Dynamics of the linuron hydrolase libA gene pool size in response to linuron application and environmental perturbations in agricultural soil and on-farm biopurification systems

libA, a gene encoding a novel type of linuron hydrolase, was recently identified in the linuron-mineralizing Variovorax sp. strain SRS16. In order to assess the contribution of libA to linuron degradation in environmental settings, libA abundance was monitored in response to the application of linuron and to environmental perturbations in agricultural soil microcosms and microcosms simulating the matrix of on-farm biopurification systems. libA numbers were measured by real-time PCR and linked to reported data of Variovorax community composition and linuron mineralization capacity. In the soil microcosms and one biopurification system setup, libA numbers responded to the application of linuron and environmental changes in congruency with the modulation of linuron mineralization capacity and the occurrence of a particular Variovorax phylotype (phylotype A). However, in another biopurification system setup, no such correlations were found. Our data suggest that in the simulated environmental settings, the occurrence of libA can be linked to the linuron mineralization capacity and that libA is primarily hosted by Variovorax phylotype A strains. However, the results also suggest that, apart from libA, other, as-yet-unknown isofunctional genes play an important role in linuron mineralization in the environment.     

Synergistic degradation of linuron by a bacterial consortium and isolation of a single linuron-degrading variovorax strain

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.     

Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil

In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the (13)C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-(13)C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from (13)C-enriched DNA and were designated "anthracene group 1." Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.     

Improvement of pesticide mineralization in on-farm biopurification systems by bioaugmentation with pesticide-primed soil

Microcosms were used to examine whether pesticide-primed soils could be preferentially used over nonprimed soils for bioaugmentation of on-farm biopurification systems (BPS) to improve pesticide mineralization. Microcosms containing a mixture of peat, straw and either linuron-primed soil or nonprimed soil were irrigated with clean or linuron-contaminated water. The lag time of linuron mineralization, recorded for microcosm samples, was indicative of the dynamics of the linuron-mineralizing biomass in the system. Bioaugmentation with linuron-primed soil immediately resulted in the establishment of a linuron-mineralizing capacity, which increased in size when fed with the pesticide. Also, microcosms containing nonprimed soil developed a linuron-mineralizing population, but after extended linuron feeding. Additional experiments showed that linuron-mineralization only developed with some nonprimed soils. Concomitant with the increase in linuron degradation capacity, targeted PCR-denaturing gradient gel electrophoresis showed the proliferation of a Variovorax phylotype related to the linuron-degrading Variovorax sp. SRS16 in microcosms containing linuron-primed soil, suggesting the involvement of Variovorax in linuron degradation. The correlation between the appearance of specific Variovorax phylotypes and linuron mineralization capacity was less clear in microcosms containing nonprimed soil. The data indicate that supplementation of pesticide-primed soil results in the establishment of pesticide-mineralizing populations in a BPS matrix with more certainty and more rapidly than the addition of nonprimed soil.     

Characterization of novel linuron-mineralizing bacterial consortia enriched from long-term linuron-treated agricultural soils

Linuron-mineralizing cultures were enriched from two linuron-treated agricultural soils in the presence and absence of a solid support. The cultures contained linuron-degrading bacteria, which coexisted with bacteria degrading either 3,4-dichloroaniline (3,4-DCA) or N,O-dimethylhydroxylamine (N,O-DMHA), two common metabolites in the linuron degradation pathway. For one soil, the presence of a solid support enriched for linuron-degrading strains phylogenetically related to but different from those enriched without support. Most linuron-degrading consortium members were identified as Variovorax, but a Hydrogenophaga and an Achromobacter strain capable of linuron degradation were also obtained. Several of the linuron-degrading isolates also degraded 3,4-DCA. Isolates that degraded 3,4-DCA but not linuron belonged to the genera Variovorax, Cupriavidus and Afipia. Hyphomicrobium spp. were involved in the metabolism of N,O-DMHA. Whereas several isolates degraded linuron independently, more efficient degradation was achieved by combining linuron and 3,4-DCA-degraders or by adding casamino acids. These data suggest that (1) linuron degradation is performed by a group of metabolically interacting bacteria rather than by individual strains, (2) there are other genera in addition to Variovorax that degrade linuron beyond 3,4-DCA, (3) linuron-degrading consortia of different origins have a similar composition, and (4) interactions between consortium members can be complex and can involve exchange of both metabolites and other nutrients.     

