The caldariella group of extreme thermoacidophile bacteria: Direct comparison of lipids in Sulfolobus,Thermoplasma, and the MT strains

It is shown that the lipids from 5 extreme thermoacidophile bacteria of the Caldariella group—2 isolates of Sulfolobus acidocaldarius, one of Thermoplasma acidophila, and 2 of the MT series—are all based on the same type of cyclic diether combining glycerol and one of a series of very unusual C₄₀ isoprenoid diols. The relative proportions of the different C₄₀ components in each isolate have been determined.     

Analysis of lipids from a hydrothermal vent methanogen and associated vent sediment by supercritical fluid chromatography

Lipids from a thermophilic methanogen and the associated hydrothermal vent sediment (Guaymas Basin, Gulf of California) were analyzed by gas chrmotography-mass spectrometry (GC-MS) and supercritical fluid chromatography (SFC). The neutral lipids of the thermophilic methagonen consisted of straight chain alkanes (nC₂₂ to nC₃₆), with nC₂₄, nC₂₈, nC₃₂ and nC₃₆ predominating and C₂₅, C₃₀ and C₃₅-isoprenoids and hydroisoprenoids. The squalene (C₃₀) series was the most abundant (95.6%). The backbone structure of the novel C₃₅-isoprenouds was tentatively identified as 2,6,10,14,19,23,27-heptamethyloctacosane. Polar lipids of the thermophilic methanogen were analyzed by SFC and consisted fo diphytanyl glyceril diether (61.6%), macrocyclic glycerol diether (15.3%), dibiphytanyl diglycerol tetraether (11.8%) and an unidentified component (11.4%).Biomarker analysis of the Guaymas Basin sediment revealed the presence of small amounts of polyunsaturated C₃₀-isoprenoids, with a distribution similar to the C₃₀-isoprenoids of the thermophilic methanogen. In addition, the sediment contained ‘free’ diphytanyl glycerol diether as predominant ether lipid. Low levels of polar ether lipids, indicative of ‘active’ archaebacteria, were also detected. Results suggest that Guaymas Basin sediment recently contained active microbial populations with a lipid profile similar to the isolated thermophilic methanogen.     

Comparison of thermophilic methanogens from submarine hydrothermal vents

An extremely thermophilic methanogen was isolated from hydrothermal vent sediment (80°–120° C) collected from the Guaymas Basin, Gulf of California, at a depth of approximately 2000 m. The isolate was a characteristic member of the genus Methanococcus based on its coccoid morphology, ability to produce methane from CO₂ and H₂, and DNA base composition (31.4 mol% G+C); it is distinguished from previously described extremely thermophilic vent methanogens by its ability to grow and produce methane from formate and in the composition of membrane lipids. The temperature range for growth was 48°–94° C (optimum near 85° C); the pH optimum was 6.0. The isolate grew autotrophically but was stimulated by selenium and growth nutrients supplied by yeast extract and trypticase. Extracted polar lipids consisted primarily of diphytanyl glycerol diether (62%), macrocyclic glycerol diether (15.3%), and dibiphytanyl glycerol tetraether (11.8%). Neutral lipids were dominated by a series of C₃₀ isoprenoids; in addition, a novel series of C₃₅ isoprenoids were detected. The isolate appears to be a close relative of the previously described Methanococcus jannaschii, isolated from the East Pacific Rise hydrothermal vent system. From the frequency of isolation, it appears that extremely thermophilic methanococci are the predominant representatives of the methanogenic archaebacteria occurring at deep sea hydrothermal vents.     

Chemical structure of the ether lipids of thermophilic acidophilic bacteria of the Caldariella group

The lipids of the Caldariella group of extremely thermophilic acidophilic bacteria are based on a 72-membered macrocyclic tetraether made up from two C₄₀ diol units and either two glycerol units or one glycerol and one nonitol. The C₄₀ components have the 16,16′-biphytanyl skeleton and the detailed structure of three of them is established.     

