Phenotypic Adaptations Help Rhodococcus erythropolis Cells during the Degradation of Paraffin Wax

During crude oil extraction, the reduction in temperature and pressure results in the precipitation of paraffin wax that contains 20–40 carbon chain hydrocarbons. The paraffin wax may accumulate inside production tubes, pipelines, and processing facilities, and also in tankers during petroleum transportation. There are few bacterial strains that are able to degrade solid substrates. In the present study, the biodegradation of paraffin is evaluated using Rhodococcus erythropolis cells. This bacterium is able to grow using paraffin wax from an oil refinery plant as the sole carbon source. The cells grow as a thick biofilm over the solid substrate, make scale‐like structures that increase the area of the initially smooth surface of paraffin, produce biosurfactants, and become more negatively charged than ethanol‐ or glucose‐grown cells. When paraffin wax is supplied as microparticles, to increase the cell–substrate contact area and to simulate paraffin precipitation, the cells also adjust the composition of the fatty acids of the phospholipids of the cellular membrane to decrease its fluidity and paraffin biodegradation increases considerably. The study suggests that the phenotypic adaptation of R. erythropolis cells may be used to degrade paraffin wax under real conditions.     

Microcalorimetry of microorganism metabolism of monosaccharides and simple aromatic compounds

Heat conduction solution enable rapid determination of the heats of aerobic and anaerobic metabolism of substrate by microorganisms. Aliquots of 1.0 ml cell suspension, 5 × 10⁹ cell/ml, were mixed with a few dozen nmol substrate contained in 0.5 ml, under a controlled atmosphere of air, O₂, or N₂. At these substrate concentration, with adapted microorganisms, metabolism and its heat generation are usually complete within 300 to 600 sec. The raw data yield ΔH_app values. The ΔH_app were determined in the range 0.001 to 0.010% substrate, and extrapolated (limit substrate concentration →0), to yield Δ⁰H?, the limiting differential molar heat of metabolism. The Δ⁰H? values express the heat generated when there is rapid metabolism but little new growth, minimal contribution by H⁺ transfer from metabolites, and maintenance of aerobicity or anaerobicity as specified. Escherichiacoli B/5 was used for aerobic and anaerobic combustion of eight sugars. Pseudomonas multivorans, and an Acinetobacter, strain B-1, were used for aerobic metabolism of benzene, toluene, naphthalene, and a methylnaphthalene. The larger heats of combustion of the hydrocarbons enable the use of aqueous solutions of hydrocarbons well below their solubility limits. The quotient Δ⁰H?/n (n = atoms carbon/molecule substrate) varies from (-)36 to (-)67 kcal/mol carbon for the sugars. The most reduced sugar yields the largest exothermic heats. The quotient varies from (-)27 to (-)81 kcal/mol carbon for the aromatic hydrocarbons. Comparison of the calorimetric heats of metabolism of those from total aerobic combustion in aquo (where available) give measure of the efficiencies with which the heat contents of the aqueous substrate are used by the bacteria.     

Genetic variation at the alcohol dehydrogenase locus in Drosophila melanogaster in relation to environmental variation: Ethanol levels in breeding sites and allozyme frequencies

Summary Ethanol levels in Drosophila breeding sites in seepages of unfortified wine inside wineries have been found to be similar to those in many decaying fruits and vegetables. Fortified wine seepages inside wineries have ethanol levels on average three times as high as other breeding sites. However there was no evidence that this variation in ethanol levels was associated with differences in Adh ^F frequencies in D. melanogaster at sites either within wineries or outside wineries. D. simulans was at lower frequencies at sites inside wineries compared to sites outside although this difference may not be related to ethanol levels. It is concluded that adaptation to natural levels of environmental ethanol by D. melanogaster does not necessarily modify Adh frequencies.     

Is conspecific substrate marking a long‐term external memory of previously colonized overwintering sites in Harmonia axyridis?

