Glycerol fermentation by (open) mixed cultures: a chemostat study

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.     

Thermal stabilty of glucokinase (GK) as influenced by the substrate glucose, an allosteric glucokinase activator drug (GKA) and the osmolytes glycerol and urea

We investigated how glycerol, urea, glucose and a GKA influence kinetics and stability of wild-type and mutant GK. Glycerol and glucose stabilized GK additively. Glycerol barely affected the TF spectra of all GKs but decreased k(cat), glucose S(0.5) and K(D) values and ATP K(M) while leaving cooperativity unchanged. Glycerol sensitized all GKs to GKA as shown by TF. Glucose increased TF of GKs without influence of glycerol on the effect. Glycerol and GKA affected kinetics and binding additively. The activation energies for thermal denaturation of GK were a function of glucose with K(D)s of 3 and 1mM without and with glycerol, respectively. High urea denatured wild type GK reversibly at 20 and 60°C and urea treatment of irreversibly heat denatured GK allowed refolding as demonstrated by TF including glucose response. We concluded: Glycerol stabilizes GK indirectly without changing the folding structure of the apoenzyme, by restructuring the surface water of the protein, whereas glucose stabilizes GK directly by binding to its substrate site and inducing a compact conformation. Glucose or glycerol (alone or combined) is unable to prevent irreversible heat denaturation above 40°C. However, urea denatures GK reversibly even at 60°C by binding to the protein backbone and directly interacting with hydrophobic side chains. It prevents irreversible aggregation allowing complete refolding when urea is removed. This study establishes the foundation for exploring numerous instability mutants among the more than 600 variant GKs causing diabetes in animals and humans.     

Valorization of biodiesel derived glycerol as a carbon source to obtain added-value metabolites: Focus on polyunsaturated fatty acids

The amount of glycerol derived from the biodiesel industry is exponentially increasing. The valorization of glycerol has acquired attention and resources with an obvious economic and environmental interest. Glycerol has the potential to improve the profitability of biodiesel in a biorefinery scenario. Added-value metabolites obtained from glycerol-based fermentations are the target of multiple research studies, primarily chemicals and biopolymers. Pigments and polyunsaturated fatty acids are exceptional examples as they have market presence as nutraceuticals. Most of the studies reviewed have been based on microalgae cultures. Depending on the strain and the engineering aspects of such cultures the final yield suffers notable variations. This is an emerging field which shows great potential from the perspective of a byproduct usage and the increasing yields (value) obtained from the bioprocess.     

酵母细胞甘油代谢与生理功能研究进展

甘油是酵母细胞生长代谢过程中常见的多元醇物质。尽管甘油的结构简单,代谢途径并不复杂,但是其在细胞内的生理功能十分重要。甘油代谢过程主要参与细胞的高渗透压生理调节和厌氧条件下的胞内氧化还原平衡调节。近年来许多学者在酵母细胞的甘油代谢及生理功能方面开展了深入的研究。在扼要介绍甘油生理代谢的基础上,重点阐述甘油代谢参与细胞高渗压甘油应答信号途径和氧化还原平衡调节的生理机制,同时就酵母细胞甘油合成的代谢工程进行归纳和评述。     

Fermentation of glycerol by Anaerobium acetethylicum and its potential use in biofuel production

Growth of biodiesel industries resulted in increased coproduction of crude glycerol which is therefore becoming a waste product instead of a valuable ‘coproduct’. Glycerol can be used for the production of valuable chemicals, e.g. biofuels, to reduce glycerol waste disposal. In this study, a novel bacterial strain is described which converts glycerol mainly to ethanol and hydrogen with very little amounts of acetate, formate and 1,2‐propanediol as coproducts. The bacterium offers certain advantages over previously studied glycerol‐fermenting microorganisms. Anaerobium acetethylicum during growth with glycerol produces very little side products and grows in the presence of maximum glycerol concentrations up to 1500 mM and in the complete absence of complex organic supplements such as yeast extract or tryptone. The highest observed growth rate of 0.116 h^?1 is similar to that of other glycerol degraders, and the maximum concentration of ethanol that can be tolerated was found to be about 60 mM (2.8 g l^?1) and further growth was likely inhibited due to ethanol toxicity. Proteome analysis as well as enzyme assays performed in cell‐free extracts demonstrated that glycerol is degraded via glyceraldehyde‐3‐phosphate, which is further metabolized through the lower part of glycolysis leading to formation of mainly ethanol and hydrogen. In conclusion, fermentation of glycerol to ethanol and hydrogen by this bacterium represents a remarkable option to add value to the biodiesel industries by utilization of surplus glycerol.     

