Growth in coculture stimulates metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the beta-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of (14)C-labeled isoproturon to (14)CO(2) and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Isolation from Agricultural Soil and Characterization of a Sphingomonas sp. Able To Mineralize the Phenylurea Herbicide Isoproturon

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the α-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.     

In-Field Spatial Variability in the Degradation of the Phenyl-Urea Herbicide Isoproturon Is the Result of Interactions between Degradative Sphingomonas spp. and Soil pH

Substantial spatial variability in the degradation rate of the phenyl-urea herbicide isoproturon (IPU) [3-(4-isopropylphenyl)-1,1-dimethylurea] has been shown to occur within agricultural fields, with implications for the longevity of the compound in the soil, and its movement to ground- and surface water. The microbial mechanisms underlying such spatial variability in degradation rate were investigated at Deep Slade field in Warwickshire, United Kingdom. Most-probable-number analysis showed that rapid degradation of IPU was associated with proliferation of IPU-degrading organisms. Slow degradation of IPU was linked to either a delay in the proliferation of IPU-degrading organisms or apparent cometabolic degradation. Using enrichment techniques, an IPU-degrading bacterial culture (designated strain F35) was isolated from fast-degrading soil, and partial 16S rRNA sequencing placed it within the Sphingomonas group. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial community 16S rRNA revealed two bands that increased in intensity in soil during growth-linked metabolism of IPU, and sequencing of the excised bands showed high sequence homology to the Sphingomonas group. However, while F35 was not closely related to either DGGE band, one of the DGGE bands showed 100% partial 16S rRNA sequence homology to an IPU-degrading Sphingomonas sp. (strain SRS2) isolated from Deep Slade field in an earlier study. Experiments with strains SRS2 and F35 in soil and liquid culture showed that the isolates had a narrow pH optimum (7 to 7.5) for metabolism of IPU. The pH requirements of IPU-degrading strains of Sphingomonas spp. could largely account for the spatial variation of IPU degradation rates across the field.     

Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon.

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the alpha-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.     

Isolation and characterization of an isoproturon mineralizing <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. strain SH from a French agricultural soil

The phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralized in an agricultural soil in France that had been periodically exposed to IPU. Enrichment cultures from samples of this soil isolated a bacterial strain able to mineralize IPU. 16S rRNA sequence analysis showed that this strain belonged to the phylogeny of the genus Sphingomonas (96% similarity with Sphingomonas sp. JEM-14, AB219361) and was designated Sphingomonas sp. strain SH. From this strain, a partial sequence of a 1,2-dioxygenase (catA) gene coding for an enzyme degrading catechol putatively formed during IPU mineralization was amplified. Phylogenetic analysis revealed that the catA sequence was related to Sphingomonas spp. and showed a lack of congruence between the catA and 16S rRNA based phylogenies, implying horizontal gene transfer of the catA gene cluster between soil microbiota. The IPU degrading ability of strain SH was strongly influenced by pH with maximum degradation taking place at pH 7.5. SH was only able to mineralize IPU and its known metabolites including 4-isopropylaniline and it could not degrade other structurally related phenylurea herbicides such as diuron, linuron, monolinuron and chlorotoluron or their aniline derivatives. These observations suggest that the catabolic abilities of the strain SH are highly specific to the metabolism of IPU.     

Biodegradation of the Phenylurea Herbicide Isoproturon and its Metabolites in Agricultural Soils

