Screening of bacterial strains capable of converting biodiesel‐derived raw glycerol into 1,3‐propanediol, 2,3‐butanediol and ethanol

The ability of bacterial strains to assimilate glycerol derived from biodiesel facilities to produce metabolic compounds of importance for the food, textile and chemical industry, such as 1,3‐propanediol (PD), 2,3‐butanediol (BD) and ethanol (EtOH), was assessed. The screening of 84 bacterial strains was performed using glycerol as carbon source. After initial trials, 12 strains were identified capable of consuming raw glycerol under anaerobic conditions, whereas 5 strains consumed glycerol under aerobiosis. A plethora of metabolic compounds was synthesized; in anaerobic batch‐bioreactor cultures PD in quantities up to 11.3 g/L was produced by Clostridium butyricum NRRL B‐23495, while the respective value was 10.1 g/L for a newly isolated Citrobacter freundii. Adaptation of Cl. butyricum at higher initial glycerol concentration resulted in a PD_max concentration of ～32 g/L. BD was produced by a new Enterobacter aerogenes isolate in shake‐flask experiments, under fully aerobic conditions, with a maximum concentration of ～22 g/L which was achieved at an initial glycerol quantity of 55 g/L. A new Klebsiella oxytoca isolate converted waste glycerol into mixtures of PD, BD and EtOH at various ratios. Finally, another new C. freundii isolate converted waste glycerol into EtOH in anaerobic batch‐bioreactor cultures with constant pH, achieving a final EtOH concentration of 14.5 g/L, a conversion yield of 0.45 g/g and a volumetric productivity of ～0.7 g/L/h. As a conclusion, the current study confirmed the utilization of biodiesel‐derived raw glycerol as an appropriate substrate for the production of PD, BD and EtOH by several newly isolated bacterial strains under different experimental conditions.     

Acetate non-utilizing mutants of Arabidopsis: evidence that organic acids influence carbohydrate perception in germinating seedlings

A phenotypic screen was employed to isolate Arabidopsis plants that are deficient in their ability to utilize or sense acetate. The screening strategy, based on resistance to the toxic acetate analogue monofluoroacetic acid, was adapted from one that has been used successfully to identify important metabolic and regulatory genes involved in acetate metabolism in fungi. Following conventions established from the fungal work, the mutants were called acn mutants for ac etate n on-utilization. Three highly resistant plant lines were the focus of genetic and physiological studies. Mutant acn1 appears to be a true acetate non-utilizing mutant, as it displays increased sensitivity to exogenous acetate. The progeny of the original acn2 mutant did not germinate, even in the presence of sucrose as an exogenous carbon source. The germination of seeds from the F3 generation depended on the sucrose concentration in the medium. Only a small proportion of seeds germinated in the absence of exogenous sucrose and in the presence of 100 mM sucrose, but up to 70% of seeds germinated on 20 mM sucrose. Mutant acn3 exhibited sensitivity to exogenous sucrose, showing significant chlorosis on medium containing 20 mM sucrose, but no chlorosis when grown in the absence of exogenous sucrose. This phenotype was alleviated if acetate was provided. The acn mutants demonstrate that disrupting organic acid utilization can have profound affects on carbohydrate metabolism.Communicated by G. Jürgens     

Purification and properties of two Swedish isolates of barley yellow dwarf virus and their serological relationships with American isolates

Two Swedish isolates of barley yellow dwarf virus (BYDV), one (39/78) transmitted much more efficiently by Rhopalosiphum padi than by Sitobion avenae and the other (27/77) transmitted specifically by Sitobion avenae, were purified with yields of 200–300 μg/100 g infected oat leaves. Three light scattering zones were obtained when isolate 39/78 was sedimented in 10–40% (w/w) sucrose density gradients but only one zone with the other isolate. The sedimentation coefficients of 39/78 particles were 94,106 and 150 S, respectively, whereas the 27/77 particles sedimented at 110 S. Four protein bands of mol. wts 31 900, 29 700, 28 000 and 15 000 were detected when disrupted 150 5 particles of the 39/78 isolate were electrophoresed in SDS-polyacrylamide gels. Only one major band of mol. wt 24 500 was detected when 94 or 106 S particles were electrophoresed under the same conditions. The 27/77 isolate yielded one major band of mol. wt 23 500. A weak serological relationship was found between isolates 39/78 and 27/77. In enzyme-linked immunosorbent assay, isolate 39/78 was serologically closely related to New York isolate PAY, whereas 27/77 was closely related to MAY.     

