Glycerol suppresses glucose consumption in trypanosomes through metabolic contest

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as “catabolite repression,” allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named “metabolic contest” for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This “metabolic contest” depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.

This study shows that Trypanosomes use a "metabolic contest" for the regulation of nutrient utilization based on the competition between two enzymes for a common substrate, instead of the well known "catabolite repression" used by most microorganisms.     

A novel pathway for lipid biosynthesis: the direct acylation of glycerol.

The acylation of glycerol-3-phosphate by acyl-CoA is regarded as the first committed step for the synthesis of the lipoidal moiety in glycerolipids. The direct acylation of glycerol in mammalian tissues has not been demonstrated. In this study, lipid biosynthesis in myoblasts and hepatocytes was reassessed by conducting pulse-chase experiments with [1,3-(3)H]glycerol. The results demonstrated that a portion of labeled glycerol was directly acylated to form monoacylglycerol and, subsequently, diacylglycerol and triacylglycerol. The direct acylation of glycerol became more prominent when the glycerol-3-phosphate pathway was attenuated or when exogenous glycerol levels became elevated. Glycerol:acyl-CoA acyltransferase activity, which is responsible for the direct acylation of glycerol, was detected in the microsomal fraction of heart, liver, kidney, skeletal muscle, and brain tissues. The enzyme from pig heart microsomes displayed optimal activity at pH 6.0 and the preference for arachidonyl-CoA as the acyl donor. The apparent K(m) values for glycerol and arachidonyl-CoA were 1.1 mM and 0.17 mM, respectively. The present study demonstrates the existence of a novel lipid biosynthetic pathway that may be important during hyperglycerolemia produced in diabetes or other pathological conditions.     

Positional specificity and substrate preference of purified Staphylococcus aureus lipase

We have studied the substrate preference and specificity, including positional specificity, of a lipase purified from Staphylococcus aureus (strain FN 37). This extracellular bacterial enzyme is relatively insensitive to product inhibition, and hydrolyzes tri-, di- and monooleoylglycerol in emulsified and micellar form at similar rates and without marked substrate preference. The lipase lacks positional specificity, and the hydrolysis of triacylglycerol proceeds rapidly to free fatty acid and glycerol without accumulation of intermediary products.     

Molar concentrations of sorbitol and polyethylene glycol inhibit the Plasmodium aquaglyceroporin but not that of E. coli: Involvement of the channel vestibules

The aquaglyceroporins of Escherichia coli, EcGlpF, and of Plasmodium falciparum, PfAQP, are probably the best characterized members of the solute-conducting aquaporin (AQP) subfamily. Their crystal structures have been elucidated and numerous experimental and theoretical analyses have been conducted. However, opposing reports on their rates of water permeability require clarification. Hence, we expressed EcGlpF and PfAQP in yeast, prepared protoplasts, and compared water and glycerol permeability of both aquaglyceroporins in the presence of different osmolytes, i.e. sucrose, sorbitol, PEG300, and glycerol. We found that water permeability of PfAQP strongly depends on the external osmolyte, with full inhibition by sorbitol, and increasing water permeability when glycerol, PEG300, and sucrose were used. EcGlpF expression did not enhance water permeability over that of non-expressing control protoplasts regardless of the osmolyte. Glycerol permeability of PfAQP was also inhibited by sorbitol, but to a smaller extent, whereas EcGlpF conducted glycerol independently of the osmolyte. Mixtures of glycerol and urea passed PfAQP equally well under isosmotic conditions, whereas under hypertonic conditions in a countercurrent with water, glycerol was clearly preferred over urea. We conclude that PfAQP has high and EcGlpF low water permeability, and explain the inhibiting effect of sorbitol on PfAQP by its binding to the extracellular vestibule. The preference for glycerol under hypertonic conditions implies that in a physiological setting, PfAQP mainly acts as a water/glycerol channel rather than a urea facilitator.     