Degradation of the phenylurea herbicide isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) and several phenylurea and aniline metabolites was studied in agricultural soils previously exposed to isoproturon. The potential for degradation of the demethylated metabolite 3-(4-isopropylphenyl)-1-methylurea in the soils was much higher compared to isoproturon. In the most active soil only 6% of added ¹⁴C-labelled isoproturon was mineralised to ¹⁴C₂ within 20 days while in the same period 45% of added ¹⁴C-labelled 3-(4-isopropylphenyl)-1-methylurea was mineralized. This indicates that the initial N-demethylation may be a limiting step in the complete mineralization of isoproturon. Repeated addition of 3-(4-isopropylphenyl)-1-methylurea to the soil and further subculturing in mineral medium led to a highly enriched mixed bacterial culture with the ability to mineralize 3-(4-isopropylphenyl)-1-methylurea.The culture did not degrade either isoproturon or the didemethylatedmetabolite 3-(4-isopropylphenyl)-urea when provided as sole source of carbon and energy. The metabolite 4-isopropyl-aniline was also degraded and utilised for growth, thus indicating that 3-(4-isopropylphenyl)-1-methylurea is degraded byan initial cleavage of the methylurea-group followed by mineralizationof the phenyl-moiety. Several attempts were made to isolate pure bacterial cultures degrading 3-(4-isopropylphenyl)-1-methylurea or 4-isopropyl-aniline,but they were not successful.     

Hydroxylation of the herbicide isoproturon by fungi isolated from agricultural soil

Several asco-, basidio-, and zygomycetes isolated from an agricultural field were shown to be able to hydroxylate the phenylurea herbicide isoproturon [N-(4-isopropylphenyl)-N',N'-dimethylurea] to N-(4-(2-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea and N-(4-(1-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea. Bacterial metabolism of isoproturon has previously been shown to proceed by an initial demethylation to N-(4-isopropylphenyl)-N'-methylurea. In soils, however, hydroxylated metabolites have also been detected. In this study we identified fungi as organisms that potentially play a major role in the formation of these hydroxylated metabolites in soils treated with isoproturon. Isolates of Mortierella sp. strain Gr4, Phoma cf. eupyrena Gr61, and Alternaria sp. strain Gr174 hydroxylated isoproturon at the first position of the isopropyl side chain, yielding N-(4-(2-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea, while Mucor sp. strain Gr22 hydroxylated the molecule at the second position, yielding N-(4-(1-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea. Hydroxylation was the dominant mode of isoproturon transformation in these fungi, although some cultures also produced traces of the N-demethylated metabolite N-(4-isopropylphenyl)-N'-methylurea. A basidiomycete isolate produced a mixture of the two hydroxylated and N-demethylated metabolites at low concentrations. Clonostachys sp. strain Gr141 and putative Tetracladium sp. strain Gr57 did not hydroxylate isoproturon but N demethylated the compound to a minor extent. Mortierella sp. strain Gr4 also produced N-(4-(2-hydroxy-1-methylethyl)phenyl)-N'-methylurea, which is the product resulting from combined N demethylation and hydroxylation.     

Hydroxylation of the Herbicide Isoproturon by Fungi Isolated from Agricultural Soil

Several asco-, basidio-, and zygomycetes isolated from an agricultural field were shown to be able to hydroxylate the phenylurea herbicide isoproturon [N-(4-isopropylphenyl)-N′,N′-dimethylurea] to N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea and N-(4-(1-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea. Bacterial metabolism of isoproturon has previously been shown to proceed by an initial demethylation to N-(4-isopropylphenyl)-N′-methylurea. In soils, however, hydroxylated metabolites have also been detected. In this study we identified fungi as organisms that potentially play a major role in the formation of these hydroxylated metabolites in soils treated with isoproturon. Isolates of Mortierella sp. strain Gr4, Phoma cf. eupyrena Gr61, and Alternaria sp. strain Gr174 hydroxylated isoproturon at the first position of the isopropyl side chain, yielding N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea, while Mucor sp. strain Gr22 hydroxylated the molecule at the second position, yielding N-(4-(1-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea. Hydroxylation was the dominant mode of isoproturon transformation in these fungi, although some cultures also produced traces of the N-demethylated metabolite N-(4-isopropylphenyl)-N′-methylurea. A basidiomycete isolate produced a mixture of the two hydroxylated and N-demethylated metabolites at low concentrations. Clonostachys sp. strain Gr141 and putative Tetracladium sp. strain Gr57 did not hydroxylate isoproturon but N demethylated the compound to a minor extent. Mortierella sp. strain Gr4 also produced N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′-methylurea, which is the product resulting from combined N demethylation and hydroxylation.     

