Glycerol Degradation Research Articles

Parameters Affecting the Formation of 1,2-Propanediol from Glycerol over Ru/SiO2 Catalyst

The production of 1,2-propanediol from renewable glycerol in hydrogen atmosphere is of high interest. In this study the reaction was performed in the presence of 5 wt %Ru/SiO2 catalyst and the effects of reaction temperature, hydrogen pressure and glycerol concentration were investigated. The catalytic results indicate that increased temperature and pressure favor glycerol conversion and propylene glycol selectivity. Glycerol conversion remains almost constant with water dilution, but 1,2-propanediol selectivity increases due to the function of water as a solvent and the suppression of side reactions, such as degradation and polymerization. At the optimum reaction conditions (T = 240 °C, P = 8 MPa H2, pure glycerol feedstock, catalyst/glycerol ratio = 0.006, reaction time = 5 h) glycerol conversion reaches the value of 21.7% along with 60.5% 1,2-propanediol selectivity. The activity of the catalyst does not change in two consecutive runs, while propylene glycol selectivity slightly decreases due to the formation of overhydrogenolysis products (propanols). The reaction pathways over Ru/SiO2 catalyst were explored using intermediate and final products as reactants. The production of 1,2-propanediol is favorable as it is formed with high selectivity from acetol and its overhydrogenolysis to propanols is very limited. The results suggest that ethylene glycol is a primary product originating from the direct degradation of glycerol.

Non-Catalytic and MgSO4 - Catalyst based Degradation of Glycerol in Subcritical and Supercritical Water Media

This research aims to study the glycerol degradation reaction in subcritical and supercritical water media. The degradation of glycerol into other products was performed both with sulphate salt catalysts and without catalyst. The reactant was made from glycerol and water with the mass ratio of 1:10. The experiments were carried out using a batch reactor at a constant pressure of 250 kgf/cm2, with the temperature range of 200-400oC, reaction time of 30 minutes, and catalyst mol ratio in glycerol of 1:10 and 1:8. The products of the non-catalytic glycerol degradation were acetaldehyde, methanol, and ethanol. The use of sulphate salt as catalyst has high selectivity to acetaldehyde and still allows the formation alcohol product in small quantities. The mechanism of ionic reaction and free radical reaction can occur at lower temperature in hydrothermal area or subcritical water. Conversion of glycerol on catalytic reaction showed a higher yield when compared with the reaction performed without catalyst

Kinetics of anaerobic biodegradation of glycerol by sulfate-reducing bacteria

A kinetic model has been developed and kinetic parameters of anaerobic degradation of glycerol, an abundant by-product of biofuel manufacturing, by a consortium of sulfate reducing bacteria (SRB) in a closed system have been determined. The following main species of SRB has been identified in the consortium: Desulfovibrio baarsii, Desulfomicrobium sp., and Desufatomaculum sp. The proposed model included processes of glycerol degradation, sulfate reduction, and inhibition by metabolic products, as well as effects of pH and temperature. The suggested equation for the anaerobic glycerol degradation was based on Edward and Andrew’s equation. The following kinetic parameters of the anaerobic glycerol degradation were obtained for the initial glycerol concentration from 0.15 to 4 ml/l and sulfate concentration of 2760 mg/l at 22°C: maximum specific growth rate of SRB μmax = 0.56 day−1, economic coefficient of ashless biomass from glycerol of 0.08 mol SRB/mol COC, and yield of ashless biomass from sulfate of 0.020 mol SRB/mol SO4. It was shown that the optimum molar ratio of $$ {{C_{Gl} } \mathord{\left/ {\vphantom {{C_{Gl} } {C_{SO_4 } }}} \right. \kern-\nulldelimiterspace} {C_{SO_4 } }} $$ for SRB growth was 0.8. Initial boundary concentration of inhibition by undissociated hydrogen sulfide was 70 mg/l. Dependence of the specific growth rate of bacteria on the temperature was approximated by the Arrhenius equation in the temperature range of 20–30°C with the goodness of fit R2 = 0.99.