Water structure in vitro and within Saccharomyces cerevisiae yeast cells under conditions of heat shock

The OH stretch mode from water and organic hydroxyl groups have strong infrared absorption, the position of the band going to lower frequency with increased H-bonding. This band was used to study water in trehalose and glycerol solutions and in genetically modified yeast cells containing varying amounts of trehalose. Concentration-dependent changes in water structure induced by trehalose and glycerol in solution were detected, consistent with an increase of lower-energy H-bonds and interactions at the expense of higher-energy interactions. This result suggests that these molecules disrupt the water H-bond network in such a way as to strengthen molecule-water interactions while perturbing water-water interactions. The molecule-induced changes in the water H-bond network seen in solution do not translate to observable differences in yeast cells that are trehalose-deficient and trehalose-rich. Although comparison of yeast with low and high trehalose showed no observable effect on intracellular water structure, the structure of water in cells is different from that in bulk water. Cellular water exhibits a larger preference for lower-energy H-bonds or interactions over higher-energy interactions relative to that shown in bulk water. This effect is likely the result of the high concentration of biological molecules present in the cell. The ability of water to interact directly with polar groups on biological molecules may cause the preference seen for lower-energy interactions.     

Specificity and glyceride synthesis by mycelial lipases of Rhizopus delemar

Mycelial lipase activity of the mould Rhizopus delemar was purified by gel filtration chromatography to three distinct proteins of notable lipase activity. The three enzymes were designated A′, B′ and C′, according to elution volumes from a Sephadex G150 column. The capacity of the three lipases to catalyse glyceride synthesis from free fatty acids and glycerol indicated a tendency towards short-chain and unsaturated fatty acids in preference to long-chain saturated fatty acids. The postional specificity of all lipases involved in such synthetic reactions indicated the formation of ester bonds at positions 1 and 3 of glycerol.     

A study of the effect of solvent composition and viscosity on the molecular dynamics of human serum albumin using Rayleigh scattering of M?ssbauer radiation

By means of RSMR changes of human serum albumen intramolecular mobility by addition of 1.5% and 7.5% of glutar dialdehyde (GD) in concentrated protein solution, heat denaturation of a protein or substitution of water by water-glycerol solvent with amount of water to glycerol: 1 to 2 were studied. It is shown that the elastic fraction for HSA is changed much less addition of GD or by heat denaturation than by substitution of water solution by water-glycerol. It seems that the observed strong influence of glycerol on intramolecular mobility of HSA is connected mostly with effective dehydration of protein (by substitution of the part of a water solvent by glycerol) and with a small volume decrease of protein (due to preference hydration effect) rather than with the increase of the solvent viscosity.     

Water structure in vitro and within Saccharomyces cerevisiae yeast cells under conditions of heat shock

The OH stretch mode from water and organic hydroxyl groups have strong infrared absorption, the position of the band going to lower frequency with increased H-bonding. This band was used to study water in trehalose and glycerol solutions and in genetically modified yeast cells containing varying amounts of trehalose. Concentration-dependent changes in water structure induced by trehalose and glycerol in solution were detected, consistent with an increase of lower-energy H-bonds and interactions at the expense of higher-energy interactions. This result suggests that these molecules disrupt the water H-bond network in such a way as to strengthen molecule–water interactions while perturbing water–water interactions. The molecule-induced changes in the water H-bond network seen in solution do not translate to observable differences in yeast cells that are trehalose-deficient and trehalose-rich. Although comparison of yeast with low and high trehalose showed no observable effect on intracellular water structure, the structure of water in cells is different from that in bulk water. Cellular water exhibits a larger preference for lower-energy H-bonds or interactions over higher-energy interactions relative to that shown in bulk water. This effect is likely the result of the high concentration of biological molecules present in the cell. The ability of water to interact directly with polar groups on biological molecules may cause the preference seen for lower-energy interactions.     