Methanogen diversity evidenced by molecular characterization of methyl coenzyme M reductase A (mcrA) genes in hydrothermal sediments of the Guaymas Basin

The methanogenic community in hydrothermally active sediments of Guaymas Basin (Gulf of California, Mexico) was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Members of the Methanomicrobiales and Methanosarcinales dominated the mcrA and 16S rRNA clone libraries from the upper 15 cm of the sediments. Within the H2/CO2- and formate-utilizing family Methanomicrobiales, two mcrA and 16S rRNA lineages were closely affiliated with cultured species of the genera Methanoculleus and Methanocorpusculum. The most frequently recovered mcrA PCR amplicons within the Methanomicrobiales did not branch with any cultured genera. Within the nutritionally versatile family Methanosarcinales, one 16S rRNA amplicon and most of the mcrA PCR amplicons were affiliated with the obligately acetate utilizing species Methanosaeta concilii. The mcrA clone libraries also included phylotypes related to the methyl-disproportionating genus Methanococcoides. However, two mcrA and two 16S rRNA lineages within the Methanosarcinales were unrelated to any cultured genus. Overall, the clone libraries indicate a diversified methanogen community that uses H2/CO2, formate, acetate, and methylated substrates. Phylogenetic affiliations of mcrA and 16S rRNA clones with thermophilic and nonthermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas sediments.     

Elucidating the key member of a linuron-mineralizing bacterial community by PCR and reverse transcription-PCR denaturing gradient gel electrophoresis 16S rRNA gene fingerprinting and cultivation

A bacterial community from Danish agricultural soil was enriched with linuron [N-(3,4-dichlorophenyl)-N'-methoxy-N'-methylurea] as the sole carbon and nitrogen source. The community mineralized [ring-U-14C]linuron completely to 14CO2 and 14C-biomass. Denaturing gradient gel electrophoresis analysis and cultivation revealed that a Variovorax sp. was responsible for the mineralization activity.     

Unique kinetic properties of phenol-degrading variovorax strains responsible for efficient trichloroethylene degradation in a chemostat enrichment culture

A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities. To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter(-1) day(-1)) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter(-1) day(-1) (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.     

The role of Variovorax and other Comamonadaceae in sulfur transformations by microbial wheat rhizosphere communities exposed to different sulfur fertilization regimes

Sulfonates are a key component of the sulfur present in agricultural soils. Their mobilization as part of the soil sulfur cycle is mediated by rhizobacteria, and involves the oxidoreductase AsfA. In this study, the effect of fertilization regime on rhizosphere bacterial asfA distribution was examined at the Broadbalk long-term wheat experiment, Rothamsted, UK, which was established in 1843, and has included a sulfur-free treatment since 2001. Direct isolation of desulfonating rhizobacteria from the wheat rhizospheres led to the identification of several Variovorax and Polaromonas strains, all of which contained the asfA gene. Rhizosphere DNA was isolated from wheat rhizospheres in plots fertilized with inorganic fertilizer with and without sulfur, with farmyard manure or from unfertilized plots. Genetic profiling of 16S rRNA gene fragments [denaturing gradient gel electrophoresis (DGGE)] from the wheat rhizospheres revealed that the level of inorganic sulfate in the inorganic fertilizer was correlated with changes in the general bacterial community structure and the betaproteobacterial community structure in particular. Community analysis at the functional gene level (asfA) showed that 40% of clones in asfAB clone libraries were affiliated to the genus Variovorax. Analysis of asfAB-based terminal restriction fragment length polymorphism (T-RFLP) fingerprints showed considerable differences between sulfate-free treatments and those where sulfate was applied. The results suggest the occurrence of desulfonating bacterial communities that are specific to the fertilization regime chosen and that arylsulfonates play an important role in rhizobacterial sulfur nutrition.     

