Michèle Gilewicz

Aerobic biodegradation of alkylated aromatic hydrocarbons by a bacterial community

The aerobic degradation of aromatic compounds using a bacterial community from the Arcachon Harbour (South West coast of France) was studied. The aromatic fraction was prepared from an Arabian light crude oil residue and was incubated with this bacterial community under oxic conditions for up to 7 days. The study was focussed onto the degradation of alkylated naphthalenes, dibenzothiophenes and phenanthrenes present in the aromatic fraction. The disappearance of the various aromatic compounds was followed by gas chromatography/mass spectrometry. This bacterial community used the aromatic compounds as the sole source of carbon (more than 70% of the compounds disappeared within 7 days). The degradation phenomenon was shown to follow first order kinetics and did not show any difference between non-sulphur aromatic compounds (phenanthrenes) and sulphur aromatic compounds (dibenzothiophenes). The degradation of methylated phenanthrenes and dibenzothiophenes did not show significant differences in terms of isomeric composition. On the contrary, major differences in biodegradation rates have been observed for the dimethylated derivatives.

Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene.

Abstract A marine bacterium isolated from a coastal hydrocarbon-polluted sediment has been described and attributed on the basis of its phenotypic and genotypic characteristics to the genus Sphingomonas sp. This strain was capable of using an alkylated phenanthrene 2-methylphenanthrene, as sole source of carbon and energy. In experiments, 2-methylphenanthrene (0.2 g/l) was added as crystals to the culture medium. After 5 days of aerobic growth at 30 °C, 70% was degraded and the complete dissipation occurred after 20 days. Furthermore, the strain could degrade various kinds of polyaromatic compounds, but failed to grow on aliphatic hydrocarbons.

Anaerobic oxidation of 1-n-heptadecene by a marine denitrifying bacterium

SummaryA denitrifying bacterium showing typical characteristics of Pseudomonas sp. (Al1) capable of growth on 1-heptadecene as the sole source of carbon and energy has been isolated from a hydrocarbon-polluted marine sediment by using classical enrichment techniques. The generation time for anaerobic growth on 1-heptadecene was 24 h, and the percentage of hydrocarbon degradation under anaerobic conditions ranged from 19 to 23%. The emulsifying capacity was observed and suggested that Al1 cultivated anaerobically on heptadecene produced surface-active agents.

Anaerobic biodegradation of pristane by a marine sedimentary bacterial and/or archaeal community

Pristane was incubated in anaerobic sediment slurries under conditions promoting or limiting nitrate reduction. Pristane was efficiently degraded (85% after 6 months) by the mixed microbial community when nitrate reducing processes were avoided. The biodegradation of pristane was accompanied by the abundant production of methane which indicated that methanogenic Archaea were involved in the degradation of the substrate. Comparison of the biodegradation rate of pristane with that of unsaturated isoprenoid alkenes indicated that, in Recent anoxic sediments, acyclic isoprenoid alkanes can also undergo relatively rapid biotransformations and, therefore, can no longer be considered as recalcitrant biomarkers.

Partial purification of cytochrome P-450 of Candida tropicalis and reconstitution of hydroxylase activity

The various animal cytochromes P-450 have been widely studied and purified [l-5] but very few results have been obtained in this field concerning the cytochromes P-450 synthesized by yeasts [6--81. The microsomal membranes of Can&& tropicalis grown on hydrocarbons catalyse the transformation of lauric acid to w-hydroxydodecanoic acid and then to 1 ,I 2-dodecandioic acid [9]. This transformation is due to a monooxygenase comprising a cytochrome P-450 and a NADPH~ytochrome c reductase on one side and to alcoholand aldehyde-dehydrogenases on the other. In this yeast, these enzymatical systems are specifically induced by alkanes [IO]. We have established that it is possible to liberate 70% of the NADPH-cytochrome c reductase by an osmotic wash of the microsomal membrane [1 11. This made it possible to purify and characterize a soluble form of this flavoprotein [ 111. This paper describes a method for the isolation and partial purification of cytochrome P-450, the other proteic conlponent of the monooxygenase induced by the alkanes, in Candida tropicalis. This method based on the solubilization of the microsomal membranes by a detergent is different from that in [I 21 where a solubilization of the cytochrome P-450 from a very particular strain of C. tropical& was obtained. Effectively, the cytochrome P-450 of this strain was liberated from the microsomal membrane by French press treatment of the cells of this strain in presence of 30% glycerol. This possibility

Degradation of phenanthrene, methylphenanthrenes and dibenzothiophene by a Sphingomonas strain 2mpII

Abstract. Strain Sphingomonas sp. (2MPII), isolated from marine sediment, was able to utilize phenanthrene (P) or 2-methylphenanthrene (2MP) as the sole carbon source. However, 9-methylphenanthrene (9MP) and dibenzothiophene (DBT) were weakly degraded. The degradation rates of 9MP and DBT increased in the presence of 2MP, whilst the degradation rate of 2MP increased in the presence of 9MP. However, the presence of DBT inhibited the degradation of 2MP. DBT sulfone, a DBT metabolite, was not assimilated by the bacteria and its presence also decreased the degradation rate of 2MP.