Chronic binge-like moderate ethanol drinking in rats results in widespread decreases in brain serotonin, dopamine, and norepinephrine turnover rates reversed by ethanol intake

This research was initiated to assess the turnover rates (TORs) of dopamine (DA), norepinephrine (NA), serotonin (5-HT), aspartate, glutamate, and GABA in brain regions during rodent ethanol/sucrose (EtOH) and sucrose (SUC) drinking and in animals with a history of EtOH or SUC drinking to further characterize the neuronal systems that underlie compulsive consumption. Groups of five male rats were used, with two trained to drink EtOH solutions, two to drink SUC and one to serve as a non-drinking control. When stable drinking patterns were obtained, rats were pulse labeled intravenously and killed 60 or 90 min later and the TORs of DA, norepinephrine, 5-HT, aspartate, glutamate, and GABA determined in brain regions. Changes in the TOR of 5-HT, DA, and NA were detected specific to EtOH drinking, SUC drinking or a history of EtOH or SUC drinking. An acute EtOH deprivation effect was detected that was mostly reversed with EtOH drinking. These results suggest that binge-like drinking of moderate amounts of EtOH produces a deficit in neuronal function that could set the stage for the alleviation of anhedonic stimuli with further EtOH intake that strengthen EtOH seeking behaviors which may contribute to increased EtOH use in at risk individuals.     

Osmoregulatory responses of bacteria isolated from fresh or composted,olive-mill waste-waters

Halotolerant bacteria isolated from raw, olive-mill waste-waters (alpechin) and from composted alpechin could grow on solid medium containing up to 10% (w/v) NaCl. Most (70%) of these halotolerant isolates could also grow in liquid minimal medium with the same NaCl concentration and three isolates from this group were chosen for further study. When grown in tryptic soy broth (TSB), two isolates responded to lowering of water activity (a _w) by addition of NaCl or sucrose in the expected manner: by increasing the proportion of membrane anionic lipids diphosphatidylglycerol or phosphatidylglycerol. Some solute-specific differences were observed. In contrast, the third isolate did not alter its membrane phospholipid composition significantly in response to growth in NaCl, whereas in sucrose there was an increase in phosphatidylethanolamine. This response is contrary to the accepted interpretation of the function of such a _w-dependent changes, as being a mechanism for preserving the membrane lipid-bilayer phase. When all three isolates were grown in the presence of alpechin, there was a decrease in the proportion of phosphatidylglycerol and a rise in the level of phosphatidylethanolamine. Quantitative and qualitative differences in compatible solute composition were observed when the three isolates were grown in TSB with NaCl or sucrose added to lower a _w. The major compatible solutes in two of the isolates were proline and betaine, whereas in the third they were proline, betaine and ectoine; one isolate also contained some trehalose when NaCl but not sucrose was the osmolyte.     

Identification and characterisation of the extracellular sucrases of Zymomonas mobilis UQM 2716 (ATCC 39676)

An investigation was conducted to isolate, and characterise the extracellular sucrases of Zymomonas mobilis UQM 2716. Levansucrase (EC 2.4.1.10) was the only extracellular sucrase produced by this organism. This enzyme was responsible for sucrose hydrolysis, levan formation, and oligosaccharide production. It had a molecular mass of 98 kDa, a Michaelis constant (K _m) of 64 mm, and a pH optimum of 5.5. It was inhibited by glucose, but not by fructose, ethanol, sorbitol, NaCl, TRIS or ethylenediaminetetraacetic acid (EDTA). The formation of levan was the principal reaction catalysed by this enzyme at low temperatures. However, levan formation was thermolabile, being irreversibly lost when levansucrase was heated to 35°C. S This did not effect sucrose hydrolysis or oligosaccharide formation, which were optimal at 45°C. Sucrose concentration greatly influenced the type of acceptor molecule used in the transfructosylation reactions catalysed by levansucrase. At low sucrose concentration, the predominant reaction catalysed was the hydrolysis of sucrose to free glucose and fructose. At high sucrose concentrations, oligosaccharide production was the major reaction catalysed.     

