Degradation of long chain n-alkanes by mucorales

Summary Extracellular oxidation products isolated from the substrates ofMortierella isabellina (CBS 224. 35) grown on n-dodecane and n-tridecane were primary and secondary isomeric alcohols, isomeric ketones, aldehydes and isomeric esters with the same numbers of carbon atoms presented in the used n-alkanes as detected by combined glass capillary gas chromatography — mass spectrometry. All esters were identified which theoretically could originate in the isomeric ketones by a reaction mechanism resembling a Baeyer-Villinger-type oxidation.     

Degradation of long-chain n-alkanes by the yeast Lodderomyces elongisporus

Summary Cells of the yeast Lodderomyces elongisporus, precultured on glycerol, were incubated with long-chain n-alkanes. The results whow that monoterminal alkane oxidation is the main pathway of alkane degradation in the investigated yeast. The amount of diterminal activity is negligible, while subterminal degradation did not occur at all.Fatty acids were the first detectable intermediates. Using different n-alkanes, in every case the fatty acids with substrate chain length predominated in the cells. The formation of radioactive fatty acids from (1-¹⁴C)-hexadecane was time-dependent and indicated that desaturation elongation and -oxidation occurred.Extracellularly, the fatty acid pattern was similar, except for the additional presence of fatty acid methyl esters and the prevalence of octadecenoic acid after growth of cells on n-hexadecane.     

Degradation of long-chain n-alkanes by the yeast Candida maltosa

Summary Microsomal membrane fractions of the yeast Candida maltosa were investigated with respect to their ability to catalyse the oxidation of n-alkanes, fatty alcohols and fatty acids. Analysis of intermediates of n-hexadecane oxidation led to the conclusion that monoterminal attack was predominant, whereas diterminal oxidation proceeded as a minor reaction. The oxidation of long-chain primary alcohols to the corresponding aldehydes occurred without addition of nicotinamide adenine dinucleotide (phosphate) [NAD(P)⁺] and was accompanied by stoichiometric oxygen consumption and hydrogen peroxide production, suggesting that an alcohol oxidase instead of an NAD(P)⁺-requiring alcohol dehydrogenase catalysed these reactions. As shown for n-hexadecane, the hydroxylation of palmitic acid was found to be carbon monoxide-dependent, indicating involvement of a cytochrome P-450 system, as in the case of n-alkane hydroxylation.     

Degradation of long-chain n-alkanes (C36 and C40) by Pseudomonas aeruginosa strain WatG

Pseudomonas aeruginosa strain WatG was unable to utilize either n-hexatriacontane (C₃₆) or n-tetracontane (C₄₀), which are both insoluble in a mineral salts medium (MSM), as a sole carbon source. However, when C₃₆ and C₄₀ were added to MSM containing crude oil, more than 25% of each of the compounds was degraded by this strain after 2 weeks at 30 °C. These results demonstrate that P. aeruginosa strain WatG has the ability to degrade long-chain alkanes up to C₄₀, when they are solubilized by crude oil components.     

Degradation of long chain n-alkanes by Mucorales

Summary The degradation pathways on n-dodecane and n-tridecane were studied in seven representative strains of five families of the order Mucorales. Using thin-layer chromatographic, gas-liquid chromatographic and mass spectrometric methods, extracellular oxidation products of the relevant n-alkanes could be isolated and identified. All strains tested exhibited a formation of isomeric primary and secondary alcohols, isomeric ketones and monoic acids with chain length equivalent to the n-alkanes in the substrates, proving that in the order Mucorales both a terminal and a subterminal oxidation pathway are realized.     

Degradation of long chain n-alkanes by Chlorococcales

Summary Four out of thirty-one algae strains belonging to the order Chlorococcales exhibited good growth on solid media containing n-alkanes. Chlorella vulgaris (397) was able to degrade n-tridecane in cooxidation. The corresponding secondary alcohols and ketones in C₂-to C₇-position could be identified in the culture broth. The same oxidation products could be found in the media of cultures grown in darkness with the addition of glucose. This demonstrates a subterminal degradation pathway of C. vulgaris.There was no indication for a mono-or diterminal oxidation of alkanes by algae.The fatty acid pattern of lipids exhibited an incease in long chain acids and a decrease in shorter chain acids. The growth rate of cells grown on alkanes increased after 72 h, but the release of autospores was retarded.     

