Production of xylitol in cell recycle fermentations of Candida tropicalis

Xylitol was produced a in two-substrate, batch fermentation with cell recycling of Candida tropicalis ATCC 13803. A series of cell-recycle experiments showed that the feeding of xylose, glucose and yeast extract in the xylitol production phase was most effective in enhancing xylitol productivity. The optimized cell recycle fermentation resulted in 0.82 g xylitol/g xylose yield, 4.94 g xylitol l^–1 h^–1 productivity, and final xylitol concentration of 189 g l^–1. These results were 1.3 times higher in volumetric xylitol productivity and 2.2 times higher in final product concentration compared with the corresponding values of the optimized two-substrate batch culture.     

Xylitol production from barley bran hydrolysates by continuous fermentation with Debaryomyces hansenii

The continuous bioconversion of xylose-containing solutions (obtained by acid hydrolysis of barley bran) into xylitol was carried out using the yeast Debaryomyces hansenii under microaerophilic conditions with or without cell recycle. In fermentations without cell recycle, the volumetric productivities ranged from 0.11–0.6 g l^–1 h^–1 were obtained for dilution rates of 0.008–0.088 h^–1. In experiments performed with cell recycle after membrane separation, the optimum xylitol productivity (2.53 g l^–1 h^–1) was reached at a dilution rate of 0.284 h^–1.     

A repeated batch process for cultivation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A repeated batch process was performed to culture Bifidobacterium longum CCRC 14634. An on-line device, oxidation-reduction potential (ORP), was used to monitor cell growth and uptake of nutrients in the culture. The ORP of the culture medium decreased substantially during fermentation until nutrients were depleted. Six cycles of batch fermentation using ORP as a control parameter were successfully carried out. As soon as ORP remained constant or increased, three-quarters of the broth was removed, and the same volume of fresh medium was fed to the fermenter for a new cycle of cultivation. Average cell concentrations of 1.9×10⁹ and 3.4×10⁹ cfu ml^–1 for repeated batch fermentation in MRS (Lactobacilli MRS broth) and WY (containing whey hydrolyzates, yeast extract, l-cysteine) medium, respectively, were achieved. Cell mass productivities for batch, fed-batch and repeated batch fermentation using MRS medium were 0.51, 0.41, and 0.64 g l^–1 h^–1, respectively, and those for batch and repeated batch using WY medium were 0.76, 0.99 g l^–1 h^–1, respectively. The results indicate a possible industrial process to culture Bifidobacteria sp.     

The production of 2,3-butanediol by fermentation of high test molasses

Summary Klebsiella oxytoca fermented 199 g·l^–1 high test or invert molasses using batch fermentation with substrate shift to produce 95.2–98.6 g 2,3-butanediol·l^–1 and 2,4–4.3 g acetoin·l^–1 with a diol yield of 96–100% of the theoretical value and a diol productivity of 1.0–1.1 g·l^–1·h^–1. Fermentation was performed numerous times with molasses in repeated batch culture with cell recovery. Such repeated batch fermentation, in addition to a high product yield, also showed a very high product concentration. For example, 118 g 2,3-butanediol·l^–1 and 2.3 g acetoin·l^–1 were produced from 280 g·l^–1 of high test molasses. The diol productivity in this fermentation amounted to 2.4 g·l^–1·h^–1 and can undoubtedly be further increased by increasing the cell concentration. Because the Klebsiella cultures ferment 2,3-butanediol at an extremely high rate once the sugar has been consumed, the culture was inhibited completely by the addition of 15 g ethanol·l^–1 and switching off aeration. Offprint requests to: A. S. Afschar     

Glucose repression of xylitol production in Candida tropicalis mixed-sugar fermentations

Glucose repressed xylose utilization inCandida tropicalis pre-grown on xylose until glucose reached approximately 0–5 g l^–1. In fermentations consisting of xylose (93 g l^–1) and glucose (47 g l^–1), xylitol was produced with a yield of 0.65 g g^–1 and a specific rate of 0.09 g g^–1 h^–1, and high concentrations of ethanol were also produced (25 g l^–1). If the initial glucose was decreased to 8 g l^–1, the xylitol yield (0.79 g g^–1) and specific rate (0.24 g g^–1 h^–1) increased with little ethanol formation (<5 g l^–1). To minimize glucose repression, batch fermentations were performed using an aerobic, glucose growth phase followed by xylitol production. Xylitol was produced under O₂ limited and anaerobic conditions, but the specific production rate was higher under O₂ limited conditions (0.1–0.4 vs. 0.03 g g^–1 h^–1). On-line analysis of the respiratory quotient defined the time of xylose reductase induction.     

