Hydrocarbon degradation by <Emphasis Type="Italic">Dietzia</Emphasis> sp. A14101 isolated from an oil reservoir model column

The hydrocarbon-degrading strain Dietzia sp. A14101 was isolated from an oil reservoir model column inoculated with oil-field bacteria. The column was continuously injected with nitrate (0.5 mM) from the start of water flooding, which lead to a gradual development of nitrate reduction in the column. Strain A14101 was able to utilize a range of aliphatic hydrocarbons as sole carbon and energy source during aerobic growth. Whole oil gas chromatography analysis of the crude oil phase from aerobic pure cultures showed that strain A14101 utilized the near complete range of aliphatic components and aromatic components toluene and xylene. Longer n-alkanes ≥C₁₇ were utilized simultaneously with the shorter C₁₀ and C₁₅. After 120 days aerobic incubation, the whole oil gas chromatography profile of the crude oil phase was similar to that of heavily biodegraded oils. Anaerobic degradation of hydrocarbons with nitrate was not observed. Nitrate reduction was, however, observed during anaerobic growth on propionate, which suggests that strain A14101 grows on fatty acids in the column rather than on hydrocarbons.     

Survey of indigenous bacterial endophytes from cotton and sweet corn

The genotypic diversity of indigenous bacterial endophytes within stems and roots of sweet corn (Zea mays L.) and cotton (Gossypium hirsutum L.) was determined in field trials throughout one growing season. Strains were isolated from surface-disinfested tissues and identified by fatty acid analysis. Gram-negative bacteria comprised 70.5% of the endophytic bacteria and 27 of the 36 genera identified. The most frequently isolated groups from sweet corn roots, were Burkholderia pickettii and Enterobacter spp.; from sweet corn stems, Bacillus megaterium. Bacterial genera present in sweet corn roots were also generally present in sweet corn stems. However, Burkholderia gladioli, Burkholderia solanacearum and Enterobacter cloacae were isolated much more frequently from sweet corn roots than stems, whereas Methylobacterium spp. were found more frequently in sweet corn stems than roots. Agrobacterium radiobacter, Serratia spp. and Burkholderia solanacearum, were the most frequently isolated groups from cotton roots; and Bacillus megaterium and Bacillus pumilus from cotton stems. Acinetobacter baumannii and Arthrobacter spp. were present in cotton stems but not in cotton roots. There were 14 taxonomic groups present in cotton roots that were not in cotton stems; all but one were Gram-negative. These included, Agrobacterium radiobacter, Bacillus megaterium, Bacillus pumilus, Enterobacter asburiae, Pseudomonas chlororaphis, Serratia spp. and Staphylococcus spp. Rhizobium japonicum and Variovorax paradoxus were isolated, almost exclusively, from the roots of both crops. Bacterial taxa present in both sweet corn and cotton early in the season were generally present late in the season. The diversity of bacteria was greater in roots than stems for each crop.     

Aroma Constituents of Roasted Coconut

The volatile components of roasted and unroasted dried coconut shreds, isolated by steam distillation, were analyzed by GC and GC-MS. Seventeen compounds were identified from the unroasted coconut, and nine of them were newly identified in coconut meat aroma. Saturated delta-C₈, C₁₀, and C₁₂ lactones were determined as the main components giving the characteristic mild, sweet, and pleasant coconut flavor. The roasted coconut gave the strong characteristic sweet and nutty aroma, and the GC-MS indicated the saturated delta-lactones as main components, and six pyrazines, two furans, and two pyrroles also found seemed to contribute greatly to the nut-like aroma of roasted coconut. The defatted and roasted meal gave a strong nut-like and burnt odor, but not the characteristic sweet aroma. A large increase in pyrazines and other Maillard reaction products and an absence of lactones and fatty acid esters were observed in the volatiles of the roasted-defatted coconut meal.     

