Polyol accumulation by two filamentous fungi grown at different concentrations of NaCl

The accumulation of polyols by Aspergillus niger (van Tiegh) strain S 1 and Penicillium chrysogenum (Thom) strain S 30 was followed during growth in media of different concentrations of NaCl. The major polyols found were glycerol, erythritol and mannitol. The total polyol pool increased in both organisms in response to raised salinity, and the proportion of glycerol and erythritol was markedly enhanced at high salinity.     

Effects of osmotic and matric potential on radial growth and accumulation of endogenous reserves in three isolates of Pochonia chlamydosporia

For the first time, the effects of varying osmotic and matric potential on fungal radial growth and accumulation of polyols were studied in three isolates of Pochonia chlamydosporia. Fungal radial growth was measured on potato dextrose agar modified osmotically using potassium chloride or glycerol. PEG 8000 was used to modify matric potential. When plotted, the radii of the colonies were found to grow linearly with time, and regression was applied to estimate the radial growth rate (mm day^?1). Samples of fresh mycelia from 25-day-old cultures were collected and the quantity (mg g^?1 fresh biomass) of four polyols (glycerol, erythritol, arabitol and mannitol) and one sugar (glucose) was determined using HPLC. Results revealed that fungal radial growth rates decreased with increased osmotic or matric stress. Statistically significant differences in radial growth were found between isolates in response to matric stress (P<0.006) but not in response to osmotic stress (P=0.759). Similarly, differences in the total amounts of polyols accumulated by the fungus were found between isolates in response to matric stress (P<0.001), but not in response to osmotic stress (P=0.952). Under water stress, the fungus accumulated a combination of different polyols important in osmoregulation, which depended on the solute used to generate the stress. Arabitol and glycerol were the main polyols accumulated in osmotically modified media, whereas erythritol was the main polyol that was accumulated in media amended with PEG. The results found that Pochonia chlamydosporia may use different osmoregulation mechanisms to overcome osmotic and matric stresses.     

Transplacental carbohydrate and sugar alcohol concentrations and their uptakes in ovine pregnancy

The concentrations of glucose, fructose, sorbitol, glycerol, and myo-inositol in sheep blood and tissues have been reported previously (1--5). However, the other polyols that are at low concentrations have not been investigated in pregnant sheep due to technical difficulties. By using HPLC and gas chromatography-mass spectrometry, seven polyols (myo-inositol, glycerol, erythritol, arabitol, sorbitol, ribitol, and mannitol) and three hexoses (mannose, glucose, and fructose) were identified and quantified in four blood vessels supplying and draining the placenta (maternal artery, uterine vein, fetal artery, and umbilical vein). Uterine and umbilical blood flows were measured, and uptakes of all the polyols and hexoses in both maternal and fetal circulations were calculated. There was a significant net placental release of sorbitol to both maternal and fetal circulations. Fructose was also taken up significantly by the uterine circulation. Maternal plasma mannose concentrations were higher than fetal concentrations, and there was a net umbilical uptake of mannose, characteristics that are similar to those of glucose. Myo-inositol and erythritol had relatively high concentrations in fetal plasma (697.8 plus minus 53 microM and 463.8 plus minus 27 microM, respectively). The ratios of fetal/maternal plasma arterial concentrations were very high for most polyols. The concentrations of myo-inositol, glycerol, and sorbitol were also high in sheep placental tissue (2489 plus minus 125 microM/kg wet tissue, 2119 plus minus 193 microM/kg wet tissue, and 3910 plus minus 369 microM/kg wet tissue), an indication that these polyols could be made within the placenta.     

Culture Age, Temperature, and pH Affect the Polyol and Trehalose Contents of Fungal Propagules

The growth and conidial physiology of the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus were studied under different conditions. The effects of culture age (up to 120 days), temperature (5 to 35(deg)C), and pH (2.9 to 11.1) were determined. Growth was optimal at pH 5 to 8 for each isolate and between 20 and 35(deg)C, depending on the isolate. The predominant polyol in conidia was mannitol, with up to 39, 134, and 61 mg g of conidia(sup-1) for B. bassiana, M. anisopliae, and P. farinosus, respectively. Conidia of M. anisopliae contained relatively small amounts of lower-molecular-weight polyols and trehalose (less than 25 mg g(sup-1) in total) in all treatments. Conidia of B. bassiana and P. farinosus contained up to 30, 32, and 25 mg of glycerol, erythritol, and trehalose, respectively, g(sup-1), depending on the treatment. Conidia of P. farinosus contained unusually high amounts of glycerol and erythritol at pH 2.9. The apparent effect of pH on gene expression is discussed in relation to the induction of a water stress response. To our knowledge, this is the first report of polyols and trehalose in fungal propagules produced over a range of temperature or pH. Some conditions and harvesting times were associated with an apparent inhibition of synthesis or accumulation of polyols and trehalose. This shows that culture age and environmental conditions affect the physiological quality of inoculum and can thereby determine its potential for biocontrol.     

