Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp.

Three strains of new mesophilic homoacetogenic bacteria were enriched and isolated from sewage sludge and from marine sediment samples with methoxyacetate as sole organic substrate in a carbonate-buffered medium under anoxic conditions. Two freshwater isolates were motile, Gram-positive, non-sporeforming rods. The marine strain was an immotile, Gram-positive rod with a slime capsula. All strains utilized only the methyl residue of methoxyacetate and released glycolic acid. They also fermented methyl groups of methoxylated aromatic compounds and of betaine to acetate with growth yields of 6–10 g dry matter per mol methyl group. H₂/CO₂, formate, methanol, hexamethylene tetramine, as well as fructose, numerous organic acids, glycerol, ethylene glycol, and glycol ethers were fermented to acetate as well. High activities of carbon monoxide dehydrogenase (0.4–2.2 U x mg protein^–1) were detected in all three isolates. The guanine-plus-cytosine-content of the DNA of the freshwater isolates was 42.7 and 44.4 mol %, with the marine isolate it was 47.7 mol %. The freshwater strains were assigned to the genus Acetobacterium as new strains of the species A. carbinolicum. One freshwater isolate, strain KoMac1, was deposited with the Deutsche Sammlung von Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.     

Desulfovibrio alcoholovorans sp. nov., a sulfate-reducing bacterium able to grow on glycerol, 1,2- and 1,3-propanediol

Desulfovibrio strain SPSN was isolated from an anaerobic industrial fermenter fed with waste water from the alcohol industry. The isolate was a gram-negative, non-spore-forming, curved organism, the motility of which is provided by a single polar flagellum. The oxidation of substrates was incomplete and included glycerol and 1,3-propanediol. Sulfate, sulfite, thiosulfate, and sulfur were utilized as electron acceptors. Pyruvate, fumarate and malate could be fermented. The DNA base composition was 64.5±0.3% G+C. Cytochrome c ₃ and desulfoviridin were present. On the basis of these characteristics and because strain SPSN could not be ascribed to any of the existing species, the isolate is established as a new species of the genus Desulfovibrio, and the name Desulfovibrio alcoholovorans is proposed.     

Ether-cleaving enzyme and diol dehydratase involved in anaerobic polyethylene glycol degradation by a new Acetobacterium sp.

A strictly anaerobic, homoacetogenic bacterium was enriched and isolated from anoxic sewage sludge with polyethylene glycol (PEG) 1000 as sole source of carbon and energy, and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The new isolate fermented ethylene glycol and PEG's with molecular masses of 106 to 1000 to acetate and small amounts of ethanol. The PEG-degrading activity was not destroyed by proteinase K treatment of whole cells. In cell-free extracts, a diol dehydratase and a PEG-degrading (ether-cleaving) enzyme activity were detected which both formed acetaldehyde as reaction product. The diol dehydratase enzyme was oxygen-sensitive and was stimulated 10–14 fold by added adenosylcobalamine. This enzyme was found mainly in the cytoplasmic fraction (65%) and to some extent (35%) in the membrane fraction. The ether-cleaving enzyme activity reacted with PEG's of molecular masses of 106 to more than 20000. The enzyme was measurable optimally in buffers of high ionic strength (4.0), was extremely oxygen-sensitive, and was inhibited by various corrinoids (adenosylcobalamine, cyanocobalamine, hydroxocobalamine, methylcobalamine). This enzyme was found exclusively in the cytoplasmic fraction. It is concluded that PEG is degraded by this bacterium inside the cytoplasm by a hydroxyl shift reaction, analogous to a diol dehydratase reaction, to form an unstable hemiacetal intermediate. The name polyethylene glycol acetaldehyde lyase is suggested for the responsible enzyme.Abbreviations EG ethylene glycol - DiEG diethylene glycol - TriEG triethylene glycol - TeEG tetraethylene glycol - PEG polyethylene glycol (molecular mass indicated)     

Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> under micro-aerobic conditions

Glycerol dehydrogenase (GDH) and 1,3-propanediol (1,3-PD) oxidoreductase had been proved two key enzymes for 1,3-PD production by Klebsiella pneumoniae. Fed-batch fermentations of the recombinant K. pneumoniae strains, over-expressing the two enzymes individually, were carried out under micro-aerobic conditions, and the behaviors of the recombinants were investigated. Results showed that over-expression of 1,3-PD oxidoreductase did not affect the concentration of 1,3-PD. However, it enhanced the molar yield from 50.6 to 64.0% and reduced the concentration of by-products. Among them, the concentrations of lactic acid, ethanol and succinic acid were decreased by 51.8, 50.6 and 47.4%, respectively. Moreover, in the recombinant the maximal concentration of 3-hydroxypropionaldehyde decreased by 73.6%. Over-expression of GDH decreased the yield of ethanol and 2,3-butanediol, meanwhile it increased the concentration of acetic acid. No significant changes were observed both in 1,3-PD yield and glycerol flux distributed to oxidative branch.     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3- propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

Production of vitamin B12 in an upflow anaerobic filter continuous reactor using Acetobacterium sp.

