Desulfurization of 2,4,6,8-tetraethyl dibenzothiophene by recombinant Mycobacterium sp. strain MR65

Recombinant Mycobacterium sp. strain MR65 harboring dszABCD genes was used to desulfurize alkyl dibenzothiophenes (C_x-DBTs) in n-hexadecane. The specific desulfurization activity for 2,4,6,8-tetraethyl DBT (C₈-DBT) by DszC enzyme was about twice that for 4,6-dipropyl DBT (C₆-DBT). However, the degradation rate of 2,4,6,8-tetraethyl DBT in n-hexadecane by resting cells of strain MR65 was only about 40% of that of 4,6-dipropyl DBT. These results indicated that the desulfurization ability for Cx-DBTs by resting cells depends on carbon number substituted at positions 4 and 6 and that the rate-limiting step in the desulfurization reaction of highly alkylated C_x-DBTs is the transfer process from the oil phase into the cell.     

Synthesis of dl-Phyllostine

“Protein-like activator (PA)” for n-alkane oxidation was formed by Pseudomonas aeruginosa S₇B₁ from long-chain n-alkanes, 1-hexadecene and cetyl alcohol but not from glucose, glycerol and palmitic acid. The molecular weight and the total amino acid residues of PA were estimated at about 14,300 and 147, respectively. PA was relatively stable to low pH and high temperature, and completely inactivated upon heating at 98°C for 45 min. The cultural fluid obtained from n-hexadecane medium stimulated the growth of the strain on n-hexadecane. The degree of the growth stimulation by the fluid depended on the amount of PA and rhamnolipid (RL) in the fluid. The heat-treated PA lost the growth-stimulaing effect and the emulsifying power on the n-hexadecane medium in the presence of RL.     

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     

Aliphatic Hydrocarbons of Cladosporium resinae Cultured on Glucose,Glutamic Acid,and Hydrocarbons

The carbon source markedly influenced the qualitative and quantitative composition of cellular hydrocarbons in Cladosporium resinae. Total lipid and hydrocarbon content was greater in cells grown on n-alkanes than in cells grown on glucose or glutamic acid. Glucose-grown cells contained a spectrum of aliphatic hydrocarbons from C₇ to C₃₆; pristane and n-hexadecane comprised 98% of the total. Cells grown on glutamic acid contained C₇ to C₂₃ hydrocarbons; n-tridecane, n-tetradecane, n-hexadecane, and pristane made up 74% of the total. n-Decane-grown cells yielded C₈ to C₃₂ compounds, and n-hexadecane (96%) was the major hydrocarbon. Cells grown on individual n-alkanes from C₁₁ to C₁₅ all contained C₁₁ to C₂₈ hydrocarbons, and cells grown on n-hexadecane contained C₁₁ to C₃₂ hydrocarbons. In n-undecane-grown cells, n-hexadecane and pristane made up 92% of the total, but in cells grown on C₁₂ to C₁₆ n-alkanes the major cellular hydrocarbon was the one on which the cells were grown. This suggests that cells cultured on n-alkanes of C₁₂ or longer accumulate n-alkanes prior to oxidizing them.     

Production of Inosine from Hydrocarbons by Corynebacterium petrophilum

Adenine-auxotrophic mutants were derived from Corynebacterium petrophilum SB 4082 by ultraviolet irradiation. The isolation of this auxotroph was carried out as follows; the adenine-auxotrophic mutants which had been derived by the UV irradiation were concentrated with penicillin. The auxotrophs were raised in concentration by recycling procedure and were separated by the ordinary method. The resultant adenine-auxotroph was cultured in the hydrocarbon medium. From its filtrate was obtained a fermentation product as crystals by means of ion-exchange resin and identified as inosine from absorption spectra and other properties.

The effects of cultural conditions on inosine production were investigated by the adenine auxotroph of Corynebacterium petrophilum SB 4082. That, the amount of adenine in the medium was very important on the inosine formation, was cleared. The addition of 10 mg of adenine and 0.5 g of yeast extract to 100 ml of medium was the best for the inosine formation. Ammonium chloride or ammonium sulphate was effective as nitrogen sources. As the carbon sources, n-C₁₀ to n-C₁₆ were utilized for the growth, but the hydrocarbons from n-C₁₂ to n-C₁₆ were the most suitable for the inosine formation. The inosine fermentation began at 24 hrs. after inoculation. The accumulation of inosine attained to the highest level after five days, the amount of which was 1.6 g per liter of the culture filtrate.     

