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.     

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.     

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.     

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.     

n-Alkane uptake and utilisation by Streptomyces strains

Streptomyces strains isolated from the Kuwait Burgan oil field were defined as S. griseoflavus, S. parvus, and S. plicatus utilised n-hexadecane, n-octadecane (purified fractions of mineral oil), kerosene, and crude oil as sole carbon and energy sources. The strains were incubated with n-alkanes and increase of the fatty acid content with chain length equivalent to the employed n-alkanes was observed. Signal transducing GTP-binding proteins (GBPs) play an important role in n-alkane uptake in streptomycetes. Specific activators of GBPs increased the uptake of hydrocarbons. Using the hydrophobic fluorescent dye diphenylhexatrien (DPH) as a probe, it was found that the microviscosity of the hydrophobic inner region of the cellular membrane is significantly lower in hydrocarbon utilisers than in non-utilisers. This difference probably reflects differences in the fatty acid composition of the strains. When cultures were grown in n-alkane containing media, electron microscopy revealed that the hydrocarbon utilisers showed less-electron dense areas as inclusions in the cytoplasm. Soil samples inoculated with Streptomyces strains eliminated hydrocarbons much faster than those not containing these strains, serving as control. When inorganic medium was supplied with n-hexadecane-1-¹⁴C as sole carbon and energy source, radioactive CO₂ was detected. Since streptomycetes have not been used until now for oil elimination, though they are known as abundant soil bacteria tolerating extreme conditions, their possible use for bioremediation of hydrocarbon contaminated soils is discussed.     

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.     

Screening of n-Alkylbenzenes Assimilating Yeasts and Identification of Oxidation Products from n-Alkylbenzenes

As a result of screening of n-alkylbenzenes assimilating yeasts, it was shown that the yeasts which grew well on n-alkane (C₁₅) showed also good growth on n-alkylbenzenes (from C₇ to C₁₉ of side chain). Among four Candida strains selected, C. tropicalis S131Y1 produced 4-(o-hydroxyphenyl)-butyric acid, o-hydroxy phenylacetic acid and phenylacetic acid from n-alkylbenzenes with even-carbon side chain and cinnamic acid from n-alkylbenzenes with odd-carbon side chain.

On the other hand, another three strains produced only phenylacetic acid from n-alkyl-benzenes with even-carbon side chain. In addition, as for the products from n-alkylbenzenes with odd-carbon side chain, two of three strains, C. parapsilosis IFO-1068 and C hydrocarbofumarica Et 15-2 produced benzoic acid.

From these oxidation products and oxygen uptake experiment, a new metabolic pathway of K-alkylbenzenes was assumed.     

Large Scale Production of Erythritol and Its Conversion to d-Mannitol Production by n-Alkane-grown Candida zeylanoides

Production of erythritol by n-alkane-grown Candida zeylanoides KY6166 was studied using large scale fermentors under conditions described in the previous report. The medium-pH was kept below 4.0 during fermentation in 5-liter or 300-liter fermentors. This strain produced about 180mg/ml of meso-erythritol and a small amount of mannitol. The yield corresponded to 90% of n-alkane consumed.

The production ratio between erythritol and mannitol was affected remarkably by the concentration of phosphate in the medium. Keeping its concentration at high level (0.04 to 0.20 mg/ml as KH₂PO₄), erythritol production was almost entirely converted to mannitol production. The yield was 63 mg/ml after 100 hr incubation in 5-liter fermentor, which corresponded to 52% of n-alkane consumed. Conversion of the production of erythritol to mannitol was also observed in several other yeasts capable of utilizing n-alkane as the sole source of carbon.     

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

Microorganisms which produced n-alkane ω,ω′-dicarboxylic acid (DC) from n-alkane were selected from natural sources. It was found that the best three producers thus obtained belonged to yeast. All of the stock cultures which are able to assimilate n-alkane and are belonged to genus Candida and Pichia were also found to produce DC from n-alkane.