Purification and properties of a nicotinamide adenine dinucleotide-linked dehydrogenase that serves an Escherichia coli mutant for glycerol catabolism.

Glycerol:NAD+2-OXIDOREDUCTASE (EC 1.1.1.6) was purified to homogeneity from a mutant of Escherichia coli K12 that uses this enzyme, instead of ATP:glycerol 3-phosphotransferase (EC 2.7.1.30), as the first enzyme for the dissimilation of glycerol. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows a subunit of 39,000 daltons. During electrophoresis under nondenaturing conditions, the protein migrates as two bands. These two forms, both of which are enzymatically active, appear to be dimers and octomers of the same subunit. The optimal pH for the oxidation of glycerol is about 10, and that for the reduction of dihydroxyacetone is about 6. Glycerol dehydrogenation is highly activated by NH4+, K+, or Rb+, but strongly inhibited by N-ethylmalemide, 8-hydroxyquinoline, 1,10-phenanthroline, Cu2+, and Ca2+. The enzyme exhibits a broad substrate specificity. In addition to glycerol, it act on 1,2-propanediol and several of its analogs.     

Mechanistic investigation of ultrasonic enhancement of glycerol bioconversion by immobilized Clostridium pasteurianum on silica support

Glycerol, the principal byproduct of biodiesel production, can be a valuable carbon source for bioconversion into diverse class of compounds. This article attempts to investigate the mechanistic aspects of ultrasound mediated bioconversion of glycerol to ethanol and 1,3‐propanediol (1,3‐PDO) by immobilized Clostridium pasteurianum cells on silica support. Our approach is of coupling experimental results with simulations of cavitation bubble dynamics and enzyme kinetics. In addition, the statistical analysis (ANOVA) of experimental results was also done. The glycerol uptake by cells was not affected by either immobilization or with ultrasonication. Nonetheless, both immobilization and ultrasonication were found to enhance glycerol consumption. The enhancement effect of ultrasound on glycerol consumption was most marked (175%) at the highest glycerol concentration of 25 g/L (271.7 mM). The highest glycerol consumption (32.4 mM) was seen for 10 g/L (108.7 mM) initial glycerol concentration. The immobilization of cells shifted the metabolic pathway almost completely towards 1,3‐PDO. No formation of ethanol was seen with mechanical shaking, while traces of ethanol were detected with ultrasonication. On the basis of analysis of enzyme kinetics parameters, we attribute these results to increased substrate‐enzyme affinity and decreased substrate inhibition for 1,3‐PDO dehydrogenase in presence of ultrasound that resulted in preferential conversion of glycerol into 1,3‐PDO. Biotechnol. Bioeng. 2013; 110: 1637–1645. © 2013 Wiley Periodicals, Inc.     

Uptake and dissimilation of glycerol by wild type and glycerol nonutilizing strains of Neurospora crassa

Summary Seven mutant strains defective for utilization of glycerol, glyceraldehyde or dihydroxyacetone were isolated. One strain was deficient for NAD-linked glycerol-3-phosphate dehydrogenase, two for glycerol kinase, and four had no detected enzymatic deficiency, although one of the latter strains was deficient in glycerol uptake. Glycerol uptake was increased by incubation in glycerol, glycerol-3-phosphate, erythritol, and propanediol, and was protein-mediated below 0.14 mM glycerol, but at higher concentrations free diffusion predominated. Glycerol uptake was decreased by cycloheximide and was more sensitive to sodium azide than to iodoacetate.     

Hyperhydration with glycerol solutions

Glycerol was tested as an agent to promote hyperhydration of male and female subjects. Series I experiments involved ingesting 0.5, 1.0, or 1.5 g glycerol/kg body wt and within 40 min drinking 0.1% NaCl, 21.4 ml/kg. In series II, 1.0 g glycerol/kg body wt was ingested at time 0, and 25.7 ml/kg of 0.1% NaCl was ingested over a 3.5-h period. Experiments were of 4-h duration and included controls without glycerol as each subject served as his/her control. Blood samples were taken at 40- or 60-min intervals for hemoglobin (Hb), hematocrit (Hct), plasma osmolality, glycerol, and multiple blood chemistry analyses. Urine was collected at 60-min intervals. Glycerol ingestion increased plasma osmolality for 2 h and reduced the total 4-h urine volume. There were no significant changes in Hb or Hct as a result of the glycerol or excess fluid intake. This study demonstrates that glycerol plus excess fluid intake can produce hyperhydration for at least 4 h.     