The accumulation of biofilm by Acetobacterium sp. during continuous culture in an upflow anaerobic filter (UAF) growing on methanol-formate was the result of space velocity and inlet concentrations of substrate and Co⁺². To achieve good development of biofilm, a space velocity of 0.38 h^–1, inlet substrate concentrations of 125 mM of both methanol and formate, and Co⁺² at 0.16 mM were required. Cell productivities in the effluent of the UAF-reactor were about 6-fold higher than in chemostat cultures (0.20 g l^–1 h^–1 for UAF and 0.035 g l^–1 h^–1 for chemostat) (previous studies), and the maximum vitamin B₁₂ specific concentration was 5.1 mg g cell^–1.     

Microbial production of 1,3-propanediol from glycerol by Klebsiella pneumoniae under micro-aerobic conditions up to a pilot scale

1,3-Propanediol (1,3-PD) can be produced from glycerol by Klebsiella pneumoniae under micro-aerobic conditions. Recently, this fed-batch fermentation process has been successfully scaled up to 1 m³. The final 1,3-PD concentration, molar yield and volumetric productivity of 72 g l⁻¹, 57% and 2.1 g l⁻¹ h⁻¹, respectively, are close to those of 75 g l⁻¹, 61%, and 2.2 g l⁻¹ h⁻¹ under anaerobic conditions. This process would be suitable for the production of 1,3-PD on a large scale.     

Fermentation of 2-methoxyethanol by Acetobacterium malicum sp. nov. and Pelobacter venetianus

Anaerobic bacteria degrading 2-methoxyethanol were enriched from freshwater sediments, and three strains were isolated in pure culture. Two of them were Grampositive non-spore-forming rods and grew strictly anaerobically by acetogenic fermentation. Optimal growth occurred at 30°C, initial pH 7.5–8.0. 2-Methoxyethanol and 2-ethoxyethanol were fermented to acetate and corresponding alcohols. Hydrogen plus carbon dioxide, formate, acetoin, l-malate, lactate, pyruvate, fructose, and methoxyl groups of 3,4,5-trimethoxybenzoate and 3,4,5-trimethoxycinnamate were fermented to acetate. 1,2-Propanediol was fermented to acetate, propionate, and propanol. Strain MuME1 was described as a new species, Actetobacterium malicum. It had a DNA base composition of 44.1 mol% guanine plus cytosine. The third strain, which was identified as Pelobacter venetianus, fermented 2-methoxyethanol to methanol, ethanol, and acetate.     

Anaerovibrio glycerini sp. nov., an anaerobic bacterium fermenting glycerol to propionate,cell matter,and hydrogen

A strictly anaerobic, Gram-negative bacterium was isolated in continuous culture from black freshwater sediment with glycerol as sole source of carbon and energy. It was present in such sediments at 10⁸ cells per ml. The isolate was highly specialized and used only glycerol and the glycerol residue of diolein as substrate, and fermented it quantitatively to propionate. During growth in mineral medium, small amounts of hydrogen were produced which corresponded exactly to the calculated amount of electrons released in cell matter formation from glycerol. Yeast extract enhanced cell yields with glycerol, but did not support growth itself. In cell-free extracts, benzylviologen-dependent hydrogenase activity as well as a b-type cytochrome and some of the enzymes of the methylmalonylCoA pathway were found. The guanine-plus-cytosine content of the DNA was 34.3±1.0 mol% and corresponded well with that of Anaerovibrio lipolytica which was found to be 31.4 mol%. The consequences of the electron balance of this glycerol fermentation are discussed with respect to glycerol fermentation by other propionic acid-forming bacteria.     