Curcumin Combats Against Cigarette Smoke and Ethanol-Induced Lipid Alterationsin Rat Lung and Liver

Background: Human population, in spite of the medical and scientific achievements, still fall as a prey to the evils of habitual smoking and alcohol, thus necessitating safer counteracting measures. Objective: To evaluate the effect of cotreatment of curcumin (Curcuma longa) in rats subjected to acute exposure to cigarette smoke (CS) and ethanol (EtOH). Methodology: Of the four groups of experimental rats, a set of rats was subjected to whole body exposure to cigarette smoke along with ethanol administration serving as a model of CS+EtOH injury. Curcumin treatment was given to two sets of rats: (i) one set receiving simultaneous CS+EtOH and (ii) one set of normal rats without any administration. The other group of rats served as control. Blood, liver and lung of rats were selected for assessment of CS+EtOH injury as well as curcumin treatment. Result: Altered lipid, lipoprotein profile and bile acid excretion were observed in CS+EtOH rats along with premalignant pathological state in tissues. In treated rats, the levels were maintained at near-normal levels along with near-normal histology. Conclusion: This biochemical picture on cotreatment with curcumin suggests that curcumin could counteract the injurious effects of combined CS and EtOH and thus might help to reduce the risk of hyperlipidemic disorders which develop due to smoking and drinking.     

Chronic Ethanol Increases the Cannabinoid Receptor Agonist Anandamide and Its Precursor N-Arachidonoylphosphatidylethanolamine in SK - N - SH Cells

Abstract : In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N-arachidonoylphosphatidylethanolamine (N-ArPE) in SK-N-SH cells that were prelabeled with [³H]arachidonic acid. The results indicate that exposure of SK-N-SH cells to EtOH (100 mM) for 72 h significantly increased levels of [³H]AnNH and [³H]N-ArPE (p < 0.05) (1.43-fold for [³H]AnNH and 1.65-fold for [³H]N-ArPE). Exposure of SK-N-SH cells to EtOH (100 mM, 24h) inhibited initially the formation of [³H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [³H]N-ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [³H]AnNH. The formation of [³H]AnNH and [³H]N-ArPE by EtOH was enhanced by the Ca²⁺ ionophore A23187 or by the depolarizing agent veratridine and the K⁺ channel blocker 4-aminopyridine. Further, the EtOH-induced formation of [³H]AnNH and [³H]N-ArPE was inhibited by exogenous AnNH, whereas only [³H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and G_i/o protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK-N-SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.     

Proteomic analyses of ethanol tolerance in Lactobacillus buchneri NRRL B‐30929

The Lactobacillus buchneri NRRL B‐30929 strain, isolated from a fuel ethanol (EtOH) production facility, exhibits high tolerance to environmental EtOH concentrations. This study aimed to identify proteins produced by B‐30929 in response to environmental EtOH. Cellular proteins expressed by B‐30929 growing in media with 10 versus 0% EtOH were compared by 2DE, followed by in‐gel digestion and MALDI‐MS analyses. Twenty EtOH responsive proteins were identified. These include a proline‐specific peptidase (Lbuc_1852); a membrane protein (Lbuc_0921), two general stress‐related proteins including a 10 kDa chaperonin (GroESL Lbuc_1359) and a 29 kDa member of the HK 97 family (Lbuc_1523); metabolic enzymes involving redox potential balances (Lbuc_2051 and Lbuc_0522) and carbohydrate fermentation (Lbuc_1319 and Lbuc_2157); nitrogen, amino acid, and fatty acid metabolism proteins (Lbuc_1994, Lbuc_0446, Lbuc_0858, Lbuc_0707, and Lbuc_0787). These changes suggested B‐30929 cells respond to EtOH by degradation of available proteins and fatty acids and increased production of specific enzymes and molecular chaperons. These results can be used to guide genetic modifications to increase EtOH tolerance in industrial biocatalysts. The data have been deposited to World‐2DPAGE ( http://world‐2dpage.expasy.org/repository/0068/ ; username liu, password 1h8d6Mg1).     

Biochemical Characterization of Helichrysum italicum (Roth) G.Don subsp. italicum (Asteraceae) from Montenegro: Phytochemical Screening,Chemotaxonomy, and Antioxidant Properties