Candida cloacae 310, a representative strain selected from natural source, was able to produce DCs having 5 to 16 carbon atoms from various n-alkanes. Among them, DCs with 5 to 9 carbon atoms were more heavily accumulated than those with more than 9, except those with the same number of carbon atoms as the substrates which were the main products from the substrates with less than 15 carbon atoms. It was also clearly demonstrated that DCs with odd carbons alone were produced from n-alkanes with odd carbons, while DCs with even carbons alone from n-alkanes with even carbons.

Then, cultural conditions of Candida cloacae 310 were studied for the production of DC-12 from n-dodecane (n-C₁₂) which showed the highest yield among the observed accumulation.

Under the optimum conditions, 2.28 g/liter of DC-12 was obtained together with 1.86 g/liter of DC-6 and 0.82 g/liter of DC-8 after 72 hr’ cultivation in a synthetic medium containing 100 ml of n-C₁₂ per liter.     

Cellular lipid accumulation by Pseudomonas aeruginosa 44T1

Summary Growth of Pseudomonas aeruginosa strain 44T1 on glucose, an n-alkane mixture or olive oil was characterized by the formation of intracellular lipid inclusions and extracellular accumulation of rhamnolipids. Maximum values of cellular lipid accumulation were obtained in olive-oil-grown cells and reached up to 38% w/w of its dry biomass. The principal fatty acids of cellular lipids drived from P. aeruginosa cultures varied with the carbon source employed. The major fatty acids detected were palmitic and trans-oleic acids. Arachidonic acid was only found in medium containing glucose or the n-alkane mixture. Offprint requests to: A. Manresa     

Formation of cobalt-porphyrin by Corynebacterium simplex grown on hydrocarbon media

A vitamin B₁₂-producing and hydrocarbon-utilizing bacterium, Corynebacterium simplex, accumulated an appreciable amount of cobalt-porphyrin in cultural filtrates when grown on a n-hexadecane medium containing sufficient amounts of cobaltous sulfate and an appropriate detergent. When grown without the detergent, the cobalt-porphyrin was found only in the cells of the organism. In the latter case, the content of cobalt-porphyrin was comparable to that of vitamin B₁₂ and 7 times lower than that of iron-porphyrin. Though the organism required cobaltous sulfate for optimal growth, the requirement could be efficiently replaced by the supplementation of cobalt-porphyrin and partly of vitamin B₁₂. The porphyrin moieties of extra- and intracellular cobalt-porphyrin were identified as coproporphyrin III in both cases.     

Intracellular Localization of Several Enzymes in Candida tropicalis Grown on Different Carbon Sources

Intracellular localization of several enzymes related to tricarboxylic acid cycle was investigated during the aerobic growth of Candida tropicalis on acetate, n-alkane and glucose. NADP-linked isocitrate dehydrogenase in acetate-grown cells was mostly found in S₂ fraction (20,000 × g supernatant fraction of protoplast lysate), whereas more than half of this activity in n-alkane-grown cells was recovered in P₂ fraction (20,000 × g pellet fraction). Large parts of NAD-linked isocitrate dehydrogenase and malate dehydrogenase were present in P₂ fraction, while NADP- and NAD-linked glutamate dehydrogenases were found preferentially in S₂ fraction, irrespective of the growth substrates used. Isocitrate lyase was detected in both fractions. Citrate synthase and aconitase in acetate-grown cells were almost particulate. Catalase activity recovered in P₂ fraction was far higher in alkane-grown cells than in acetate- or glucose-grown cells.     

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.     

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.     

Tetradecane 1,14-Dicarboxylic Acid Production from n-Hexadecane by Candida cloacae

The better condition of cultivation for tetradecane 1,14-dicarboxylic acid (DC-16) production from n-hexadecane (n-C₁₆) by Candida cloacae MR-12 was investigated by using acetic acid as carbon source for the growth. In general, the condition suitable for the growth was also favorable for the production of DC-16. The change of pH during cultivation, the use of NaOH solution as pH controlling agent after pH-change and the addition of antifoam stimulated the production of DC-16.

