Appl Microbiol Biotechnol (2011) 90:257–267 DOI 10.1007/s00253-010-3015-3
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Production of arabitol from glycerol: strain screening and study of factors affecting production yield
Srujana Koganti & Tsung Min Kuo & Cletus P. Kurtzman & Nathan Smith & Lu-Kwang Ju
Received: 26 August 2010 /Revised: 10 November 2010 /Accepted: 15 November 2010 /Published online: 3 December 2010 # Springer-Verlag 2010
Abstract Glycerol is a major by-product from biodiesel Keywords Arabitol . Xylitol . Biodiesel . Glycerol . production, and developing new uses for glycerol is Osmotolerant yeast . Debaryomyces hansenii imperative to overall economics and sustainability of the biodiesel industry. With the aim of producing xylitol and/or arabitol as the value-added products from glycerol, 214 Introduction yeast strains, many osmotolerant, were first screened in this study. No strains were found to produce large amounts of Biodiesel motor fuel produced from renewable sources such xylitol as the dominant metabolite. Some produced polyol as vegetable oil and animal fat is an attractive alternative to mixtures that might present difficulties to downstream petroleum-derived fuel (Krawczyk 1996). In biodiesel separation and purification. Several Debaryomyces hansenii production using transesterification of triglycerides, glycerol strains produced arabitol as the predominant metabolite is the major by-product produced: About 1 kg of glycerol is with high yields, and D. hansenii strain SBP-1 (NRRL Y- formed for every 9 kg of biodiesel produced (Dasari et al. 7483) was chosen for further study on the effects of several 2005). Biodiesel consumption in the USA has increased growth conditions. The optimal temperature was found to dramatically from 75 million gallons in 2005 to 450 million be 30°C. Very low dissolved oxygen concentrations or gallons in 2007. Accompanying this increase was the anaerobic conditions inhibited polyol yields. Arabitol yield production of around 45 million gallons of glycerol in improved with increasing initial glycerol concentrations, 2007 (National Biodiesel Board 2007). Refined glycerol has reaching approximately 50% (w/w) with 150 g/L initial numerous applications in the food, drug, textile, and glycerol. However, the osmotic stress created by high salt cosmetic industries whereas crude glycerol produced from concentrations (≥50 g/L) negatively affected arabitol pro- the biodiesel industry is of low value because of impurities duction. Addition of glucose and xylose improved arabitol such as spent catalyst, salts after neutralization, residual production while addition of sorbitol reduced production. methanol, methyl esters, and free fatty acids (Liu et al. 2002; Results from this work show that arabitol is a promising Bournay et al. 2005). The economics of the biodiesel value-added product from glycerol using D. hansenii SBP- industry is strongly influenced by the value of its by- 1 as the producing strain. products, and developing new uses for biodiesel glycerol is imperative to the sustainability of the biodiesel industry : S. Koganti L.-K. Ju (*) (Demirbas 2003; Haas et al. 2006). Department of Chemical and Biomolecular Engineering, In this study, the biodiesel by-product glycerol was used as The University of Akron, the substrate for production of arabitol, a polyhydric alcohol. Akron, OH 44325-3906, USA e-mail: [email protected] A study by the Department of Energy identified arabitol, and : : its enantiomer xylitol, as one of the top 12 biomass-derivable T. M. Kuo C. P. Kurtzman N. Smith building block chemicals. Arabitol and xylitol can be trans- Foodborne Pathogens and Mycology Research Unit, National formed into several groups of chemicals like arabonic/ Center for Agricultural Utilization Research, ARS, USDA, 1815 N. University Street, arabinoic acid, xylaric/xylonic acid, propylene glycol, and Peoria, IL 61604-3999, USA ethylene glycol (Werpy and Petersen 2004). Arabitol and 258 Appl Microbiol Biotechnol (2011) 90:257–267 xylitol have melting points of 103°C and 93°C, respectively. Media Both are highly soluble in water and both form white crystals when purified (Le Tourneau 1966; Talja and Roos 2001). The yeasts were maintained on YM agar slants (5.0 g The catabolism of arabitol by Escherichia coli involves the peptone, 10.0 g glucose, 3.0 g yeast extract, 3.0 g of malt formation of arabitol phosphate which induces the synthesis extract, and 20.0 g of agar in 1 L deionized water). Cultures of compounds that inhibit bacterial metabolism (Scangos and were grown for inoculation in YM broth (without agar). Reiner 1979). This property makes it possible to use arabitol The medium used in the screening study and in later studies as a sweetener that reduces dental caries. Also, the caloric on cell growth and arabitol production by Debaryomyces value of arabitol is 0.2 kcal/g, whereas it is 2.4 kcal/g for hansenii SBP-1 (NRRL Y-7483) had the following compo- xylitol (McCormick and Touster 1961;Hucketal.2004; sition (per liter of solution): yeast extract, 3 g; (NH4)2SO4, Mitchell 