Thermodesulfobacterium geofontis sp. nov., a hyperthermophilic, sulfate-reducing bacterium isolated from Obsidian Pool, Yellowstone National Park

A novel sulfate-reducing bacterium designated OPF15^T was isolated from Obsidian Pool, Yellowstone National Park, Wyoming. The phylogeny of 16S rRNA and functional genes (dsrAB) placed the organism within the family Thermodesulfobacteriaceae. The organism displayed hyperthermophilic temperature requirements for growth with a range of 70–90 °C and an optimum of 83 °C. Optimal pH was around 6.5–7.0 and the organism required the presence of H₂ or formate as an electron donor and CO₂ as a carbon source. Electron acceptors supporting growth included sulfate, thiosulfate, and elemental sulfur. Lactate, acetate, pyruvate, benzoate, oleic acid, and ethanol did not serve as electron donors. Membrane lipid analysis revealed diacyl glycerols and acyl/ether glycerols which ranged from C_14:0 to C_20:0. Alkyl chains present in acyl/ether and diether glycerol lipids ranged from C_16:0 to C_18:0. Straight, iso- and anteiso-configurations were found for all lipid types. The presence of OPF15^T was also shown to increase cellulose consumption during co-cultivation with Caldicellulosiruptor obsidiansis, a fermentative, cellulolytic extreme thermophile isolated from the same environment. On the basis of phylogenetic, phenotypic, and structural analyses, Thermodesulfobacterium geofontis sp. nov. is proposed as a new species with OPF15^T representing the type strain.     

Signature Lipids and Stable Carbon Isotope Analyses of Octopus Spring Hyperthermophilic Communities Compared with Those of Aquificales Representatives

The molecular and isotopic compositions of lipid biomarkers of cultured Aquificales genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring. Thermocrinis ruber, Thermocrinis sp. strain HI 11/12, Hydrogenobacter thermophilus TK-6, Aquifex pyrophilus, and Aquifex aeolicus all contained glycerol-ether phospholipids as well as acyl glycerides. The n-C_20:1 and cy-C₂₁ fatty acids dominated all of the Aquificales, while the alkyl glycerol ethers were mainly C_18:0. These Aquificales biomarkers were major constituents of the lipid extracts of two Octopus Spring samples, a biofilm associated with the siliceous vent walls, and the well-known pink streamer community (PSC). Both the biofilm and the PSC contained mono- and dialkyl glycerol ethers in which C₁₈ and C₂₀ alkyl groups were prevalent. Phospholipid fatty acids included both the Aquificales n-C_20:1 and cy-C₂₁, plus a series of iso-branched fatty acids (i-C_15:0 to i-C_21:0), indicating an additional bacterial component. Biomass and lipids from the PSC were depleted in ¹³C relative to source water CO₂ by 10.9 and 17.2‰, respectively. The C_20–21 fatty acids of the PSC were less depleted than the iso-branched fatty acids, 18.4 and 22.6‰, respectively. The biomass of T. ruber grown on CO₂ was depleted in ¹³C by only 3.3‰ relative to C source. In contrast, biomass was depleted by 19.7‰ when formate was the C source. Independent of carbon source, T. ruber lipids were heavier than biomass (+1.3‰). The depletion in the C_20–21 fatty acids from the PSC indicates that Thermocrinis biomass must be similarly depleted and too light to be explained by growth on CO₂. Accordingly, Thermocrinis in the PSC is likely to have utilized formate, presumably generated in the spring source region.     

Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus

Eighteen fatty acids identified in the cuticle of three insect species representing differing susceptibilities to C. coronatus infection, were tested for effects on the in vitro growth and pathogenicity of the parasitic fungus. At all applied concentrations (0.1-0.0001% w/v) growth was inhibited by C_16:0, C_16:1, C_18:0, C_18:1, C_18:2, C_18:3, C_20:0 and C_20:1. At high concentrations spore germination was inhibited by C_7:0, C_8:0, C_9:0, C_10:0, C_12:0, C_18:2 and C_18:3 and hyphal growth was merely retarded by C_5:0, C_6:0, C_6:2, C_14:0, C_16:0, C_16:1, C_18:0, C_18:1, C_20:0 and C_20:1. The presence of C_15:0 at the 0.1% concentration stimulated growth of C. coronatus. Sporulation was inhibited by all concentrations of C_16:0 and C_18-20 fatty acids. Low concentrations of C_5:0, C_6:0, C_6:2 and C_7:0 enhanced sporulation. Fatty acids C_5-12 as well as C_18:3, C_20:0 and C_20:1 decreased the ability of fungal colonies to infect G. mellonella while C_16:1 elevated it thus suggesting that C_16:1 may stimulate production of enzymes involved in the host invasion. Toxicity of metabolites released into incubation medium decreased with varying degrees in the presence of C_6:0, C_6:2, C_7:0, C_9:0, C_12:0, C_16:1, C_18:2, C_18:3, C_20:0 and C_20:1; other fatty acids had no effect. Further work is needed to analyse the effects of exogenous fatty acids on the C. coronatus enzymes implicated in fungal pathogenicity as well as on the production of insecticidal metabolites.     

Incorporation of labelled glycerols into ether lipids in Caldariella acidophila

The lipids of Caldariella acidophila, an extreme thermophile member of the new archaebacteria group, are macrocyclic tetraethers. They are made up of two glycerol molecules (or one glycerol and one nonitol) bridged through ether linkages by two C₄₀16,16′-biphytanyl chains. To elucidate the biosynthesis of the glycerol moiety of these tetraethers and the mechanism of glycerol ether assembly, labelled [U-¹⁴C, 1(3)-³H]glycerol and [U-¹⁴C, 2-³H]glycerol, were fed to C. acidophila. Both precursors were selectively incorporated with high efficiency, and without any change in the ³H/¹⁴C ratio, in the glycerol moiety of tetraethers. These results suggest that the ether forming step in the biosynthesis of tetraether lipids of C. acidophila, occurs without any loss of hydrogen from any of the glycerol carbons which in turn could be directly alkylated by geranylgeranyl pyrophosphate. The incorporation of radioactivity in the isoprenoid chains and into nonitol is also analysed.     

Purification and characterization of mononocardomycoloylglycerol from Nocardia rhodochrous

A component of the acetone-soluble lipids of Nocordia rhodochrous grown on glycerol, was purified by column chromatography on silicic acid and characterized by infrared, nuclear magnetic resonance spectroscopy, optical rotation measurement and product identification after alkaline hydrolysis. Glycerol was the sole water-soluble component and nocardomycolic acids with chain lengths ranging from C₄₀ to C₄₄ were the constituent fatty acids identified. On the basis of the evidence obtained, the substance isolated from N. rhodochrous is identified as a mixture of mononocardomycoloylglycerols in which nocardomycolic acids are bound to one of the primary hydroxyl groups of the glycerol molecule.     

Long-Chain Glycerol Diether and Polyol Dialkyl Glycerol Triether Lipids of Sulfolobus acidocaldarius

Cells of Sulfolobus acidocaldarius contain about 2.5% total lipid on a dry-weight basis. Total lipid was found to contain 10.5% neutral lipid, 67.6% glycolipid, and 21.7% polar lipid. The lipids contained C(40)H(80) isopranol glycerol diethers. Almost no fatty acids were present. The glycolipids were composed of about equal amounts of the glycerol diether analogue of glucosyl galactosyl diglyceride and a glucosyl polyol glycerol diether. The latter compound contained an unidentified polyol attached by an ether bond to the glycerol diether. The polar lipids contained a small amount of sulfolipid, which appeared to be the monosulfate derivative of glucosyl polyol glycerol diether. About 40% of the lipid phosphorus was found in the diether analogue of phosphatidyl inositol. The remaining lipid phosphorus was accounted for by approximately equal amounts of two inositol monophosphate-containing phosphoglycolipids, inositolphosphoryl glucosyl galactosyl glycerol diether and inositolphosphoryl glucosyl polyol glycerol diether.     