Glycerol degradation in single-chamber microbial fuel cells

Glycerol degradation with electricity production by a pure culture of Bacillus subtilis in a single-chamber air cathode of microbial fuel cell (MFC) has been demonstrated. Steady state polarization curves indicated a maximum power density of 0.06 mW/cm(2) with an optimal external resistance of 390Ω. Analysis of the effect of pH on MFC performance demonstrated that electricity generation was sustained over a long period of time under neutral to alkaline conditions. Cyclic voltammetry exhibited the increasing electrochemical activity with the increase of pH of 7, 8 and 9. Voltammetric studies also demonstrated that a two-electron transfer mechanism was occurring in the reactor. The low Coulombic efficiency of 23.08% could be attributed to the loss of electrons for various activities other than electricity generation. This study describes an application of glycerol that could contribute to transformation of the biodiesel industry to a more environmentally friendly microbial fuel cell-based technology.

HPLC-MS Determination of Acrolein and Acetone Generated from 13C3 -Labeled Glycerol Added to Cigarette Tobacco Using Two Machine-Smoking Regimes

Abstract The extent of blend glycerol degradation in a burning cigarette to form acrolein and acetone has been quantitatively determined by the addition of glycerol-13C3 to three styles of a leading commercial cigarette brand. Multiple Cambridge pads soaked with a solution of 2,4-dinitrophenylhydrazine (DNPH) were employed to trap hydrazone derivatives of low molecular weight carbonyl compounds in both mainstream and sidestream smoke. High performance liquid chromatography coupled with negative ion mass spectrometry was used to isolate DNPH derivatives of the volatile carbonyl products of combustion and to ascertain their concentration. Acrolein, acetone, and propionaldehyde were the principal compounds of interest. The DNPH derivatives of acrolein-13C3 and acetone-13C3 were independently synthesized, and they served as external standards for absolute quantitation. The cost of fully labeled propionaldehyde precluded its use in this study. The brand styles selected for study represent the cigarette design features that are most prevalent in the U.S. market today and afford a representative range of standardized “tar” yields (14, 10, and 5 mg/cig, respectively by the Cambridge Filter Method). The brand styles studied are part of a commercial cigarette brand family that does not contain additives to the tobacco blend, including glycerol. Mainstream smoke was generated by an automated smoking machine employing the standard Cambridge Filter Smoking Regime and a more intense regime requiring larger, more frequent puffs and 100% vent blocking that is specified for regulatory purposes by the Canadian federal government. The research indicated that only a small fraction of added glycerol (~0.25%-0.30%, w/w) was converted to the two compounds of interest, with the larger portion generally observed in sidestream smoke. Less than 0.1% of the added glycerol was converted to acrolein in mainstream smoke for all cigarette designs and smoking regimes studied.

Hydrothermal Electrolysis of Glycerol Using a Continuous Flow Reactor

Hydrothermal electrolysis reactions of glycerol were investigated under various operating conditions to determine the effects of applied DC current, electrolysis time, and alkali concentration on the decomposition mechanism of glycerol. In addition, intermediate products were identified, possible reaction schemes for both hydrothermal electrolysis and hydrothermal degradation of glycerol based on experimental data were clarified, and detailed product analysis was conducted using high performance liquid chromatography (HPLC), gas chromatography with a flame ionization detector (GC-FID), and gas chromatography with a thermal conductivity detector (GC-TCD). For the present study, a continuous flow reactor equipped with titanium electrodes (as cathode and anode), an electric furnace, a heater, a pump, a heat exchanger, a back pressure regulator, and DC supply was used. The main gaseous product was hydrogen, whereas glycolaldehyde, lactic acid, and formic acid were the main liquid products. Results indicate that greater than 92% of the glycerol could be decomposed under optimum conditions by hydrothermal electrolysis using the continuous flow reactor.

The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.

Anomalous Low Friction Under Boundary Lubrication of Steel Surfaces by Polyols

We present here anomalous low friction obtained with highly polished steel on steel hard contact lubricated by glycerol under severe mixed and boundary regimes (λ ratio below 1). We investigated the effects of contact pressure, sliding speed, and temperature on friction coefficient and electrical contact resistance. The mechanism of low friction (typically below 0.02) is thought to have two origins: first a contribution of an ultrathin EHL film of glycerol providing easy shear under pressure, second the chemical degradation of glycerol inside the contact when more severe conditions are attained, generating a nanometer-thick film containing shear-induced water molecules. This new mechanism, called “H-bond Network model”, is completely different from the well-accepted “Monolayer” model working with polar molecules containing long aliphatic chains. Moreover, we show outstanding superlubricity (friction coefficient below 0.01) of steel surfaces directly lubricated by a solution of myo-inositol (also called vitamin Bh) in glycerol at ambient temperature (25 °C) and high contact pressure (0.8 GPa) in the absence of any long chain polar molecules. Mechanism is still unknown but could be associated with friction-induced dissociation of inositol and H-bond interactions network of water-like species with steel surface.

