Appl Microbiol Biotechnol (2005) 69: 341349 DOI 10.

1007/s00253-005-1987-1

APPLIED MICROBIAL AND CELL PHY SIOLO GY

H. Djelal . A. Amrane . F. Lahrer . G. Martin

Effect of medium osmolarity on the bioproduction of glycerol and ethanol by Hansenula anomala growing on glucose and ammonium
Received: 7 December 2004 / Revised: 23 March 2005 / Accepted: 31 March 2005 / Published online: 19 April 2005 # Springer-Verlag 2005

Abstract The osmotolerant yeast Hansenula anomala survives in media at low water activity resulting from increasing NaCl concentrations in the culture medium by producing compatible solutes. High salinity resulted in the use of a large part of the assimilated carbon substrate (glucose) for cell maintenance (28%), required for intracellular synthesis compounds and for osmotic cell regulation. The maintenance coefficient for non-growth-associated glucose consumption was found to be 0.38 mmol glucose g biomass1 h1. For decreasing water activity, there is a competition between the pathways leading to glycerol and ethanol production, until an experimental ethanol/total glycerol ratio reached a value 3.4 for 2 mol l1 NaCl (close to the theoretical value of 4)illustrating the osmodependent channelling of carbon towards polyols production. This competition leads to a cessation of ethanol production during the stationary state before that of glycerol. Since osmotic adjustment occurred mainly during growth, glycerol production during stationary state can be clearly related to another mechanism other than osmotic: it was excreted by a fermentative mechanism to ensure energy for cell maintenance.

Introduction
Polyols have numerous industrial applications, particularly in pharmaceutical, food and cosmetics industries (Wang et al. 2001). Natural polyols can be found in plants, e.g. in Rosacae fruits. However, due to low concentrations their extraction remains difficult. Amongst the available polyols, glycerol is the most attractive for industrial use. Glycerol is a by-product in fat and oil industries and can be chemically obtained from propylene via various methods (Taherzadeh
H. Djelal . A. Amrane (*) . F. Lahrer . G. Martin LARCIP (Universit de Rennes 1ENSCR) ENSCR, Avenue du Gnral Leclerc, 35700 Rennes, France e-mail: abdeltif.amrane@univ-rennes1.fr Tel.: +33-22-3235755 Fax: +33-22-3238120

et al. 2002). However, micro-organisms originating from natural and renewable resources can also be used for polyol production (Elseviers and Roper 1997; Duflot et al. 2000). In 1918, it was shown (Neuberg and Reinfurth 1918) that adding sodium sulphite in the culture medium increased glycerol production by Saccharomyces cerevisiae. Indeed, under these conditions, the normal pathway for NADH reoxydation is not available, because acetaldehyde is unable to accept hydrogen. NAD+ is regenerated through the reduction of triose phosphate into glycerophosphate. However, the high amounts of sulphite added and ethanol produced inhibited growth. In 1943, the production of glycerol without sulphite adjunction was shown in a culture of Zygosaccharomyces acidifacens in the presence of 20% glucose (Nickerson 1943; Blomberg 2000; Taherzadeh et al. 2002). Methylotrophic yeasts were also shown to produce polyols. Hansenula ofunaensis produces glycerol from methanol as a carbon source (Suryadi et al. 2000). Candida boidinii produces sorbitol when grown on multiple substrates, methanolglucose or methanolfructose (Tani and Vongsuvanlert 1987). H. polymorpha only produces arabitol when grown on glucose (Escalante et al. 1990). Filamentous microorganisms can also produce polyols (Lewis and Smith 1967). Extremophile species to low water activity, such as Xeromyces bisporus, accumulate similar amounts of glycerol as xerotolerant yeasts (Brown 1978). The reaction of some Aspergillus strains to salinity was shown to be controlled by osmotic pressure, without effect of the ions present in the medium (Rahmani 1989). Glycerol, the polyol mainly produced by stressed cells, was excreted into the extracellular medium. Glycerol production by microalgae from the genus Dunaliella was also studied (Thakur et al. 2000). Microalgae accumulate glycerol and -carotene to counterbalance the osmotic pressure due to the hypersalinity of the surrounding medium (Phadwal and Singh 2003). Use of osmotolerant yeasts for polyol production, particularly glycerol, is always an attractive option (Patil et al. 2002; Liu et al. 2003). Reports on the adaptation of the marine yeast Debaromyces hansenii to osmotic pressure