Improving water stress tolerance of the biocontrol yeast Candida sake grown in molasses-based media by physiological manipulation

The biocontrol agent Candida sake was cultured on either an unmodified molasses-based medium (water activity, a(w) 0.996) or on water stressed media produced by the addition of glycerol, glucose, NaCl, sorbitol, or proline to 0.98, and 0.96 a(w) for 24, 48, and 72 h, to study their impact on subsequent cell viability, and on concentrations of endogenous sugars (trehalose and glucose) and polyols (glycerol, erythritol, arabitol, and mannitol). The viability of cells of different ages cultured on these media was evaluated on NYDA medium with freely available water (a(w) 0.995), and on medium modified with polyethylene glycol to a(w) 0.95. Regardless of solute used, viable counts of cells grown on molasses-based medium (a(w) 0.98) were equal to or higher than those obtained from the medium with water freely available. The amino acid proline stimulated growth at 10% concentration. In contrast, water stress induced by addition of NaCl, glucose, or sorbitol at a(w) 0.96 caused a significant reduction in viable counts. Older cultures were more resistant to water stress. Glycerol and arabitol were the main solutes accumulated by C. sake cells in response to lowered a(w). Intracellular concentration of these polyols depended more on the solute used to adjust the a(w) than on the a(w) itself. Candida sake was more resistant to water stress with higher intracellular concentration of glycerol and erythritol.     

Geographic differences on accumulation of sugars and polyols in locust eggs in response to cold acclimation

The accumulation of low molecular weight sugars and polyols is one of major mechanisms hypothesized to increase cold tolerance in overwintering insects. But little is known about whether these sugars and polyols are involved in geographic variation of cold tolerance. In this study, we investigated accumulation patterns of eight low molecular weight sugars and polyols of eggs in tropical and temperate populations of the migratory locust, which exhibits between-population variation in cold tolerance, in response to cold acclimation (5, 0 and −5 °C). Excluding erythritol, the other seven carbohydrates were identified as possible cryoprotectants in locust eggs. Basal maximal and minimal concentrations were 45 μg/g wet weight for trehalose and 0.59 μg/g wet weight for glycerol. Most sugars and polyols were elevated after a −5 °C exposure. In a tropical population, fructose, glucose, sorbitol and myo-inositol were significantly accumulated by low temperature treatments, but glycerol was not. In the temperate population, glycerol, glucose, mannitol, sorbitol, myo-inositol were significantly accumulated but trehalose did not increase. Our results suggest different accumulation patterns of these carbohydrates of locust eggs between tropical and temperate populations and highlighted possible roles for them in geographic variation of cold tolerance in the migratory locust.     

Resistance of food spoilage yeasts to sorbic acid

Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were grown on Sabouraud Dextrose Agar (SDA) modified with KCl to give a range of water activity (a_w) from 0.938 to 0.998. Growth of all three species was optimal at 0.983 a_w and growth occurred over the a_w range tested. Acyclic sugar alcohol (polyol) and trehalose content of conidia was determined by HPLC and found to vary with species and a_w. Conidia of B. bassiana and P. farinosus were found to contain totals of 1.5% and 2.3% polyols respectively at 0.998 a_w, and double these amounts at <0.950 a_w. Conidia of M. anisopliae contained from 5.7% to 6.8% polyols at each a_w tested. In conidia of all three species the predominant polyol was mannitol. The lower molecular weight polyols, arabitol and erythritol, were found to accumulate at reduced a_w. Small amounts of glycerol were present in conidia of each species; <15% total polyols. Conidia of B. bassiana and M. anisopliae contained about 0.5% trehalose from 0.970 to 0.998 a_w, but only trace amounts below 0.950 a_w. Conidia of P. farinosus contained 2.1% trehalose at 0.998 a_w and this decreased to <0.1% below 0.950 a_w. Potential to manipulate the endogenous reserves of conidia of these biological control agents to enhance viability and desiccation toierance is discussed     