Citric acid production from glycerol-containing waste of biodiesel industry by Yarrowia lipolytica in batch, repeated batch, and cell recycle regimes

Yarrowia lipolytica A-101-1.22 produces high citric acid (112 g l⁻¹) with a yield of 0.6 g g⁻¹ and a productivity of 0.71 g l⁻¹ h⁻¹ during batch cultivation in the medium with glycerol-containing waste of biodiesel industry. However, it was observed that the specific citric acid production rate, which was maximal at the beginning of the biosynthesis, gradually decreases in the late production phase and it makes continuation of the process over 100 h pointless. The cell recycle and the repeated batch regimes were performed as ways for prolongation of citric acid synthesis by yeast. Using cell recycle, the active citric acid biosynthesis (96–107 g l⁻¹) with a yield of 0.64 g g⁻¹ and a productivity of 1.42 g l⁻¹ h⁻¹ was prolongated up to 300 h. Repeated batch culture remained stable for over 1000 h; the RB variant of 30% feed every 3 days showed the best results: 124.2 g l^-1 citric acid with a yield of 0.77 g g^-1 and a productivity of 0.85 g l^-1 h^-1.     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Direct alcoholic fermentation of starchy biomass using amylolytic yeast strains in batch and immobilized cell systems

Summary Direct alcoholic fermentation of dextrin or soluble starch with selected amylolytic yeasts was studied in both batch and immobilized cell systems. In batch fermentations, Saccharomyces diastaticus was capable of fermenting high dextrin concentrations much more efficiently than Schwanniomyces castellii. From 200 g·l^–1 of dextrin S. diastaticus produced 77 g·l^–1 of ethanol (75% conversion efficiency). The conversion efficiency decreased to 59% but a higher final ethanol concentration of 120 g·l^–1 was obtained with a medium containing 400 g·l^–1 of dextrin. With a mixed culture of S. diastaticus and Schw. castellii 136 g·l^–1 of ethanol was produced from 400 g·l^–1 of dextrin (67% conversion efficiency). S. diastaticus cells attached well to polyurethane foam cubes and a S. diastaticus immobilized cell reactor produced 69 g·l^–1 of ethanol from 200 g·l^–1 of dextrin, corresponding to an ethanol productivity of 7.6g·l^–1·h^–1. The effluent from a two-stage immobilized cell reactor with S. diastaticus and Endomycopsis fibuligera contained 70 g·l^–1 and 80 g·l^–1 of ethanol using initial dextrin concentrations of 200 and 250 g·l^–1 respectively. The corresponding values for ethanol productivity were 12.7 and 9.6 g·l^–1·h^–1. The productivity of the immobilized cell systems was higher than for the batch systems, but much lower than for glucose fermentation.     

Evaluation of solvents for extractive butanol fermentation with Clostridium acetobutylicum and the use of poly(propylene glycol) 1200

Summary In an effort to improve the viability of acetone-butanol-ethanol fermentation by extractive fermentation, 63 organic solvents, including alkanes, alcohols, aldehydes, acids, and esters, were experimentally evaluated for biocompatibility with Clostridium acetobutylicum by observing gas evolution from cultures in contact with candidate solvents. Thirty-one of these solvents were further tested to determine their partition coefficient for butanol in fermentation medium. The biocompatible solvent with the highest partition coefficient for butanol (4.8), was poly(propylene glycol) 1200, which was selected for fermentation experiments. This is the highest partition coefficient reported to date for a biocompatible solvent. Extractive fermentations using concentrated feeds were observed to produce up to 58.6 g·l^–1 acetone and butanol in 202 h, the equivalent of three control fermentations in a single run. Product yields (based on total solvent products and glucose consumed) of 0.234 g·g^–1 to 0.311 g·g^–1 and within run solvent productivities of 0.174 g·l^–1·h^–1 to 0.290 g·l^–1·h^–1 were consistentwith conventional fermentations reported in the literature. The extended run-time of the fermentation resulted in an overall improvement in productivity by reducing the fraction of between-run down-time for fermentor cleaning and sterilization.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