Isolation and Characterization of a Neutral Glycosphingolipid from the Epimastigote Form of Trypanosoma mega1

ABSTRACT. A glycosphingolipid fraction from Trypanosoma mega was isolated after acetylation and was further purified on a silicic acid column. Final purification was by preparative thin-layer chromatography. The carbohydrate components of the glycolipid were fucose and galactose in approximately equimolar amounts. The neutral glycolipid of T. mega has a sphingosine base composition that consists of sphingosine and traces of dihydrosphingosine. Fatty acids forming amide groups with the sphingosine bases were analyzed by gas-liquid chromatography-mass spectrometry and are a mixture of normal and α-hydroxy fatty acids. Normal C_16:0, C_18:0, and 2-hydroxy C_18:0 are the predominant fatty acids.     

Degradation and mineralization of petroleum by two bacteria isolated from coastal waters

Within the framework of a study on the oil biodegradation potential of the sea the ability of a Flavobacterium sp. and Brevibacterium sp. to metabolize a paraffinic crude oil and a chemically defined hydrocarbon mixture was investigated. Major components of the crude oil were identified by combination gas chromatography and mass spectrometry. The rate and extent of total hydrocarbon biodegradation was measured. In addition, CO₂ evolution from the crude oil was continuously monitored in a shaker-mounted gas train arrangement. Degradation started after a 2 to 4 day lag period, and reached its maximum within two weeks. At this time up to 60% of the crude oil and 75% of the model hydrocarbon mixture, each added at the level of 1 ml per 100 ml artificial sea water, were degraded. Mineralization(conversion to CO₂) was slightly lower due to formation of products and bacterial cell material. n-Paraffins were preferentially degraded as compared to branched chain hydrocarbons. Biodegradation of n-paraffins in the range of C₁₂ to C₂₀ was simultaneous; no diauxie effects were observed.     

Secretory expression and characterization of a highly Ca-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca²⁺. L2 lipase showed a preference for medium to long chain triacylglycerols (C₁₀–C₁₆), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.     

The interactions of plant growth regulators and H<Subscript>2</Subscript>O<Subscript>2</Subscript> during germination improvement of sweet corn seed through spermidine application

Low seed vigor was the main constraint on the production of sweet corn in China. Spermidine (Spd) was proved to enhance sweet corn seed germination. However, little was known about the metabolisms and interactions of plant growth regulators (PGRs) and H₂O₂ in the enhancement of Spd upon sweet corn seed germination. Spd, GA, C₂H₄ and H₂O₂ soaking treatments significantly enhanced seed vigor; while their respective biosynthesis inhibitors and ABA significantly declined seed vigor. Besides, as compared with control, seed vigor showed no significant difference in Spd+ProG (prohexadione-calcium, the inhibitor of GA), however it decreased significantly in Spd+ABA. The seed vigor treated by Spd+AVG (aviglycine hydrochloride, the inhibitor of C₂H₄) and Spd+NAC (n-acetyl-l-cysteine, a scavenger of H₂O₂) were significantly lower than those soaked in Spd solution, but still significantly higher than the control. Spd+NAC with significantly lower H₂O₂ content still up-regulated GA and C₂H₄ contents and down-regulated ABA content during seed germination. The results suggested that it was Spd rather than H₂O₂ (produced through Spd) made a direct effect on PGRs metabolism regulation in seed germination enhancement by Spd. The metabolism of GA and ABA played crucial rolesas compared with C₂H₄ and H₂O₂. Besides, complicated PGRs interactions and crosstalk between H₂O₂ and PGRs existed during sweet corn seed germination after Spd soaking, and ABA might be a key hormone in this process.     

Comparative studies on simple lipids ofSclerotium cepivorum,the causal agent of white rot of onion,and other fungi of the class Deuteromycetes

Summary The simple lipids ofSclerotium cepivorum, the causal agent of white rot of onion and nine other fungal species of the same class were investigated. The fatty acid composition of the simple lipids of these fungi were determined by GLC. The main fatty acids common to these fungal species were C₁₆ (saturated) and C₁₈ (unsaturated) acids. The sterol fraction was isolated by column chromatography and its components were detected by GLC and mass spectrometry. Ergosterol and γ-Ergostenol were found mostly in all fungal species under investigation. However, two fungal species namelyAlternaria alternata andScolecobasidium constrictum showed no Ergosterol.     