Fps1p channel is the mediator of the major part of glycerol passive diffusion in Saccharomyces cerevisiae: artefacts and re-definitions

Glycerol has been shown to cross the plasma membrane of Saccharomyces cerevisiae through (1) a H(+)/symport detected in cells grown on non-fermentable carbon sources, (2) the constitutively expressed Fps1p channel and (3) by passive diffusion. The Fps1p channel has been named a facilitator for mediating glycerol low affinity transport of the facilitated diffusion type. We present experimental evidence that this kinetic is an artefact created by glycerol kinase activity. Instead, the channel is shown to mediate the major part of glycerol's passive diffusion. This is not incompatible with Fps1p's major role in vivo, which has been previously shown to be the control of glycerol export under osmotic stress or in reaction to turgor changes. We also verified that FPS1 overexpression caused an increase in H(+)/symport V(max). Furthermore, yfl054c and fps1 mutants were equally affected by exogenously added ethanol, being the correspondent passive diffusion stimulated. For the first time, to our knowledge, a phenotype attributed to the functioning of YFL054c gene is presented. Glycerol passive diffusion is thus apparently channel-mediated. This is discussed according to glycerol's chemical properties, which contradict the widely spread concept of glycerol's liposoluble nature. The discussion considers the multiple roles that the intracellular levels of glycerol and its pathway regulation might play as a central key to metabolism control.     

Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B12-dependent glycerol and diol dehydratases.

Glycerol and diol dehydratases are inducible, coenzyme B12-dependent enzymes found together in Klebsiella pneumoniae ATCC 25955 during anaerobic growth on glycerol. Mutants of this strain isolated by a novel procedure were separately constitutive for either dehydratase, showing the structural genes for the two enzymes to be under independent control in vivo. Glycerol dehydratase and a trimethylene glycol dehydrogenase were implicated as members of a pleiotropic control system that includes glycerol dehydrogenase and dihydroxyacetone kinase for the anaerobic dissimilation of glycerol (the "dha system"). The dehydratase and dehydrogenases were induced by dihydroxyacetone and were jointly constitutive in mutants isolated as constitutive for either the dha system or glycerol dehydratase. These data and the stimulation of growth by Co2+ suggested that glycerol dehydratase and trimethylene glycol dehydrogenase are obligatory enzymes for anaerobic growth on glycerol as the sole carbon source.     

Effects of modulation of glycerol kinase expression on lipid and carbohydrate metabolism in human muscle cells.

Glycerol is taken up by human muscle in vivo and incorporated into lipids, but little is known about regulation of glycerol metabolism in this tissue. In this study, we have analyzed the role of glycerol kinase (GlK) in the regulation of glycerol metabolism in primary cultured human muscle cells. Isolated human muscle cells exhibited lower GlK activity than fresh muscle explants, but the activity in cultured cells was increased by exposure to insulin. [U-(14)C]Glycerol was incorporated into cellular phospholipids and triacylglycerides (TAGs), but little or no increase in TAG content or lactate release was observed in response to changes in the medium glycerol concentration. Adenovirus-mediated delivery of the Escherichia coli GlK gene (AdCMV-GlK) into muscle cells caused a 30-fold increase in GlK activity, which was associated with a marked rise in the labeling of phospholipid or TAG from [U-(14)C]glycerol compared with controls. Moreover, GlK overexpression caused [U-(14)C]glycerol to be incorporated into glycogen, which was dependent on the activation of glycogen synthase. Co-incubation of AdCMV-GlK-treated muscle cells with glycerol and oleate resulted in a large accumulation of TAG and an increase in lactate production. We conclude that GlK is the limiting step in muscle cell glycerol metabolism. Glycerol 3-phosphate is readily used for TAG synthesis but can also be diverted to form glycolytic intermediates that are in turn converted to glycogen or lactate. Given the high levels of glycerol in muscle interstitial fluid, these finding suggest that changes in GlK activity in muscle can exert important influences on fuel deposition in this tissue.     

Microbial utilization of crude glycerol for the production of value-added products

Energy fuels for transportation and electricity generation are mainly derived from finite and declining reserves of fossil hydrocarbons. Fossil hydrocarbons are also used to produce a wide range of organic carbon-based chemical products. The current global dependency on fossil hydrocarbons will not be environmentally or economically sustainable in the long term. Given the future pessimistic prospects regarding the complete dependency on fossil fuels, political and economic incentives to develop carbon neutral and sustainable alternatives to fossil fuels have been increasing throughout the world. For example, interest in biodiesel has undergone a revival in recent times. However, the disposal of crude glycerol contaminated with methanol, salts, and free fatty acids as a by-product of biodiesel production presents an environmental and economic challenge. Although pure glycerol can be utilized in the cosmetics, tobacco, pharmaceutical, and food industries (among others), the industrial purification of crude glycerol is not economically viable. However, crude glycerol could be used as an organic carbon substrate for the production of high-value chemicals such as 1,3-propanediol, organic acids, or polyols. Microorganisms have been employed to produce such high-value chemicals and the objective of this article is to provide an overview of studies on the utilization of crude glycerol by microorganisms for the production of economically valuable products. Glycerol as a by-product of biodiesel production could be used a feedstock for the manufacture of many products that are currently produced by the petroleum-based chemical industry.     