Production of erythritol and mannitol by Yarrowia lipolytica yeast in media containing glycerol

Glycerol is a by-product generated in large amounts during the production of biofuels. This study presents an alternative means of crude glycerol valorization through the production of erythritol and mannitol. In a shake-flasks experiment in a buffered medium, nine Yarrowia lipolytica strains were examined for polyols production. Three strains (A UV'1, A-15 and Wratislavia K1) were selected as promising producers of erythritol or/and mannitol and used in bioreactor batch cultures and fed-batch mode. Pure and biodiesel-derived crude glycerol media both supplemented (to 2.5 and 3.25?%) and not-supplemented with NaCl were applied. The best results for erythritol biosynthesis were achieved in medium with crude glycerol supplemented with 2.5?% NaCl. Wratislavia K1 strain produced up to 80.0?g?l(-1) erythritol with 0.49?g?g(-1) yield and productivity of 1.0?g?l(-1)?h(-1). Erythritol biosynthesis by A UV'1 and A-15 strains was accompanied by the simultaneous production of mannitol (up to 27.6?g?l(-1)). Extracellular as well as intracellular erythritol and mannitol ratios depended on the glycerol used and the presence of NaCl in the medium. The results from this study indicate that NaCl addition to the medium improves erythritol biosynthesis, and simultaneously inhibits mannitol formation.     

Optimization of erythritol and glycerol accumulation in conidia of Beauveria bassiana by solid-state fermentation, using response surface methodology

Entomopathogenic fungi are widely produced for use as mycoinsecticides. Therefore, improvement of the shelf life of fungal propagules under good and adverse conditions should be a pre-requisite of their production. In order to improve conidial physiology as well as mycoinsecticide efficiency, culture conditions may be varied. The Doehlert design was used to generate response surfaces with an estimation of the parameters of the quadratic model allowing the study of three different factors at a different number of levels. This experimental design was applied to optimize water activity (a _w), pH, and fermentation time for Beauveria bassiana conidial production and accumulation of polyols in solid-state fermentation. Thus, it was possible to identify the region in the experimental range in which the optimum values of these parameters were simultaneously achieved. Maximal conidia production was achieved at pH 5–6 and a _w=0.999. Under these conditions, polyol accumulation was 3 mg erythritol/g conidia and 29.6 mg glycerol/g conidia. However, maximal polyol accumulation was achieved at pH 4.5 and a _w 0.950; erythritol production increased 33-fold and glycerol production 4.5-fold. Under these conditions conidia production was 1,000 times lower. The possibilities of increasing the quality of the biocontrol agent without neglecting yield are discussed.     

Drought acclimation and lipid composition in Folsomia candida: implications for cold shock,heat shock and acute desiccation stress

Many of the physiological adaptations evolved in terrestrial invertebrates to resist desiccation have also been shown to enhance the survival of low temperatures. In this study we have examined temporal changes in the physiology of the collembolan Folsomia candida during acclimation to mild desiccation stress (98.2% RH), and how physiological changes correlate with resistance to subsequent cold shock, heat shock and acute desiccation stress. Drought-acclimation increased the resistance to cold and acute drought but reduced the resistance to heat shock. The composition of membrane phospholipid fatty acids (PLFA) changed during acclimation resulting in a higher degree of unsaturation by the end of the 192-h acclimation period. This resembles typical membrane alterations seen in ectothermic animals exposed to cold. Only small changes were seen in the neutral lipid fraction. The temporal changes in cold resistance and drought resistance correlated well with changes in PLFA composition and accumulation of sugars and polyols (’cryoprotectives’). It is proposed that the drought-induced PLFA desaturation, combined with the membrane protecting accumulation of cryoprotectives, are important physiological adaptations providing tolerance to both desiccation and cold.     