2006;Crick1961). It is highly possible that arabitol 2g;K2HPO4, 2.4 g; KH2PO4, 1.6 g; MgSO4⋅7H2O, 1 g; can be used in many of the known applications of xylitol, as and glycerol, 100 g (unless specified otherwise). The a natural sweetener, a dental caries reducer, and a sugar glycerol used in this study was a crude glycerol from a substitute for diabetic patients (Gare 2003). If desirable, biodiesel plant without a glycerol refinery (provided by arabitol can also be converted to xylitol, for example, by Biodiesel Systems, Madison, WI, USA). The crude glycerol using Glucanobacter oxydans (Suzuki et al. 2002). This had 88% glycerol. To make 100 g/L glycerol in the culture bacterium was capable of oxidizing D-arabitol to D-xylulose medium, 113 g/L of the crude glycerol was added. The using the membrane-bound D-arabitol dehydrogenase and medium had an initial pH of 6.6–6.8. Glycerol (and other then converting D-xylulose to D-xylitol using the membrane- carbon sources used in some studies, i.e., glucose, xylose, bound D-xylitol dehydrogenase. Xylitol yield of around 25% and sorbitol) was autoclaved separately from other medium has been reported (Sugiyama et al. 2003). components. Arabitol is known to be produced by osmophilic yeast species such as Debaryomyces (Nobre and Costa 1985), Candida (Bernard et al. 1981), Pichia (Bisping et al. 1996), Wickerhamomyces (Hansenula; Van Eck et al. 1989), and Table 1 Genera and number of strains screened Saccharomycopsis (Endomycopsis;Hajny1964). When Genera # of strains exposed to osmotic stress, the yeast accumulates compat- ible solutes such as arabitol, glycerol, xylitol, erythritol, and Debaryomyces 67 mannitol to balance the osmotic pressure across the cell Geotrichum 41 membrane. An objective of this study was to select the Metschnikowia 37 yeast strains that produce large amounts of arabitol with Candida 24 high yields and minimal other polyols (for easier down- Dipodascus 14 stream separation) using glycerol as substrate. Experiments Pichia 5 characterized the most promising strains for growth and Trigonopsis 4 arabitol production under different culture conditions. Galactomyces 4 Zygosaccharomyces 2 Citeromyces 1 Materials and methods Saccharomycopsis 1 Hyphopichia 1 Yeast strain screening Wickerhamia 1 Lachancea 1 Extensive culture screening of 214 strains from 25 genera Torulaspora 1 was conducted for arabitol production from glycerol. The Naumovozyma 1 following five genera contained the largest numbers of Kodamaea 1 strains screened: Debaryomyces, Geotrichum, Metschniko- Sugiyamaella 1 wia, Candida, and Dipodascus. A complete list of the Hanseniaspora 1 genera and the numbers of screened strains from each genus Cephaloascus 1 is given in Table 1. All of these strains were obtained from Botryozyma 1 the Agricultural Research Service Culture Collection Trichomonascus 1 (NRRL) at National Center for Agricultural Utilization Sporopachydermia 1 Research, United States Department of Agriculture, Peoria, Endomyces 1 IL, USA. NRRL accession numbers for the species Schizoblastosporion 1 considered is shown in Table 2. Appl Microbiol Biotechnol (2011) 90:257–267 259
Table 2 Strains producing at least 5 g/L of total polyols, listed in the reaction time, the flasks were removed from the incubator/ alphabetical order shaker and processed for analysis of glycerol and polyol Species NRRL # SBP # Total polyol (g/L) product concentrations.
Candida quercitrusa Y-5392 118 6 Culture conditions for D. hansenii SBP-1 (NRRL Y-7483) Debaryomyces hansenii Y-7483 1 10 D. hansenii Y-1015 2 11 Inoculum was prepared by transferring a loop of cells from D. hansenii Y-10452 3 9 an agar plate to 50 mL YM broth in a 250 mL flask covered D. hansenii Y-1448 5 5 with cheese cloth. The culture was grown at room D. hansenii Y-7426 7 5 temperature (22±1°C) for 24 h under vigorous magnetic D. hansenii Y-1454 8 5 stirring; 2.5 mL of the inoculum thus prepared was added to D. hansenii Y-10150 15 5 each flask in the subsequent study of culture conditions, D. coudertii Y-5984 33 5 which were made with a 50-mL medium volume in 250 mL Galactomyces reesei Y-17566 167 8 flasks shaken at 200 rpm. The temperature used in these Geotrichum candidum Y-552 12 14 studies was 30°C except in the study of temperature effects. G. candidum Y-714 181 8 Multiple samples were taken during cultivation to establish G. candidum Y-1282 182 5 the profiles of cell growth, substrate consumption, and G. candidum Y-17010 188 9 product formation. G. candidum Y-2071 189 15 G. cucujoidarum Y-27731 194 19 Effect of medium volume in shaker flasks G. cucujoidarum Y-27732 219 13 G. fermentans Y-17567 169 10 Shake flasks are not very suitable for studying the effects G. fragrans Y-17571 177 7 of dissolved oxygen concentrations (DO) on cell growth G. histeridarum Y-27729 195 10 and product formation. Nonetheless, to obtain an indica- G. klebahnii Y-12820 197 9 tion of culture sensitivity to low DO or anaerobic G. silvicola Y-27641 199 6 conditions, a study was done with D. hansenii SBP-1 in Geotrichum sp. Y-7525 201 8 250 mL shake flasks containing the following different Geotrichum sp. Y-5422 204 9 medium volumes: 30, 50, 75, 100, and 150 mL. Under the Geotrichum sp. YB-3552 210 7 same shake speed (200 rpm), the flasks with smaller Geotrichum sp. YB-376 211 9 volumes were expected to have better oxygen transfer Geotrichum sp. YB-503 215 6 efficiency via surface aeration, resulting in higher broth Geotrichum sp. YB-5154 216 8 DO for the cultures of similar cell concentrations reached G. suaveolens Y-6852 217 8 in the N-limited culture medium. G. vulgare Y-27915 218 8 Analytical methods Metschnikowia zobellii Y-5387 14 5