Under the optimum conditions where the culture medium contained 15% (v/v) n-C₁₆, 1.4% (w/v) acetic acid, inorganic salts and growth factors, and pH was changed from 6.5 to 7.75 at 16 hr after the inoculation, the highest level of DC-16 production was attained after about 72 hr cultivation and the amount of the product accumulated was 61.5 g per liter of the medium.

When a mixture of various n-alkanes was used as starting material, DCs corresponding to the respective n-alkanes were produced as mixture.     

Production of Fluorescent Compounds from Guanine by Microorganisms

One species of hydrocarbon utilizing bacteria was isolated from soil. This strain was named as Achromobacter petrophilum No. 4017. This bacterial species utilizes normal hydrocarbons with carbon chains of nC₁₀ to nC₁₈, but does not utilize glucose or other carbohydrates. Achromobacter petrophilum forms small amounts of green-yellow, green-blue and violet fluorescent compounds in the medium containing n-hexadecane (nC₁₆) as a carbon source. The mutant strain, No. 4510, which requires hypoxanthine and thiamine for growth, was obtained from Achromobacter petrophilum No. 4017 by ultraviolet irradiation and formed considerable amounts of green-yellow fluorescent compound by the addition of guanine to the n-hexadecane medium. This fluorescent compound was crystallized from culture broth.     

Investigation of saturated and aromatic hydrocarbon resistance mechanisms in Pseudomonas aeruginosa IBBML1

Pseudomonas aeruginosa IBB_ML1, isolated from Poeni petroleum sludge, was able to tolerate and degrade both saturated (n-hexane, n-heptane, n-hexadecane, cyclohexane) and aromatic (benzene, ethylbenzene, propylbenzene, xylene isomers, styrene) hydrocarbons. Molecular studies have revealed that the high hydrocarbon resistance of Pseudomonas aeruginosa IBB_ML1 could be due to the action of members of the HAE1 (hydrophobe/amphiphile efflux 1) family of transporters. It is further possible that additional mechanisms may account for the tolerance of Pseudomonas aeruginosa IBB_ML1 to hydrocarbons, and a combination of short-term and long-term mechanisms may act together in the adaptation of Pseudomonas aeruginosa IBB_ML1 cells to saturated and aromatic hydrocarbons. β-galactosidase activity measurements revealed that there was significant induction of the lacZ gene in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of either 5% and 10% (v/v) saturated or aromatic hydrocarbons, compared with control (cells incubated without hydrocarbons). Rhodamine 6G accumulation in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of 5% and 10% (v/v) saturated hydrocarbons was higher than rhodamine 6G accumulation in cells grown in the presence of 5% and 10% (v/v) aromatic hydrocarbons. The study of cellular and molecular modifications to Pseudomonas aeruginosa IBB_ML1 induced by 5% and 10% (v/v) saturated and aromatic hydrocarbons revealed a complex response of bacterial cells to the presence of different hydrophobic substrates in the culture medium.     

Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production

An organic solvent-tolerant lipase from newly isolated Pseudomonas aeruginosa LX1 has been purified by ammonium sulfate precipitation and ion-exchange chromatography leading to 4.3-fold purification and 41.1% recovery. The purified lipase from P. aeruginosa LX1 was homogeneous as determined by SDS-PAGE, and the molecular mass was estimated to be 56 kDa. The optimum pH and temperature for lipase activity were found to be 7.0 and 40 °C, respectively. The lipase was stable in the pH range 4.5–12.0 and at temperatures below 50 °C. Its hydrolytic activity was found to be highest towards p-nitrophenyl palmitate (C16) among the various p-nitrophenol esters investigated. The lipase displayed higher stability in the presence of various organic solvents, such as n-hexadecane, isooctane, n-hexane, DMSO, and DMF, than in the absence of an organic solvent. The immobilized lipase was more stable in the presence of n-hexadecane, tert-butanol, and acetonitrile. The transesterification activity of the lipase from P. aeruginosa LX1 indicated that it is a potential biocatalyst for biodiesel production.     

Intensification of surfactant synthesis in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 cultivated on hexadecane

Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP⁺-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 μmol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5–1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3–5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.