Degradation of long chain n-alkanes by disrupted cells ofChlorella vulgaris

Summary The alkane oxidation byChlorella vulgaris is improved by disruption of the cells. Although living cells are not able to attack n-dodecane, disrupted cells produced detectable amounts of oxidation products. The amount of isomeric alcohols and ketones of n-tridecane was nearly double the sum found in living cells, whereas the equilibrium was shifted to the ketones. With n-tetradecane and n-pentadecane only the amount of ketones increased.     

Anaerobic oxidation of long-chain n-alkanes by the hyperthermophilic sulfate-reducing archaeon,Archaeoglobus fulgidus

The thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain VC-16 (DSM 4304), which is known to oxidize fatty acids and n-alkenes, was shown to oxidize saturated hydrocarbons (n-alkanes in the range C₁₀–C₂₁) with thiosulfate or sulfate as a terminal electron acceptor. The amount of n-hexadecane degradation observed was in stoichiometric agreement with the theoretically expected amount of thiosulfate reduction. One of the pathways used by anaerobic microorganisms to activate alkanes is addition to fumarate that involves alkylsuccinate synthase as a key enzyme. A search for genes encoding homologous enzymes in A. fulgidus identified the pflD gene (locus-tag AF1449) that was previously annotated as a pyruvate formate lyase. A phylogenetic analysis revealed that this gene is of bacterial origin and was likely acquired by A. fulgidus from a bacterial donor through a horizontal gene transfer. Based on three-dimensional modeling of the corresponding protein and molecular dynamic simulations, we hypothesize an alkylsuccinate synthase activity for this gene product. The pflD gene expression was upregulated during the growth of A. fulgidus on an n-alkane (C₁₆) compared with growth on a fatty acid. Our results suggest that anaerobic alkane degradation in A. fulgidus may involve the gene pflD in alkane activation through addition to fumarate. These findings highlight the possible importance of hydrocarbon oxidation at high temperatures by A. fulgidus in hydrothermal vents and the deep biosphere.     

Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes

A thermophilic Bacillus strain NG80-2 growing within the temperature range of 45–73°C (optimum at 65°C) was isolated from a deep subterranean oil-reservoir in northern China. The strain was able to utilize crude oil and liquid paraffin as the sole carbon sources for growth, and the growth with crude oil was accompanied by the production of an unknown emulsifying agent. Further examination showed that NG80-2 degraded and utilized only long-chain (C15–C36) n-alkanes, but not short-chain (C8–C14) n-alkanes and those longer than C40. Based on phenotypic and phylogenic analyses, NG80-2 was identified as Geobacillus thermodenitrificans. The strain NG80-2 may be potentially used for oily-waste treatment at elevated temperature, a condition which greatly accelerates the biodegradation rate, and for microbial enhancing oil recovery process.Lei Wang, Yun Tang and Shuo Wang contributed equally to this study.     

Sexuality in mucorales

Using varied strains of Blakeslea trispora it is shown that, contrary to earlier reports, a limiting role in hormone synthesis in mated cultures cannot be distinctively assigned to either the plus or the minus partner.     

Degradation of long-chain n-alkanes by the yeast Candida maltosa

Summary The yeast Candida maltosa precultivated on liquid n-alkanes utilized different solid n-alkanes (especially C₂₀–C₂₅) in the presence of pristane as an organic phase with rates comparable to, or somewhat larger than, those of liquid n-alkanes. Analysis of cellular fatty acids indicated an assimilation of solid n-alkanes via monoterminal oxidation. The resulting fatty acids with substrate chain length were chain-shortened by C₂ units down to an optimal range of chain length from C₁₆ to C₁₈ and incorporated into cellular, lipids directly or after desaturation. The intermediates of chain-shortening with numbers of carbon atoms higher than C₁₈, as well as the unusually long-chain fatty acids of substrate chain length, were detected in trace amounts only. Even-carbon-numbered and odd-numbered fatty acids predominated in experiments with evenchain and odd-chain n-alkanes, respectively. Studies with cerulenin indicated that de novo synthesis of fatty acids was negligible. Oxidation of solid n-alkanes by the yeast C. maltosa yielded fatty acid patterns similar to those of cells grown on liquid n-alkanes.     