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(Adler and Gustafsson, 1980; Nobre and Da Costa 1985) showed that this yeast produces glycerol when grown on a highly salted medium. Batch and discontinuous fermentation modes were compared (Vijaikishore and Karanth 1984), as well as free and immobilised cells of Pichia farinosa growing on media containing 20% glucose (Bisping et al. 1990). Glycerol is the main compatible solute in S. cerevisiae (Tokuoka et al. 1992; Nevoigt and Stahl 1997). Pichia sorbitophila is a halotolerant yeast capable of surviving extracellular NaCl concentrations of up to 4 mol l1 in a mineral medium when glucose or glycerol is the only carbon and energy source (Lages and Lucas 1995). In response to high osmotic pressures of the surrounding medium, Hansenula anomala was observed to grow and produce glycerol, arabitol and ethanol (Van Eck et al. 1989; Bellinger and Larher 1991). Glycerol production by a culture of H. anomala on melasse, an economically attractive carbon source, was also demonstrated (Parekh and Pandey 1985; Patil and Sastri 1996). Even if the molecular details of the metabolic adaptations of yeast cells to osmotic stress is rather well known (Blomberg 2000), there is a lack of information concerning the effect of nutrients on glycerol (and ethanol) production by osmotolerant yeast (Wang et al. 2001). It was therefore the aim of this work, carried out using H. anomala growing on glucose and ammonium, to shed more light on this particular topic. Particular attention was paid to the effect of NaCl concentration responsible for water activity decrease.

as carbon and nitrogen sources, as well as an EDTA mineral solution, derived from the Wikerham medium (Wickerham 1951) (mg l1): CaCl2, 6H2O, 150; FeSO4, 7H2O, 100; ZnSO4, 7H2O, 30; CuSO4, 5H2O, 0.79; H3BO3, 15; KI, 2; NaMoO4, 2H2O, 5; MnSO4, H2O, 32; CoCl2, 6H2O, 5.6; EDTA, 100, as well as KH2PO4 and MgSO4, 7H2O at the required concentrations. NaCl was used to achieve the required medium osmolarity. Media were sterilised by filtration on a 0.2-m membrane (Sartorius, Goettingen, Germany). KOH (1 mol l1) was used to adjust the pH to 5.5. Composition of the culture media is presented in Table 1. Culture conditions Two modes of culture were used: In 500-ml Erlenmeyer flasks, cultures were propagated on an orbital shaker (Unimax 2010; Heidolph, Kelheim, Germany). The working volume was 200 ml. A 2-l fermentor (Biolafitte SA, Saint Germain-en-Laye, France) was also used. pH (Mettler-Toledo, Wilmington, MA, USA) was maintained at 5.5 by automatic addition of 1 mol l1 KOH, and the dissolved oxygen was monitored via a pO2 probe (Mettler-Toledo). The working volume was 1,200 ml. Agitation speed was 1,000 rpm and aeration rate was 1.4 vvm.

Materials and methods

Microorganism The osmotolerant yeast H. anomala var. anomala (CBS 5759, Delft, Netherlands) was used. Stock cultures (glucose 20 g l1, peptone 20 g l1, yeast extract 10 g l1 and agar 20 g l1) were maintained on a gelified medium and were stored at 4C. Inoculum preparation Ten drops of a yeast suspension in KCl 150 mM was added to 25 ml of seed culture medium (g l1): glucose 20, peptone 20 and yeast extract 10, contained in a 250-ml Erlenmeyer flask. Cells were pre-cultivated at 28C and 180 rpm until they reached a turbidity value of 1 unit at 610 nm. After centrifugation (4C, 1,800 g and 5 mn), cells were harvested, resuspended in 25 ml KCl 150 mM and recentrifuged under similar conditions. The suspension obtained after cells were harvested and resuspended in 10 ml KCl 150 mmol l1 was used to inoculate culture media. Culture media Vitamins were not required by H. anomala for its growth (Burkholder et al. 1944). A synthetic medium was used for medium optimisation; it contained glucose and ammonium