Chemical characterization of the lipophilic extract of Hydrilla verticillata: a widely spread aquatic weed

The composition of the lipophilic extract from Hydrilla verticillata, the common aquatic weed collected from Sasthamkotta Lake, the largest freshwater lake in Kerala, south of the west coast of India, was investigated. The lake is a designated wetland of international importance under the Ramsar Convention since 2002. GC-MS study of the unsaponifiable lipophilic extract of H. verticillata confirmed the presence of 3,5,11,15-tetramethyl 1- hexadecen-3-ol (C₂₀H₄₀O) and phytol (C₂₀H₄₀O) as major components, and their structures were elucidated. Phytol and 3,5,11,15-tetramethyl 1- hexadecen3-ol are the two isomers of the diterpeneol (C₂₀H₄₀O) found in the unsaponifible lipophilic extract of H. verticillata and are formed by the hydrolysis of the alcohol moiety of chlorophyll. On quantification, an appreciable concentration of phytol (6.39 g?Kg^?1) was estimated. The feasibility to utilize H. verticillata to produce phytol is to be addressed by further studies since H. verticillata is considered as one of the world’s fast widely spread aquatic weeds on account of its numerous mechanisms of vegetative reproductions.     

Complex lipids of Rhodomicrobium vannielii

Eight components, seven of which contained phosphorus, were found in the phospholipid fraction of Rhodomicrobium vannielii. The major components were lipoamino acid (o-ornithine ester of phosphatidyl glycerol, 46.5%) and phosphatidyl choline (26.5%). The other six components were phosphatidyl glycerol (9.7%), bisphosphatidic acid (6.7%), phosphatidyl ethanolamine (4.5%), phosphatidic acid (1.8%), lysophosphatidyl glycerol-o-ornithine ester (3.2%), and N,N-ornithine amide of unidentified fatty acid (0.95%). Total phospholipid accounted for 4.2% of cell dry weight. The major fatty acid was vaccenic acid, C_18:1, which accounted for approximately 90% of the total fatty acids of the complex lipid fraction. The other four fatty acids were C_16:0 (6.25%), C_18:0 (3.8%), C_14:0 (0.7%), and C_16:1 (0.35%). The sulfolipid content was 0.01% of the cell dry weight or 0.14 μmoles per g of dried cells, assuming that its fatty acid component is vaccenic acid. No steroids were detected.     

The composition of external lipids from adult whiteflies,Bemisia tabaci and Trialeurodes vaporariorum

The total surface lipids, including the wax particles, of the adult whiteflies of Bemisia tabaci and Trialeurodes vaporariorum were characterized. At eclosion, there were similar amounts of long-chain hydrocarbons, aldehydes, alcohols and wax esters. Within a few hours post-eclosion, long-chain aldehydes and long-chain alcohols were the dominant surface lipid components, C₃₄ on B. tabaci and C₃₂ on T. vaporariorum. Hydrocarbons, mainly n-alkanes, were minor components of the surface lipids. The major wax esters were C₄₆ on B. tabaci and C₄₂ on T. vaporariorum. The major acid and alcohol moieties in the wax esters of B. tabaci were C₂₀ and C₂₆, respectively, and of T. vaporariorum were C₂₀ and C₂₂, respectively. Both B. tabaci and T. vaporariorum had a minor wax ester composed of the fatty acid C_18:1 esterified to the major alcohols, C₃₄ and C₃₂, respectively. Bemisia were readily distinguished from Trialeurodes based on the composition of their wax particles and/or their wax esters; however, no differentiating surface lipid components were detected between biotypes A and B of B. tabaci.     

Gibberellins in Embryo-Suspensor of Phaseolus coccineus Seeds at the Heart Stage of Embryo Development

Gibberellins (GAs) in suspensors and embryos of Phaseolus coccineus seeds at the heart stage of embryo development were analyzed by combined gas chromatography-mass spectrometry (GC-MS). From the suspensor four C₁₉-GAs, GA₁, GA₄, GA₅, GA₆, and one C₂₀ GA, GA₄₄, were identified. From the embryo, five C₁₉-GAs GA₁, GA₄, GA₅, GA₆, GA₆₀ and two C₂₀ GAs, GA₁₉ and GA₄₄ were identified. The data, in relation to previous results, suggest a dependence of the embryo on the suspensor during early stages of development.     