Glycerol kinase activity and glycerol metabolism of rat granular pneumocytes in primary culture

Glycerol kinase activity and glycerol utilization by rat granular pneumocytes were determined in order to investigate the rate-limiting step for glycerol incorporation into lung lipids. Granular pneumocytes were isolated in primary culture following trypsinization of rat lungs. Glycerol kinase activity was 8.2 nmol/h per 10(6) cells. Incorporation of [1,3-14C]glycerol into total cell lipids was 0.29 nmol/h per 10(6) cells. In the presence of saturating glycerol concentration, production of 3H2O from [2-3H]glycerol was 13 times greater than incorporation of [14C]glycerol into lipids. Glycerol phosphate dehydrogenase activity in isolated cells was approximately 10 times glycerol kinase activity. In the presence of 5.6 mM glucose, glycerol incorporation into lipids was decreased 79% and detritiation of glycerol was decreased 34%. This effect of glucose was due to a 25% increase in cell glycerol 3-phosphate content, resulting in dilution of the precursor pool and possible inhibition of glycerol phosphorylation. These results indicate that the relatively limited incorporation of glycerol into surfactant phospholipids by lung epithelial cells reflects the relatively high rate of glycerol 3-phosphate oxidation.     

Control of glycerol production by rainbow smelt (Osmerus mordax) to provide freeze resistance and allow foraging at low winter temperatures

The rainbow smelt (Osmerus mordax) is a small anadromous fish that actively feeds under the ice at temperatures as low as the freeze point of seawater. Freezing is avoided through the production of both non-colligative antifreeze protein (AFP) and glycerol that acts in a colligative manner. Glycerol is constantly lost across the gills and skin, thus glycerol production must continue on a sustained basis at low winter temperatures. AFP begins to accumulate in early fall while water temperatures are still high. Glycerol production is triggered when water temperatures decrease to about 5 degrees C. Glycerol levels rapidly increase with carbon flow from dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) to glycerol. Glucose/glycogen serves as the initial carbon source for glycerol accumulation with amino acids contributing thereafter. The period of glycerol accumulation is associated with increases in GPDH mRNA and PEPCK mRNA followed by elevations in protein synthesis and enzyme activities. Plasma glycerol levels may reach in excess of 500 mM in winter. The high freeze resistance allows rainbow smelt to invade water of low temperature and forage for food. The lower the temperature, the higher the glycerol must be, and the higher the glycerol the greater the loss to the environment through diffusion. During the winter, rainbow smelt feed upon protein rich invertebrates with glycerol production being fueled in part by dietary amino acids via the gluconeogenic pathway. At winter temperatures, glycerol is quantitatively more important than AFP in providing freeze resistance of blood; however, the importance of AFPs to other tissues is yet to be assessed. Glycerol levels rapidly plummet in the spring when water temperature is still close to 0 degrees C. During this period, freeze resistance must be provided by AFP alone. Overall, the phenomenon of glycerol production by rainbow smelt reveals an elegant connection of biochemistry to ecology that allows this species to exploit an otherwise unavailable food resource.     

Improvement of the catalytic performance of glycerol kinase from Bacillus subtilis by chromosomal site-directed mutagenesis

Biotechnology Letters - Glycerol kinase is the key enzyme in glycerol metabolism, and its catalytic efficiency has an important effect on glycerol utilization. Based on an analysis of the glycerol...     

Glycerol: a neglected variable in metabolic processes?

Glycerol is a small and simple molecule produced in the breakdown of glucose, proteins, pyruvate, triacylglycerols and other glycerolipid, as well as release from dietary fats. An increasing number of observations show that glycerol is probably involved in a surprising variety of physiopathologic mechanisms. Glycerol has long been known to play fundamental roles in several vital physiological processes, in prokaryotes and eukaryotes, and is an important intermediate of energy metabolism. Despite some differences in the details of their operation, many of these mechanisms have been preserved throughout evolution, demonstrating their fundamental importance. In particular, glycerol can control osmotic activity and crystal formation and then act as a cryoprotective agent. Furthermore, its properties make it useful in numerous industrial, therapeutic and diagnostic applications. Few studies have focussed directly on glycerol, however, and while its metabolism is increasingly well documented, much of the details remain unknown. Considering the importance of glycerol in multiple vital physiological processes, its study could help unlock important physiopathological mechanisms.     