Cell concentration
Culture methods Cell concentrations were mostly determined from the intracellular protein concentrations measured using the Screening Bradford protein assay kit II (Bio-Rad Laboratories, Hercules, CA, USA). A 5-mL broth sample was centrifuged Three milliliters of YM broth in a sterile screw top test tube at 8,000 rpm for 10 min (Sorvall RC 5c, DuPont, was inoculated with yeast cells from a YM slant. The tube Wilmington, DE, USA). The supernatant was collected was then placed in an Innova 4430 incubator/shaker (New and frozen for future analyses of substrate and product Brunswick Scientific) set at 25°C and 200 rpm for 24 h. (arabitol) concentrations. The cell pellet was washed twice Next, 100 μL of YM/yeast culture was pipetted into 10 mL with deionized water and then lysed by addition of 5 mL of of the screening medium in a 50-mL flask. The flask was 0.2 N NaOH and heating at 100°C for 20 min. The protein then placed in an Innova 4430 or an Innova 4335 incubator concentration of the lysate was measured according to the shaker set at 30°C and 200 rpm for 72 h. Initial screening Bradford assay, with the absorbance at 595 nm measured was completed with a single replicate of each yeast strain using a UV/VIS spectrophotometer (Model UV-1601, tested. The results from these experiments were later Shimadzu Corporation, Columbia, MD, USA). The rela- confirmed by re-screening in duplicate. After 72 h of tionship between the intracellular protein concentration and 260 Appl Microbiol Biotechnol (2011) 90:257–267 the cell dry-weight concentration was established with the at a flow rate of 1.0 mL/min. The sorbitol peak appeared at samples taken during the exponential growth phase of two a retention time of about 16.3 min. repeated batch fermentation experiments. The following With retention times of 23.3 and 23.5 min, mannitol and relationship was obtained (R2=0.92): arabitol could not be reliably separated by the HPLC method used in the screening study. When a broader (or double/ ðÞ= Cell dry weight concentration g L shouldering) mannitol/arabitol HPLC peak was detected, a ¼ Intracellular protein concentrationðÞ g=L 12:42: GC analysis was done to determine the ratio of mannitol and arabitol. This ratio was then used to quantify the two polyols from the peak in the HPLC analysis. For GC analysis, the Substrate and product concentrations samples (50 μL) contained in test tubes were first dried in a heat block (Thermolyne type 17600 Dri-Bath) at 60°C under For culture samples taken during the screening study, air using a Pierce model 18780 Reacti-Vap evaporating unit. 10 g/L ribitol or rhamnose was added as internal standard. Then, 1 mL of 45 g/L hydroxylamine hydrochloride in The samples were then centrifuged at 12,000 rpm, 10°C pyridine was added to each sample, vortexed, and then placed for 15 min using a Beckman Coulter model J2-21 in the heat block for 10 min. The samples were vortexed again centrifuge to collect the supernatant. The supernatant and placed back into the heat block for another 10 min. Next, was concentrated about twofold using a speed vacuum the samples were removed from the heat block and cooled (Thermo Electron Corporation, Waltham, MA, USA) set at under cold running water to near room temperature. Then, 65°C. After being passed through a pre-swollen DE52 1 mL of acetic anhydride was added to each test tube and the (diethylaminoethyl cellulose, Whatman) anionic exchange above vortexing and 10-min heating in the heat block were resin in Bio-Rad mini-columns, the sample was filtered repeated twice. After being cooled again under cold running and analyzed by high-performance liquid chromatography water to near room temperature, the samples were completely (HPLC; Shimadzu) with a Waters 410 differential refrac- dried under air. Following this treatment, each test tube tive index detector. Used at a flow rate of 0.6 mL/min, the received 3 mL of water and 2 mL of ethyl acetate, was mobile phase was 100% water generated by filtering vortexed thoroughly, and then left standing for phase deionized water through an ELGA PURELAB Ultra separation. The top layer (ethyl acetate phase) was collected system. The main column used was a Rezex RCM- into a 1-dram vial. Another 2 mL of ethyl acetate was added to Monosaccharide column (300×7.8 mm, 8 μm, Phenom- each test tube, and the above extraction procedure was enex, Torrance, CA, USA) maintained at 85–90°C by a repeated. The ethyl acetate extract was again collected and column heater. Three guard columns