Shifts in the carbohydrate,polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in flesh flies (<Emphasis Type="Italic">Sarcophaga crassipalpis</Emphasis>): a metabolomic comparison

Flesh flies can enhance their cold hardiness by entering a photoperiod-induced pupal diapause or by a temperature-induced rapid cold-hardening process. To determine whether the same or different metabolites are involved in these two responses, derivatized polar extracts from flesh flies subjected to these treatments were examined using gas chromatography–mass spectrophotometry (GC–MS). This metabolomic approach demonstrated that levels of metabolites involved in glycolysis (glycerol, glucose, alanine, pyruvate) were elevated by both treatments. Metabolites elevated uniquely in response to rapid cold-hardening include glutamine, cystathionine, sorbitol, and urea while levels of β-alanine, ornithine, trehalose, and mannose levels were reduced. Rapid cold-hardening also uniquely perturbed the urea cycle. In addition to the elevated metabolites shared with rapid cold-hardening, leucine concentrations were uniquely elevated during diapause while levels of a number of other amino acids were reduced. Pools of two aerobic metabolic intermediates, fumarate and citrate, were reduced during diapause, indicating a reduction of Krebs cycle activity. Principal component analysis demonstrated that rapid cold-hardening and diapause are metabolically distinct from their untreated, non-diapausing counterparts. We discuss the possible contribution of each altered metabolite in enhancing the overall cold hardiness of the organism, as well as the efficacy of GC–MS metabolomics for investigating insect physiological systems.     

Enhanced Trehalose Accumulation and Desiccation Survival of Entomopathogenic Nematodes Through Cold Preacclimation

Limited storage stability is a major obstacle to further expansion of the use of entomopathogenic nematodes for pest control. Progress has been made that Steinernema carpocapsae can now be stored under partial anhydrobiosis for up to 6 months at 25°C and 10 months at 5°C in a water-dispersible granular (WG) formulation. However, other species have been more difficult to store in the WG formulation due to migration of nematodes out of the granules and sensitivity of some species to desiccation directly at cold temperatures. As acclimation to cold induces trehalose accumulation (a major cryo- and desiccation protectant) in many invertebrates, it was hypothesized that cold preacclimation of entomopathogenic nematodes will enhance their survival in the WG formulation at cold temperatures. This hypothesis was tested using a temperate species Steinernema feltiae , a subtropical species S. carpocapsae , and a tropical species Steinernema riobrave possessing different thermal niche breadths and reproduction temperature optima. Cold acclimation of infective juveniles increased trehalose accumulation in all three species and the amount of trehalose accumulated was both temperature and species dependent. Trehalose content reached at its peak after 6 days at 5°C in S. feltiae (82.28 μg/mg dry weight), after 10 days at 10°C in S. carpocapsae (94.16 μg/mg dry weight) and after 6 days at 15°C in S. riobrave (47.58 μg/mg dry weight). Cold preacclimation at 5°C for 2 days enhanced desiccation survival of S. feltiae in 25% glycerol (osmotic desiccation) at both 5 and 25° and of S. carpocapsae and S. riobrave only at 5°C. Non-cold acclimated S. carpocapsae and S. riobrave were extremely sensitive to desiccation directly at 5°C in 25% glycerol, resulting in over 98% mortality within 6 days, but S. feltiae was more sensitive to desiccation at 25°C than at 5°C. Cold preacclimation increased survival of all the three species in the WG formulation at both 5 and 25°C. The survival of S. riobrave at 5°C in the WG formulation was positively correlated with the length of preacclimation period at 5°C (R 2 = 0.99) and with the amount of trehalose accumulated during cold preacclimation (R 2 = 0.81). These results support the hypothesis that cold preacclimation enhances desiccation survival of entomopathogenic nematodes at cold temperatures and the increased survival correlates well with the increased trehalose accumulation. Results also demonstrate that cold preacclimation can be used as a tool to enhance survival of nematodes in the formulations with reduced water activity.     

A comparative study on the formation of citric acid and polyols and on morphological changes of three strains of free and immobilized Aspergillus niger

Summary The formation of citric acid, oxalic acid, erythritol and glycerol by three strains of Aspergillus niger immobilized in calcium alginate was investigated and compared with that of free cells when cultivated in shake flasks under phosphate limitation. Morphological changes were followed using an electron microscope. The production of acids and polyols, the consumption of glucose and fructose, and also the morphological changes were strain-dependent. The results also reflected the influence of long storage of a strain on productivity, morphological behaviour and phosphate consumption. Offprint requests to: H.-J. Rehm     

Roles of Sugar Alcohols in Osmotic Stress Adaptation. Replacement of Glycerol by Mannitol and Sorbitol in Yeast