In-field spatial variability in the degradation of the phenyl-urea herbicide isoproturon is the result of interactions between degradative Sphingomonas spp. and soil pH

Substantial spatial variability in the degradation rate of the phenyl-urea herbicide isoproturon (IPU) [3-(4-isopropylphenyl)-1,1-dimethylurea] has been shown to occur within agricultural fields, with implications for the longevity of the compound in the soil, and its movement to ground- and surface water. The microbial mechanisms underlying such spatial variability in degradation rate were investigated at Deep Slade field in Warwickshire, United Kingdom. Most-probable-number analysis showed that rapid degradation of IPU was associated with proliferation of IPU-degrading organisms. Slow degradation of IPU was linked to either a delay in the proliferation of IPU-degrading organisms or apparent cometabolic degradation. Using enrichment techniques, an IPU-degrading bacterial culture (designated strain F35) was isolated from fast-degrading soil, and partial 16S rRNA sequencing placed it within the Sphingomonas group. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial community 16S rRNA revealed two bands that increased in intensity in soil during growth-linked metabolism of IPU, and sequencing of the excised bands showed high sequence homology to the Sphingomonas group. However, while F35 was not closely related to either DGGE band, one of the DGGE bands showed 100% partial 16S rRNA sequence homology to an IPU-degrading Sphingomonas sp. (strain SRS2) isolated from Deep Slade field in an earlier study. Experiments with strains SRS2 and F35 in soil and liquid culture showed that the isolates had a narrow pH optimum (7 to 7.5) for metabolism of IPU. The pH requirements of IPU-degrading strains of Sphingomonas spp. could largely account for the spatial variation of IPU degradation rates across the field.     

Isolation and characterization of three <Emphasis Type="Italic">Sphingobium</Emphasis> sp. strains capable of degrading isoproturon and cloning of the catechol 1,2-dioxygenase gene from these strains

Three strains of bacteria (designated as YBL1, YBL2, YBL3 respectively) capable of degrading isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea, were isolated from the soils of two herbicide plants. Based on the comparative analysis of the 16S rRNA gene, and phenotypic and biochemical characterization, these strains were identified as Sphingobium sp. The optimum conditions for isoproturon degradation by these strains were pH 7.0, and temperature 30°C. Mg²⁺ (1 mM) enhanced the isoproturon degradation rate, while Ni²⁺ and Cu²⁺ (1 mmol l⁻¹) inhibited isoproturon degradation significantly. These three strains also showed the ability to remove the residues of other phenylurea herbicides such as chlorotoluron, diuron and fluometuron in mineral salt culture medium. The N-demethylation was the first step of degradation of dimethylurea-substituted herbicides. Strain YBL1 was found capable of degrading both dimethylurea-substituted herbicides and methoxymethylphenyl-urea herbicides i.e. linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea). Using the PCR method, partial sequences of the catechol 1,2-dioxygenase gene were obtained from these strains.     

Genetic labelling and application of the isoproturon-mineralizing Sphingomonas sp. strain SRS2 in soil and rhizosphere

AIMS: To construct a luxAB-labelled Sphingomonas sp. strain SRS2 maintaining the ability to mineralize the herbicide isoproturon and usable for monitoring the survival and distribution of strain SRS2 on plant roots in laboratory systems. METHODS AND RESULTS: We inserted the mini-Tn5-luxAB marker into strain SRS2 using conjugational mating. In the transconjugant mutants luciferase was produced in varying levels. The mutants showed significant differences in their ability to degrade isoproturon. One luxAB-labelled mutant maintained the ability to mineralize isoproturon and was therefore selected for monitoring colonization of barley roots. CONCLUSIONS: We successfully constructed a genetically labelled isoproturon-mineralizing-strain SRS2 and demonstrated its ability to survive in soil and its colonization of rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction of a luxAB-labelled strain SRS2 maintaining the degradative ability, provides a powerful tool for ecological studies serving as the basis for evaluating SRS2 as a bioremediation agent.     

