Phenanthrene degradation, emulsification and surface tension activities of apseudomonas putidastrain isolated from a coastal oil contaminated microbial mat

Rontani J., Mouzdahir A., Michotey V., Caumette P., Bonin P.

ABSTRACT This paper describes the production of 5,9,13-trimethyltetradeca-4 E ,8 E ,12-trienyl-5,9,13-trimethyltetradeca-4 E ,8 E ,12-trienoate during the aerobic degradation of squalene by a Marinobacter strain, 2Asq64, isolated from the marine environment. A pathway involving initial cleavage of the C 10 -C 11 or C 14 -C 15 double bonds of the squalene molecule is proposed to explain the formation of this polyunsaturated isoprenoid wax ester. The isoprenoid wax ester content reached 1.1% of the degraded squalene at the mid-exponential growth phase and then decreased during the stationary phase. The wax ester content increased by approximately threefold in N-limited cultures, in which the ammonium concentration corresponds to conditions often found in marine sediments. This suggests that the bacterial formation of isoprenoid wax esters might be favored in such environments. The bacterial strain is then characterized as a member of a new species, for which we propose the name Marinobacter squalenivorans sp. nov.

Nawab A.

Soil samples were taken from different agricultural fields and analyzed for organochlorine pesticide residues by gas chromatography. The analysis indicated that the soil samples contained some common organochlorine pesticides DDT, DDD, DDE, HCH and Aldrin. gamma-HCH was detected as 47.35 ppb whereas the concentrations of alpha-HCH, beta-HCH, p('),p(')-DDE, o('),p(')-DDT were 38.81, 1.79, 7.10 and 13.30 ppb, respectively, in the same soil. Two Pseudomonas strains isolated from agricultural soil were found to possess gamma-hexachlorocyclohexane degrading ability when the isolates were grown in a mineral salt medium containing gamma-HCH as the sole source of carbon and a number of metabolites were produced and detected by the gas chromatography. These bacterial isolates were further tested for carbohydrate and amino acid utilization as well as for their susceptibility against 10 commonly used antibiotics namely amoxycillin, chloramphenicol, cloxacillin, doxycycline, methicillin, nalidixic acid, neomycin, nitrofurantoin, streptomycin and tetracycline. Both the isolates were also screened for plasmid DNA and found to harbour a single plasmid.

Mohan S.V., Sistla S., Guru R.K., Prasad K.K., Kumar C.S., Ramakrishna S.V., Sarma P.N.

Pseudomonas (PI2) capable of degrading pyridine was isolated from the mixed population of the activated sludge unit which was being used for treating complex effluents, the strain was characterized. Aerobic degradation of pyridine was studied with the isolated strain and the growth parameters were evaluated. Pyridine degradation was further conformed by chromatography (HPLC) analysis. The process parameters like biomass growth and dissolved oxygen consumption were monitored during pyridine degradation. In order to conform with the plasmid capability to degrade pyridine, the requisite plasmid was isolated and transferred to DH 5alpha Escherichia coli. The subsequent biodegradation studies revealed the ability of the transformed plasmid capability to degrade the pyridine.

Dean-Ross D., Moody J., Cerniglia C.E.

Belhaj A., Desnoues N., Elmerich C.

Pseudomonas aeruginosa strains that grow on crude oil as the sole source of carbon and energy were isolated from an environment in Morocco polluted by petroleum refinery effluents. The twenty isolates grew on saturated alkanes from C12 to C22. Three of the isolates were also able to grow on low molecular weight C6 to C10 n-alkanes, but the other 17 strains were not. The strains were tested for alkB and a/kB-related genes encoding alkane-1-monooxygenase (alkane hydroxylase). Oligonucleotide primers specific for the alkB gene of strain P. putida (GPo1 ) and for the alkB1 and alkB2 genes of P. aeruginosa strain PAO1 allowed amplification from the P. aeruginosa isolates of fragments similar to alkB1 and alkB2 genes of strain PAO1. Only 3 strains carried an alkB gene very similar to that of strain GPo1, and these strains were the same ones that could utilise C6 to C10 n-alkanes.

Samanta S.K., Singh O.V., Jain R.K.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic to microorganisms as well as to higher systems including humans. Although various physicochemical methods have been used to remove these compounds from our environment, they have many limitations. Xenobiotic-degrading microorganisms have tremendous potential for bioremediation but new modifications are required to make such microorganisms effective and efficient in removing these compounds, which were once thought to be recalcitrant. Metabolic engineering might help to improve the efficiency of degradation of toxic compounds by microorganisms. However, efficiency of naturally occurring microorganisms for field bioremediation could be significantly improved by optimizing certain factors such as bioavailability, adsorption and mass transfer. Chemotaxis could also have an important role in enhancing biodegradation of pollutants. Here, we discuss the problems of PAH pollution and PAH degradation, and relevant bioremediation efforts.

Holden P.A., LaMontagne M.G., Bruce A.K., Miller W.G., Lindow S.E.