Data consistency,yield, maintenance,and hysteresis in batch cultures of Candida lipolytica cultured on n-hexadecane

Candida lipolytica was cultured batchwise using n-hexadecane as the main carbon source. Biomass production, n-hexadecane consumption, oxygen consumption, and carbon dioxide evolution were measured to follow the fermentation. The consistency of the measured data was examined using integrated and instantaneous available electron and carbon balances. Values of the “true” growth yield, η_max, and maintenance coefficient, m_e were estimated using three different sets of data (biomass and n-hexadecane, oxygen and biomass, and CO₂ and biomass), and the results were compared with estimates obtained from literature data. Hysteresis patterns were observed in plots of specific rates of oxygen consumption and carbon dioxide evolution versus specific growth rate.     

n-Alkane Chain Length Alters Dietzia sp. Strain DQ12-45-1b Biosurfactant Production and Cell Surface Activity

Upon growth on n-hexadecane (C₁₆), n-tetracosane (C₂₄), and n-hexatriacontane (C₃₆), Dietzia sp. strain DQ12-45-1b could produce different glycolipids, phospholipids, and lipopeptides. Interestingly, cultivation with C₃₆ increased cell surface hydrophobic activity, which attenuated the negative effect of the decline of the emulsification activity. These results suggest that the mechanisms of biosurfactant production and cell surface hydrophobicity are dependent upon the chain lengths of the n-alkanes used as carbon sources.     

Anaerobic oxidation of saturated hydrocarbons to CO2 by a new type of sulfate-reducing bacterium

n-Hexadecane added as electron donor and carbon source to an anaerobic enrichment culture from an oil production plant or to anoxic marine sediment samples allowed dissimilatory sulfate reduction to sulfide. The enrichment from the oil field was purified via serial dilutions in liquid medium under a hexadecane phase and in agar medium with caprylate. A pure culture of a sulfate-reducing bacterium, strain Hxd3, with relatively tiny cells (0.4–0.5 by 0.8–2 m) was isolated that grew anaerobically on hexadecane without addition of further organic substrates. Most of the cells were found to adhere to the hydrocarbon phase. It was verified that neither organic impurities in hexadecane nor residual oxygen were responsible for growth. Strain Hxd3 was grown with n-hexadecane of high purity (99.5%) in anoxic glass ampoules sealed by fusion. Of 0.4 ml hexadecane added per l (1.4 mmol per l), 90% was degraded with concomitant reduction of sulfate. Controls with pasteurized cells or a common Desulfovibrio species neither consumed hexadecane nor reduced sulfate. Incubation of cell-free medium with low reducing capacity and a redox indicator showed that the ampoules were completely oxygen-tight. Measured degradation balances and enzyme activities suggested a complete oxidation of the alkane to CO₂ via the carbon monoxide dehydrogenase pathway. However, the first step in anaerobic alkane oxidation is unknown. On hexadecane, strain Hxd3 produced as much as 15 to 20 mM H₂S, but growth was rather slow; with 5% inoculum, cultures were fully grown after 5 to 7 weeks. The new sulfate reducer grew on alkanes from C₁₂ to C₂₀, 1-hexadecene, 1-hexadecanol, 2-hexadecanol, palmitate and stearate. Best growth occurred on stearate (doubling time around 26 h). Growth on soluble fatty acids such as caprylate was very poor. Alkanes with chains shorter than C₁₂, lactate, ethanol or H₂ were not used. Strain Hxd3 is the first anaerobe shown to grow definitely on saturated hydrocarbons.Abbreviations CO dehydrogenase carbon monoxide dehydrogenase - DTE 1,4-dithioerythritol - Tris tris(hydroxymethyl)-aminomethane Dedicated to Dr. Ralph S. Wolfe on occasion of his 70th birthday     

Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes

Strain M-l which was derived from Candida cloacae 310 as a mutant unable to assimilate dicarboxylic acid (DC) produced large amount of DCs from n-alkanes, as expected. It produced DCs with the same number of carbon atoms as those of n-alkanes used (9 to 18 carbon atoms). Among DCs produced, n-tetradecane ω,φ′-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) was most abundantly accumulated and the highest level of DC-16, i.e., 29.3g/liter was obtained by resting cells.

On the other hand, since the growth rate of strain M-l on n-alkane markedly decreased in comparison with that of the parent strain, other carbon source which supported the growth of strain M-l was necessary for the production of DC from n-alkane by growing cells. When acetic acid was used as carbon source for the growth in DC-16 production from n-C₁₆, the highest level of DC-16, i.e., 21.8 g/liter was obtained ofter 3 days' cultivation.     

Production of Dicarboxylic Acids by Candida cloacae Mutant Unable to Assimilate n-Alkane

Strain MR-12 which was derived from Candida cloacae M-l as a mutant unable to assimilate n-alkane showed marked increase in dicarboxylic acid (DC) productivity from n-alkane.

Resting cells of strain MR-12 produced 42.7g/liter of n-tetradecane 1,14-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) after 72 hr’ incubation. DC degradation activities of strain M-1 and MR-12 were found to be markedly reduced and their activities against DC-16 decreased to 40% and 10% of that of the parent strain, respectively.