Glycerol-3-phosphatase of Corynebacterium glutamicum

Formation of glycerol as by-product of amino acid production by Corynebacterium glutamicum has been observed under certain conditions, but the enzyme(s) involved in its synthesis from glycerol-3-phosphate were not known. It was shown here that cg1700 encodes an enzyme active as a glycerol-3-phosphatase (GPP) hydrolyzing glycerol-3-phosphate to inorganic phosphate and glycerol. GPP was found to be active as a homodimer. The enzyme preferred conditions of neutral pH and requires Mg2? or Mn2? for its activity. GPP dephosphorylated both L- and D-glycerol-3-phosphate with a preference for the D-enantiomer. The maximal activity of GPP was estimated to be 31.1 and 1.7 U mg?1 with K(M) values of 3.8 and 2.9 mM for DL- and L-glycerol-3-phosphate, respectively. For physiological analysis a gpp deletion mutant was constructed and shown to lack the ability to produce detectable glycerol concentrations. Vice versa, gpp overexpression increased glycerol accumulation during growth in fructose minimal medium. It has been demonstrated previously that intracellular accumulation of glycerol-3-phosphate is growth inhibitory as shown for a recombinant C. glutamicum strain overproducing glycerokinase and glycerol facilitator genes from E. coli in media containing glycerol. In this strain, overexpression of gpp restored growth in the presence of glycerol as intracellular glycerol-3-phosphate concentrations were reduced to wild-type levels. In C. glutamicum wild type, GPP was shown to be involved in utilization of DL-glycerol-3-phosphate as source of phosphorus, since growth with DL-glycerol-3-phosphate as sole phosphorus source was reduced in the gpp deletion strain whereas it was accelerated upon gpp overexpression. As GPP homologues were found to be encoded in the genomes of many other bacteria, the gpp homologues of Escherichia coli (b2293) and Bacillus subtilis (BSU09240, BSU34970) as well as gpp1 from the plant Arabidosis thaliana were overexpressed in E. coli MG1655 and shown to significantly increase GPP activity.     

Solubilization and hydrophobicity test by Triton X-114-partitioning of NADPH-protochlorophyllide oxidoreductase from the unicellular alga Scenedesmus obliquus, mutant C-2A'

NADPH-protochlorophyllide oxidoreductase (PChilde reductase, EC 1.3.1.33), a key enzyme in light-dependent greening and the conversion of etioplasts into chloroplasts was investigated in the the greening mutant C-2A' of the unicellular green alga Scenedesmus obliquus. In the absence of detergent, the solubilization of the enzyme increased with high glycerol concentrations in the buffer. Solubilization capacities of 4 non-ionic or zwitterionic detergents, Triton X-100, CHAPS, octylglucoside and decyl-maltopyranoside, were compared. Due to the addition of these detergents, the enzyme activity in the soluble fraction was increased severalfold. Hydrophobicity of the enzyme was analyzed by Triton X-114 phase partitioning. The protein had a preference for the aqueous phase, but its distribution was strongly influenced by the glycerol concentration of the buffer. These results indicate that the PChlide reductase of the green alga Scenedesmus obliquus is a hydrophobic, membrane-associated enzyme, but not an integral membrane protein.     

Complete denitration of nitroglycerin by bacteria isolated from a washwater soakaway

Four axenic bacterial species capable of biodegrading nitroglycerin (glycerol trinitrate [GTN]) were isolated from soil samples taken from a washwater soakaway at a disused GTN manufacturing plant. The isolates were identified by 16S rRNA gene sequence homology as Pseudomonas putida, an Arthrobacter species, a Klebsiella species, and a Rhodococcus species. Each of the isolates utilized GTN as its sole nitrogen source and removed nitro groups sequentially from GTN to produce glycerol dinitrates and mononitrates (GMN), with the exception of the Arthrobacter strain, which achieved removal of only the first nitro group within the time course of the experiment. The Klebsiella strain exhibited a distinct preference for removal of the central nitro group from GTN, while the other five strains exhibited no such regioselectivity. All strains which removed a second nitro group from glycerol 1,2-dinitrate showed regiospecific removal of the end nitro group, thereby producing glycerol 2-mononitrate. Most significant was the finding that the Rhodococcus species was capable of removing the final nitro group from GMN and thus achieved complete biodegradation of GTN. Such complete denitration of GTN has previously been shown only in mixed bacterial populations and in cultures of Penicillium corylophilum Dierckx supplemented with an additional carbon and nitrogen source. Hence, to the best of our knowledge, this is the first report of a microorganism that can achieve complete denitration of GTN.     