ABSTRACT Low pollutant substrate bioavailability limits hydrocarbon biodegradation in soils. Bacterially produced surface-active compounds, such as rhamnolipid biosurfactant and the PA bioemulsifying protein produced by Pseudomonas aeruginosa , can improve bioavailability and biodegradation in liquid culture, but their production and roles in soils are unknown. In this study, we asked if the genes for surface-active compounds are expressed in unsaturated porous media contaminated with hexadecane. Furthermore, if expression does occur, is biodegradation enhanced? To detect expression of genes for surface-active compounds, we fused the gfp reporter gene either to the promoter region of pra , which encodes for the emulsifying PA protein, or to the promoter of the transcriptional activator rhlR . We assessed green fluorescent protein (GFP) production conferred by these gene fusions in P. aeruginosa PG201. GFP was produced in sand culture, indicating that the rhlR and pra genes are both transcribed in unsaturated porous media. Confocal laser scanning microscopy of liquid drops revealed that gfp expression was localized at the hexadecane-water interface. Wild-type PG201 and its mutants that are deficient in either PA protein, rhamnolipid synthesis, or both were studied to determine if the genetic potential to make surface-active compounds confers an advantage to P. aeruginosa biodegrading hexadecane in sand. Hexadecane depletion rates and carbon utilization efficiency in sand culture were the same for wild-type and mutant strains, i.e., whether PG201 was proficient or deficient in surfactant or emulsifier production. Environmental scanning electron microscopy revealed that colonization of sand grains was sparse, with cells in small monolayer clusters instead of multilayered biofilms. Our findings suggest that P. aeruginosa likely produces surface-active compounds in sand culture. However, the ability to produce surface-active compounds did not enhance biodegradation in sand culture because well-distributed cells and well-distributed hexadecane favored direct contact to hexadecane for most cells. In contrast, surface-active compounds enable bacteria in liquid culture to adhere to the hexadecane-water interface when they otherwise would not, and thus production of surface-active compounds is an advantage for hexadecane biodegradation in well-dispersed liquid systems.

Tuleva B.K., Ivanov G.R., Christova N.E.

Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN. The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp. Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l-1. When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m-1 and formed stable and compact emulsions with emulsifying activity of 69%

Yamamoto S., Kasai H., Arnold D.L., Jackson R.W., Vivian A., Harayama S.

Phylogenetic analysis of the genus Pseudomonas: was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas: (a total of 125 strains). Pseudomonas: strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the 'P: aeruginosa complex', which included P: aeruginosa, P: alcaligenes, P: citronellolis, P: mendocina, P: oleovorans and P: pseudoalcaligenes, and the 'P: stutzeri complex', which included P: balearica and P: stutzeri. IGC II was further split into three subclusters that were designated the 'P: putida complex', the 'P: syringae complex' and the 'P: fluorescens complex'. The 'P: putida complex' included P: putida and P: fulva. The 'P: syringae complex' was the cluster of phytopathogens including P: amygdali, P: caricapapayae, P: cichorii, P: ficuserectae, P: viridiflava and the pathovars of P. savastanoi and P. syringae. The 'P. fluorescens complex' was further divided into two subpopulations, the 'P. fluorescens lineage' and the 'P. chlororaphis lineage'. The 'P. fluorescens lineage' contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the 'P. chlororaphis lineage' included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the 'P. fluorescens complex'.

Beal R., Betts W.B.

Cuny P., Faucet J., Acquaviva M., Bertrand J.-., Gilewicz M.

Hyuk Choi D., Hori K., Tanji Y., Unno H.

An Acinetobacter species was isolated and found to be able to grow on crude oil n-alkanes and solid alkanes at room temperature as the sole carbon source. The growth of the isolate on n-heneicosane dissolved in non-biodegradable pristane has been studied. A kinetic model of the growth of microorganism on the hydrophobic substrate dissolved in non-biodegradable oil droplet assuming direct contact of cell with oil droplet was developed and validated with a model system of crude oil biodegradation. The model was focused on the substrate transport to the cell being contact with the surface of droplet. The high value of saturation constant of n-heneicosane, Ks = 0.086 kg m−3, and the maximum specific growth rate, μm = 0.60 h−1, were obtained. The transport limitation was considered and estimated. The high value of attached cell fraction was reasonable to explain the observed growth rate by the direct contact model and varied with time till it reached a plateau at the stationary growth phase. By considering the direct contact of the cells with the surface of pristane and the transport of n-heneicosane to the cell, the degradation of hydrophobic substrate in the oil phase could be elucidated.

Gilewicz M., Not Available N.A., Nadalig T., Budzinski H., Doumenq P., Michotey V., Bertrand J.C.

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.

Pahlman R., Pelkonen O.