Substrate specificity of the epoxidation reaction in sex pheromone biosynthesis of the Japanese giant looper (Lepidoptera: Geometridae)

Female moths of the Japanese giant looper (Ascotis selenaria cretacea, Lepidoptera: Geometridae) secrete (Z,Z)-6,9-cis-3,4-epoxynonadecadiene as a sex pheromone component. To the pheromone glands of the decapitated females, [19,19,19-D₃](Z,Z,Z)-3,6,9-nonadecatriene was applied after an injection of pheromone biosynthesis activating neuropeptide. GC-MS analysis of the gland extract showed its specific conversion into the pheromonal cis-3,4-epoxide indicating that the C₁₉ triene which had been identified in the gland was a precursor of the pheromone. In order to examine the substrate specificity of the enzyme catalyzing this epoxidation step, several unsaturated hydrocarbons not occurring in the gland were applied to it. Not only (Z,Z,Z)-3,6,9-trienes with varying chain lengths (C₁₇, C₁₈ and C₂₀ to C₂₂) but (Z,Z)-3,6-dienes (C₁₇, C₁₉ and C₂₀) were converted into the corresponding cis-3,4-epoxides in a rather good yield, while no 6,7- and 9,10-epoxides could be detected. (Z)-3-Nonadecene was also changed to the cis-epoxide, but (E)-3-, (Z)-2- and (Z)-4-double bonds in the C₁₉ chain were not oxidized. These in vivo experiments revealed that the monooxygenase regiospecifically attacked the (Z)-3-double bond of straight chain hydrocarbons regardless of their length and degree of unsaturation.     

Ribonuclease activities in bean roots

Vicia faba meristematic and elongating root cells (zones 0–4 and 10–20 mm) contained one nuclease (A₁) and four ribonucleases (A₂, A₃, C₁, C₂). When the overall activity of each enzyme was expressed per cell, the elongating cells contained 4-, 4-, 4-, 10- and 17-fold more activity than meristematic cells for A₁, C₁, C₂, A₂ and A₃, respectively.     

Qualitative and Quantitative Analyses of Gibberellins in Vegetative Shoots of Normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 Seedlings of Zea mays L

Gibberellins A₁₂ (GA₁₂), GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₂₉, GA₁, and GA₈ have been identified from extracts of vegetative shoots of normal (wild type) maize using full scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Seven of these gibberellins (GAs) have been quantified by capillary gas chromatography-selected ion monitoring using internal standards of [¹⁴C₄]GA₅₃, [¹⁴C₄]GA₄₄, [²H₂] GA₁₉, [¹³C₁]GA₂₀, [¹³C₁]GA₂₉, [¹³C₁]GA₁, and [¹³C₁]GA₈. Quantitative data from extracts of normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 seedlings support the operation of the early 13-hydroxylation pathway in vegetative shoots of Zea mays. These data support the positions in the pathway blocked by the mutants, previously assigned by bioassay data and metabolic studies. The GA levels in dwarf-2, dwarf-3, and dwarf-5 were equal to, or less than, 2.0 nanograms per 100 grams fresh weight, showing that these mutants are blocked for steps early in the pathway. In dwarf-1, the level of GA₁ was very low (0.23 nanograms per 100 grams fresh weight) and less than 2% of that in normal shoots, while GA₂₀ and GA₂₉ accumulated to levels over 10 times those in normals; these results confirm that the dwarf-1 mutant blocks the conversion of GA₂₀ to GA₁. Since the level of GAs beyond the blocked step for each mutant is greater than zero, each mutated gene probably codes for an altered gene product, thus leading to impaired enzyme activities.     

Final structure of caulerpicin,a toxin mixture from the green alga Caulerpa racemosa

Caulerpicin from the green alga Caulerpa racemosa is shown to consist of a mixture of ceramides derived from 2S, 3R-sphinganine with C₁₈ (32%), C₂₀(2%), C₂₂(6%), C₂₄(35%) and C₂₆(25%) saturated fatty acid     

Some variations in the composition of suberin from the cork layers of higher plants

