6View ArticleGoogle Scholar
- Leandro MJ, Fonseca C, Gonçalves P: Hexose and pentose transport in ascomycetous yeasts: an overview. FEMS Yeast Res 2009, 9: 511-525. 10.1111/j.1567-1364.2009.00509.xView ArticleGoogle Scholar
- Mogi N, Sugai E, Fuse Y, Funazukuri T: Infinite dilution binary diffusion coefficients for six sugars at 0.1 MPa and temperatures from (273.2 to 353.2) K. J Chem Eng Data 2006, 52: 40-43.View ArticleGoogle Scholar
- Yaws CL: Yaws’ Handbook of Thermodynamic and Physical Properties of Chemical Compounds. Knovel; 2003. [http://app.knovel.com/hotlink/toc/id:kpYHTPPCC4/yaws-handbook-thermodynamic] Google Scholar
- Wilke CR, Chang P: Correlation of diffusion coefficients in dilute solutions. AIChE J 1955, 1: 264-270. 10.1002/aic.690010222View ArticleGoogle Scholar
- Cheong SH, Park JK, Kim BS, Chang HN: Microencapsulation of yeast cells in the calcium alginate membrane. Biotechnol Tech 1993, 7: 879-884. 10.1007/BF00156366View ArticleGoogle Scholar
- Ylitervo P, Franzén CJ, Taherzadeh M: Impact of furfural on rapid ethanol production using a membrane bioreactor. Energies 2013, 6: 1604-1617. 10.3390/en6031604View ArticleGoogle Scholar
- Jordan DB, Braker JD, Bowman MJ, Vermillion KE, Moon J, Liu ZL: Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural. Biochim Biophys Acta (BBA) 2011, 1814: 1686-1694. 10.1016/j.bbapap.2011.08.011View ArticleGoogle Scholar
- Kostesha NV, Almeida JRM, Heiskanen AR, Gorwa-Grauslund MF, Hahn-Hägerdal B, Emnéus J: Electrochemical probing of in vivo 5-hydroxymethyl furfural reduction in Saccharomyces cerevisiae . Anal Chem 2009, 81: 9896-9901. 10.1021/ac901402mView ArticleGoogle Scholar
- Liu ZL, Moon J: A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. Gene 2009, 446: 1-10. 10.1016/j.gene.2009.06.018View ArticleGoogle Scholar
- Gietz RD, Woods R: Yeast transformation by the LiAc/SS carrier DNA/PEG method. In Methods in Molecular Biology. Volume 313 Yeast Protocol. Edited by: Wei X. Totowa: Humana Press; 2006:107-120.Google Scholar
- Verduyn C, Postma E, Scheffers WA, Van Dijken JP: Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 1992, 8: 501-517. 10.1002/yea.320080703View ArticleGoogle Scholar
- Taherzadeh MJ, Lidén G, Gustafsson L, Niklasson C: The effects of pantothenate deficiency and acetate addition on anaerobic batch fermentation of glucose by Saccharomyces cerevisiae . Appl Microbiol Biotechnol 1996, 46: 176-182. 10.1007/s002530050801View ArticleGoogle Scholar
- Verduyn C, Postma E, Scheffers WA, van Dijken JP: Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J Gen Microbiol 1990, 136: 395-403. 10.1099/00221287-136-3-395View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.