Irrespective of the mode of culture, 2 ml seed culture per litre of culture medium was used as an inoculum, and the temperature was maintained at 28C, the optimal value for H. anomala growth (Burkholder et al. 1944). Growth was indirectly monitored via off-line turbidimetric measurements (Spectronic 20, Spectronic Analytical Instruments, Leeds, UK) at 610 nm, after calibration by dry cellular weight measurement. Analysis Glucose and ammonium were colorimetrically determined by the Somogyi (Somogyi 1945) and the Mann (Mann 1963)
Table 1 Culture media composition used (also containing the EDTA mineral solution given in Materials and methods) KH2PO4 (mmol l1) NaCl MgSO4, NH4Cl Glucose 7H2O (mol l1) (mol l1) (mmol l1) (mmol l1) 5 4 4 4 4 5 00.51 10 1.52 012 3.3 0 2 2 2 01.53 1530 3.310 10 10 0.56 0.28 0.28 0.28 0.56 0.56 Figures

38 3.7 3.7 3.7 3.7 38

Fig. 1 Figs. 2 and 3 Fig. 4 Fig. 5 Fig. 5 Fig. 6

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methods, respectively. Glycerol was enzymatically determined (Wieland 1963). Metabolite identification and determination were carried out by HPLC involving an ion exclusion column HPX-87H (3007.8 mm, Bio-Rad, Hercules, CA, USA), maintained at 45C (Oven Croco-Cil; Cluzeau-Info-Labo, Ste Foy La Grande, France). Elution was performed at a flow rate of 0.6 ml mn1 (Waters Pump, Milford, MA, USA) using 0.01 N sulphuric acid. An ERC 7512 refractometer (Saitama, Japan) was used for the detection of the different compounds. The standard solution contained glucose, glycerol, ethanol, arabitol and acetic acid, at 5 mmol l1 each.

Results
Both intra- and extracellular glycerol concentrations increased when medium salinity was increased in the range of 02.0 mol l1 NaCl (Fig. 1). Conversely, ethanol production decreased when NaCl concentration was increased from 0.5 to 2.0 mol l1 in the culture medium (Fig. 1). Figure 2 highlights a decrease in glucose assimilation for increasing amounts of NaCl in the medium: glucose consumption was 20, 18 and 16% at the end of the exponential growth phase and its consumption continued during stationary state until 46, 30 and 32% of glucose was assimilated after 80 h of culture on media containing 0, 1.0 and 2.0 mol l1 NaCl, respectively. Irrespective of the NaCl concentration in the culture medium, the nitrogen source (NH4Cl) was assimilated within 20 h of culture (Fig. 2). For all experiments, the end of ammonium assimilation corresponded with the end of exponential growth (Fig. 2), clearly showing that the available nitrogen source was the limiting growth factor. High salinity did not favour growth. Indeed, the end of exponential growth, always observed for similar turbidity values (about 2 absorbance units), was recorded later for increasing NaCl concentrations in the culture medium, leading to a decrease in the rate of ammonium assimilation, from 0.22 to 0.15 mmol l1 h1 for 0 and 2.0 mol l1 NaCl, respectively (Fig. 2).
Fig. 1 Effect of initial NaCl additions on the accumulation (closed bars) and excretion (open bars) of glycerol, as well as ethanol production (dense grid lines), by H. anomala growing in shake cultures on 0.56 mol l1 glucose and 10 mmol l1 NH4Cl (Table 1)