The chemical composition and antioxidant properties of the essential oil and EtOH extract of immortelle (Helichrysum italicum (Roth) G.Don subsp. italicum, Asteraceae) collected in Montenegro were evaluated. The essential oil was characterized by GC/MS analysis, and the content of total phenolics and flavonoids in the EtOH extract was determined using the Folin? Ciocalteu reagent. The free‐radical‐scavenging capacity (RSC) of both the essential oil and the EtOH extract was assessed with the 2,2‐diphenyl‐1‐pycrylhydrazyl (DPPH) method. Moreover, the inhibition of hydroxyl radical (^.OH) generation by the EtOH extract of immortelle was evaluated for the first time here. Neryl acetate (28.2%) and γ‐curcumene (18.8%) were the main compounds in the essential oil, followed by neryl propionate (9.1%) and ar‐curcumene (8.3%). The chemical composition of the oils of the examined and additional 16 selected Helichrysum italicum taxa described in literature were compared using principal component (PCA) and cluster (CA) analyses. The results of the statistical analyses implied the occurrence of at least four different main and three subchemotypes of essential oils. Considering the antioxidant properties, the EtOH extract of immortelle exhibited similar potential as propyl gallate and quercetin, while the essential oil exhibited relatively weak DPPH^.‐scavenging capacity.     

Aflatoxin formation and the dual phenomenon in Aspergillus flavus link

Trials were performed with three aflatoxin-forming isolates of Aspergillus flavus from formic acid-treated materials containing aflatoxin, one A. flavus strain isolated from mouldy barley kept for two months in an anaerobic jar and one non-toxic A. flavus strain from the culture collection at our Department. The nontoxic strain and one aflatoxin producer were cultured in salts-sugar-asparagine substrate (SLM) for aflatoxin production and in a specially prepared grass substrate (GS). Formic acid and ammonium formate were added to both substrates, and sucrose in a low amount was added to the grass substrate. The aflatoxin-forming isolate segregated on the grass substrate into two different lines, one with high aflatoxin production and one with very low aflatoxin-forming ability, higher growth rate and reduced sporulation, on the SLM substrate. When exposed to sucrose in grass substrate and ammonium formate in SLM, one toxic and one non-toxic strain were provoked to increased aflatoxin formation. The A. flavus isolate from the anaerobic jar also segregated on the grass substrate, and these segregants were more sensitive to a high dose of formic acid. In these A. flavus strains there seems to be a continuous variation between different lines, depending on cultivation conditions. In the two aflatoxin-forming isolates left, such segregation tendencies were not very marked on any substrate.     

Isolation of Intracytoplasmic Membrane from the Methanotrophic Bacterium Methylomicrobium album BG8

Methane-oxidizing bacteria, including Methylomicrobium album BG8, form an intracytoplasmic membrane in addition to the cytoplasmic and outer membranes of the cell envelope. Techniques to isolate the intracytoplasmic membrane of M. album BG8 were developed. An intracytoplasmic membrane fraction was separated from a cell envelope fraction on the basis of sedimentation velocity in sucrose density gradients. Proteins associated with the particulate methane monooxygenase were found in both membrane fractions. Received: 27 July 1999 / Accepted: 30 August 1999     

Myelin subfractions isolated from mouse brain. Studies of normal mice during development,quaking mutants,and three brain regions

Myelin isolated from three areas of mouse brain, from whole brain at several ages in normal mice, and from whole brain of adult quaking mutant mice was separated into seven bands and a pellet on discontinuous density gradients using 0.32, 0.45, 0.55, 0.60, 0.70, 0.75, and 0.85 M sucrose. The distribution of myelin in the subfractions was independent of homogenization and shocking conditions employed to isolate the myelin preparations, but was related to the type of myelin applied to the gradient. Compared to myelin isolated from older animals, myelin isolated from 18–24 day old mice displayed a distribution pattern with greater proportions of material banding at lesser sucrose densities. Similarly, myelin obtained from hindbrain contained proportionately more material layering at lesser sucrose densities compared to myelin isolated from cerebral cortex. Myelin subfraction patterns observed for 8–12 day old control mice and quaking mutants were unlike each other or any other myelin preparation examined. In the 18–90 day old animals, the markers studied were not uniformly distributed among the myelin subfractions. The pellet and the layer banding at the 0.75/0.85 M sucrose interface contained the highest specific concentrations of sialic acid, nucleic acid, and total adenosine triphosphatase activity. In contrast, the specific activity of 2′,3′ -cyclicnucleotide-3′-phosphohydrolase was lowest in the pellet as well as the three bands obtained above 0.60 M sucrose and was highest in the fraction banding at the 0.65/0.70 M sucrose interface. The results obtained were not consistent with an artifactual origin of the myelin subfractions, but instead suggested that the subfractions have physiological significance. One explanation for the different banding patterns observed between young and mature myelin may be the different amount of myelin in various brain regions during development.     