The multicoloured Asian ladybeetle, Harmonia axyridis (Pallas), aggregates inside dwellings during winters to survive the cold. This beetle uses chemical cues coming from congeners to select an overwintering site. Recent research has shown that they preferentially gather at places where conspecifics previously laid a substrate marking made up of saturated and unsaturated hydrocarbons. Some authors have reported that H. axyridis colonizes the same overwintering sites from 1 year to another. Herein, the hypothesis that this substrate marking is used by H. axyridis to settle in the same aggregation sites from one winter to another was tested. To this aim, the temporal modification in the chemical profile of the hydrocarbon marking was studied by performing chromatographic analyses. After 1 year, the overall profile was modified qualitatively and quantitatively: the unsaturated hydrocarbons were no longer detected while some saturated hydrocarbons were still present in large quantities. In a behavioural assay conducted in the laboratory, the 12‐month‐old marking did not induce the aggregation of H. axyridis. This result indicates that the chemical markings left by conspecifics during a previous aggregation period in an overwintering site are not sufficient to induce the gathering of the newly arriving individuals.     

Restoration of a Mediterranean forest after a fire: bioremediation and rhizoremediation field‐scale trial

Forest fires pose a serious threat to countries in the Mediterranean basin, often razing large areas of land each year. After fires, soils are more likely to erode and resilience is inhibited in part by the toxic aromatic hydrocarbons produced during the combustion of cellulose and lignins. In this study, we explored the use of bioremediation and rhizoremediation techniques for soil restoration in a field‐scale trial in a protected Mediterranean ecosystem after a controlled fire. Our bioremediation strategy combined the use of Pseudomonas putida strains, indigenous culturable microbes and annual grasses. After 8 months of monitoring soil quality parameters, including the removal of monoaromatic and polycyclic aromatic hydrocarbons as well as vegetation cover, we found that the site had returned to pre‐fire status. Microbial population analysis revealed that fires induced changes in the indigenous microbiota and that rhizoremediation favours the recovery of soil microbiota in time. The results obtained in this study indicate that the rhizoremediation strategy could be presented as a viable and cost‐effective alternative for the treatment of ecosystems affected by fires.     

Chemical Studies on Components of Dried Bonito, “Katsuobushi”

Volatile components obtained by the extraction of “Katsuobushi” with 80% ethanol and by the subsequent steam distillation of the extract were fractionated by the usual methods, and the resulting hydrocarbon fraction was investigated. Gas chromatographic study on this fraction originated from “Katsuobushi” of bonito (Katsuwonus pelamis) revealed 9 hydrocarbons, including n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane, n-nonadecane, n-eicosane, n-heneicosane and n-docosane, which were tentatively identified by the retention times with the aid of authentic hydrocarbons. n-Pentadecane and n-heptadecane that were main components among these hydrocarbons were identified further by NMR and IR spectrometry. “Katsuobushi” of frigate mackerel (Auxis thazard), mackerel (Scomber Japonicus Houttuyn) or muroaji (Decapterus muroadsi) also contained n-penta-decane and n-heptadecane in large amounts, but did other hydrocarbons in negligible amounts.

Possible mechanisms of the hydrocarbon formation during the processing of “Katsuobushi” were discussed.     

Micro-evolution in a wine cellar population: An historical perspective

The population ofDrosophila melanogaster inside the wine cellar of Chateau Tahbilk of Victoria, Australia was found by McKenzie and Parsons (1974) to have undergone microevolution for greater alcohol tolerance when compared to the neighboring population outside the cellar. This triggered additional studies at Tahbilk, and at other wine cellars throughout the world. The contributions and interactions of researchers and the development of ideas on the ecology and genetics of this unique experimental system are traced. Although the ADH-F/ADH-S polymorphism was found to be maintained by selection in the Tahbilk populations, the selection is not significantly associated with alcohol tolerance. The environment inside the Tahbilk wine cellar is not as rich in ethanol as was originally anticipated, and selection that affects the alcohol dehydrogenase polymorphism may be more concerned with the relative efficiency with which ethanol is used as a nutrient byD. melanogaster. The synthesis and modification of lipids, particularly in membranes, appears to be important to alcohol tolerance. The studies of the Tahbilk population are at a crossroad. New experimental approaches promise to provide the keys to the selection that maintains the alcohol dehydrogenase polymorphism, and to factors that are important to alcohol tolerance and stress adaptation. From these research foundations at Tahbilk very significant contributions to our future understanding of the genetic processes of evolution can be made.     