A survey for isoenzymes of glucosephosphate isomerase,phosphoglucomutase, glucose-6-phosphate dehydrogenase and 6-Phosphogluconate dehydrogenase in C3-, C4-and crassulacean-acid-metabolism plants,and green algae

Two isoenzymes each of glucosephosphate isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) were separated by (NH₄)₂SO₄ gradient solubilization and DEAE-cellulose ion-exchange chromatography from green leaves of the C₃-plants spinach (Spinacia oleracea L.), tobacco (Nicotiana tabacum L.) and wheat (Triticum aestivum L.), of the Crassulacean-acid-metabolism plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen, and from the green algae Chlorella vulgaris and Chlamydomonas reinhardii. After isolation of cell organelles from spinach leaves by isopyenic centrifugation in sucrose gradients one of two isoenzymes of each of the four enzymes was found to be associated with whole chloroplasts while the other was restricted to the soluble cell fraction, implying the same intracellular distribution of these isoenzymes also in the other species.Among C₄-plants, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in only one form in corn (Zea mays L.), sugar cane (Saccharum officinarum L.) and Coix lacrymajobi L., but as two isoenzymes in Atriplex spongiosa L. and Portulaca oleracea L. In corn, the two dehydrogenases were mainly associated with isolated mesophyll protoplasts while in Atriplex spongiosa they were of similar specific activity in both mesophyll protoplasts and bundle-sheath strands. In all five C₄-plants three isoenzymes of glucosephosphate isomerase and phosphoglucomutase were found. In corn two were localized in the bundle-sheath strands and the third one in the mesophyll protoplasts. The amount of activity of the enzymes was similar in each of the two cell fractions. Apparently, C₄ plants have isoenzymes not only in two cell compartments, but also in physiologically closely linked cell types such as mesophyll and bundle-sheath cells. New address: Institut für Pflanzenphyiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a, D-1000 Berlin 33     

Anaerobic oxidation of alkanes by newly isolated denitrifying bacteria

The capacity of denitrifying bacteria for anaerobic utilization of saturated hydrocarbons (alkanes) was investigated with n-alkanes of various chain lengths and with crude oil in enrichment cultures containing nitrate as electron acceptor. Three distinct types of denitrifying bacteria were isolated in pure culture. A strain (HxN1) with oval-shaped, nonmotile cells originated from a denitrifying enrichment culture with crude oil and was isolated with n-hexane (C₆H₁₄). Another strain (OcN1) with slender, rod-shaped, motile cells was isolated from an enrichment culture with n-octane (C₈H₁₈). A third strain (HdN1) with oval, somewhat pleomorphic, partly motile cells originated from an enrichment culture with aliphatic mineral oil and was isolated with n-hexadecane (C₁₆H₃₄). Cells of hexane-utilizing strain HxN1 grew homogeneously in the growth medium and did not adhere to the alkane phase, in contrast to the two other strains. Quantification of substrate consumption and cell growth revealed the capacity for complete oxidation of alkanes under strictly anoxic conditions, with nitrate being reduced to dinitrogen. Received: 3 August / Accepted: 6 October 1999     

Studies on the Changes of Meat Fats by Various Processings

Data are presented to show the gas chromatographic identification of a total of 18 saturated aliphatic γ- and δ-lactones obtained from melted beef depot fat, namely, δ-C₆, γ-C₇, γ-C₈, γ-C₉, and a homologous series of γ- and δ-lactones of the even-carbon numbers C₁₀ to C₁₆ and of smaller amount of the odd-carbon numbers C₁₁ to C₁₅. These lactones were isolated by steam distillation and silicic acid adsorption chromatography, and identified through gas chromatography and infrared spectroscopy.

Lactones obtained had a peach-like flavor, and it was suggested that lactones were important in heated beef fat as the flavor compounds.     