Strain M-1 and MR-12 produced DC from the various oxidized derivatives of n-alkane such as alcohol, diol, aldehyde, fatty acid and methyl- or ethylester of fatty acid other than n-alkane.

The carbon balance in n-C₁₆ oxidation was determined by using resting cells of strain MR-12 and about 60% of utilized carbon was recovered as DC-16 and about 40% was recovered as CO₂.     

Production of Inosine from Hydrocarbons by Corynebacterinm petrophilum

Using the adenine auxotroph of hydrocarbonoclastic microorganism, Corynebacterium petrophilum, the effects of glucose on the inosine productivity were investigated. The mutant did not produce inosine from glucose as the sole source of carbon. Production of inosine in n-C₁₆ medium was found to be inhibited by the addition of glucose. To obtain information on such effect of glucose, several characters were compared between the cells grown in glucose medium and those grown in n-C₁₆ medium. Intracellular content of UV-absorbing materials of the glucose-cells was higher than that of hydrocarbon-cells. The glucose-celle could not grow in media containing adenosine or 5′-AMP. On the other hand, hydrocarbon-cells were able to achieve growth, with adenine, adenosine and 5′-AMP contained in the hydrocarbon medium, but, in the case of glucose medium, the cells could grow only in the presence of adenine. Furthermore, the growth of this mutant in n-C₁₆ medium was found to be inhibited by a larger amount of adenine than that required for the maximum growth, and this inhibition was overcome by the addition of guanine. The significance of the effect of guanine was discussed.     

Formation of Rhamnolipid by Pseudomonas aeruginosa and its Function in Hydrocarbon Fermentation

Screening test for obtaining growth stimulant (GS) produced by a hydrocarbon-utilizing bacterium, Pseudomonas aeruginosa S₇B₁, was carried out. In consequence, the anthrone positive substance was most effective on the growth of this strain. Although the growth of this strain on glucose medium had no relation with the addition of GS, the growth on n-hexadecane medium was remarkably stimulated by the addition of GS. This effect of GS seemed to be specific on the growth of P. aeruginosa. GS which had a strong surface activity and emulsifying power was comfirmed to be rhamnolipid.     

Protein-like Activator for n-Alkane Oxidation by Pseudomonas aeruginosa S7B1

During the study of the n-alkane oxidation by Pseudomonas aeruginosa S₇B₁, a nondialysable activator for n-alkane oxidation was discovered in the culture broth of the strain. The activator was purified, as judged by cellulose acetate membrane electrophoresis, by ammonium sulfate precipitation and chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-75 columns.

The purified activator, which was positive in protein color reactions, remarkable stimulated only the oxidation of n-hexadecane, though it was not observed in case of palmitic acid or glucose oxidation.

Co-operative action between the activator and rhamnolipid, which had been isolated as a growth stimulant of P. aeruginosa S₇B₁ on n-hexadecane by the authors, was observed not only in the oxidation of n-hexadecane but also in the growth on n-hexadecane.     

A Hypothetical Cyclic Pathway for the Metabolism of Odd-carbon n-Alkanes or Propionyl-CoA via Seven-carbon Tricarboxylic Acids in Yeasts

An extensive study has been undertaken to elucidate the physiological significance of threo-D_s-2-methylisocitric acid produced mainly from odd-carbon n-alkanes by a mutant strain of Candida lipolytica. The mutant strain showed slower growth responses to odd-carbon n-alkanes, especially of shorter chain-length, and failed to utilize this acid as sole carbon source, whereas the parent strain and many other yeasts tested were able to utilize this acid. About one half of yeasts tested accumulated this acid extracellularly. Under a thiamine-deficient condition, amounts of pyruvate produced by the parent strain from odd-carbon n-alkanes were ten times as large as those from even-carbon n-alkanes. A scheme for the partial oxidation of propionyl-CoA to pyruvate via C₇-tricarboxylic acid by yeasts was supposed. This scheme may offer suggestion on the metabolism of propionyl-CoA by other living organisms. A hypothetical pathway of citrate accumulation from odd-carbon n-alkane was also presented.     

Excretion of Ribitol and Sucrose by Green Algae into the Culture Medium

Among 8 strains of algae grown with C¹⁴O₂ as a sole source of carbon, two species of Trebouxia produced appreciable amounts of two photosynthetic products in the culture medium. One of them was identified as sucrose by cochromatography and by acid hydrolysis. The other compound was identified as ribitol by paper chromatography, paper electrophoresis, periodic acid oxidation, recrystallization with authentic ribitol and finally by the enzymatic method with ribitol dehydrogenase.     