Constitutive mineralization of low concentrations of the herbicide linuron by a Variovorax sp. strain

The mineralization of the herbicide linuron at concentrations of μg and mg L⁻¹ was studied in liquid batch experiments with Variovorax sp. strain SRS16. The strain was highly efficient at mineralizing a range of linuron concentrations (0.002–10 mg L⁻¹) with 20–60% of the added ¹⁴C-ring-labeled linuron metabolized to ¹⁴CO₂ within hours to days depending on the initial linuron concentration and incubation period. At mg L⁻¹ linuron concentrations the mineralization activity by SRS16 was inducible and a shift to constitutive mineralization activity was apparent with a reduction in the linuron concentration to μg L⁻¹ levels. This study revealed that strain SRS16 is a promising candidate for bioaugmentation of water or soil resources contaminated with low linuron concentrations.     

An untapped bacterial cellulolytic community enriched from coastal marine sediment under anaerobic and thermophilic conditions

A bacterial community with strong cellulose [filter paper (FP) and microcrystalline cellulose] degradation ability was isolated from the coastal marine environment. They were isolated under thermophilic (60?°C) and anaerobic cultivation conditions. The library of 16S rRNA gene clones revealed a total of 16 operational taxonomic units after 50 clones were surveyed. Sixty percent of the clones were most related to the type strain of Clostridium thermocellum with 16S rRNA gene identity around 87-89%. All of them showed extremely low sequence similarities and were novel at least in species level. The gene clone libraries of glycosyl hydrolase family 48 showed low gene and amino acid sequence similarities around 70-72%. The results indicated that the cellulose degradation systems in the specific environment have not been well studied. The enrichment could disrupt FP within 3?days in a basal medium. The cellulase activity of the community was comparable to that of C.?thermocellum LQR1. The main fermentation products were ethanol, acetic acid and butyric acid. This work identified a novel microbial resource with a potential in lignocellulose conversion and biofuel production.     

Identification and quantification of uncultivated Proteobacteria associated with pyrene degradation in a bioreactor treating PAH-contaminated soil

Uncultivated bacteria associated with the degradation of pyrene in a bioreactor treating soil contaminated with polycyclic aromatic hydrocarbons (PAH) were identified by DNA-based stable-isotope probing (SIP) and quantified by real-time quantitative PCR. Most of the 16S rRNA gene sequences recovered from (13)C-enriched DNA fractions clustered phylogenetically within three separate groups of beta- and gamma-Proteobacteria unassociated with described genera and were designated "Pyrene Groups 1, 2 and 3". One recovered sequence was associated with the Sphingomonas genus. Pyrene Groups 1 and 3 were present in very low numbers in the bioreactor but represented 75% and 7%, respectively, of the sequences recovered from 16S rRNA gene clone libraries constructed from (13)C-enriched DNA. In a parallel time-course incubation with unlabelled pyrene, there was between a 2- and 4-order-of-magnitude increase in the abundance of 16S rRNA genes from Pyrene groups 1 and 3 and from targeted Sphingomonas spp. over a 10 day incubation. Sequences from Pyrene Group 2 were 11% of the SIP clone libraries but accounted for 14% of the total bacterial 16S rRNA genes in the bioreactor community. However, the abundance of this group did not increase significantly in response to pyrene disappearance. These data indicate that the primary pyrene degraders in the bioreactor were uncultivated, low-abundance beta- and gamma-Proteobacteria not previously associated with pyrene degradation.     