红曲菌利用甘油促进色素产生及其机制的研究进展

红曲色素(Monascus pigments,MPs)是红曲菌(Monascus sp.)的主要次生代谢产物,作为食品着色剂在我国已有上千年的应用历史。甘油,特别是生物柴油等生产过程中产生的甘油,作为一种相对廉价的碳源可被红曲菌等微生物利用,并可促进红曲色素的产生。本文对甘油促进红曲菌色素产生的相关研究进行综述,并对其机制进行分析。     

Optimization of medium compositions favoring butanol and 1,3-propanediol production from glycerol by Clostridium pasteurianum

Medium compositions favoring butanol and 1,3-propanediol (1,3-PDO) production from glycerol by Clostridium pasteurianum DSM525 were investigated using statistical experimental designs. Medium components affecting butanol and 1,3-PDO production were screened using a fractional factorial experimental design. Among the six tested variables (phosphate buffer, MnSO4·H2O, MgSO4·7H2O, FeSO4·7H2O, (NH4)2SO4, and yeast extract), FeSO4·7H2O, (NH4)2SO4, and yeast extract were found to be significant variables for further optimization of medium using a Box-Behnken design. Optimal butanol (0.98 g/L/h) and 1,3-PDO (1.19 g/L/h) productivities were predicted by the corresponding quadratic model for each product and the models were validated experimentally under optimized conditions. The optimal medium composition for butanol production was significantly different from that for 1,3-PDO production (0.06 vs. 0 g/L for FeSO4·7H2O, 7.35 vs. 0 g/L for (NH4)2SO4, and 5.08 vs. 8.0 g/L for yeast extract), suggesting that the product formation from glycerol by C. pasteurianum DSM525 can be controlled by changing medium compositions.     

Metabolic flux and robustness analysis of glycerol metabolism in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The knowledge of the mechanism of flux distribution will benefit understanding cell physiology and regulation of metabolism. In this study, the measured fluxes obtained under steady-state conditions were used to estimate intracellular fluxes and identify the robustness of branch points of the anaerobic glycerol metabolism in Klebsiella pneumoniae for the production of 1,3-propanediol by metabolic flux analysis. The biomass concentration increased as NADH₂/NAD⁺ decreased at low initial concentration and inversed at high initial glycerol concentration. The flux distribution revealed that the branch points of glycerol and dihydroxyacetonephosphate were rigid to the environmental conditions. However, the pyruvate and acetyl coenzyme A metabolisms gave cells the flexibility to regulate the energy and intermediate fluxes under various environmental conditions. Additionly, it was found that the formation rate of ethanol and the ratio of pyruvate dehydrogenase to pyruvate formate lyase appeared visible fluctuations at high glycerol uptake rate.     

Clostridium halophilium sp. nov. and C. litorale sp. nov., an obligate halophilic and a marine species degrading betaine in the Stickland reaction

Two new mesophilic, sporeforming, gram-positive, strictly anaerobic, rod-shaped bacteria were isolated which utilized betaine in the Stickland reaction. Strain M1 was obtained from pasteurized hypersaline sediments. Cells were motile rods and formed spherical terminal spores. Betaine was used with hydrogen and several amino acids as electron donors. In addition, several carbohydrates served as substrates. Growth required 1.5% NaCl with an optimum at 6.0% NaCl. The guanine plus cytosine content of the DNA was 26.9%. This strain is described as a new species, Clostridium halophilum.Strain W6 was isolated from marine sediments. Cells were motile rods and formed ovoid, subterminal spores. Betaine was used with hydrogen and several amino acids as electron donors. Carbohydrates were not fermented. Growth optimum was at 1.0% NaCl. The guanine plus cytosine content of the DNA was 26.1%. This strain is described as a new species, Clostridium litorale.Non standard abbreviations DMG N,N-dimethylglycine - TMA trimethylamine - PY peptone-yeast extract - PYG peptone-yeast extract-glucose     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> VPI 3266 using a synthetic medium and raw glycerol

Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l^–1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.     

Fermentation of raw glycerol to 1,3-propanediol by new strains ofClostridium butyricum

Industrial glycerol obtained through the transesterification process using rapeseed oil did not support growth of several strains ofClostridium butyricum obtained from bacterial culture collections. Ten new strains ofC. butyricum were obtained from mud samples from a river, a stagnant pond, and a dry canal. These new isolates fermented the commercial glycerol and produced 1,3-propanediol as a major fermentation product with concomitant production of acetic and butyric acids. Four of the ten isolates were able to grow on industrial glycerol obtained from rapeseed oil. One strain,C. butyricum E5, was very resistant to high levels of glycerol and 1,3-propanediol. Using fed-batch fermentation, 109 g L^–1 of industrial glycerol were converted into 58 g of 1,3-propanediol, 2.2 g of acetate and 6.1 g of butyrate per liter.     