The monomeric composition of the suberins from 16 species of higher plants was determined by chromatographic methods following depolymerization of the isolated extractive-free cork layers with sodium methoxide-methanol. 1-Alkanols (mainly C₁₈C₂₈), alkanoic (mainly C₁₆C₃₀), α,ω-alkanedioic (mainly C₁₆C24), ω-hydroxyalkanoic (mainly C₁₆C₂₁), dihydroxyhexadecanoic (mainly 10,16-dihydroxy- and 16-dihydroxyhexadecanoic), monohydroxyepoxyalkanoic (9,10-epoxy-18-hydroxyoctadecanoic), trihydroxyalkanoic (9,10, 18-trihydroxyoctadecanoic), epoxyalkanedioic (9,10-epoxyoctadecane-1,18-dioic) and dihydroxyalkanedioic (9,10-dihydroxyoctadecane-1 18-dioic) acids were detected in all species. The suberins differed from one another mainly in the relative proportions of these monomer classes and in the homologue content of their 1-alkanol, alkanoic, α,ω-alkanedioic and ω-hydroxyalkanoic acid fractions. C₁₈ epoxy and vic-diol monomers were major components (32–59%) of half of the suberins examined (Quercus robur, Q. ilex, Q. suber, Fagus sylvatica, Castanea sativa, Betula pendula, Acer griseum, Fraxinus excelsior) where as ω-hydroxyalkanoic and α,ω-alkanedioic acids predominated in those that contained smaller quantities of such polar C₁₈ monomers (Acer pseudoplatanus, Ribes nigrum, Euonymus alatus, Populus tremula, Solanum tuberosum, Sambucus nigra, Laburnum anagyroides, Cupressus leylandii). All species, however, contained substantial amounts (14–55 %) of ω-hydroxyalkanoic acids, the most common homologues being 18:1 (9) and 22: 0. The dominant α,ω-alkanedioic acid homologues were 16: 0 and 18: 1 (9) whereas 22: 0, 24: 0 and 26: 0, and 20: 0, 22: 0 and 24: 0 were usually the principal homologues in the 1-alkanol and alkanoic acid fractions, respectively. The most diagnostic feature of the suberins examined was the presence of monomers greater than C₁₈ in chain length; most of the C₁₆ and C₁₈ monomers identified in the suberins also occur in plant cutins emphasizing the close chemical similarity between the two anatomical groups of lipid biopolymer.     

Anaerobic Fermentation of Glycerol in Paenibacillus macerans: Metabolic Pathways and Environmental Determinants