Redesign of coenzyme B(12) dependent diol dehydratase to be resistant to the mechanism-based inactivation by glycerol and act on longer chain 1,2-diols

Coenzyme B(12) dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co-C bond. Bachovchin et al. [Biochemistry16, 1082-1092 (1977)] reported that glycerol bound in the G(S) conformation, in which the pro-S-CH(2) OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)-1,2-propanediol, i.e. in the G(S) conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. k(inact) of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. k(cat) /k(inact) showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols. Database Structural data are available in the Protein Data Bank under the accession number 3AUJ. Structured digital abstract ? Diol dehydrase gamma subunit, Diol dehydrase beta subunit and Diol dehydrase alpha subunit physically interact by X-ray crystallography (View interaction).     

Location and orientation of serotonin receptor 1a agonists in model and complex lipid membranes

Magic angle spinning (MAS) NMR has been used to investigate the location and orientation of five serotonin receptor 1a agonists (serotonin, buspirone, quipazine, 8-OH-DPAT, and LY-163,165) in single component model lipid and brain lipid membranes. The agonist locations are probed by monitoring changes in the lipid proton chemical shifts and by MAS-assisted nuclear Overhauser enhancement spectroscopy, which indicates the orientation of the agonists with respect to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids. In the single component bilayer, the membrane agonists are found predominantly in the top of the hydrophobic chain or in the glycerol region of the membrane. Most of the agonists orient approximately parallel to the membrane plane, with the exception of quipazine, whose piperazine ring is found in the glycerol region, whereas its benzene ring is located within the lipid hydrophobic chain. The location of the agonist in brain lipid membranes is similar to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayers; however, many of the agonists appear to locate close to the cholesterol in the membrane in preference to the phospholipids.     

A DNA endonuclease isolated from yeast nuclear extract.

We have isolated and partially purified a DNA endonuclease from nuclei of the yeast Saccharomyces cerevisiae. Although purified on the basis of its ability to degrade denatured DNA, the enzyme can also attack native DNA. Denatured oligonucleotide products of the enzyme are sensitive to venom phosphodiesterase (EC3.1.4.1.) but not to bovine spleen phosphodiesterase (EC3.1.4.18). The enzyme has an estimated molecular weight of 6.6--7.5 X 10(4), more than twice as large as the endonucleases involved in DNA repair in Escherichia coli. When analyzed on glycerol gradients, the endonuclease sedimented as a single activity against both denatured DNA and closed circular DNA duplexes. The enzyme showed a 10-fold preference for denatured over native T7 DNA substrate, and appears to produce random nicks in a supercoiled replicative form of phiX174 DNA (RFI) with no discernable preference for the unpaired bases in the supercoiled duplex. The endonuclease appears to be distinct from the yeast endonucleases previously described.     

Complete Denitration of Nitroglycerin by Bacteria Isolated from a Washwater Soakaway

Four axenic bacterial species capable of biodegrading nitroglycerin (glycerol trinitrate [GTN]) were isolated from soil samples taken from a washwater soakaway at a disused GTN manufacturing plant. The isolates were identified by 16S rRNA gene sequence homology as Pseudomonas putida, an Arthrobacter species, a Klebsiella species, and a Rhodococcus species. Each of the isolates utilized GTN as its sole nitrogen source and removed nitro groups sequentially from GTN to produce glycerol dinitrates and mononitrates (GMN), with the exception of the Arthrobacter strain, which achieved removal of only the first nitro group within the time course of the experiment. The Klebsiella strain exhibited a distinct preference for removal of the central nitro group from GTN, while the other five strains exhibited no such regioselectivity. All strains which removed a second nitro group from glycerol 1,2-dinitrate showed regiospecific removal of the end nitro group, thereby producing glycerol 2-mononitrate. Most significant was the finding that the Rhodococcus species was capable of removing the final nitro group from GMN and thus achieved complete biodegradation of GTN. Such complete denitration of GTN has previously been shown only in mixed bacterial populations and in cultures of Penicillium corylophilum Dierckx supplemented with an additional carbon and nitrogen source. Hence, to the best of our knowledge, this is the first report of a microorganism that can achieve complete denitration of GTN.     

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used ³¹P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.     