Effects of oxygen on Pseudomonas nautica growth on n-alkane with or without nitrate

The denitrifying marine bacterium, Pseudomonas nautica 617, can grow on lactate aerobically or anaerobically in presence of nitrate with generation times of 1.5 and 3 h respectively. The growth on heptadecane occurs only in presence of oxygen whatever its concentration with a genrration time of 8.5 h. The influence of oxygen, carbon sources (lactate or heptadecane) and nitrate was examined on O2, NO3-, NO2- consumption, on nitrate and nitrite reductases activities, on cell yields, and on the ratio of CO2 produced per unit of biomass. Pseudomonas nautica metabolizes hydrocarbons under denitrifying conditions in the presence of oxygen. Nitrate and nitrite are used during growth on lactate and heptadecane up to oxygen concentrations corresponding to 50 and 30% of air-saturation, respectively. When growth on n-alkane was not oxygen-limited (above 50% of air-saturation) the catabolism decreases in favour of carbon incorporation into the cell. Nitrate and nitrite reductases were strongly inhibited after 20% of airsaturation in the presence of lactate as growth substrate. With n-alkane, only the nitrate reductase activity was greatly reduced.

Purified detergent-solubilized NADPH-cytochrome c (P-450) reductase from Candida tropicalis grown on alkanes

Abstract A microsomal NADPH-cytochrome c reductase from yeast was purified by column chromatography on Phenyl-Sepharose CL-4B, DEAE cellulose, hydroxylapatite and Sephadex G-150 in the presence of sodium cholate and Mulgofen BC-720, a non ionic detergent. On SDS-polyacrylamide gel electrophoresis, the purified enzyme gives a single band of 76,000 MW. FMN and FAD were present in approximately equal amounts. Contrary to the enzyme obtained by osmotic wash, the cholate — solubilized reductase is able to transfer the electrons from NADPH to cytochrome P-450 and may thus be considered as a NADPH-cytochrome c (P-450) reductase.

Salt requirements in the denitrifying bacterium Pseudomonas nautica 617

Pseudomonas nautica 617, which was isolated from superficial marine sediment, was found to require sodium for growth. Growth also appeared to be sensitive to the divalent cation, Mg2+, the presence of which, together with that of Na+, was necessary for achieving maximal growth. We investigated cell capacity to resist lysis after washing with either 0.05 M MgCl2 or 0.5 M NaCl, by monitoring suspension optical density changes as well as the release of ultraviolet absorbing material. Mg2+ turned out to play a significant role in stabilizing the structure of the cell envelope. Respiratory activity was also sensitive to ionic environment. With cells washed with 0.05 M MgCl2 and suspended in 0.05 M Tris buffer, the respiration rate, assessed by N2O evolution, was 15% of that measured in artificial sea water. Upon addition of 0.5 M Na+, nitrous oxide production rose to 32% of the reference level. The dinitrification rate was fully restored by further addition of 0.05 M Mg2+. K+ alone had almost no effect, but when added with Na+, the rate of denitrification increased to 45%.

Development of a Protocol to Study Aerobic Bacterial Degradation of Polycyclic Aromatic Hydrocarbons: Application to Phenanthrenes

Abstract A method was developed and validated to study the degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria under oxic conditions. Studies of toxicity of acetonitrile used as dissolution solvent have allowed to determine the quantity of acetonitrile necessary to obtain a good PAH dissolution without toxic effects. The degradation of a mixture of phenanthrene (P), 2-methylphenanthrene (2MP) and 9-methylphenanthrene (9MP) by a bacterial community was studied. The results obtained show a difference of degradation rates between the three compounds. P and 2MP were more rapidly degraded than 9MP. Degradation of methylphenanthrenes in petroleum was also investigated. P was more degraded than substituted phenanthrenes and no selectivity of degradation was observed between the methylphenanthrenes. Identification of 2MP metabolites has shown two metabolic pathways. The first step of one way is the hydroxylation of the methylgroup, the second way is initiated by the double hydroxylation of one aromatic ring.