During exponential growth, low amounts of extracellular glycerol were produced: less than 1.5 mmol l1 for 0 and 1.0 mol l1 NaCl in the culture medium and 2.6 mmol l1 for 2.0 mol l1 NaCl. In contrast, an increase in the rate of glycerol production was observed during the stationary state for all runs, until 5.7, 8.2 and 11.0 mmol l1 of glycerol were produced at the end of culture (80 h) on media containing 0, 1.0 and 2.0 mol l1 NaCl, respectively (Fig. 2). Time-courses of product (glycerol) on carbon substrate (glucose) yield YP/S are shown in Fig. 3. In the absence of NaCl in the culture medium, YP/S remained below 1.5% throughout culture. For 1.0 mol l1 NaCl supplementation, YP/S increased throughout culture; whereas, for 2.0 mol l1 NaCl supplementation, higher YP/S were always obtained. At a high salinity of the culture medium (2.0 mol l1 NaCl), the YP/S yield decreased during exponential growth (from 6 to 32 h of culture) due to intracellular glycerol accumulation, as illustrated in Fig. 6. Maximum YP/S yields, always recorded at the end of culture (80 h), increased from 1 to 6% when NaCl concentration was increased incrementally from 0 to 2.0 mol l1 NaCl (Fig. 3). Above 15 mmol l1, no effect on growth of a further increase in NH4Cl can be observed (Fig. 4). Therefore, two ammonium concentrations (3.3 and 10 mmol l1) in the range 015 mmol l1 were tested in fermentor (Table 1). The lowest ammonium concentration (3.3 mmol l1) corresponded to the lowest maximum biomass1.0 absorbance unit (AU) against 1.2 AU for 10 mmol l1 ammonium supplementation (Fig. 5a)and also to the lowest nitrogen and carbon substrate consumption rates. Indeed, ammonium became exhausted after 30 and 75 h of culture (Fig. 5b), and 67.5 and 98% of the available glucose was assimilated at the end of culture (150 h) for 3.3 and 10 mmol l1 ammonium supplementation, respectively (Fig. 5c). Glycerol was produced throughout culture; whereas, after 125 h culture, a decrease in ethanol concentration was recorded for both media. For the highest ammonium concentration (10 mmol l1), maximum glycerol and ethanol productions were two- and threefold the level recorded for 3.3 mmol l1 NH4Cl supplementation; and the ratio of the maximum concentrations of ethanol on glycerol were ap-

344 Fig. 2 Time-courses of logarithmic growth (a), ammonium (b) and glucose (c) consumption, as well as glycerol excretion (d) during shake cultures of H. anomala on medium (Table 1) containing 0 (), 1.0 () and 2.0 () mol l1 NaCl

Fig. 3 Time-courses of product (glycerol) on carbon substrate (glucose) yield YP/S for initial additions of 0 (), 1.0 () and 2.0 () mol l1 NaCl in the culture medium (Table 1) of H. anomala

Fig. 4 Time-courses of logarithmic growth during shake cultures of H. anomala growing on (Table 1) 0.28 mol l1 glucose and 0 (), 1.5 (), 3.0 (), 15.0 () and 30.0 () mmol l1 NH4Cl

345 Fig. 5 Time-courses of logarithmic growth (a), ammonium (b) and glucose (c) consumption as well as glycerol (d) and ethanol (e) excretion during fermentor cultures of H. anomala on media (Table 1) containing 0.28 mol l1 glucose and 3.3 () or 10 () mmol l1 NH4Cl, or containing 0.56 mol l1 glucose and 10 mmol l1 NH4Cl ()

proximately 5 and 10 for 3.3 and 10 mmol l1 NH4Cl, respectively (Fig. 5d and e). For 0.28 mol l1 glucose supplementation, 98% of the glucose was assimilated after 150 h of culture (Fig. 5c). According to Adler et al. (1985), after glucose exhaustion, the excreted glycerol can be subsequently assimilated by the yeast D. hansenii. To avoid this problem, in case of a similar behaviour for H. anomala, a culture fermentor on medium supplemented with 0.56 mol l1 glucose (Table 1) was carried out and compared to the culture mentioned above (0.28 mol l1 glucose). For 0.56 mol l1 glucose, after 40 h, exponential growth ceased, followed by a clear deceleration growth phase (Fig. 5a). Ammonium was completely exhausted after 50 h of culture (75 h for 0.28 mol l1 glucose, Fig. 5b), corresponding to the beginning of the stationary state (Fig. 5a). After nitrogen source exhaustion, the rate of glucose assimilation decreased but continued during the stationary state until 70% of the available glucose was assimilated at the end of culture (Fig. 5c).