Saponified palm kernel oil and its major free fatty acids as carbon substrates for the production of polyhydroxyalkanoates in Pseudomonas putida PGA1

The synthesis of polyhydroxyalkanoates (PHA) by Pseudomonas putida PGA1, using saponified palm kernel oil (SPKO), was investigated. The PHA produced from SPKO was compared with those produced by the major free fatty acids found in the palm kernel oil. Owing to the absence of lipase activity in P.␣putida, palm kernel oil did not support cell growth. However, SPKO could support cell growth and produced relatively high yield of both dry cells and PHA. The polyester produced was similar in properties to those derived from lauric (C_12:0) and myristic (C_14:0) acids, while oleic acid (C_18:1) gave rise to PHA that was sticky and of broader molecular mass distribution. Nuclear magnetic resonance and gas chromatography showed that these PHA were copolymers consisting mainly of n-alkanoate monomers ranging from C₆ to C₁₄, with C₈ as the predominant component. PHA derived from SPKO and oleic acid also contained a small amount of unsaturated monomers. Received: 25 March 1996 / Received last revision: 30 September 1996 / Accepted: 18 October 1996     

Degradation of Raspberry Suberin by Fusarium solani f. sp. Pisi and Armillaria mellea

When submers cultures of Fusarium solani f. sp. pisi and Armillaria mellea were grown in a medium supplemented with 0.5 % suberin isolated from raspberry periderm, hydrolytic enzymes were produced and measured by a spectrophotometric assay using p-nitrophenyl butyrate as substrate. The enzymatic activity in the culture fluids reached its peak after 32 to 44 days of incubation. In a gas-chromatographic assay of the enzymatic degradation of suberin, concentrated culture fluids of suberin-grown fungi were incubated with raspberry suberin. The culture fluids of F. solani and A. mellea catalyzed the release of chloroform-soluble products, which were analyzed by gas-liquid chromatography. Suberin monomers like fatty alcohols and acids with chain-lengths from C₁₆ to C₂₆ as well as C₁₆ and C₁₈ω-hy-droxyacids could be identified as products. The suberin-induced enzymes showed catalytic properties similar to cutin-hydrolyzing enzymes previously isolated from different fungi.     

Isolation and characterization of fatty acid methyl ester (FAME)‐producing Streptomyces sp. S161 from sheep (Ovis aries) faeces

An actinomycete producing oil‐like mixtures was isolated and characterized. The strain was isolated from sheep faeces and identified as Streptomyces sp. S161 based on 16S rRNA gene sequence analysis. The strain showed cellulase and xylanase activities. The ¹H nuclear magnetic resonance (NMR) spectra of the mixtures showed that the mixtures were composed of fatty acid methyl esters (52·5), triglycerides (13·7) and monoglycerides (9·1) (mol.%). Based on the gas chromatography–mass spectrometry (GC‐MS) analysis, the fatty acid methyl esters were mainly composed of C14‐C16 long‐chain fatty acids. The results indicated that Streptomyces sp. S161 could produce fatty acid methyl esters (FAME) directly from starch. To our knowledge, this is the first isolated strain that can produce biodiesel (FAME) directly from starch.

Significance and Impact of the Study

Nowadays, production of biodiesel is based on plant oils, animal fats, algal oils and microbial oils. Lipid mostly consists of triacylglycerols (TAG), and conversion of these lipids into fatty acid short‐chain alcohol esters (methanol or ethanol) is the final step in biodiesel production. In this study, an oil‐producing Streptomyces strain was isolated from sheep faeces. The oil was composed of C₁₄‐C₁₆ long‐chain fatty acid methyl esters, triglycerides and monoglycerides. This is the first isolated strain‐producing biodiesel (FAME) directly from starch. Due to showing cellulase and xylanase activities, the strain would be helpful for converting renewable lignocellulose into biodiesel directly.     

Studies on the Essential Oils of Tobacco Leaves

An investigation was conducted on the neutral fraction in the essential oil of Virginia tobacco leaves. α-Pyrryl methyl ketone, ethyl alcohol, ethyl acetate and furfuryl alcohol were isolated and identified. An alcohol, C₄, and an ester of benzoic acid were also separated. Hydrocarbons were separated from the total neutral fraction by liquid chromatography, prior to any further procedure; and a straight chain paraffin near C₃₀ and an unsaturated hydrocarbon resembling myrcene were also isolated and their contents were determined. A technique of chromatostrip was found to be very useful for detecting liquid chromatographic separation.     

Uptake of hydrocarbons by the marine diatomCyclotella cryptica

The accumulation of exogenous hydrocarbons by the marine diatomCyclotella cryptica grown in culture has been studied using gas chromatography. Exposure of the alga to paraffins for 10 days results in accumulation of n-alkanes having between C₁₃ and C₁₆ carbon atoms. The C₁₆ level in the accumulated fraction is twice as high as that in the original oil.     