Dependence of polycyclic aromatic hydrocarbon (PAH)-induced mutagenicity on the bay region of the molecule and on the activating cytochrome P-450 enzyme was studied. Eleven PAHs with and six without a bay region were activated by postmitochondrial supernatants from control and 3-methylcholanthrene (MC)-pretreated C57BL/6 mice and from control, MC- and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-pretreated DBA/2 mice and from control and MC-pretreated Sprague-Dawley and Lewis rats. S-9 fractions from MC- or TCDD-treated animals induced more mutagenicity with PAHs with a bay region compared with S-9 fractions from control animals or MC-treated D2 mice. Mutagenicities of PAHs without a bay region were largely independent of the source of activating enzyme. There were three exceptions, namely benzo[e]pyrene, phenanthrene and perylene (each possessing a bay region), which were not mutagenic. These studies support the notion that the Ah-locus-controlled induction of cytochrome P1-450 activating PAHs into reactive intermediates at the bay region of the hydrocarbon molecule is of prime importance in the mutagenicity of PAHs. Qualitative correspondence to carcinogenicity is also apparent.

White K.L.

Barros D., Oliveira P., Pascoal C., Cássio F.

Fungi, particularly aquatic hyphomycetes, have been recognized as playing a dominant role in microbial decomposition of plant litter in streams. In this study, we used a microcosm experiment with different levels of fungal diversity (species number and identity) using monocultures and combinations with up to five aquatic hyphomycete species (Articulospora tetracladia, Tricladium splendens, Heliscus submersus, Tetrachaetum elegans and Flagellospora curta) to assess the effects of ethanol and phenanthrene on three functional measures: plant litter decomposition, fungal biomass accrual and reproduction. Alder leaves were conditioned by fungi for 7days and then were exposed to phenanthrene (1mgL(-1)) dissolved in ethanol (0.1% final concentration) or ethanol (at the concentration used to solubilise phenanthrene) for further 24days. Exposure to ethanol alone or in combination with phenanthrene decreased leaf decomposition and fungal reproduction, but increased fungal biomass produced. All aspects of fungal activity varied with species number. Fungal activity in polycultures was generally higher than that expected from the sum of the weighted performances of participating species in monoculture, suggesting complementarity between species. However, the activity of fungi in polycultures did not exceed the activity of the most productive species either in the absence or presence of ethanol alone or with phenanthrene.

Djeridi I., Militon C., Grossi V., Cuny P.

The potential for surfactant production by the extreme halophilic archaeon Haloferax sp. MSNC14 in the presence of individual hydrocarbon substrates was studied. This strain was selected for its ability to grow on different types of hydrocarbons at high NaCl concentrations. Linear (n-heptadecane or C17) and isoprenoid (pristane) alkanes, a polyaromatic hydrocarbon (phenanthrene) and ammonium acetate (highly water-soluble control compound) were used as growth substrates. The adherence potential was demonstrated by the ability of the cells to adhere to liquid or solid hydrocarbons. The biosurfactant production was indicated by the reduction of the surface tension (ST) and by the emulsification activity (EA) of cell-free supernatants. Growth on acetate was accompanied by a low EA (lower than 0.1) and a high ST (~70 mN/m), whereas an important EA (up to 0.68 ± 0.08) and a reduction of ST (down to 32 ± 2.3 mN/m) were observed during growth on the different hydrocarbons. Both ST and EA varied with the growth phase. The adhesion to hydrocarbons was higher when cells were grown on C17 (by 60–70 %) and pristane (by 30–50 %) than on phenanthrene (~25 %). The results demonstrated that strain MNSC14 was able to increase the bioavailability of insoluble hydrocarbons, thus facilitating their uptake and their biodegradation even at high salt concentration.

Moreirinha C., Duarte S., Pascoal C., Cássio F.

Urbanization and industrial activities have contributed to widespread contamination by metals and polycyclic aromatic hydrocarbons, but the combined effects of these toxics on aquatic biota and processes are poorly understood. We examined the effects of cadmium (Cd) and phenanthrene on the activity and diversity of fungi associated with decomposing leaf litter in streams. Leaves of Alnus glutinosa were immersed for 10 days in an unpolluted low-order stream in northwest Portugal to allow microbial colonization. Leaves were then exposed in microcosms for 14 days to Cd (0.06–4.5 mg L−1) and phenanthrene (0.2 mg L−1) either alone or in mixture. A total of 19 aquatic hyphomycete species were found sporulating on leaves during the whole study. The dominant species was Articulospora tetracladia, followed by Alatospora pulchella, Clavatospora longibrachiata, and Tetrachaetum elegans. Exposure to Cd and phenanthrene decreased the contribution of A. tetracladia to the total conidial production, whereas it increased that of A. pulchella. Fungal diversity, assessed as denaturing gradient gel electrophoresis fingerprinting or conidial morphology, was decreased by the exposure to Cd and/or phenanthrene. Moreover, increased Cd concentrations decreased leaf decomposition and fungal reproduction but did not inhibit fungal biomass production. Exposure to phenanthrene potentiated the negative effects of Cd on fungal diversity and activity, suggesting that the co-occurrence of these stressors may pose additional risk to aquatic biodiversity and stream ecosystem functioning.

Abdel-Mawgoud A.M., Lépine F., Déziel E.

Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-Rha-C10-C10), α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoate (Rha-Rha-C10), as well as their mono-rhamnolipid congeners Rha-C10-C10 and Rha-C10. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.