Fig. 6 Time-course of intracellular glycerol production during shake culture of H. anomala growing on (Table 1) 0.56 mol l1 glucose, 10 mmol l1 NH4Cl and 2.0 M NaCl

346 Fig. 7 Carbon conversion yields of glucose into the various products (mass of carbon of the considered product on the mass of carbon from the assimilated glucose) resulting from the metabolic activity of H. anomala cultivated during 150 h on 2.0 mol l1 NaCl, 0.56 mol l1 glucose and 10 mmol l1 NH4Cl (Table 1)

The amount of glycerol excreted increased throughout culture until a final concentration of 80 mmol l1, which is twice of that produced for 0.28 mol l1 glucose in the medium (Fig. 5d). Ethanol was produced until about 75 h of culture before it remained constant, at a similar value to the maximum rate recorded for 0.28 mol l1 glucose (Fig. 5e). Intracellular glycerol accumulation was clearly related to growth (Fig. 5a), because the maximum rate was recorded after 50 h of culture of H. anomala for 0.56 mol l1 glucose in the medium (displayed as an example in Fig. 6). During the stationary state, the amount of intracellular glycerol decreased (Fig. 6). Assimilated glucose was converted into various products; the carbon conversion yields are shown as an example for the experiment carried out with 0.56 mol l1 glucose (10 mol l1 ammonium and 2.0 mol l1 NaCl) in the medium

(Fig. 7). To determine carbon yield, final concentrations of various components are needed: arabitol 34.3 mmol l1, acetic acid 25.8 mmol l1; glucose, ethanol and glycerol concentrations are given in Fig. 5. To determine the yield carbon from biomass on carbon from glucose, final biomass concentration and its carbon content are needed, which were 3.6 g l1 and 50%, respectively. It was also assumed that 5 mol CO2 was formed for 4 mol ethanol produced (Fig. 8).

Discussion
The high salinity of the culture medium did not favour carbon and nitrogen substrate consumption by H. anomala (Fig. 2), in sharp contrast to glycerol on glucose yield YP/S

Fig. 8 Competition between metabolic pathways for glycerol and ethanol productions

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(Fig. 3). Similar behaviour was also previously recorded for P. farinosa (Vijaikishore and Karanth 1984). A high salinity inhibits the mechanism of glucose transport in the yeast D. hansenii (Lindman 1981); and the use of increasing amount of carbon substrate used for cell maintenance accounts for the diminution of the specific growth rate of S. cerevisiae for high NaCl concentrations (Watson 1970). Increasing the ammonium concentration had a positive impact on biomass concentration, glucose consumption and both glycerol and ethanol productions; glycerol production rate was less affected by ammonium concentration than ethanol production rate (Fig. 5). Similar behaviour was previously reported for P. farinosa (Vijaikishore and Karanth 1984). It was assumed that the accumulated intracellular products were used for cell maintenance: energy for cell maintenance was used for intracellular synthesis compounds that were continuously degraded (turnover) and for osmotic cell regulation (Sinclair and Kristiansen 1987). As shown in Fig. 7, the main part of the assimilated glucose was used for ethanol and carbon dioxide productions (45.6%), which were preponderant compared to glycerol and arabitol productions (17.6%). Confirming the assumption cited above, a significant part of the assimilated glucose (28.3%0.11 mol l1) was used for cell maintenance. The salt tolerance of yeast is a function of its capacity to use energy to excrete Na+ and uptake K+ (Onishi 1990). The amount of glucose used by S. cerevisiae for proton transport at a pressure of 9.00 MPa was seven times higher than that used at 0.28 MPa (Brown 1990). In the presence of NaCl in the medium ( 4.80 MPa at 30C), S. cerevisiae required nine times more glucose per milligram of yeast formed than that in the absence of salt in the medium (Brown 1990). The decrease (28%, Fig. 5a and c) in biomass on carbon substrate yield YX/S in response to an increase in glucose concentration from 0.28 to 0.56 mol l1 was in agreement with previous results (Engasser et al. 1986), which showed that for low glucose concentrations the carbon substrate was assimilated at a low rate but at a maximal YX/S yield. Intracellular glycerol accumulation occurred mainly during growth (Figs. 5a and 6). However, during the stationary state, glucose consumption continued as well as extracellular glycerol production, whereas the amount of intracellular compatible solutes in cells decreased (Fig. 6). As osmotic adjustment occurred mainly during growth, polyol production during the stationary state can be clearly related to another mechanism other than the osmotic. During the stationary state, polyols were probably produced by a fermentative process to ensure energy for cell maintenance. The kinetic of carbon substrate consumption involved two terms, one growth-associated (g) and the other related to cell maintenance (m):     ds ds ds (1) dt dt g dt m where s is the glucose concentration.