Superlubricity and tribochemistry of polyhydric alcohols

The anomalous low friction of diamondlike carbon coated surfaces lubricated by pure glycerol was observed at $80\text{ }\ifmmode^\circ\else\textdegree\fi{}\text{C}$. Steel surfaces were coated with an ultrahard 1 \textmu{}m thick hydrogen-free tetrahedral coordinated carbon (ta-C) layer produced by physical vapor deposition. In the presence of glycerol, the friction coefficient is below 0.01 at steady state, corresponding to the so-called superlubricity regime (when sliding is then approaching pure rolling). This new mechanism of superlow friction is attributed to easy glide on triboformed OH-terminated surfaces. In addition to the formation of OH-terminated surfaces but at a lower temperature, we show here some evidence, by coupling experimental and computer simulations, that superlow friction of polyhydric alcohols could also be associated with triboinduced degradation of glycerol, producing a nanometer-thick film containing organic acids and water. Second, we show outstanding superlubricity of steel surfaces directly lubricated by a solution of myo-inositol (also called vitamin Bh) in glycerol at ambient temperature $(25\text{ }\ifmmode^\circ\else\textdegree\fi{}\text{C})$. For the first time, under boundary lubrication at high contact pressure, friction of steel is below 0.01 in the absence of any long chain polar molecules. The mechanism is still unknown but could be associated with friction-induced dissociation of glycerol and interaction of waterlike species with steel surface.

Metabolism of halophilic archaea

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-008-0138-x) contains supplementary material, which is available to authorized users.

Glycerol metabolism and bitterness producing lactic acid bacteria in cidermaking

Several lactic acid bacteria were isolated from bitter tasting ciders in which glycerol was partially removed. The degradation of glycerol via glycerol dehydratase pathway was found in 22 out of 67 isolates. The confirmation of glycerol degradation by this pathway was twofold: showing their glycerol dehydratase activity and detecting the presence of the corresponding gene by a PCR method. 1,3-propanediol (1,3-PDL) and 3-hydroxypropionic acid (3-HP) were the metabolic end-products of glycerol utilization, and the accumulation of the acrolein precursor 3-hydroxypropionaldehyde (3-HPA) was also detected in most of them. The strain identification by PCR-DGGE rpoB showed that Lactobacillus collinoides was the predominant species and only 2 belonged to Lactobacillus diolivorans. Environmental conditions conducting to 3-HPA accumulation in cidermaking were studied by varying the fructose concentration, pH and incubation temperature in L. collinoides 17. This strain failed to grow with glycerol as sole carbon source and the addition of fructose enhanced both growth and glycerol degradation. Regarding end-products of glycerol metabolism, 1,3-PDL was always the main end-product in all environmental conditions assayed, the only exception being the culture with 5.55 mM fructose, where equimolar amounts of 1,3-PDL and 3-HP were found. The 3-HPA was transitorily accumulated in the culture medium under almost all culture conditions, the degradation rate being notably slower at 15 °C. However, no disappearance of 3-HPA was found at pH 3.6, a usual value in cider making. After sugar exhaustion, L. collinoides 17 oxidated lactic acid and/or mannitol to obtain energy and these oxidations were accompanied by the removal of the toxic 3-HPA increasing the 1,3-PDL, 3-HP and acetic acid contents.

Dihydroxyacetone metabolism in Salinibacter ruber and in Haloquadratum walsbyi

The extremely halophilic bacterium Salinibacter ruber inhabits saltern crystallizer ponds worldwide, together with the square archaeon Haloquadratum walsbyi. Cultures of Salinibacter have been shown to convert up to 20% of the glycerol added to a not previously characterized overflow product. We here identify this product of incomplete glycerol oxidation by Salinibacter as dihydroxyacetone. Genomic information suggests that H. walsbyi possesses an efficient uptake system for dihydroxyacetone, and we show here that dihydroxyacetone is indeed metabolized by Haloquadratum cultures, as well as by the heterotrophic prokaryotic community of the saltern crystallizer ponds in Eilat, Israel, dominated by Haloquadratum-like cells. In the absence of glycerol, Salinibacter also takes up dihydroxyacetone. Degradation of glycerol, produced in hypersaline lakes as an osmotic solute by the green alga Dunaliella salina may thus involve dihydroxyacetone as an intermediate, which can then be taken up by different types of heterotrophs present in the environment.