Application of Biofilm-Forming Bacteria on the Enhancement of Organophosphorus Fungicide Degradation

This study aimed to develop technology enhancing the biodegradation efficacy against organophosphorus fungicide with biofilm-forming bacteria in situ. Using the crystal violet staining method, two bacterial strains having biofilm formation capability were isolated and identified as Pseudomonas sp. C7 and Bacillus sp. E5. Compared with the culture of tolclofos-methyl degrader Sphingomonas sp. 224, biofilm formation was improved by co-inoculation with biofilm-forming bacterium Bacillus sp. E5. Evaluated in liquid culture conditions, this two-species mixed consortium was observed to degrade tolclofos-methyl more effectively than Sphingomonas sp. 224 alone, with an approximately 90% degradation efficiency within 48 h of dosing. The improved effectiveness of the consortium biofilm was reflected using soil in situ with an approximately 7% increased degradation ratio over Sphingomonas sp. 224 alone. This is the first report demonstrating improved bioremediation degradation efficacy against tolclofos-methyl exhibited by a consortium biofilm. This work presents a possible effective bioremediation strategy using a specific biofilm composition against pollutants containing organophosphorus compounds in situ.     

Rapid mineralization of the phenylurea herbicide diuron by Variovorax sp. strain SRS16 in pure culture and within a two-member consortium

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of herbicide formulations, and consequently, it is frequently detected as a major water contaminant in areas where there is extensive use. We constructed a linuron [N-(3,4-dichlorophenyl)-N-methoxy-N-methylurea]- and diuron-mineralizing two-member consortium by combining the cooperative degradation capacities of the diuron-degrading organism Arthrobacter globiformis strain D47 and the linuron-mineralizing organism Variovorax sp. strain SRS16. Neither of the strains mineralized diuron alone in a mineral medium, but combined, the two strains mineralized 31 to 62% of the added [ring-U-(14)C]diuron to (14)CO(2), depending on the initial diuron concentration and the cultivation conditions. The constructed consortium was used to initiate the degradation and mineralization of diuron in soil without natural attenuation potential. This approach led to the unexpected finding that Variovorax sp. strain SRS16 was able to mineralize diuron in a pure culture when it was supplemented with appropriate growth substrates, making this strain the first known bacterium capable of mineralizing diuron and representatives of both the N,N-dimethyl- and N-methoxy-N-methyl-substituted phenylurea herbicides. The ability of the coculture to mineralize microgram-per-liter levels of diuron was compared to the ability of strain SRS16 alone, which revealed the greater extent of mineralization by the two-member consortium (31 to 33% of the added [ring-U-(14)C]diuron was mineralized to (14)CO(2) when 15.5 to 38.9 mug liter(-1) diuron was used). These results suggest that the consortium consisting of strains SRS16 and D47 could be a promising candidate for remediation of soil and water contaminated with diuron and linuron and their shared metabolite 3,4-dichloroaniline.     

Characterization of Two Novel Propachlor Degradation Pathways in Two Species of Soil Bacteria

Propachlor (2-chloro-N-isopropylacetanilide) is an acetamide herbicide used in preemergence. In this study, we isolated and characterized a soil bacterium, Acinetobacter strain BEM2, that was able to utilize this herbicide as the sole and limiting carbon source. Identification of the intermediates of propachlor degradation by this strain and characterization of new metabolites in the degradation of propachlor by a previously reported strain of Pseudomonas (PEM1) support two different propachlor degradation pathways. Washed-cell suspensions of strain PEM1 with propachlor accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain PEM1 grew on propachlor with a generation time of 3.4 h and a K_s of 0.17 ± 0.04 mM. Acinetobacter strain BEM2 grew on propachlor with a generation time of 3.1 h and a K_s of 0.3 ± 0.07 mM. Incubations with strain BEM2 resulted in accumulation of N-isopropylacetanilide, N-isopropylaniline, isopropylamine, and catechol. Both degradative pathways were inducible, and the principal product of the carbon atoms in the propachlor ring was carbon dioxide. These results and biodegradation experiments with the identified metabolites indicate that metabolism of propachlor by Pseudomonas sp. strain PEM1 proceeds through a different pathway from metabolism by Acinetobacter sp. strain BEM2.     