Production and characterization of poly‐3‐hydroxybutyrate from biodiesel‐glycerol by Burkholderia cepacia ATCC 17759

Glycerol, a byproduct of the biodiesel industry, can be used by bacteria as an inexpensive carbon source for the production of value‐added biodegradable polyhydroxyalkanoates (PHAs). Burkholderia cepacia ATCC 17759 synthesized poly‐3‐hydroxybutyrate (PHB) from glycerol concentrations ranging from 3% to 9% (v/v). Increasing the glycerol concentration results in a gradual reduction of biomass, PHA yield, and molecular mass (M_n and M_w) of PHB. The molecular mass of PHB produced utilizing xylose as a carbon source is also decreased by the addition of glycerol as a secondary carbon source dependent on the time and concentration of the addition. ¹H‐NMR revealed that molecular masses decreased due to the esterification of glycerol with PHB resulting in chain termination (end‐capping). However, melting temperature and glass transition temperature of the end‐capped polymers showed no significant difference when compared to the xylose‐based PHB. The fermentation was successfully scaled up to 200 L for PHB production and the yield of dry biomass and PHB were 23.6 g/L and 7.4 g/L, respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effects of glycerol on VLDL secretion by the isolated rat liver.

Stimulation of VLDL production by increasing fatty acid availability is now well established. However, a possible regulatory role of glycerol, another lipid precursor, in VLDL synthesis by the liver has not yet been substaniated. The present experiments investigate this problem using the isolated perfused rat liver. [14C] Glycerol uptake and metabolism were studied at two different glycerol concentrations: 1 mumol/perfusate (control) or 1.6 mmol/perfusate. VLDL production and lipid synthesis were investigated using [14C]leucine and several labelled fatty acids as precursors in control and glycerol-overloaded livers. Neoglycogenesis and lipogenesis from glycerol carbons are negligible in our conditions. The absolute amount of glycerol, but not the precentage, taken up by the liver, increased after raising its concentration in the perfusate. A major part of exogenous (plasmatic) glycerol was esterified with endogenous (non plasmatic) fatty acids. Incorporation of radioactive fatty acids into liver or plasma lipids was lower than in the the control group. Significant differences were observed between saturated and unsaturated fatty acids used as lipid precursors. Production of VLDL as assessed by radioactive leucine and fatty acid incorporation in the VLDL of the perfusate was depressed by glycerol. Glycerol partly inhibits the normal stimulation of VLDL production by plasmatic fatty acid overload.     

Physiological studies on regulation of glycerol utilization by the phosphoenolpyruvate:sugar phosphotransferase system in Enterococcus faecalis.

In vitro studies with purified glycerol kinase from Enterococcus faecalis have established that this enzyme is activated by phosphorylation of a histidyl residue in the protein, catalyzed by the phosphoenolpyruvate-dependent phosphotransferase system (PTS), but the physiological significance of this observation is not known. In the present study, the regulation of glycerol uptake was examined in a wild-type strain of E. faecalis as well as in tight and leaky ptsI mutants, altered with respect to their levels of enzyme I of the PTS. Glycerol kinase was shown to be weakly repressible by lactose and strongly repressible by glucose in the wild-type strain. Greatly reduced levels of glycerol kinase activity were also observed in the ptsI mutants. Uptake of glycerol into intact wild-type and mutant cells paralleled the glycerol kinase activities in extracts. Glycerol uptake in the leaky ptsI mutant was hypersensitive to inhibition by low concentrations of 2-deoxyglucose or glucose even though the rates and extent of 2-deoxyglucose uptake were greatly reduced. These observations provide strong support for the involvement of reversible PTS-mediated phosphorylation of glycerol kinase in the regulation of glycerol uptake in response to the presence or absence of a sugar substrate of the PTS in the medium. Glucose and 2-deoxyglucose were shown to elicit rapid efflux of cytoplasmic [14C]lactate derived from [14C]glycerol. This phenomenon was distinct from the inhibition of glycerol uptake and was due to phosphorylation of the incoming sugar by cytoplasmic phosphoenolpyruvate. Lactate appeared to be generated by sequential dephosphorylation and reduction of cytoplasmic phosphoenolpyruvate present in high concentrations in resting cells. The relevance of these findings to regulatory phenomena in other bacteria is discussed.