Hot compressed water as reaction medium and reactant: 2. Degradation reactions

Hot compressed water (HCW, here water above 200 °C) owns interesting properties. The impact of the unique properties is discussed exemplary for the thermal degradation of tert-butylbenzene and the oxidation of methanol. Both reactions have been conducted not only in HCW but also in other high-pressure media and from the comparison the impact of the special properties of HCW can be settled. In addition the degradation of glycerol, a model substance for carbohydrates and biomass in HCW was studied. This reaction shows a strong dependence on the properties of HCW. The examples picture an increased specific impact of HCW with rising polarity of the reactants and intermediates. The studies also points to higher importance of microscopic properties for understanding reactions in HCW than assumed in the past.

Development of the first potent and specific inhibitors of endocannabinoid biosynthesis

Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases α and β (DAGLα and β) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLα to screen new synthetic substances as DAGLα inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLα and MAGL activities were assessed by using sn-1-[ 14C]-oleoyl-2-arachidonoyl-glycerol and 2-[ 3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat®) (IC 50 ∼ 60 nM), the most potent inhibitors of DAGLα were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC 50 = 500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC 50 = 160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB 1 and CB 2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl- O- p-nitro-phenyl oleylphosphonate] inhibited both DAGLα and MAGL with similar potencies (IC 50 = 0.8–0.1 and 3.7–3.2 μM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.

New Functions for Parts of the Krebs Cycle in Procyclic Trypanosoma brucei, a Cycle Not Operating as a Cycle

We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.

Glycerol catabolism by Pediococcus pentosaceus isolated from beer

Among the lactic acid bacteria isolated from beer at different stages of elaboration, Pediococcus pentosaceus was the predominant species and the only that used glycerol as sole carbon source. Its utilization was studied in CAg strain growing on glycerol or on glycerol and limited concentration of glucose. Glycerol kinase and glycerol dehydratase pathways were responsible for glycerol degradation. On glycerol alone, the enzymatic activities of both pathways were expressed simultaneously and after glycerol consumption, the main products were acetate, 2,3-butanediol (2,3-BD) and 1,3-propanediol (1,3-PD). When the carbon sources were glycerol and glucose, glycerol was firstly degraded by the reductive pathway and after glucose consumption the activities of the glycerol kinase pathway were expressed. In this condition, glycerol was transformed into lactate, acetate, 2,3-BD and 1,3-PD. According to the enzymatic activities and fermentation balances, the production of acetate, providing ATP, can be correlated to lactate degradation, and the NAD required by these transformations could be obtained by the formation of 1,3-PD and 2,3-BD. Glycerol degradation should be correlated with the sensitivity of a beer to bacterial spoilage, and P. pentosaceus CAg considered as spoilage strain because it produces high volatile acidity and aroma compounds from glycerol conferring unacceptable flavour to beer.

Ionic reactions and pyrolysis of glycerol as competing reaction pathways in near- and supercritical water

Experimental results of the decomposition of glycerol in near- and supercritical water are presented considering measurements in the temperature range of 622–748 K, at pressures of 25, 35, or 45 MPa, reaction times from 32 to 165 s, and different initial concentrations. The reaction was carried out in a tubular reactor and a conversion between 0.4 and 31% was observed. The main products of the glycerol degradation are methanol, acetaldehyde, propionaldehyde, acrolein, allyl alcohol, ethanol, formaldehyde, carbon monoxide, carbon dioxide, and hydrogen. The results are compared with the studies of other working groups. The non-Arrhenius behavior of the overall degradation, as well as the pressure dependence of the reaction rate, and furthermore, the product distribution indicates the occurrence of two competing reaction pathways. One pathway consists of ionic reaction steps, which are preferred at higher pressures and/or lower temperatures. The second reaction pathway is a free radical degradation and dominates at lower pressures and/or higher temperatures. For reaction modeling, both mechanisms, the ionic and the free radical reaction network are compiled into one reaction model. The computer software package chemkin was used for the model calculations. The reaction model and the kinetic parameters were optimized in order to describe the experimental results for glycerol and the main products at 450 bar and all temperatures. This reaction model, consisting of the ionic and the free radical sub-mechanism satisfactorily describes the complex reaction at 450 bar.