The utilization of the mixtures of monosaccharides by some mucorales

Summary The utilization of mixtures of monosaccharides byBlakeslea trispora Thaxter,Choanephora circinans (Naganish &Kawakami)Hesseltine &Benjamin,Gilbertella persicaria var.indica Mehrotra &Mehrotra andHelicostylum piriforme Bainier was studied. The effect of sorbose on the utilization of other sugars present in the mixtures was also studied. It was found that all the mixtures of sugars in combination with asparagine or ammonium chloride were valueless for all the organisms exceptHelicostylum piriforme. Growth ofHelicostylum piriforme on the mixtures with asparagine as the nitrogen source was better than on the mixtures with ammonium chloride as the source of nitrogen. Asparagine being a favourable source counteractecd sorbose inhibition, while ammonium chloride failed to do so. On the other hand, both of the nitrogen sources failed to counteract sorbose inhibition in the rest of the organisms. None of the organisms could finish sorbose and rhamnose from any of the mixtures within the specified period.     

Degradation of oil tank sludge using long-chain alkane-degrading bacteria

Bacteria degrading a very long-chain alkane, n-tetracosane, were isolated from enrichment culture of soil in Okinawa. Phylogenetic analysis of their16S rRNA sequences revealed that they belong to classes Gammaproteobacteria and Actinomycetes. Three isolates belonging to the genera Acinetobacter sp., Pseudomonas sp., and Gordonia sp. showed a stable growth on n-tetracosane and had a wide range of assimilation of aliphatic hydrocarbons from C₁₂ to C₃₀, while not on alkanes shorter than C₈. Of the isolates, Gordonia sp. degraded oil tank sludge hydrocarbons efficiently by solving the sludge in a hydrophobic solvent, while Acinetobacter sp. showed little degradation, possibly due to the difference in the mechanism of hydrophobic substrate incorporation between proteobacteria and actinobacteria. The data suggested that non-heme di-iron monooxygenases of the AlkB-type, not bacterial CYP153 type cytochrome P450 alkane hydroxylase, was involved in the alkane degradation.     

Degradation of n-alkanes by Candida parapsilosis and Penicillium frequentans immobilized on granular clay and aquifer sand

Summary The immobilization intensity of cells of Penicillium frequentans and Candida parapsilosis on materials such as granular clay, granular clay + aquifer sand and aquifer sand alone, was followed by scanning electron microscopy (SEM). The results demonstrate that the granular clay was the best adsorbent for both organisms, followed by the mixture of both granular clay and aquifer sand. Poor adhesion of cells was detected on using aquifer sand alone with C. parapsilosis.The highest degree of degradation of the alkane mixture (C₁₂–C₁₈) used was achieved by cells immobilized on granular clay, followed by those cells adsorbed on clay and sand. The weakest degradation was observed with cells immobilized on the sand alone.     

铜绿假单胞菌SJTD-2降解长链烷烃与原油的特性

摘要:【目的】石油污染严重威胁生态系统和生物圈,微生物修复被认为是一种安全有效可代替物化方法来治理石油污染的办法。本文对我们从石油污染土壤中分离获得的一株可分解正烷烃和原油的革兰氏阴性菌SJTD-2的理化性质和降解效能进行了研究。【方法】利用菌株表型和生理性质、16S rRNA序列比较分析与进化树绘制,确定新分离菌株SJTD-2的种属;测定菌株SJTD-2的生长曲线,确定其利用不同长度烷烃和原油为单一碳源的效能;利用GC-MS检测烷烃类物质的残留量,确定菌株SJTD-2降解烷烃和原油SJTD-2的降解效率和降解周期。【结果】菌株表型与16S rRNA序列比较及进化树比对分析结果显示,菌株SJTD-2与假单胞菌属的亲缘关系十分接近,为铜绿假单胞菌。菌株SJTD-2 可有效分解C10到C26的中链和长链烷烃及原油,利用它们作为其单一碳源生长;该菌株对长链烷烃(C18－C22)的利用效果较中链烷烃好,其中正二十二烷被认为是其最佳碳源。48 h内,该菌株可完全降解500 mg/L正二十二烷;72h 后,2 g/L的正二十二烷可几乎被菌株全部分解利用。此外,菌株SJTD-2在7 d内可将2 g/L的原油分解88%以上。【结论】与现有其它烷烃降解菌相比,铜绿假单胞菌SJTD-2具有突出的长链烷烃与原油降解效能及耐受能力,该菌株的发现与研究将有助于烷烃降解机制的研究和环境修复的进程。     