By introducing the expressions for growth- and nongrowth-associated glucose consumptions in the above equation, rs ds  x mx dt YX =S (2)

where rs is the substrate consumption rate, YX/S is the biomass on glucose yield, x is the biomass concentration, is the specific growth rate and m is the maintenance coefficient. Specific consumption rate can then be deduced: qs ds  m x dt YX =S (3)

Specific growth rate becomes negligible during stationary state; the rate of glucose consumption becomes: ds m x dt (4)

By considering the final biomass concentration (3.6 g l1) and the constant rate of glucose consumption recorded between 75 and 150 h (1.4 mmol l1 h1, Fig. 5c) of culture on medium containing 0.56 mol l1 glucose (Table 1), the maintenance coefficient (m=(ds/xdt)) was deduced to be 0.38 mmol glucose g biomass1 h1. The high energy required for cell maintenance was in agreement with previous results recorded for S. Cerevisiae (Watson 1970). Indeed, it was shown that in the presence of NaCl (1 mol l1) in the culture medium, S. cerevisiae growing under anaerobic conditions required ten times more maintenance energy than in the absence of NaCl. The most probable explanation was that a significant part of the maintenance energy was used for the osmoadaptation of the yeast. In 1956, Spencer and Sallans (1956) showed that, for S. cerevisiae and Torulopsis magnoliae, the Embden Meyerhof pathway is used for both glycerol and ethanol synthesis. Glycerol biosynthesis is therefore mostly accompanied by ethanol biosynthesis. During glycolysis, glucose is converted into pyruvate, which is in turn converted into acetaldehyde by means of pyruvate decarboxylase; acetaldehyde is then reduced into ethanol by means of alcohol dehydrogenase. This reduction requires NADH, resulting from the conversion of glyceraldehyde 3-phosphate into biphospho-1, 3-glycerate in the presence of NAD+. NADH is therefore essential for both ethanol and glycerol synthesis (Fig. 8), leading to a competition between both pathways, as illustrated in Fig. 1: in decreasing water activity resulting from increasing NaCl concentrations in the medium, both intra- and extracellular glycerol concentrations increased, whilst ethanol production decreased. The ratio between the amount of ethanol produced and the total (the sum of intra and extracellular) glycerol produced decreased from 47.7 to 3.4 for increasing NaCl concentrations from 0

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to 2 mol l1 (Fig. 1). This result illustrates the osmodependent channelling of carbon towards polyol production, until an experimental ratio of ethanol on total glycerol reached 3.4, which is close to the theoretical value of 4 (Fig. 8), and has to be brought together with the relatively low decrease in glucose consumption (from 46 to 32%; Fig. 2) for increasing NaCl concentrations (from 0 to 2 mol l1) to highlight the positive effect of decreasing water activity on glycerol production. The decrease in ethanol production for increasing NaCl concentrations (Fig. 1) was also previously reported for P. farinosa (Vijaikishore and Karanth 1984). It also agreed with the increase in arabitol production due to the increase in glyceradehyde-3-phosphate dehydrogenase activity when D. hansenii was cultivated on highly salted media (Adler et al. 1985), as well as the decrease in alcohol dehydrogenase activity when S. cerevisiae grew on medium containing 0.7 mol l1 NaCl (Blomberg and Adler 1989). Cessation of ethanol production during the stationary state can not be attributed to its inhibitory effect, since more than 0.3 mol l1 was produced for 10 mmol l1 NH4Cl in the medium, whilst only about 0.1 mol l1 was generated for 3.3 mmol l1 NH4Cl (Fig. 5e). These concentrations were below the threshold tolerance of yeast found in the literature, 0.87 mol l1 (Engasser 1993). The competition between ethanol and glycerol production (Fig. 8) led to a cessation of ethanol production, during the stationary state, before that of glycerol (Fig. 5d and e), which continued to be produced to ensure sufficient energy for cell maintenance. This, in turn, leads to the major portion of glycerol being produced during the stationary state (76%). The above results clearly showed that glycerol production was improved by the following: Decreasing water activity induced by increasing NaCl concentrations in the culture medium. Indeed, cellular osmoadaptation led to a channelling from ethanol production towards glycerol production. Nitrogen (ammonium) limitation. In this case, assimilation of the carbon substrate (glucose) during the stationary state resulted in the extracellular production of glycerol to ensure energy for cell maintenance. In contrast, during growth, intracellular glycerol was accumulated for cellular osmoadaptation.

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