Assimilation of glycerol by a strain of Lactobacillus collinoides isolated from cider

A collection of glycerol-degrading lactic acid bacteria from spoiled cider was isolated. The most widespread species was Lactobacillus collinoides, a heterofermentative species which mainly produced the D -lactic acid isomer from glucose. Conditions of glycerol assimilation by such bacteria were investigated for strain Lb. collinoides IOEB 9527. It could not be cultivated with glycerol alone as carbon source. Sugars were necessary for growth, and fructose was preferred to glucose. Glycerol was degraded, mainly to 1,3-propanediol. Small amounts of acrolein, produced from 3-hydroxypropion-aldehyde (3-HPA) during distillation of samples, were detected. When Lb. collinoides was cultivated with glucose or fructose alone, it produced the usual compounds of the heterofermentative pathway, notably D -lactic acid and small amounts of L -lactic acid, acetic acid and ethanol. When glycerol was present, the amounts of lactic acid were surprisingly much lower and acetic acid + ethanol much higher than expected. Oxidation of D -lactic acid, and to a lesser extent of L -lactic acid, was coupled to degradation of glycerol to 1,3-propanediol. This co-metabolism leading to acetic acid provided energy. Consequently, in fermented beverages spoiled by such strains, glycerol is degraded and 3-HPA plus acetic acid are produced. Both components are undesirable for health and sensorial quality.

Anaerobic degradation of glycerol by desulfovibrio fructosovorans and D. carbinolicus and evidence for glycerol-dependent utilization of 1,2-propanediol

The degradation of glycerol by Desulfovibrio carbinolicus and Desulfovibrio fructosovorans was tested in pure culture with sulfate and in coculture with Methanospirillum hungatei. Desulfovibrio carbinolicus degraded glycerol into 3-hydroxypropionate with the formation of sulfide in pure culture and methane in the coculture. The maximum growth rates were 0.063 h-1 in pure culture and 0.014 h-1 in coculture (corresponding growth yields: 8.9 and 6.0 g dry weight/mol glycerol). With D. fructosovorans, the pathway of glycerol degradation depended upon the terminal electron acceptor. Acetate and sulfide were produced in the presence of sulfate, while 3-hydroxypropionate and methane were formed by the syntrophic association with M. hungatei. The maximum growth rates were 0.057 h-1 in pure culture and 0.020 h-1 in coculture (corresponding growth yields: 8.9 and 6.0 g dry weight/mol glycerol). In a medium containing both glycerol and 1,2-propanediol but no sulfate, D. carbinolicus and D. fructosovorans degraded both substrates. A drop in the concentration of 1,3-propanediol was observed, and propionate and n-propanol production was recorded. Putative biochemical pathways of 1,2-propanediol degradation by D. carbinolicus and D. fructosovorans indicated that the enzymes involved in this metabolism are present only when the strains are grown on a mixture of 1,2-propanediol and glycerol without sulfate.

Organic solutes in freezing tolerance.

The accumulation of high levels of low-molecular-weight solutes (polyhydric alcohols, saccharides) provides cryoprotection to freeze-tolerant animals by minimizing, via colligative effects, the percentage of body water converted to extracellular ice and the extent of cell volume reduction. Many freeze-tolerant insects accumulate high levels of polyols during autumn cold hardening, whereas freeze-tolerant frogs respond to ice formation in peripheral tissues by synthesizing large amounts of glucose in the liver and rapidly distributing the sugar throughout the body. Seasonal patterns of enzymatic change occur in cold-hardy insects; activities associated with cryoprotectant synthesis rise in the fall, whereas enzymes associated with polyol degradation dominate in the spring. Enzyme profiles also revealed the route of glycerol degradation via polyol dehydrogenase and the novel enzyme, glyceraldehyde kinase. Proton magnetic resonance imaging of freezing and thawing in whole frogs showed a new adaptive effect of the very high glucose levels in core organs; during thawing, organs such as liver and heart melted first, allowing recovery of their vital functions to begin while the rest of the frog thawed. New studies have examined signal transduction in the stimulation of glucose production by wood frog liver, revealing the key role of beta-adrenergic receptors and cAMP-mediated activation of glycogenolysis for cryoprotectant synthesis. The seasonal elevation of plasma membrane glucose transporters was also shown to be key to cryoprotectant distribution during freezing. Other new work has shown that frog freeze tolerance probably grew out of preexisting mechanisms of amphibian dehydration tolerance and that both freeze-tolerant and -intolerant frogs show a hyperglycemic response to desiccation at 5 degrees C.