Purification and Properties of Mesophyll and Bundle Sheath Cell alpha-Glucan Phosphorylases from Zea mays L. : Equivalence of the Enzymes with the Cytosol and Plastid Phosphorylases from Spinach

Two major α-glucan phosphorylases (I and II) from leaves of the C₄ plant corn (Zea mays L.) were previously shown to be compartmented in mesophyll and bundle sheath cells, respectively (C Mateyka, C Schnarrenberger 1984 Plant Sci Lett 36: 119-123). The two enzymes were separated by chromatography on DEAE-cellulose and purified to homogeneity by affinity chromatography on immobilized starch, according to published procedures, as developed for the cytosol and chloroplast phosphorylase from the C₃ plant spinach. The two α-glucan phosphorylases have their pH optimum at pH 7. The specificity for polyglucans was similar for soluble starch and amylopectin, however, differed for glycogen (K_m = 16 micrograms per milliliter for the mesophyll cell and 250 micrograms per milliliter for the bundle sheath cell phosphorylase). Maltose, maltotriose, and maltotetraose were not cleaved by either phosphorylase. If maltopentaose was used as substrate, the rate was about twice as high with the bundle sheath cell phosphorylase, than with the mesophyll cell phosphorylase. The phosphorylase I showed a molecular mass of 174 kilodaltons and the phosphorylase II of 195 kilodaltons for the native enzyme and of 87 and of 53 kilodaltons for the SDS-treated proteins, respectively. Specific antisera raised against mesophyll cell phosphorylase from corn leaves and against chloroplast phosphorylase from spinach leaves implied high similarity for the cytosol phosphorylase of the C₃ plant spinach with mesophyll cell phosphorylase of the C₄ plant corn and of chloroplast phosphorylase of spinach with the bundle sheath cell phosphorylase of corn.     

Trypsin inhibitors in plants

Trypsin inhibitors were found in several food plants. Potato and sweet corn were the most inhibitory, while fruits had negligible activity. Intermediate in activity were sweet potato, spinach, broccoli, Brussels sprouts and cucumber. The trypsin inhibitor of sweet corn was isolated by extraction in dilute salt solution, ammonium sulfate fractionation, chromatography with Sephadex G75 and CM-cellulose and lyophilization. Two components were demonstrated by disc gel electrophoresis. The inhibitor was heat stable. It had little inhibitory activity against papain but was moderately active against chymotrypsin.     

Biodegradation of slop oil from a petrochemical industry and bioreclamation of slop oil contaminated soil

Slop oil, i.e. waste oil from a petrochemical complex, contains at least 240 hydrocarbon components, of which 54% are from C₅ to C₁₁ and the rest from C₁₂ to C₂₃. Of 22 isolated bacterial cultures that were able to degrade slop oil, seven could each degrade about 40% of the slop oil, and a mixture of all seven could degrade 50% in liquid medium. Bioaugmentation of soil contaminated with slop oil with the mixed bacterial culture gave up to 70% degradation of slop oil after 30 days. This compares with 40% degradation without bioaugmentation. Bioaugmentation led to a significant increase in counts of bacteria able to degrade slop oil. Wheat sown on bioaugmented soil germinated and grew better than on non-augmented soil and led to increased degradation of slop oil (up to 80%). This indicates the potential of mixed culture for bioremediation.     

Production of fumonisins B1, B2 and B3 byFusarium proliferatum Isolated from rye grains

Using the seed- plate technique, we have isolated a strain ofF. proliferatum from rye grains that produces 3 fumonisins, fumonisin B₁ (FB₁), FB₂ and FB₃ on inoculated rice and corn. Inoculated corn and rice were extracted with an aqueous methanol solution and fumonisin concentrations estimated using high performance liquid chromatography. Production of all 3 fumonisins (FB₁, FB₂ and FB₃) was much higher on rice than corn; ranging from 3816, 1068 and 985 ppm to 1643, 350 and 162 ppm respectively. We conclude that all natural substrates whereF. proliferatum is present as a component of the mycoflora should be monitored for the presence of fumonisins.