Gloeocapsopsis AAB1, an extremely desiccation-tolerant cyanobacterium isolated from the Atacama Desert

The comprehensive study of microorganisms that evolved in the Atacama Desert, the driest and oldest on earth, may help to understand the key role of water for life. In this context, we previously characterized the microenvironment that allows colonization of the underside of quartzes in the Coastal Range of this desert by hypolithic microorganisms (Azua-Bustos et al. Microb Ecol 58:568–581, 2011). Now, we describe the biodiversity composition of these biofilms and the isolation from it of a new cyanobacterial strain. Based on morphologic and phylogenetic analyses, this isolate (AAB1) was classified as a new member of the Gloeocapsopsis genus. Physiological, morphological and molecular responses by isolate AAB1 show that this strain is extremely tolerant to desiccation. Our results also indicate that the isolate biosynthesizes sucrose and trehalose in response to this stressful condition. We identified two candidate genes involved in sucrose synthesis, namely sucrose 6-phosphate synthase and sucrose 6-phosphate phosphatase. Thus, the Gloeocapsopsis isolate AAB1 may represent a suitable model for understanding tolerance to low water availability.     

Characterization of an O-desmethylangolensin-producing bacterium isolated from human feces

A bacterium that converted daidzein to O-desmethylangolensin was isolated from the feces of healthy humans. It was an obligately anaerobic, nonsporeforming, nonmotile and Gram-positive rod. The isolate used glucose, sucrose, raffinose, maltose, and fructose as carbon sources. It did not hydrolyze gelatin, esculin, or starch. The strain was urease, acid phosphatase, and arginine dihydrolase positive. It was catalase, oxidase, H₂S, and indole negative. The major products of glucose fermentation were butyrate and lactate. Its mol% G+C was 51.2. The major cellular fatty acids were C_16:0 DMA, C_16:0, and C_16:0 aldehyde. The structural type of cell wall peptidoglycan was suggested to be A1γ. The isolate was susceptible to β-lactam, cefem, and macrolide antibiotics and resistant to aminoglycoside and quinolone antibiotics. The bacterium was related to Eubacterium ramulus ATCC29099^T, Eubacterium rectale ATCC33656^T, and species of the genus Roseburia, but the highest 16S rRNA gene similarity to these described species was only 94.4%, consistent with its being classified as a novel genus. Based on the above, the isolate, named strain SY8519, was identified as belonging to a novel genus in the Clostridium rRNA cluster XIVa.     

Chemically defined medium for high yields of sterigmatocystin

Isolate of Aspergillus versicolor strain produced 138 g/ml of sterigmatocystin in a complete synthetic medium containing sucrose, salts, 1-phenylalanine, and Ca-pantothenate. The SSP (sucrose salts phenylalanine) medium apparently provided all necessary ingredients for the production of high levels of sterigmatocystin. For optimal sterigmatocystin formation, the amounts of sucrose and 1-phenylalanine were found to be 200 g and 5 g per liter, respectively. When Ca-pantothenate (0.01 g per liter) added, much higher amounts of sterigmatocystin were recovered, whereas CaCl₂ addition (0.01%) drastically reduced the yield. The high levels of sterigmatocystin were recovered in the cultures which incubated stationarily at 26 to 29 °C for over 12 days. Seven strains or isolates tested yielded high levels of sterigmatocystin in the SSP medium, whereas in each other media such as YES medium and rice medium only one isolate yielded highest amount of sterigmatocystin was found.     

Biological treatments for the control of decay in tree wounds.: Field tests

In an attempt to control fungi causing decay a range of micro-organisms, selected after laboratory tests, was inoculated into wounds made in the trunks of beech trees. The most successful micro-organism was an isolate of Trichoderma viride, which, over a period of 4 years, maintained significantly lower colonisation by decay fungi at both sites - c. 15% of the level found in uninoculated wounds. T. viride could be reisolated from the majority of wounds into which it had been inoculated up to 4 years later. No other antagonist persisted very effectively. Inoculation of T. viride in glycerol improved its establishment but malt extract and sucrose had no such effect. Both the addition of glycerol to wounds and the wrapping of them in black PVC sheet increased natural colonisation by T. viride. The use of PVC but not glycerol reduced numbers of decay fungi but not through the agency of T. viride present by natural infection, as this proved generally ineffective in the control of decay fungi. Another isolate of T. viride persisted considerably better when covered with a wound sealant.