Methanogen Diversity Evidenced by Molecular Characterization of Methyl Coenzyme M Reductase A (mcrA) Genes in Hydrothermal Sediments of the Guaymas Basin

The methanogenic community in hydrothermally active sediments of Guaymas Basin (Gulf of California, Mexico) was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Members of the Methanomicrobiales and Methanosarcinales dominated the mcrA and 16S rRNA clone libraries from the upper 15 cm of the sediments. Within the H₂/CO₂- and formate-utilizing family Methanomicrobiales, two mcrA and 16S rRNA lineages were closely affiliated with cultured species of the genera Methanoculleus and Methanocorpusculum. The most frequently recovered mcrA PCR amplicons within the Methanomicrobiales did not branch with any cultured genera. Within the nutritionally versatile family Methanosarcinales, one 16S rRNA amplicon and most of the mcrA PCR amplicons were affiliated with the obligately acetate utilizing species Methanosaeta concilii. The mcrA clone libraries also included phylotypes related to the methyl-disproportionating genus Methanococcoides. However, two mcrA and two 16S rRNA lineages within the Methanosarcinales were unrelated to any cultured genus. Overall, the clone libraries indicate a diversified methanogen community that uses H₂/CO₂, formate, acetate, and methylated substrates. Phylogenetic affiliations of mcrA and 16S rRNA clones with thermophilic and nonthermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas sediments.     

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.     

Reconstruction of ribosomal RNA genes from metagenomic data

Direct sequencing of environmental DNA (metagenomics) has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.     

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.     

石油污染盐碱土壤翅碱蓬根围的细菌多样性及耐盐石油烃降解菌筛选

为了更好地了解石油污染盐碱土壤翅碱蓬根围的细菌多样性,采用16S rRNA基因克隆文库方法对其进行分析,在此基础上采用富集培养方法从该生境中分离筛选耐盐石油烃降解菌.16S rRNA基因克隆文库分析结果表明,海杆菌属(Marinobacter)、食烷菌属(Alcanivorax)和假单胞菌属(Pseudomonas)是该生境中的优势菌.他们可能在石油污染盐碱土壤翅碱蓬植物修复过程中起重要作用.进一步采用富集培养方法,从该生境中分离得到8株耐盐石油烃降解菌,可以耐受6％-10％浓度的NaCl,石油烃降解率在32.3％-57.0％之间.经16S rRNA基因序列分析,8株菌隶属于戈登氏菌属(Gordonia)、无色杆菌属(Achromobacter)、迪茨菌属(Dietzia)、芽胞杆菌属(Bacillus)和假单胞菌属(Pseudomonas).他们可能参与石油污染盐碱土壤翅碱蓬植物修复过程中的石油烃降解.     

Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles

The effect of three phenyl urea herbicides (diuron, linuron, and chlorotoluron) on soil microbial communities was studied by using soil samples with a 10-year history of treatment. Denaturing gradient gel electrophoresis (DGGE) was used for the analysis of 16S rRNA genes (16S rDNA). The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analysing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the herbicide-treated and nontreated soils were significantly different. Moreover, the bacterial diversity seemed to decrease in soils treated with urea herbicides, and sequence determination of several DGGE fragments showed that the most affected species in the soils treated with diuron and linuron belonged to an uncultivated bacterial group. As well as the 16S rDNA fingerprints, the substrate utilization patterns of the microbial communities were compared. Principal-component analysis performed on BIOLOG data showed that the functional abilities of the soil microbial communities were altered by the application of the herbicides. In addition, enrichment cultures of the different soils in medium with the urea herbicides as the sole carbon and nitrogen source showed that there was no difference between treated and nontreated soil in the rate of transformation of diuron and chlorotoluron but that there was a strong difference in the case of linuron. In the enrichment cultures with linuron-treated soil, linuron disappeared completely after 1 week whereas no significant transformation was observed in cultures inoculated with nontreated soil even after 4 weeks. In conclusion, this study showed that both the structure and metabolic potential of soil microbial communities were clearly affected by a long-term application of urea herbicides.