Determination of carbohydrates, sugar alcohols, and glycols in cell cultures and fermentation broths using high-performance anion-exchange chromatography with pulsed amperometric detection

Cell cultures and fermentation broths are complex mixtures of organic and inorganic compounds. Many of these compounds are synthesized or metabolized by microorganisms, and their concentrations can impact the yields of desired products. Carbohydrates serve as carbon sources for many microorganisms, while sugar alcohols (alditols), glycols (glycerol), and alcohols (methanol and ethanol) are metabolic products. We used high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) to simultaneously analyze for carbohydrates, alditols, and glycerol in growing yeast (Saccharomyces cerevisiae) cultures and their final fermentation broths. Both cultures were grown on complex undefined media, aliquots centrifuged to remove particulates, and the supernatants diluted and directly injected for analysis. Pulsed amperometry allowed a direct detection of the carbohydrates, alditols, and glycols present in the cultures and fermentation broths with very little interference from other matrix components. The broad linear range of three to four orders of magnitude allowed samples to be analyzed without multiple dilutions. Peak area RSDs were 2-7% for 2, 3-butanediol, ethanol, glycerol, erythritol, rhamnose, arabitol, sorbitol, galactitol, mannitol, arabinose, glucose, galactose, lactose, ribose, raffinose, and maltose spiked into a heat-inactivated yeast culture broth supernatant that was analyzed repetitively for 48 h. This method is useful for directly monitoring culture changes during fermentation. The carbohydrates in yeast cultures were monitored over 1 day. A yeast culture with medium consisting primarily of glucose and trace levels of trehalose and arabinose showed a drop in sugar concentration over time and an increase in glycerol. Yeast growing on a modified culture medium consisting of multiple carbohydrates and alditols showed preference for specific carbon sources and showed the ability to regulate pathways leading to catalysis of alternative carbon sources.     

Studies of the genetics, function, and kinetic mechanism of TagE, the wall teichoic acid glycosyltransferase in Bacillus subtilis 168

The biosynthetic enzymes involved in wall teichoic acid biogenesis in gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis.     

Microbial ecology of an Antarctic hypersaline lake: genomic assessment of ecophysiology among dominant haloarchaea

Deep Lake in Antarctica is a cold, hypersaline system where four types of haloarchaea representing distinct genera comprise >70% of the lake community: strain tADL ∼44%, strain DL31 ∼18%, Halorubrum lacusprofundi ∼10% and strain DL1 ∼0.3%. By performing comparative genomics, growth substrate assays, and analyses of distribution by lake depth, size partitioning and lake nutrient composition, we were able to infer important metabolic traits and ecophysiological characteristics of the four Antarctic haloarchaea that contribute to their hierarchical persistence and coexistence in Deep Lake. tADL is characterized by a capacity for motility via flagella (archaella) and gas vesicles, a highly saccharolytic metabolism, a preference for glycerol, and photoheterotrophic growth. In contrast, DL31 has a metabolism specialized in processing proteins and peptides, and appears to prefer an association with particulate organic matter, while lacking the genomic potential for motility. H. lacusprofundi is the least specialized, displaying a genomic potential for the utilization of diverse organic substrates. The least abundant species, DL1, is characterized by a preference for catabolism of amino acids, and is the only one species that lacks genes needed for glycerol degradation. Despite the four haloarchaea being distributed throughout the water column, our analyses describe a range of distinctive features, including preferences for substrates that are indicative of ecological niche partitioning. The individual characteristics could be responsible for shaping the composition of the haloarchaeal community throughout the lake by enabling selection of ecotypes and maintaining sympatric speciation.     

Metabolic modelling of syntrophic-like growth of a 1,3-propanediol producer, Clostridium butyricum, and a methanogenic archeon, Methanosarcina mazei, under anaerobic conditions

Clostridium butyricum can convert glycerol into 1,3-propanediol, thereby generating unfortunately a high amount of acetate, formate and butyrate as inhibiting by-products. We have proposed a novel mixed culture comprising C. butyricum and a methane bacterium, Methanosarcina mazei, to relieve the inhibition and to utilise the by-products for energy production. In order to examine the efficiency of such a mixed culture, metabolic modelling of the culture system was performed in this work. The metabolic networks for the organisms were reconstructed from genomic and physiological data. Several scenarios were analysed to examine the preference of M. mazei in scavenging acetate and formate under conditions of different substrate availability, including methanol as a co-substrate, since it may exist in glycerol solution from biodiesel production. The calculations revealed that if methanol is present, the methane production can increase by 130%. M. mazei can scavenge over 70% of the acetate secreted by C. butyricum.