were used in series: a combined with the previous extract in the 1-dram vial. The Phenomenex Security Guard with Carbo-Ca (4×3 mm) extract in the vial was completely dried under nitrogen using cartridge and two Phenomenex Rezex RCM (50×7.8 mm, an Organomation N-EVAP analytical evaporator and then 8 μm) guard columns. The resulting chromatograms were redissolved in 300 μL of ethyl acetate. After being transferred compared to the chromatograms of known standards and into a GC vial, the sample was analyzed on a Hewlett Packard calibration curves for identification and quantification of HP 6890 series GC with a Phenomenex Zebron ZB-5 column. the polyols present. The following were representative The resulting chromatograms were compared to chromato- HPLC retention times (in minutes) observed for the grams of known polyol standards mainly for determination of internal standards and relevant polyols: rhamnose—16.8, the ratios of mannitol and arabitol, which could not be ribitol—20, glycerol—21.6, mannitol—23.3, arabitol— separated by the HPLC method used. The following were 23.5, and xylitol—27.2. The typical standard errors in representative retention times (in minutes) observed in the GC the analyzed polyol concentrations were 1–7%. analysis: glycerol—6.8, ribitol (internal standard)—17.1, For samples taken in the later studies with D. hansenii arabitol—17.4, xylitol—17.8, and mannitol—22.1. SBP-1, the glycerol, glucose, xylose, and arabitol concen- trations were similarly measured by HPLC (Shimadzu) with a refractive index detector, using a different carbohydrate Results column (Supelco column H, 250×4.6 mm, with a guard column, 50×4.6 mm) maintained at ambient temperature. Screening for arabitol production from glycerol The mobile phase used was 0.1% H3PO4 at a flow rate of 0.17 mL/min. The representative retention times for Among the cultures screened (Table 1), the genera glucose, xylose, arabitol, and glycerol were 14.2, 15.3, Debaryomyces and Geotrichum had the largest numbers 17.2, and 20.7 min, respectively. Sorbitol concentration was of strains that produced noticeable amounts (≥5 g/L) of analyzed using a Supelcosil LC-NH2 column (250× polyols from glycerol, after 3 days of cultivation in the 4.6 mm) with 75:25 acetonitrile/water as the mobile phase shake flasks (Table 2). Debaryomyces and Metschnikowia Appl Microbiol Biotechnol (2011) 90:257–267 261 strains tended to produce predominantly arabitol whereas compared in Fig. 2. All of these strains showed maximal Geotrichum strains produced significant amounts of man- arabitol production at 30°C. D. hansenii (SBP-1) was found nitol, in addition to arabitol. Examples for the distribution particularly sensitive to higher temperature, giving negligi- of different polyols produced are compared in Table 3 for ble arabitol production at 35°C. Arabitol production by M. several strains. Selected strains from these genera, specif- zobellii (SBP-14) was, on the other hand, similar at 30°C ically D. hansenii (SBP-1, NRRL Y-7483), Geotrichum and 35°C. candidum (SBP-12, NRRL Y-552), Geotrichum cucujoida- rum (SBP-219, NRRL Y-27732), and Metschnikowia Effect of initial glycerol concentration zobellii (SBP-14, NRRL Y-5387), were examined further for the effects of some cultivation conditions. More Arabitol production is associated with osmophilic yeasts thorough studies were done with D. hansenii (SBP-1) (Blakley and Spencer 1962). The effects of glycerol and because the minimal amount of non-arabitol polyols salt concentrations, both of which can provide osmotic produced by this strain was expected to significantly pressure to the cells, are described in this and the next simplify the downstream arabitol purification process. sections, respectively. Shown in Fig. 3a are the cell concentrations of D. Effect of culture volume in shake flasks hansenii SBP-1 at 0, 72, and 120 h in the systems with 50, 90, 120, and 150 g/L of glycerol in the initial media. The The different medium volumes (30, 50, 75, 100, and cell concentrations were comparable, reaching 17–20 g/L, 150 mL) used in the systems studied were expected to presumably because all were limited by the same N-source cause different profiles (varying with time) of dissolved concentration in the media. Glycerol was not completely oxygen concentrations in the broth. DO profiles were, exhausted in any systems at 120 h (glycerol concentration however, difficult to follow in shake flask cultures. Instead, data not shown). The profiles of arabitol production in these the concentrations of D. hansenii SBP-1 cells, arabitol systems are shown in Fig. 3b. In the system with 50 g/L produced, and glycerol consumed were compared in Fig. 1 glycerol initially, the arabitol production essentially stopped to show the possible effects of DO. The cell concentrations after 72 h (when the remaining glycerol concentration were measured at 80 h because the preliminary study had dropped below 20 g/L). Arabitol production continued after shown that the cultures would typically have reached the 72 h in the systems with higher initial glycerol concen- stationary