Aeration alleviates ethanol inhibition and glycerol production during fed-batch ethanol fermentation

In this study, we investigated the effects of aeration on ethanol inhibition and glycerol production during fed-batch ethanol fermentation. When aeration was conducted at 0.13, 0.33, and 0.8 vvm, the ethanol productivity, specific ethanol production rate, and ethanol yield in the presence of greater than 100 g/L of ethanol were higher than when aeration was not conducted. In addition, estimation of the parameters (α and β) in a model equation of ethanol inhibition kinetics indicated that aeration alleviated ethanol inhibition against the specific growth rate and the specific ethanol production rate. Specifically, when aeration was conducted, the glycerol yield and specific glycerol production rate decreased approximately 50 and 70%, respectively. Finally, the results of this study indicated that aeration during fed-batch ethanol fermentation may improve the ethanol concentration in the final culture broth, as well as the ethanol productivity.     

The Relationship Between Tobacco Yield and Time of Infection with Meloidogyne javanica

Yield of tobacco was related to the amount of infection by Meloidogyne javanica during the first month after transplanting. Six nematicidal treatments significantly reduced infection during this period and subsequently increased yield. However, during the second month after transplanting, infection in plots treated with O-ethyl S,S-dipropyl phosphorodithioate (V-C 9-104) and a mixture of 80% chlorinated C₃ hydrocarbons + 20% methyl isothioeyanate (DD + MENCS) was not significantly different from infection in untreated plots. After 3 months, root-knot indices in plots treated with V-C 9-104, DD + MENCS, O,O-diethyl O-[p-(methylsulfinyl) phenyl] phosphorothioate (B-25141), and 1,3-dichloropropene, 1,2-dichloropropane (DD) were not significantly different from those in untreated plots; reduced infection was present only in plots treated with ethylene dibromide (EDB) and 2-methyl-2 (methylthio) propionaldehyde O-(methylcarbamoyl) oxime (aldicarb). At the end of harvest (4 months after transplanting), root-knot indices in all plots were essentially equal.     

Indole-based assay to assess the effect of ethanol on <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1 dioxygenase activity

Toluene dioxygenase (TDO) is ubiquitous in nature and has a broad substrate range, including benzene, toluene, ethylbenzene and xylenes (BTEX). Pseudomonas putida F1 (PpF1) induced on toluene is known to produce indigo from indole through the activity of TDO. In this work, a spectrophotometric assay previously developed to measure indole to indigo production rates was modified to characterize the effects of various ethanol concentrations on toluene aerobic biodegradation activity and assess catabolite repression of TDO. Indigo production rate by cells induced on toluene alone was 0.0012 ± 0.0006 OD₆₁₀ min⁻¹. The presence of ethanol did not fully repress TDO activity when toluene was also available as a carbon source. However, indigo production rates by PpF1 grown on ethanol:toluene mixtures (3:1 w/w) decreased by approximately 50%. Overall, the proposed spectrophotometric assay is a simple approach to quantify TDO activity, and demonstrates how the presence of ethanol in groundwater contaminated with reformulated gasoline is likely to interfere with naturally occurring microorganisms from fully expressing their aerobic catabolic potential towards hydrocarbons bioremediation.     

Development of Autotrophy and Tolerance to Acclimatization of Myrtus Communis Transplants Cultured In Vitro under Different Aeration

The behaviour of myrtle (Myrtus communis L.) plantlets during the last phase of in vitro culture before transplanting was studied. Myrtle plants were sampled from Mediterranean shrubland vegetation. In vitro growth of myrtle microcuttings was evaluated during the rooting phase using 500 cm³ containers fitted with two different types of closures. The number of gas exchanges and time in which aerated and closed vessels lose half of their gas content were calculated. Both types of vessel closure allowed photosynthetic activity in myrtle cultures even though the higher aeration rate induced higher net photosynthetic rate (P_N) during all the culture. In vitro morphogenetic and rooting of myrtle microcuttings were affected by the different environment conditions inside the culture vessels: plantlet growth and root formation of myrtle explants increased in aerated vessels in comparison with closed ones. The well developed root system, the higher P_N and dry mass accumulation during the pre-acclimatization phase in aerated vessels induced a better ability to face the transplant stress.     