Inducible hydroxylation and demethylation of the herbicide isoproturon by Cunninghamella elegans

A screening of 27 fungal strains for degradation of the phenylurea herbicide isoproturon was performed and yielded 15 strains capable of converting the herbicide to polar metabolites. The zygomycete fungus Cunninghamella elegans strain JS/2 isolated from an agricultural soil converted isoproturon to several known hydroxylated metabolites. In addition, unknown metabolites were produced in minor amounts. Inducible degradation was indicated by comparing resting cells pregrown with or without isoproturon. This shows that strain JS/2 is capable of partially degrading isoproturon and that one or more of the enzymes involved are inducible upon isoproturon exposure.     

Biodegradation of the herbicide propanil, and its 3,4-dichloroaniline by-product in a continuously operated biofilm reactor

The persistence of propanil in soil and aquatic environments along with the possible accumulation of toxic degradation products, such as chloroanilines, is of environmental concern. In this work, a continuous small-scale bioprocess to degrade the herbicide propanil, its main catabolic by-product, 3,4-dichloroaniline (3,4-DCA), and the herbicide adjuvants is carried out. A microbial consortium, constituted by nine bacterial genera, was selected. The isolated strains, identified by amplification and sequencing of their 16S rDNA, were: Acidovorax sp., Luteibacter (rhizovicinus), Xanthomonas sp., Flavobacterium sp., Variovorax sp., Acinetobacter (calcoaceticus), Pseudomonas sp., Rhodococcus sp., and Kocuria sp. The ability of the microbial consortium to degrade the herbicide was evaluated in a biofilm reactor at propanil loading rates ranging from 1.9 to 36.8 mg L^?1 h^?1. Complete removal of propanil, 3,4-DCA, chemical oxygen demand and total organic carbon was obtained at propanil loading rates up to 24.9 mg L^?1 h^?1. At higher loading rates, the removal efficiencies decayed. Four of the identified strains could grow individually in propanil, and 3,4-DCA: Pseudomonas sp., Acinetobacter calcoaceticus, Rhodococcus sp., and Xanthomonas sp. The Kokuria strain grew on 3,4-DCA, but not on propanil. The first three bacteria have been related to biodegradation of phenyl urea herbicides or chlorinated anilines. Although some strains of the genera Xanthomonas and Kocuria have a role in the biodegradation of several xenobiotic compounds, as far as we know, there are no reports about degradation of propanil by Xanthomonas or 3,4-DCA by Kocuria species.     

Culture Independent Detection of Sphingomonas sp. EPA 505 Related Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons (PAHs)

The Sphingomonas genus hosts many interesting pollutant-degrading strains. Sphingomonas sp. EPA505 is the best studied polycyclic aromatic hydrocarbon (PAH)-degrading Sphingomonas strain. Based on 16S rRNA gene sequence analysis, Sphingomonas sp. strain EPA505 forms a separate branch in the Sphingomonas phylogenetic tree grouping exclusively PAH-degrading isolates. For specific PCR detection and monitoring of Sphingomonas sp. EPA505 and related strains in PAH-contaminated soils, a new 16S rRNA gene-based primer set was designed. The new primer set was shown to be highly selective for Sphingomonas sp. strain EPA505 as it only amplified DNA from strain EPA505 and not from other tested Sphingomonas strains or soil bacteria not belonging to the Sphingomonas genus. Using DNA extracts of a variety of inoculated PAH-contaminated soils, the primer pair was able to detect EPA505 in concentrations as low as 10² cells per gram of soil. Applying the new primer set, 16S rRNA gene fragments which were 99–100% similar to the corresponding gene of strain EPA505 were amplified from four of five PAH-contaminated soils. On the other hand, no PCR products were obtained from any of five tested uncontaminated soils. The preferential presence of EPA505 related Sphingomonas strains in PAH-contaminated soils with very different contamination profiles and different origin suggests an important role of this type of Sphingomonas in the natural Sphingomonas community colonizing PAH-contaminated sites.     