phase by 80 h. Arabitol and glycerol concen- trations. The concentrations of arabitol produced and trations were measured at 120 h, to allow ample time for glycerol consumed at 120 h are summarized in Fig. 3c. arabitol production. The systems with 30, 50, and 75 mL The arabitol production in the three systems with high medium were found to have comparable results for all three initial glycerol concentrations (≥90 g/L) appeared to be concentrations (cells, arabitol, and glycerol), with p>0.05 comparable whereas the glycerol consumption decreased from one-way analysis of variance using Minitab (Minitab, with increasing initial glycerol concentrations. The resultant Inc., State College, PA, USA). The systems with 100 and arabitol yields from the consumed glycerol at 120 h were 150 mL medium reached lower cell and arabitol concen- shown in Fig. 3d. The arabitol yield increased with the trations and consumed less glycerol, presumably due to the increase in initial glycerol concentration, particularly from insufficient oxygen transfer in these larger-volume systems. 50 to 90 g/L. The arabitol yield reached about 50% in the More importantly, the yields of arabitol from consumed system with 150 g/L of initial glycerol. The findings glycerol remained about 20% (19–22%) in the three suggested that certain glycerol concentration (and/or its systems with lower volumes but decreased to 10% and associated osmotic pressure) was required for arabitol 5% as the volume increased to 100 and 150 mL, respec- synthesis by the osmophilic yeast. tively. The results indicated that the 50-mL volume used in the initial screening study was suitable. The same volume Effect of salt concentration was used in all the subsequent shake flask studies. The results also suggested that very low or zero DO, The above results also indicated that certain concentrations corresponding to the systems of larger medium volumes, of glycerol would remain unconsumed when the arabitol was not good for arabitol production. production by D. hansenii SBP-1 became very slow or stopped. The remaining glycerol would complicate the Effect of temperature downstream collection and purification of arabitol. It was thought that salt (NaCl) might offer the necessary osmotic The concentrations of arabitol produced at different temper- stress for complete conversion of glycerol to arabitol. D. atures by D. hansenii (SBP-1), G. candidum (SBP-12), and hansenii was reported to tolerate high salt concentrations, M. zobellii (SBP-14), after 3 days of cultivation, were up to 4 M NaCl (Larsson et al. 1990). 262 Appl Microbiol Biotechnol (2011) 90:257–267
Table 3 Percentages of different polyols produced by some osmotolerant yeast strains
Species SBP # NRRL # Total polyol (g/L) Polyol distribution (%)
Arabitol Xylitol Mannitol Ribitol
Debaryomyces hansenii 1 Y-7483 10 97.8 1.6 ND 0.6 D. hansenii 2 Y-1015 11 97.4 2.6 ND ND Geotrichum candidum 12 Y-552 14 65.3 1.0 33.7 ND G. cucujoidarum 194 Y-27731 19 59.0 0.8 39.4 0.8 G. cucujoidarum 219 Y-27732 13 71.7 0.8 25.9 1.6 Metschnikowia zobellii 14 Y-5387 5 94.9 ND ND 5.1
ND not detectable
The study was made in media containing 100 g/L of Effects of NaCl addition (25, 50, and 100 g/L) on glycerol and 0, 50, 100, and 150 g/L of NaCl, respectively. arabitol production were also evaluated with other strains, The cell growth was not affected by addition of 50 and including D. hansenii SBP-2 and SBP-5, G. candidum 100 g/L NaCl but was slowed significantly in the system SBP-12, and G. cucujoidarum SBP-194 and SBP-219. Salt with 150 g/L NaCl (Fig. 4). Arabitol production was more addition was found to have similar negative effects on sensitive to the salt addition (Fig. 4). Presence of even 50 g/ arabitol production by these strains (data not shown). L NaCl caused significantly poorer arabitol production. The system with 150 g/L NaCl produced less than 1 g/L of Effects of addition of other carbon substrates arabitol. To separate the effect of NaCl addition on arabitol Since salt addition could not be used to promote more production from that on cell growth, a subsequent study complete conversion of glycerol to arabitol, the effects of was made with the salt being added after 2 days of cell addition of a second carbon source (glucose, xylose, or growth in the medium with 100 g/L glycerol. Three sorbitol), along with glycerol, on arabitol production by D. systems, with 0 (control), 100, and 150 g/L NaCl, hansenii SBP-1 were investigated. The study was made in respectively, were included for comparison. Delaying the four systems. The culture was first grown in the medium salt addition successfully minimized the negative effect on with an initial glycerol concentration of 30 g/L. After 74 h cell growth (data not shown). Arabitol production was, (when the cultures were in the early stationary phase), 30 g/ however, completely stopped after the salt addition (Fig. 5). L glucose, xylose, or sorbitol plus 50 g/L glycerol were It is therefore concluded that high salt concentrations have added to three of the systems, and 80 g/L glycerol was negative effects on