Glycerol production by Candida krusei employing NaCl as an osmoregulator in batch and continuous fermentations

Addition of 40 g NaCl l^–1 to a chemically defined medium containing 140 g glucose l^–1 in shake-flask culture improved glycerol production by Candida krusei from 16.5 g l^–1 to 47.7 g l^–1. With 40 g NaCl l^–1 at a dilution rate of 0.065 h^–1, glycerol concentration, glycerol yield (based on glucose consumed), and productivity in a four-stage cascade bioreactor were higher by 240%, 27% and 28%, respectively, than in a single-stage continuous culture system.     

Xylitol production from aspenwood hemicellulose hydrolysate by Candida guilliermondii

The production of xylitol by the yeast Candida guilliermondii was investigated in batch fermentations with aspenwood hemicellulose hydrolysate and compared with results obtained in semi-defined media with a mixture of glucose and xylose. The hemicellulose hydrolysate had to be supplemented by yeast extract and the maximum xylitol yield (0.8 g g^–1) and productivity (0.6 g l^–1 h^–1) were reached by controlling oxygen input.     

Effects of different growth forms of Mucor indicus on cultivation on dilute-acid lignocellulosic hydrolyzate, inhibitor tolerance, and cell wall composition

The dimorphic fungus Mucor indicus was grown in different forms classified as purely filamentous, mostly filamentous, mostly yeast-like and purely yeast-like, and the relationship between morphology and metabolite production, inhibitor tolerance and the cell wall composition was investigated. Low concentrations of spores in the inoculum with subsequent aeration promoted filamentous growth, whereas higher spore concentrations and anaerobic conditions promoted yeast-like growth. Ethanol was the main metabolite with glycerol next under all conditions tested. The yields of ethanol from glucose were between 0.39 and 0.42 g g⁻¹ with productivities of 3.2–5.0 g l⁻¹ h⁻¹. The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0 g l⁻¹ h⁻¹ by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6 g l⁻¹ furfural and 10 g l⁻¹ acetic acid and assimilate the sugars, although with different consumption rates. The cell wall content of the fungus measured as alkali insoluble materials (AIM) of the purely yeast-like cells was 26% of the biomass, compared to 8% of the pure filaments. However, the chitosan concentration of the filaments was 29% of the AIM, compared to 6% of the yeast-like cells.     

Continuous ethanol production byZymomonas mobilis andSaccharomyces cerevisiae in biofilm reactors