Residues effects of isoproturon in mature earthworm (Aporrectodea caliginosa) under laboratory conditions

This study was conducted to investigate the residues of isoproturon and its metabolites, 1-(4-isopropylphenyl)-3-methylurea, 1-(4-isopropylphenyl) urea, and 4-isopropylanilin in soil and mature earthworms under laboratory conditions. Mature earthworms (Aporrectodea caliginosa) were exposed for various durations (7, 15, 30, and 60 days) to soils contaminated with isoproturon concentrations (2, 4, 6, 8, and 10 mg.kg(-1) soil). The decrease in isoproturon concentration in the soil depended on initial concentration it was slower at higher concentrations. The isoproturon and its metabolites accumulated in earthworms it increased during the first 15 days and decreased thereafter. Acute toxicity of isoproturon was determined together with total soluble protein content and glycogen of worms. These parameters were related to isoproturon concentration in soil and earthworms. No lethal effect of isoproturon was observed even at the concentration 1000 mg.kg(-1) soil after 60 days of exposure. A reduction of total soluble protein was observed in all treated worms (maximum 59.54%). This study is suggesting the use of the total soluble protein content and glycogen of earthworms as biomarker of exposure to isoproturon.     

Biodegradation of bisphenol A by cells and cell lysate from <Emphasis Type="Italic">Sphingomonas </Emphasis>sp. strain AO1

The capacity and pathway of bisphenol A [BPA; 2,2-bis(4-hydroxyphenyl)propane] degradation in Sphingomonassp. strain AO1, which was isolated from the soil of a vegetable-growing field in Japan, were investigated. The bacterial strain was able to grow in a basal mineral salt medium containing BPA as the sole carbon source (BSMB medium), and was able to degrade 115 gml^–1 BPA in 6h in L medium. Several BPA metabolites were detected in the culture supernatant by HPLC and then identified by GC-MS and LC-MS-MS. These compounds were confirmed to be the same as those reported for other BPA-degrading bacteria. BPA degradation by cells in the basal mineral salt medium was induced by BPA, and activity was detected only in the intracellular soluble fraction in the presence of coenzymes, such as NADH, NAD⁺ , NADPH or NADP⁺. The addition of metyrapone, a cytochrome P450 inhibitor, to BSMB medium resulted in a decrease in BPA degradation and cell growth. The BPA-degradation activity of the intracellular soluble fraction was also inhibited by the cytochrome P450 inhibitor. Carbon monoxide difference spectra indicated that cytochrome P450 was present in the cells and that the amount of cytochrome P450 corresponded to the cellular BPA-degradation activity. Our results provide evidence that the cytochrome P450 system is involved in BPA metabolism in Sphingomonassp. strain AO1.     

Bacterial endophyte Sphingomonas sp. LK11 produces gibberellins and IAA and promotes tomato plant growth

Plant growth promoting endophytic bacteria have been identified as potential growth regulators of crops. Endophytic bacterium, Sphingomonas sp. LK11, was isolated from the leaves of Tephrosia apollinea. The pure culture of Sphingomonas sp. LK11 was subjected to advance chromatographic and spectroscopic techniques to extract and isolate gibberellins (GAs). Deuterated standards of [17, 17-²H₂]-GA₄, [17, 17-²H₂]-GA₉ and [17, 17-²H₂]-GA₂₀ were used to quantify the bacterial GAs. The analysis of the culture broth of Sphingomonas sp. LK11 revealed the existence of physiologically active gibberellins (GA₄: 2.97 ± 0.11 ng/ml) and inactive GA₉ (0.98 ± 0.15 ng/ml) and GA₂₀ (2.41 ± 0.23). The endophyte also produced indole acetic acid (11.23 ± 0.93 μM/ml). Tomato plants inoculated with endophytic Sphingomonas sp. LK11 showed significantly increased growth attributes (shoot length, chlorophyll contents, shoot, and root dry weights) compared to the control. This indicated that such phyto-hormones-producing strains could help in increasing crop growth.