Degradation of n-alkanes and polycyclic aromatic hydrocarbons in petroleum by a newly isolated Pseudomonas aeruginosa DQ8

A bacterial isolate, designated as DQ8, was found capable of degrading diesel, crude oil, n-alkanes and polycyclic aromatic hydrocarbons (PAHs) in petroleum. Strain DQ8 was assigned to the genus Pseudomonas aeruginosa based on biochemical and genetic data. The metabolites identified from n-docosane as substrate suggested that P. aeruginosa DQ8 could oxidize n-alkanes via a terminal oxidation pathway. P. aeruginosa DQ8 could also degrade PAHs of three or four aromatic rings. The metabolites identified from fluorene as substrate suggested that P. aeruginosa DQ8 may degrade fluorene via two pathways. One is monooxygenation at C-9 of fluorene, and the other is initiated by dioxygenation at C-3 and C-4 of fluorene. P. aeruginosa DQ8 should be of great practical significance both in bioremediation of oil-contaminated soils and biotreatment of oil wastewater.     

Heavy oils,principally long-chain n-alkanes secreted by Aureobasidium pullulans var. melanogenum strain P5 isolated from mangrove system

In this study, the yeast strain P5 isolated from a mangrove system was identified to be a strain of Aureobasidium pullulans var. melanogenum and was found to be able to secrete a large amount of heavy oil into medium. After optimization of the medium for heavy oil production and cell growth by the yeast strain P5, it was found that 120.0 g/l of glucose and 0.1 % corn steep liquor were the most suitable for heavy oil production. During 10-l fermentation, the yeast strain P5 produced 32.5 g/l of heavy oil and cell mass was 23.0 g/l within 168 h. The secreted heavy oils contained 66.15 % of the long-chain n-alkanes and 26.4 % of the fatty acids, whereas the compositions of the fatty acids in the yeast cells were only C_16:0 (21.2 %), C_16:1(2.8 %), C_18:0 (2.9 %), C_18:1 (39.8 %), and C_18:2 (33.3 %). We think that the secreted heavy oils may be used as a new source of petroleum in marine environments. This is the first report of yeast cells which can secrete the long-chain n-alkanes.     

Diversity of dioxygenases that catalyze the first step of oxidation of long-chain n-alkanes in Acinetobacter sp. M-1

Abstract Three kinds of enzymes, designated A, B and C, involved in n -alkane oxidation were found in the cytoplasm of n -alkanegrown Acinetobacter sp. M-1. All catalyzed the dioxygenation of n -alkanes to the corresponding n -alkyl hydroperoxides. Purified enzyme A consisted of four identical subunits having a molecular mass of 72 kDa. The enzyme was strongly inhibited by several iron-chelating agents, such as o -phenanthroline, 8-hydroxyquinoline and α,α'-dipyridyl, and could be distinguished from enzyme C, a Cu²⁺-requiring flavoprotein. Enzyme B was relatively unstable on purification. The three enzymes used n -alkanes, n -alkenes, and aryl compounds with longer alkyl side chains as substrates. Enzymes B and C were more active toward relatively short n -alkanes (C_12–16). Enzyme A oxidized solid n -alkanes well, the most preferable substrate being tetracosane (C₂₄). Enzyme A is responsible for about 80% of the total activity found in the soluble fraction of n -alkane-grown Acinetobacter sp. M-1, indicating that the enzyme plays a major role during growth on solid n -alkanes.