Long-Chain Aldehyde Dehydrogenase That Participates in n-Alkane Utilization and Wax Ester Synthesis in Acinetobacter sp. Strain M-1

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C₄ to C₁₄), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Δ) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Δ strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.     

Hydrocarbon uptake in hydrocarbon fermentations

Candida lipolytica (strain ATCC 8662) was grown on a simple defined medium with n-hexadecane as the main carbon Source under batch fermentation conditions. The relative importance of the cells growing in the aqueous phase on the overall kinetics was studied. The effect of interfacial tension, unoccupied interfacial area, and pseudosolubility on the specific growth was also studied. Results are presented and discussed here.     

Production of poly(3-hydroxyalkanoates) by a bacterium of the genus Alcaligenes utilizing long-chain fatty acids

Summary The synthesis of poly(3-hydroxyalkanoates) [P(3HA)] by a new Alcaligenes species was investigated. The new species was grown on various carbon sources such as n-alkanoic acids of carbon numbers ranging from C₂ to C₂₂, plant oils and animal fats, and accumulated P(3HA) within the cells. When the bacterium was cultured in mineral media containing sodium salts of n-alkanoic acids, the homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from n-alkanoates of even carbon numbers, whereas the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate units [P(3HB-co-3HV)] was produced from n-alkanoates of odd carbon numbers. Relatively high yields of both dry cells and P(3HA) were obtained by the use of n-alkanoates from C₁₂ to C₁₆ as the sole carbon source. Correspondence to: Y. Doi     

The effects of carbon sources and micronutrients in fermented whey on the biodegradation of n-hexadecane in diesel fuel contaminated soil

Fermented whey has previously been shown to stimulate biodegradation of n-hexadecane in diesel contaminated soils. The proposed explanation for the stimulatory effect is that fermented whey provides easily accessible carbon and micronutrients, which give rise to an increased degrading biomass.The objective of this work has been to investigate the role of the different carbon sources and vitamins in fermented whey on the microbial degradation of n-hexadecane in soil.The effects of lactose, lactate, vitamins and free amino acids were tested in combinations according to a full factorial design experiment, at concentrations corresponding to those present in fermented whey. The target substance was ¹⁴C-labeled n-hexadecane in nutrient amended soil microcosms contaminated with 5000 mg diesel fuel kg⁻¹ dw. Biodegradation was monitored by determination of evolved ¹⁴CO₂.Significant effects on the biodegradation of n-hexadecane were observed for lactate and amino acids additions in a sandy soil. Lactate showed both an inhibitory effect in the early phase of the experiment and a stimulatory effect in the later phase. The effect of amino acids was slightly stimulatory, mainly evident as a shortening of the lag time.The degree of n-hexadecane degradation at the end of the experiment was correlated with the total concentration of organic compounds added to the soil.

Scientific relevance

There are a handful papers describing the potential of using organic amendments (often industrial by-products) with a content of both easily accessible carbon and micronutrients, to enhance the bioremediation of polluted soils. Enhanced biodegradation is often reported and the proposed explanations are that the combination of easily accessible carbon and micronutrients increases the degrading biomass.In this paper, we examine the effect of fermented whey on the degradation of n-hexadecane and correlate the observed effects on the biodegradation with the main components lactate, amino acids, lactose and B-vitamins. This has to our knowledge never been done before.     

Enhancement of lipase production from hydrocarbons by mutation of Trichosporon fermentans

Lipase high-producing mutants with petroleum products as carbon sources were successfully induced from Trichosporon fermentans WU-C12 by ultraviolet (UV) light irradiation. In the first mutation step, one mutant strain, PU-30, derived from strain WU-C12 was selected. The productivity of extracellular lipase of PU-30 reached 58 units (U)/ml in the medium containing kerosene, being approximately twice the productivity of the parental strain WU-C12. In the second mutation step, the mutant strain 2PU-18 was induced from strain PU-30. In medium containing kerosene, gas oil and liquid paraffin, the 2PU-18 produced 70 U/ml, 62 U/ml and 60 U/ml of extracellular lipase, respectively. When various n-alkanes (C₈-C₁₈) were used as carbon sources, the parental strain WU-C12 produced more than 20 U/ml of lipase only from C₉-C₁₂ alkanes, but 2PU-18 could produce more than 50 U/ml of lipase from C₈-C₁₈ alkanes. When cultivated for 3 days in medium containing liquid paraffin, the activity ratios of extracellular lipase to total lipase and the values of extracellular lipase activity per dry-cell weight were 0.44 and 0.65 U/mg for WU-C12, and 0.62 and 1.82 U/mg for 2PU-18, respectively. These results indicate that the mutant strain 2PU-18 is superior in both total lipase productivity and permeability of lipase to the parental strain WU-C12 when petroleum products are used as carbon sources. Correspondence to: S. Usami