For many organisms there is a correlation between increases of metabolites and osmotic stress tolerance, but the mechanisms that cause this protection are not clear. To understand the role of polyols, genes for bacterial mannitol-1-P dehydrogenase and apple sorbitol-6-P dehydrogenase were introduced into a Saccharomyces cerevisiae mutant deficient in glycerol synthesis. Sorbitol and mannitol provided some protection, but less than that generated by a similar concentration of glycerol generated by glycerol-3-P dehydrogenase (GPD1). Reduced protection by polyols suggested that glycerol had specific functions for which mannitol and sorbitol could not substitute, and that the absolute amount of the accumulating osmoticum might not be crucial. The retention of glycerol and mannitol/sorbitol, respectively, was a major difference. During salt stress, cells retained more of the six-carbon polyols than glycerol. We suggest that the loss of >98% of the glycerol synthesized could provide a safety valve that dissipates reducing power, while a similar high intracellular concentration of retained polyols would be less protective. To understand the role of glycerol in salt tolerance, salt-tolerant suppressor mutants were isolated from the glycerol-deficient strain. One mutant, sr13, partially suppressed the salt-sensitive phenotype of the glycerol-deficient line, probably due to a doubling of [K(+)] accumulating during stress. We compare these results to the "osmotic adjustment" concept typically applied to accumulating metabolites in plants. The accumulation of polyols may have dual functions: facilitating osmotic adjustment and supporting redox control.     

The effects of doxycycline administration on amino acid neurotransmitters in an animal model of neonatal hypoxia-ischemia

Neonatal hypoxia-ischemia (HI) is a major contributor to many neurological, psychiatric and behavioral disorders. Previous studies in our laboratory have shown that a one-time dose of doxycycline (DOXY), even when given 3 h after HI insult, was neuroprotective and significantly reduced microglial activation and cleaved caspase-3 protein expression in the immature brain. In light of these data, the goal of this study was to investigate the effects of DOXY administration on amino acid neurotransmitters. Post-natal-day 7 rats received DOXY (10 mg/kg) or vehicle (VEH) concomitant with the onset of HI, and were euthanized 30 min, 1, 2 or 4 h post-HI (n ≥ 6). Extracted brains were either immediately dissected for frontal cortex, striatum and hippocampal regions, or removed in their entirety and flash frozen in isopentane for histological analyses. Dissected regions were homogenized and aliquots were prepared for high performance liquid chromatography (HPLC) analyses of amino acid levels and brain levels of DOXY. HPLC extraction revealed that systemic administration of DOXY resulted in mean drug levels of 867.1 ± 376.1 ng/g of brain tissue. Histological analyses revealed microglial activation, caspase-3 activation and neuronal degeneration consistent with a mild injury in the regions most vulnerable to HI. We found that HI caused significant, time-dependent, regional changes in brain amino acids including glutamate, GABA, alanine, aspartate, asparagine, serine, glutamine, glycine and taurine. HI significantly increased glutamate levels in the hippocampus (HI + VEH = 15.8 ± 3.1 ng/μg versus control = 11.8 ± 1.4 ng/μg protein) 4 h post-HI (p < 0.05). Pups treated with DOXY had lower glutamate levels (13.1 ± 2.4 ng/μg) when compared to VEH-treated pups (15.8 ± 3.1 ng/μg), however these values failed to reach significance. In addition, DOXY-treated pups had significantly lower alanine (HI + VEH = 1.1 ± 0.2 ng/μg versus HI + DOXY = 0.5 + 0.1 ng/μg) and serine (HI + VEH = 1.4 ± 0.4 ng/μg versus HI + DOXY = 0.7 + 0.1 ng/μg) levels in the hippocampus, 4 h post-HI. Similar normalizations and significant reductions in alanine and serine were seen in the cortex and striatum. These results show that in addition to its previously reported and well-documented anti-inflammatory and anti-apoptotic properties, DOXY has significant effects on amino acid neurotransmitters.     

Directing enzyme devolution for biosynthesis of alkanols and 1,n-alkanediols from natural polyhydroxy compounds

Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as “specialists”. However, some enzymes are “generalists” that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols, including 1,2-butanediol, 1,2,4-butanetriol, erythritol, 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays, and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals.     