arabitol production by D. hansenii SBP- added to the fourth (control) system. All of the systems 1. It is infeasible to use salt addition to apply osmotic reached similar maximum cell concentrations (about 16 g/ pressure for complete conversion of glycerol to arabitol. L, data not shown). The resultant arabitol concentration profiles are shown in Fig. 6a. Before the addition of more carbon substrates at 74 h, all of the systems produced about 2 g/L arabitol. The subsequent addition of 80 g/L glycerol (in the control system) did not lead to much more arabitol production (Fig. 6a). Such a two-step addition of glycerol (30 and then 80 g/L) appeared to be less favorable for arabitol production, when compared to the addition of all the glycerol in the initial medium (see the arabitol profiles for the systems with 90 and 120 g/L of initial glycerol concentrations in Fig. 3b and the profiles for the control systems in Figs. 4 and 5). The addition of sorbitol along with glycerol also did not give good arabitol production (Fig. 6a). On the other hand, additions of glucose and xylose significantly improved the arabitol production. Fig. 1 Concentrations of arabitol produced, glycerol consumed, and Concentrations of the potential second C-source (glucose, cells grown in the systems with different initial culture volumes. Arabitol and glycerol concentrations were measured at 120 h; cell xylose, or sorbitol) and glycerol consumed after the concentrations were measured at 80 h addition in the stationary phase (during 74–145 h) are Appl Microbiol Biotechnol (2011) 90:257–267 263
Fig. 2 Arabitol produced by selected strains of Debaryomy- ces, Geotrichum, and Metschni- kowia at different temperatures. Samples were taken after 3 days of cultivation
summarized in Fig. 6b. More glycerol was consumed in the Discussion control system (added with only glycerol) than in the other three systems. Little sorbitol was consumed. The lower The screening study conducted in this work showed that the glycerol consumption in this system might be caused by the species from different genera produced different polyols or lower added glycerol concentration (50 g/L, as compared to polyol mixtures from glycerol. Using glucose as the 80 g/L in the control) or by the inhibition of sorbitol. On substrate, others have reported the production of various the other hand, glucose and xylose were simultaneously or polyols such as erythritol, xylitol, and mannitol by different preferentially consumed by the yeast. It should also be species (Nozaki et al. 2003; Groleau et al. 1995; Saha et al. noted that arabitol remained the only major metabolite 2007; Spencer 1968). D. hansenii SBP-1 was chosen for detected in all of the systems. Addition of these other more detailed evaluations in this work because it produced carbon substrates did not shift the culture metabolism to arabitol as the predominant polyol and with high concen- synthesize other major metabolites. trations (>10 g/L in shake flasks).
Fig. 3 Effects of different initial glycerol concentrations on D. hansenii SBP-1 fermentation: a cell growth profiles, b arabitol production profiles, c concen- trations of glycerol consumed and arabitol produced at 120 h, and d arabitol yield (from con- sumed glycerol) at 120 h 264 Appl Microbiol Biotechnol (2011) 90:257–267
Fig. 4 Effects of different salt concentrations on D. hansenii SBP-1 fermentation with 100 g/L of initial glycerol concentration: cell Fig. 6 Effects of addition of 30 g/L glucose, xylose, or sorbitol as a growth and arabitol production profiles potential second carbon source, along with 50 g/L glycerol, on arabitol production by stationary-phase D. hansenii SBP-1. Cells were grown for 74 h in media containing 30 g/L glycerol before the second C- D. hansenii is known to tolerate high concentrations of source and glycerol were added. The control system was added with 80 g/L glycerol. Arabitol production profiles are compared in a. sugar and salt (Tokuoka 1993). It was reported to tolerate Concentrations of the second C-source and glycerol consumed by the salt stress up to 240 g/L although the cell growth rate stationary-phase cultures (during 74–145 h) are shown in b. Although decreased at NaCl concentrations above 160 g/L (Norkrans not shown, the standard deviations of the consumed concentrations in 1966). In the current study, the growth of D. hansenii SBP- b were in the range of 13–22% 1 was observed not to be affected by the presence of 100 g/ L NaCl, but the growth rate decreased significantly at Zygosaccharomyces rouxii was found to produce glyc- 150 g/L NaCl. erol and arabitol in glucose-based media (Onishi and Shiromaru 1984). The glycerol and arabitol distribution in the polyol mixture produced depended on the salt and glucose concentrations used. Less arabitol (10 g/L) than glycerol (29 g/L) was produced when the initial medium contained 180 g/L NaCl and 100 g/L glucose. On the other hand, the concentrations of arabitol and glycerol produced became similar (about 32 g/L) when the medium had a higher glucose concentration (300 g/L) and a low NaCl concentration (0.1 g/L). According to the study results, high osmotic pressures (generated by high salt and/or sugar concentrations) induced this yeast to produce glycerol while high sugar and low salt concentrations were more favorable for arabitol production. These findings were largely Fig. 5 Effects of salt addition on arabitol production by D. hansenii SBP-1 in media with 100 g/L of initial glycerol concentration. The salt consistent with the results of the current study. In the was added after 2 days of growth current study, high initial glycerol concentrations (≥90 g/L) Appl Microbiol Biotechnol (2011) 90:257–267 265 and no or low NaCl concentrations (≪50 g/L) were found to and erythritol (Bernard et al. 1981). With glucose as the be favorable for arabitol production and yield. High salt substrate, two routes have been reported for Z. rouxii (Saha concentrations, on the other hand, tended to inhibit yeast et al. 2007; Ingram and Wood 1965; Blakley and Spencer growth and, particularly, arabitol production. Cell growth 1962): Glucose is converted to ribulose 5-phosphate, which was affected at salt concentrations higher than 100 g/L; is then converted either to ribulose by ribulokinase or to arabitol production was inhibited even at 50 g/L NaCl. xylulose 5-phosphate by ribulose 5-phosphate epimerase. In another study with a different strain of D. hansenii, Ribulose is reduced to arabitol by an NADPH-dependent the intra- and extracellular contents of polyols, i.e., glycerol arabitol dehydrogenase. Xylulose 5-phosphate is dephos- and arabitol, were measured in response to increasing phorylated to xylulose by xylulokinase and then salinity in a medium containing only 5 g/L glucose (Adler reduced to arabitol by an NADH-dependent arabitol and Gustafsson 1980). It was found that in the medium dehydrogenase. containing 181 g/L NaCl, the yeast accumulated intracellu- Arabitol synthesis from xylose also may follow two lar glycerol and released extracellular glycerol (without possible ways (Fig. 7), as reported in the studies with Z. production of either intra- or extracellular arabitol) while rouxii and Aerobacter aerogenes (Wilson and Mortlock glucose was available; after glucose was depleted, both 1973; Saha et al. 2007). In the first route, xylose is reduced intra- and extracellular glycerols were consumed and the to xylitol and then to xylulose. In the second route, xylose intracellular arabitol content increased (still without pro- is directly converted to xylulose by xylose isomerase. The duction of extracellular arabitol). While high salt concen- xylulose formed from either route is then reduced to trations were again found to cause glycerol production from arabitol by arabitol dehydrogenase. sugar, the sugar concentration was too low (5 g/L) in this Arabitol syntheses from sorbitol and glycerol, if occur- study to allow production of extracellular arabitol. ring, are expected to follow similar routes as the synthesis Arabitol production was found to be improved by from glucose after they are converted to glucose-6- addition of glucose and xylose (but not sorbitol). Arabitol phosphate (Fig. 7). Sorbitol is first converted to fructose- is synthesized via the pentose phosphate pathways (Saha et 6-phosphate via fructose or sorbitol-6-phosphate. Fructose- al. 2007). The possible routes are summarized in Fig. 7. 6-phosphate is then converted to glucose-6-phosphate. As Ribulose-5-phosphate is considered as an important for glycerol, the metabolic pathway in yeasts like Candida precursor for production of polyols like arabitol, xylitol, utilis and Saccharomyces cerevisiae is initiated by glycerol
Sorbitol Glucose
Hexokinase Sorbitol dehydrogenase Glucose-6-phosphate PPP Ribulose-5-phosphate Sorbitol-6-P Fructose Hexokinase Fructose-6-phosphate Sorbitol-6-phosphate Xylulose-5-phosphate Ribulose dehydrogenase Arabitol Fructose 1,6 bis- dehydrogenase phosphate DHAP Arabitol dehydrogenase Xylulose dehydrogenase Arabitol DHAP Glyceraldehyde -3- phosphate Glycerrol-3-phosphate Xylitol Xyloseisomerase dehydrogenase dehydrogenase Glycerol 3-phosphate Xylitol Xylose Xylose reductase Glycerol kinase % identity of nucleotide sequence in SBP-1 Glycerol TCA cycle 76% 80% 92% 96% 0%
Fig. 7 Possible pathways for the conversion of various substrates to arabitol (DHAP: Dihydroxyacetone phosphate, PPP: Pentose phosphate pathway) 266 Appl Microbiol Biotechnol (2011) 90:257–267 kinase and a mitochondrial sn-glycerol 3-phosphate dehy- strains produced mannitol along with arabitol. The optimal drogenase (Gancedo et al. 1968). An alternative pathway in temperature for arabitol production by D. hansenii SBP-1 yeasts lacking glycerol kinase is indicated by the presence of was 30°C. Very low DO or anaerobic conditions inhibited NAD-dependent glycerol dehydrogenase and dihydroxyace- arabitol production. High initial glycerol concentrations tone kinase (Babel and Hofmann 1982). Dihydroxyacetone improved the yield of arabitol, reaching approximately phosphate, formed in the above routes, is converted to 50% (w/w) at about 150 g/L initial glycerol. Though the