Four novel yeasts from decaying organic matter: Blastobotrys robertii sp. nov., Candida cretensis sp. nov., Candida scorzettiae sp. nov. and Candida vadensis sp. nov

Four novel yeast species are described, two from decaying mushrooms, viz. Candida cretensis and Candida vadensis, and two from rotten wood, viz. Blastobotrys robertii and Candida scorzettiae. Accession numbers for the CBS and ARS Culture Collections, and GenBank accession numbers for the D1/D2 domains of the large subunit of ribosomal DNA are: B. robertii CBS 10106^T, NRRL Y-27775, DQ839395; C. cretensis CBS 9453^T, NRRL Y-27777, AY4998861 and DQ839393; C. scorzettiae CBS 10107^T, NRRL Y-27665, DQ839394; C. vadensis CBS 9454^T, NRRL Y-27778, AY498863 and DQ839396. The GenBank accession number for the ITS region of C. cretensis is AY498862 and that for C. vadensis is AY498864. C. cretensis was the only species of the four that displayed fermentative activity. All four type strains grew on n-hexadecane. C. scorzettiae is the only one of the new species that assimilates some phenolic compounds, viz. 3-hydroxy derivatives of benzoic, phenylacetic and cinnamic acids, but not the corresponding 4-hydroxy acids. This is indicative of an operative gentisate pathway.     

New species of psychrophilic acetogens: Acetobacterium bakii sp. nov., A. paludosum sp. nov., A. fimetarium sp. nov.

Three strains of new acetogenic bacteria were isolated from several low temperature environments. Cells were gram-positive, oval-shaped flagellated rods. The organisms fermented H₂/CO₂, CO, formate, lactate, and several sugars to acetate. Strains Z-4391 and Z-4092 grew in the temperature range from 1 to 30°C with an optimum at 20°C; strain Z-4290 grew in the range from 1 to 35°C with an optimum at 30°C. The DNA G+C content of strains Z-4391, Z-4092, and Z-4290 was 42.1, 41.7, and 45.8 mol% respectively.     

Enhancement of bioremediation by Ralstonia sp. HM-1 in sediment polluted by Cd and Zn

In this study, the potential for the application of the bioaugmentation to Cd and Zn contaminated sediment was investigated. A batch experiment was performed in the lake sediments augmented with Ralstonia sp. HM-1. The degradation capacity of 18.7 mg-DOC/l/day in the treatment group was bigger than that of the blank group (4.4 mg-DOC/l/day). It can be regarded as the result of the reduction of the metal concentration in the liquid phase due to adsorption into the sediments, with the increased alkalinity resulting from the reduction of sulfate by sulfate reducing bacteria (SRB). The removal efficiency of cadmium and zinc in the treatment group was both 99.7% after 35 days. Restrain of elution to water phase from sediment in the Ralstonia sp. HM-1 added treatment group was also shown. In particular, the observed reduction of the exchangeable fraction and an increase in the bound to organics or sulfide fraction in the treatment group indicate its role in the prevention of metal elution from the sediment. Therefore, for bioremediation and restrain of elution from the sediment polluted by metal, Ralstonia sp. augmentation with indigenous microorganism including SRB, sediment stabilization and restrain of elution to surface water is recommended.     

Candida alocasiicola sp. nov., Candida hainanensis sp. nov., Candida heveicola sp. nov. and Candida musiphila sp. nov., novel anamorphic, ascomycetous yeast species isolated from plants

In a taxonomic study on the ascomycetous yeasts isolated from plant materials collected in tropical forests in Yunnan and Hainan Provinces, southern China, four strains isolated from tree sap (YJ2E(T)) and flowers (YF9E(T), YWZH3C(T) and YYF2A(T)) were revealed to represent four undescribed yeast species. Molecular phylogenetic analysis based on the large subunit (26S) rRNA gene D1/D2 domain sequences showed that strain YJ2E(T) was located in a clade together with Candida haemulonii and C. pseudohaemulonii. Strain YF9E(T) was most closely related to C. azyma and strain YWZH3C(T) to C. sorbophila and C. spandovensis. Strain YYF2A(T) was clustered in a clade containing small-spored Metschnikowia species and related anamorphic Candida species. The new strains differed from their closely related described species by more than 10% mismatches in the D1/D2 domain. No sexual states were observed for the four strains on various sporulation media. The new species are therefore assigned to the genus Candida and described as Candida alocasiicola sp. nov. (type strain, YF9E(T) = AS 2.3484(T) = CBS 10702(T)), Candida hainanensis sp. nov. (type strain, YYF2A(T) = AS 2.3478(T) = CBS 10696(T)), Candida heveicola sp. nov. (type strain, YJ2E(T) = AS 2.3483(T) = CBS 10701(T)) and Candida musiphila sp. nov. (type strain, YWZH3C(T) = AS 2.3479(T) = CBS 10697(T)).