Cloning, heterologous expression, and characterization of three aquaglyceroporins from Trypanosoma brucei

Trypanosoma brucei, causative for African sleeping sickness, relies exclusively on glycolysis for ATP production. Under anaerobic conditions, glucose is converted to equimolar amounts of glycerol and pyruvate, which are both secreted from the parasite. As we have shown previously, glycerol transport in T. brucei occurs via specific membrane proteins (Wille, U., Schade, B., and Duszenko, M. (1998) Eur. J. Biochem. 256, 245-250). Here, we describe cloning and biochemical characterization of the three trypanosomal aquaglyceroporins (AQP; TbAQP1-3), which show a 40-45% identity to mammalian AQP3 and -9. AQPs belong to the major intrinsic protein family and represent channels for small non-ionic molecules. Both TbAQP1 and TbAQP3 contain two highly conserved NPA motifs within the pore-forming region, whereas TbAQP2 contains NSA and NPS motifs instead, which are only occasionally found in AQPs. For functional characterization, all three proteins were heterologously expressed in yeast and Xenopus oocytes. In the yeast fps1Delta mutant, TbAQPs suppressed hypoosmosensitivity and rendered cells to a hyper-osmosensitive phenotype, as expected for unregulated glycerol channels. Under iso- and hyperosmotic conditions, these cells constitutively released glycerol, consistent with a glycerol efflux function of TbAQP proteins. TbAQP expression in Xenopus oocytes increased permeability for water, glycerol and, interestingly, dihydroxyacetone. Except for urea, TbAQPs were virtually impermeable for other polyols; only TbAQP3 transported erythritol and ribitol. Thus, TbAQPs represent mainly water/glycerol/dihydroxyacetone channels involved in osmoregulation and glycerol metabolism in T. brucei. This function and especially the so far not investigated transport of dihydroxyacetone may be pivotal for the survival of the parasite survival under non-aerobic or osmotic stress conditions.     

Physiology of Aspergillus niger in oxygen‐limited continuous cultures: Influence of aeration,carbon source concentration and dilution rate

In industrial production of enzymes using the filamentous fungus Aspergillus niger supply of sufficient oxygen is often a limitation, resulting in the formation of by‐products such as polyols. In order to identify the mechanisms behind formation of the different by‐products we studied the effect of low oxygen availability, at different carbon source concentrations and at different specific growth rates, on the metabolism of A. niger, using continuous cultures. The results show that there is an increase in the production of tricarboxylic acid (TCA) cycle intermediates at low oxygen concentrations. Indeed, at these conditions, a decrease in the mitochondrial respiratory chain activity leads to an accumulation of NADH and to a decreased ATP production which uncouples catabolism and anabolism, influences the intracellular pH and leads to production and excretion of organic acids. Moreover, mannitol is being produced in order to ensure reoxidation of NADH, and this is the main cellular response to balance the ratio NADH/NAD at low oxygen availability. Mannitol production is also coupled to low specific growth rate, which suggests a control of carbon catabolite repression on the mannitol pathway. The roles of two other polyols, erythritol and glycerol, were also investigated. Both compounds are known to accumulate intracellularly, at high osmotic pressure, in order to restore the osmotic balance, but we show that the efficiency of this system is affected by a leakage of polyols through the membrane. Biotechnol. Bioeng. Biotechnol. Bioeng. 2009;103: 956–965. © 2009 Wiley Periodicals, Inc.     

Comparison of citric acid production from glycerol and glucose by different strains of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

A wild type strain A-101 of Y. lipolytica and its three acetate-negative mutants (Wratislavia 1.31, Wratislavia AWG7, and Wratislavia K1) were compared for the production of citric acid from glucose and glycerol (pure and crude) in batch cultures. The substrates were used either as single carbon sources or as mixtures of glucose and pure or crude glycerol. The kinetic parameters, i.e., the volumetric citric acid production rate and yield obtained in the study show that the Wratislavia 1.31 and Wratislavia AWG7 strains produced the highest amount of citric acid from glycerol, with a yield from 0.40 to 0.53 g g⁻¹. This substrate was found to be a better carbon source for the biosynthesis of citric acid than glucose. The results obtained with the same strains have shown low content of isocitric acid and polyols, such as erythritol and mannitol. Y. lipolytica A-101 strain produced the highest amount of isocitric acid, from 13.8 to 21% isocitric acid in the sum of citric acids. However, the highest concentrations of erythritol were found in cultures with Y. lipolytica Wratislavia K1, from 18.1 to 30 g l⁻¹, for glucose and pure glycerol, respectively.