glyceraldehyde-3-phosphate and, subsequently via gluconeo- growth of D. hansenii SBP-1 was not noticeably affected genesis pathway, to glucose-6-phosphate. C. utilis has been in media with up to 100 g/L NaCl, arabitol production by reported to utilize glycerol faster than S. cerevisiae (Gancedo the yeast was negatively affected by high salt concen- et al. 1968). There seem to be no reports on the uptake trations. Presence of glucose and xylose, in addition to transport system of glycerol in C. utilis,althoughglycerol glycerol, in the medium improved the arabitol production. transport by simple diffusion has been described for S. Sorbitol, on the other hand, was not a good substrate for cerevisiae (Lages and Lucas 1997). the yeast and replacing a portion of glycerol with sorbitol The nucleotide sequences for the relevant enzymes led to poorer arabitol production. A study to further reported have been searched and compared with the D. improve the arabitol production is being conducted, by hansenii genome (NC_006048) using the National Center optimizing the operating parameters such as pH, dissolved for Biotechnology Information’s Basic Local Alignment oxygen concentration, choice of limiting nutrient, and Search Tool. The matching percentages are indicated in medium composition. Fig. 7 by different arrow styles. This yeast’s ability to convert sorbitol to fructose-6-phosphate is noticeably less Acknowledgment The work was supported by a grant from the certain, potentially responsible for the insignificant sorbitol United Soybean Board (Projects 7435, 8435, and 9435). We thank Karen Ray for technical advice. utilization observed in this study (Fig. 6b). As mentioned in the “Introduction”, xylitol (but not arabitol) has many known or existent commercial appli- References cations. It may therefore be desirable to produce xylitol from arabitol. Xylitol is currently produced by chemical Adler L, Gustafsson L (1980) Polyhydric alcohol production and reduction of xylose derived from wood hydrolysate intracellular amino acid pool in relation to halotolerance of (Melaja and Hamalainen 1977). The chemical process the yeast Debaryomyces hansenii. Arch Microbiol 124(2):123– uses an expensive catalyst at high pressure (50 atm) and 130 temperature (80–140°C). Xylitol production from xylose Babel W, Hofmann KH (1982) The relation between the assimilation of methanol and glycerol in yeasts. Arch Microbiol 132(2):179– by biological processes has also been explored (Buhner 184 and Agblevor 2004; Kastner et al. 2003; Kim et al. 2002; Bernard EM, Christiansen KJ, Tsang SF, Kiehn TE, Armstrong D Leathers and Dien 2000). Yeasts can convert xylose to (1981) Rate of arabinitol production by pathogenic yeast species. xylitol using NAD(P)H-coupled xylose reductase. Unfor- J Clin Microbiol 14(2):189–194 Bisping B, Baumann U, Simmering R (1996) Effect of immobilization tunately, the xylitol produced tends to be oxidized to on polyol production by Pichia farinosa. Prog Biotechnol + xylulose by NAD -coupled xylitol dehydrogenase. Good 11:395–401 xylitol yields from such a process require tightly con- Blakley ER, Spencer JF (1962) Studies on the formation of D- trolled, high intracellular NAD(P)H/NAD+ ratios. This arabitol by osmophilic yeasts. Can J Biochem Physiol 40:1737– 1748 control is not an easy task in large-scale industrial Bournay L, Casanave D, Delfort B, Hillion G, Chodorge JA (2005) operations where the environment (particularly the dis- New heterogeneous process for biodiesel production: a way to solved oxygen concentrations) inside the large bioreactors improve the quality and the value of the crude glycerin produced is non-homogeneous. Furthermore, both of the above by biodiesel plants. Cat Today 106(1–4):190–192 Buhner J, Agblevor FA (2004) Effect of detoxification of dilute-acid chemical and biological processes require expensive corn fiber hydrolysate on xylitol production. Appl Biochem separation of xylose from the complex sugar mixtures in Biotechnol 119(1):13–30 the biomass hydrolysate. The alternative approach of Crick R (1961) Improvements in or relating to sweetening agents for producing arabitol and then converting the arabitol to food. vol. US patent 884,961 Dasari MA, Kiatsimkul PP, Sutterlin WR, Suppes GJ (2005) Low- xylitol has the potential of being more economically pressure hydrogenolysis of glycerol to propylene glycol. Appl attractive. Catal A Gen 281(1–2):225–231 In summary, strains belonging to the genera Debaryo- Demirbas A (2003) Biodiesel fuels from vegetable oils via catalytic myces and Geotrichum produced total polyol concentra- and non-catalytic supercritical alcohol transesterifications and – ≥ other methods: a survey. Energy Convers Manag 44(13):2093 tions 5 g/L after 3 days of cultivation among the cultures 2109 screened. However, while Debaryomyces strains were able Gancedo C, Gancendo JM, Sols A (1968) Pathways of utilization and to produce arabitol as the main metabolite, Geotrichum production. Eur J Biochem 5(2):165–172 Appl Microbiol Biotechnol (2011) 90:257–267 267
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