Continuous ethanol fermentations were performed in duplicate for 60 days withZymomonas mobilis ATCC 331821 orSaccharomyces cerevisiae ATCC 24859 in packed-bed reactors with polypropylene or plastic composite-supports. The plastic composite-supports used contained polypropylene (75%) with ground soybean-hulls (20%) and zein (5%) forZ. mobilis, or with ground soybean-hulls (20%) and soybean flour (5%) forS. cerevisiae. Maximum ethanol productivities of 536 gL^–1 h^–1 (39% yield) and 499 gL^–1 h^–1 (37% yield) were obtained withZ. mobilis on polypropylene and plastic composite-supports of soybean hull-zein, respectively. ForZ. mobilis, and optimal yield of 50% was observed at a 1.92h^–1 dilution rate for soybean hull-zein plastic composite-supports with a productivity of 96gL^–1h^–1, whereas with polypropylene-supports the yield was 32% and the productivity was 60gL^–1h^–1. With aS. cerevisiae fermentation, the ethanol production was less, with a maximum productivity of 76gL^–1h^–1 on the plastic composite-support at a 2.88h^–1 dilution rate with a 45% yield. Polypropylene-support bioreactors were discontinued due to reactor plugging by the cell mass accumulation. Support shape (3-mm chips) was responsible for bioreactor plugging due to extensive biofilm development on the plastic composite-supports. With suspensionculture continuous fermentations in continuously-stirred benchtop fermentors, maximum productivities of 5gL^–1h^–1 were obtained with a yield of 24 and 26% withS. cerevisiae andZ. mobilis, respectively. Cell washout in suspensionculture continuous fermentations was observed at a 1.0h^–1 dilution rate. Therefore, for continuous ethanol fermentations, biofilm reactors out-performed suspension-culture reactors, with 15 to 100-fold higher productivities (gL^–1h^–1) and with higher percentage yields forS. cerevisiae andZ. mobilis, respectively. Further research is needed with these novel supports to evaluate different support shapes and medium compositions that will permit medium flow, stimulate biofilm formation, reduce fermentation costs, and produce maximum yields and productivities.This is Journal Paper No. J-16357 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3253     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Hydrolysate detoxification with activated charcoal for xylitol production by Candida guilliermondii

A detoxification method using activated charcoal with concentrated rice straw hemicellulosic hydrolysate improved the conversion of xylose to xylitol by the yeast Candida guilliermondii by 22%. This was achieved when the hydrolysate:charcoal ratio was 40 g g^–1, resulting in removal of 27% of phenolic compounds. Under this condition, the xylitol yield factor (0.72 g g^–1) and volumetric productivity (0.61 g l^–1 h^–1) were close to those attained in a semi-defined medium simulating hydrolysate sugars.     

Production of acetone,butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping

We examined the effect of gas-stripping on the in situ removal of acetone, butanol, and ethanol (ABE) from batch reactor fermentation broth. The mutant strain (Clostridium beijerinckii BA101) was not affected adversely by gas stripping. The presence of cells in the fermentation broth affected the selectivities of ABE. A considerable improvement in the productivity and yield was recorded in this work in comparison with the non-integrated process. In an integrated process of ABE fermentation-recovery using C. beijerinckii BA101, ABE productivities and yield were improved up to 200 and 118%, respectively, as compared to control batch fermentation data. In a batch reactor C. beijerinckii BA101 utilized 45.4 g glucose l^–1 and produced 17.7 g total ABE l^–1, while in the integrated process it utilized 161.7 g glucose l^–1 and produced total ABE of 75.9 g l^–1. In the integrated process, acids were completely converted to solvents when compared to the non-integrated process (batch fermentation) which contained residual acids at the end of fermentation. In situ removal of ABE by gas stripping has been reported to be one of the most important techniques of solvent removal. During these studies we were able to maintain the ABE concentration in the fermentation broth below toxic levels.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

Xylitol production by recombinant Corynebacterium glutamicum under oxygen deprivation

Wild-type Corynebacterium glutamicum produced 0.6 g l⁻¹ xylitol from xylose at a productivity of 0.01 g l⁻¹ h⁻¹ under oxygen deprivation. To increase this productivity, the pentose transporter gene (araE) from C. glutamicum ATCC31831 was integrated into the C. glutamicum R chromosome. Consequent disruption of its lactate dehydrogenase gene (ldhA), and expression of single-site mutant xylose reductase from Candida tenuis (CtXR (K274R)) resulted in recombinant C. glutamicum strain CtXR4 that produced 26.5 g l⁻¹ xylitol at 3.1 g l⁻¹ h⁻¹. To eliminate possible formation of toxic intracellular xylitol phosphate, genes encoding xylulokinase (XylB) and phosphoenolpyruvate-dependent fructose phosphotransferase (PTS^fru) were disrupted to yield strain CtXR7. The productivity of strain CtXR7 increased 1.6-fold over that of strain CtXR4. A fed-batch 21-h CtXR7 culture in mineral salts medium under oxygen deprivation yielded 166 g l⁻¹ xylitol at 7.9 g l⁻¹ h⁻¹, representing the highest bacterial xylitol productivity reported to date.