Paenibacillus macerans is one of the species with the broadest metabolic capabilities in the genus Paenibacillus, able to ferment hexoses, deoxyhexoses, pentoses, cellulose, and hemicellulose. However, little is known about glycerol metabolism in this organism, and some studies have reported that glycerol is not fermented. Despite these reports, we found that several P. macerans strains are capable of anaerobic fermentation of glycerol. One of these strains, P. macerans N234A, grew fermentatively on glycerol at a maximum specific growth rate of 0.40 h⁻¹ and was chosen for further characterization. The use of [U-¹³C]glycerol and further analysis of extracellular metabolites and proteinogenic amino acids via nuclear magnetic resonance (NMR) spectroscopy allowed identification of ethanol, formate, acetate, succinate, and 1,2-propanediol (1,2-PDO) as fermentation products and demonstrated that glycerol is incorporated into cellular components. A medium formulation with low concentrations of potassium and phosphate, cultivation at acidic pH, and the use of a CO₂-enriched atmosphere stimulated glycerol fermentation and are proposed to be environmental determinants of this process. The pathways involved in glycerol utilization and synthesis of fermentation products were identified using NMR spectroscopy in combination with enzyme assays. Based on these studies, the synthesis of ethanol and 1,2-PDO is proposed to be a metabolic determinant of glycerol fermentation in P. macerans N234A. Conversion of glycerol to ethanol fulfills energy requirements by generating one molecule of ATP per molecule of ethanol synthesized. Conversion of glycerol to 1,2-PDO results in the consumption of reducing equivalents, thus facilitating redox balance. Given the availability, low price, and high degree of reduction of glycerol, the high metabolic rates exhibited by P. macerans N234A are of paramount importance for the production of fuels and chemicals.Although many microorganisms can metabolize glycerol in the presence of external electron acceptors (respiratory metabolism), few are able to do so fermentatively (i.e., in the absence of electron acceptors). Fermentative metabolism of glycerol has been reported in species of the genera Klebsiella, Citrobacter, Enterobacter, Clostridium, Lactobacillus, Bacillus, Propionibacterium, and Anaerobiospirillum but has been studied more extensively in a few species of the family Enterobacteriaceae, namely, Citrobacter freundii and Klebsiella pneumoniae (6, 9). Glycerol fermentation in these organisms is mediated by a two-branch pathway, which results in the synthesis of the glycolytic intermediate dihydroxyacetone (DHA) phosphate (DHAP) and the fermentation product 1,3-propanediol (1,3-PDO) (Fig. ​(Fig.1A)1A) (6). In the oxidative branch, glycerol is dehydrogenated to DHA by a type I NAD-linked glycerol dehydrogenase (glyDH). DHA is then phosphorylated by ATP- or phosphoenolpyruvate (PEP)-dependent DHA kinases (DHAKs) to generate DHAP. In the parallel reductive branch, glycerol is dehydrated by glycerol dehydratase, and 3-hydroxypropionaldehyde (3-HPA) is formed. 3-HPA is then reduced to the major fermentation product 1,3-PDO by an NADH-linked 1,3-PDO dehydrogenase (1,3-PDODH), thereby regenerating NAD⁺ (Fig. ​(Fig.1A).1A). Organisms that lack the capacity to synthesize 1,3-PDO have been deemed unable to utilize glycerol in a fermentative manner (6, 9, 10). The metabolism of glycerol in these organisms is thought to require an electron acceptor and takes place through a respiratory pathway that involves a glycerol kinase and two respiratory (aerobic and anaerobic) glycerol-3-phosphate dehydrogenases (G3PDHs) (6, 7, 24, 29, 35, 38) (Fig. ​(Fig.1B).1B). A recent development in this area is the finding that Escherichia coli, an organism that is unable to produce 1,3-PDO, can indeed ferment glycerol in the absence of external electron acceptors (15, 26). In this model, synthesis of the fermentation products 1,2-PDO and ethanol enables glycerol fermentation by facilitating redox balance and ATP generation, respectively (Fig. ​(Fig.1C)1C) (15). A type II glyDH and a PEP-dependent DHAK mediate the conversion of glycerol to glycolytic intermediates. glyDH also catalyzes the last step in the synthesis of the key fermentation product 1,2-PDO (Fig. ​(Fig.1C1C).Open in a separate windowFIG. 1.Glycerol metabolism in bacteria. (A) 1,3-PDO model for the fermentative utilization of glycerol. (B) Respiratory metabolism of glycerol (i.e., metabolism in the presence of an electron acceptor). (C) 1,2-PDO-ethanol model for the fermentative utilization of glycerol. Dashed lines indicate multiple steps. glyD, glycerol dehydratase; glyDH-I, type I glyDH; GK, glycerol kinase; ae-G3PDH, aerobic G3PDH; an-G3PDH, anaerobic G3PDH; QH₂, reduced quinone; glyDH-II, type II glyDH; FHL, formate hydrogen lyase; ADH, alcohol/acetaldehyde dehydrogenase.Paenibacillus macerans, previously called Bacillus macerans and Bacillus acetoethylicum, is a gram-positive, spore-forming bacterium belonging to the genus Paenibacillus (17) that is capable of fermentative metabolism of hexoses, deoxyhexoses, pentoses, cellulose, and hemicellulose (33, 39, 40, 41). Glycerol, however, is considered a nonfermentable carbon source for P. macerans. The “nonfermentable status” of glycerol has been used to determine whether certain electron acceptors, such as fumarate, trimethylamine N-oxide, nitrate, and nitrite, can mediate anaerobic respiration in this organism (34).In this study we found that several P. macerans strains are able to ferment glycerol in the absence of external electron acceptors. The fermentation of glycerol by one of these strains, P. macerans N234A, occurred at high metabolic rates and in the absence of an active 1,3-PDO pathway. Therefore, the environmental and metabolic determinants of glycerol fermentation in P. macerans N234A were investigated.