Metabolite profiles of the biocontrol yeast<Emphasis Type="Italic"> Pichia anomala</Emphasis> J121 grown under oxygen limitation

The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (a), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (a), but continued to be produced in (b). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (b). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (b) than in (a), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.     

Taxonomic value of the property of fungi to assimilate hydrocarbons

A wide range of fungi were tested for their ability to assimilate a series of hydrocarbons, which includedn-paraffins, aromatic hydrocarbons and petroleum fractions.The property is not evenly distributed among the various fungal classes, but is to be found mainly in two orders, the Mucorales and the Moniliales. Within the latter order, the generaAspergillus andPenicillium are rich in hydrocarbon-assimilating strains. In other genera, the property of assimilating hydrocarbons is relatively rare.Hydrocarbon assimilation is not necessarily common to related species, nor proper to one species, but more the property of individual strains. Different strains belonging to the same species differ in metabolic activity when they are tested against a series of hydrocarbons. The property of assimilating hydrocarbons appears to lack taxonomic value. Species of the same genus show only a tendency to behave in a similar way, e.g.Penicillium strains usually assimilaten-decane and light gas oil whereasAspergillus strains seldom do so. Aspergillus species sporulate better on long chainn-paraffins. On some hydrocarbons, they develop particular pigments. n-Paraffins with at least ten carbon atoms support better growth than petroleum fractions. Individual strains are very sensitive to minor changes in hydrocarbon composition or structure. Only sparse delayed growth is observed on aromatic hydrocarbons.n-Heptane, petroleum ether, naphtha and kerosene are often toxic whereas aromatic hydrocarbons are usually non-toxic.     

Enzymes and genes involved in the aerobic biodegradation of methyl tert-butyl ether (MTBE)

Fuel oxygenates, mainly methyl tert-butyl ether (MTBE) but also ethyl tert-butyl ether (ETBE), are added to gasoline in replacement of lead tetraethyl to enhance its octane index. Their addition also improves the combustion efficiency and therefore decreases the emission of pollutants (CO and hydrocarbons). On the other hand, MTBE, being highly soluble in water and recalcitrant to biodegradation, is a major pollutant of water in aquifers contaminated by MTBE-supplemented gasoline during accidental release. MTBE was shown to be degraded through cometabolic oxidation or to be used as a carbon and energy source by a few microorganisms. We have summarized the present state of knowledge about the microorganisms involved in MTBE degradation and the MTBE catabolic pathways. The role of the different enzymes is discussed as well as the rare and recent data concerning the genes encoding the enzymes involved in the MTBE pathway. The phylogeny of the microorganisms isolated for their capacity to grow on MTBE is also described.     

Ovulate cone,pollination drop,and pollen capture in Sequoiadendron (Taxodiaceae)

In Sequoiadendron ovules are borne inside the ovulate cone, and pollination drops secreted from these ovules collect pollen. We examined: (1) the relation between ovular position and pollen capture; (2) pollen behavior when in contact with a pollination drop; and (3) ultrastructure of ovules during pollination drop secretion. During wet periods a water sheet forms on the surface of the cone due to bract shape and wettability. Pollination drops persist inside the wetted cone, and pollen capture resumes immediately after drying. Pollen landing on a pollination drop is taken inside the drop and carried into the micropyle when the drop contracts. Several notable ultrastructural features appear in the nucellus, integument, chalaza, and bract lamina during pollination-drop secretion. The abaxial surface of the lamina is covered by a membrane that may contribute to the wettable nature of the surface.     

Induction of Catalase Activity by Hydrocarbons in Candida tropicalis рK 233

1. The catalase activity of Candida tropicalis pK 233 was induced by hydrocarbons but not by glucose, galactose, ethanol, acetate or lauryl alcohol.

2. The induction of the catalase activity depending upon hydrocarbons was sensitive to cycloheximide but not to chloramphenicol.

3. Glucose repressed strongly the induction of the catalase activity by hydrocarbons but galactose did not affect seriously.

4. When C. tropicalis was incubated with hydrocarbons, the appearance of microbodies was observed electronmicroscopicaliy.

     

Role of alcohol dehydrogenase activity and the acetaldehyde in ethanol- induced ethane and pentane production by isolated perfused rat liver.

The volatile hydrocarbons ethane and n-pentane are produced at increased rates by isolated perfused rat liver during the metabolism of acutely ethanol. The effect is half-maximal at 0.5 mM-ethanol, and its is not observed when inhibitors of alcohol dehydrogenase such as 4-methyl- or 4-propyl-pyrazole are also present. Propanol, another substrate for the dehydrogenase, is also active. Increased alkane production can be initiated by adding acetaldehyde in the presence of 4-methyl- or 4-propyl-pyrazole. An antioxidant, cyanidanol, suppresses the ethanol-induced alkane production. The data obtained with the isolated organ demonstrate that products known to arise from the peroxidation of polyunsaturated fatty acids are formed in the presence of ethanol and that the activity of alcohol dehydrogenase is required for the generation of the active radical species. The mere presence of ethanol, e.g. at binding sites of special form(s) of cytochrome P-450, it not sufficient to elicit an increased production of volatile hydrocarbons by rat liver.     

Production of Higher-quality Plastein from a Crude Single-cell Protein

From defatted n-paraffin-assimilating yeast cells, a crude protein was obtained by alkaliextraction followed by acid-precipitation. Then the protein was treated with ether until extractable substances were removed exhaustively at this stage. However, at the next stage where the ether-treated protein had been partially hydrolyzed with pepsin, when the hydrolysate was retreated with ether, it was found that ether-extractable substances totalling 270 mg/100 g were obtainable additionally. Chromatographic investigations demonstrated that the substances included significant amounts of aliphatic and aromatic hydrocarbons, some indoles, and a ubiquinone (n = 8).

From the protein hydrolysate (substrate) after the above ether-treatment, a plastein was synthesized with Bioprase under the specific conditions. The plastein was obtained as a precipitate when the whole reaction mixture was treated with aqueous ethanol or acetone. The quantity and quality (nitrogen content) of the plastein depended on the ethanol or acetone concentration. Roughly speaking, the higher the concentration, the more the plastein quantity. The converse relation held for the quality; a plastein precipitated by treatment solely with water showed a higher quality than any other case.     

Oxidation of anthracene in water/solvent mixtures by the white-rot fungus,Bjerkandera sp. strain BOS55

Polycyclic aromatic hydrocarbons (PAH) are persistent priority pollutants of soil and sediments. The use of white-rot fungi has been proposed as a means of bioremediating PAH-polluted sites. However, higher PAH compounds of low bioavailability in polluted soil are biodegraded slowly. In order to enhance their bioavailability, PAH solubilization, can be increased in water/solvent mixtures. The oxidation of a model PAH compound, anthracene, in the presence of cosolvents by the white-rot fungus, Bjerkandera sp. strain BOS55 was investigated. Acetone and ethanol at 5% were toxic to this fungus when added at the time of inoculation. However, when solvents up to 20% (v/v) were added to 9-day-old cultures, ligninolytic activity as indicated by Poly R-478 dye decolorization and anthracene oxidation was evident for several days. Since 20% solvent was toxic to cells, the oxidation of anthracene can be attributed to extracellular peroxidases, which were shown to tolerate the solvent. Solvent additions of 11%–21% (v/v) acetone or ethanol increased the rate of anthracene bioconversion to anthraquinone in liquid medium by a factor of 2–3 compared to fungal cultures receiving 1%–3% solvent.