Evidence for shifts in the structure and abundance of the microbial community in a long-term PCB-contaminated soil under bioremediation.

Although the impact of bioremediation of PCB-contaminated sites on the indigenous microbial community is a key question for soil restoration, it remains poorly understood. Therefore, a small-scale bioremediation assay made of (a) a biostimulation treatment with carvone, soya lecithin and xylose and (b) two bioaugmentation treatments, one with a TSZ7 mixed culture and another with a Rhodococcus sp. Z6 pure strain was set up. Changes in the structure of the global soil microbial community and in the abundances of different taxonomic phyla were monitored using ribosomal intergenic spacer analysis (RISA) and real-time PCR. After an 18-month treatment, the structure of the bacterial community in the bioremediated soils was significantly different from that of the native soil. The shift observed in the bacterial community structure using RISA analysis was in accordance with the monitored changes in the abundances of 11 targeted phyla and classes. Actinobacteria, Bacteriodetes and α- and γ-Proteobacteria were more abundant under all three bioremediation treatments, with Actinobacteria representing the dominant phylum. Altogether, our results indicate that bioremediation of PCB-contaminated soil induces significant changes in the structure and abundance of the total microbial community, which must be addressed to implement bioremediation practices in order to restore soil functions.

Taxonomic and functional diversity of atrazine-degrading bacterial communities enriched from agrochemical factory soil.

AIMS: To characterize atrazine-degrading potential of bacterial communities enriched from agrochemical factory soil by analysing diversity and organization of catabolic genes. METHODS AND RESULTS: The bacterial communities enriched from three different sites of varying atrazine contamination mineralized 65-80% of (14) C ring-labelled atrazine. The presence of trzN-atzBC-trzD, trzN-atzABC-trzD and trzN-atzABCDEF-trzD gene combinations was determined by PCR. In all enriched communities, trzN-atzBC genes were located on a 165-kb plasmid, while atzBC or atzC genes were located on separated plasmids. Quantitative PCR revealed that catabolic genes were present in up to 4% of the community. Restriction analysis of 16S rDNA clone libraries of the three enrichments revealed marked differences in microbial community structure and diversity. Sequencing of selected clones identified members belonging to Proteobacteria (α-, ß- and γ-subclasses), the Actinobacteria, Bacteroidetes and TM7 division. Several 16S rRNA gene sequences were closely related to atrazine-degrading community members previously isolated from the same contaminated site. CONCLUSIONS: The enriched communities represent a complex and diverse bacterial associations displaying heterogeneity of catabolic genes and their functional redundancies at the first steps of the upper and lower atrazine-catabolic pathway. The presence of catabolic genes in small proportion suggests that only a subset of the community has the capacity to catabolize atrazine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insights into the genetic specificity and the repertoire of catabolic genes within bacterial communities originating from soils exposed to long-term contamination by s-triazine compounds.

Genetic potential, diversity and activity of an atrazine-degrading community enriched from a herbicide factory effluent.

AIMS: To characterize an atrazine-degrading bacterial community enriched from the wastewater of a herbicide factory. METHODS AND RESULTS: The community mineralized 81.4 +/- 1.9% of [(14)C-ring]atrazine and 31.0 +/- 1.8% of [(14)C-ethyl]atrazine within 6 days of batch cultivation in mineral salts medium containing atrazine as the sole nitrogen source. Degradation activity of the community towards different chloro- and methylthio-substituted s-triazine compounds was also demonstrated. Restriction analysis of amplified 16S rDNA revealed high diversity of bacterial populations forming the community, with Pseudomonas species dominating in the clone library. Atrazine-degrading genetic potential of the community determined by PCR revealed the presence of trzN, atzB, atzC and trzD genes. The trzN, atzB and atzC genes were shown to be located on a plasmid of 322 kb. Quantitative PCR showed that relative abundances of atzB, atzC and trzD genes were approx. 100-fold lower than 16S rDNA. CONCLUSIONS: The enriched community represents a complex bacterial association expressing substantial atrazine-mineralizing activity and a broad specificity towards a range of s-triazine compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study is beginning to yield insights into the richness, genetic potential and density of functional atrazine-mineralizing community that could be a potential bioaugmentation agent for improving biotransformation processes in wastewaters bearing different s-triazine compounds.

Possible interactions within a methanotrophic-heterotrophic groundwater community able to transform linear alkylbenzenesulfonates.

The relationships and interactions within a methanotrophic-heterotrophic groundwater community were studied in a closed system (shake culture) in the presence of methane as the primary carbon and energy source and with the addition of the pure linear alkylbenzenesulfonate (LAS) congener 2-[4-(sulfophenyl)]decan as a cometabolic substrate. When cultured under different conditions, this community was shown to be a stable association, consisting of one obligate type II methanotroph and four or five heterotrophs possessing different nutritional and physiological characteristics. The results of experiments examining growth kinetics and nutritional relationships suggested that a number of complex interactions existed in the community in which the methanotroph was the only member able to grow on methane and to cometabolically initiate LAS transformation. These growth and metabolic activities of the methanotroph ensured the supply of a carbon source and specific nutrients which sustained the growth of four or five heterotrophs. In addition to the obligatory nutritional relationships between the methanotroph and heterotrophs, other possible interactions resulted in the modification of basic growth parameters of individual populations and a concerted metabolic attack on the complex LAS molecule. Most of these relationships conferred beneficial effects on the interacting populations, making the community adaptable to various environmental conditions and more efficient in LAS transformation than any of the individual populations alone.

Growth characteristics and metabolic activities of the methanotrophic-heterotrophic groundwater community.

In this work the growth characteristics and metabolic activities of the methanotrophic-heterotrophic groundwater community (culture MM1) as well as of individual community members were studied. When growing in shake flasks, under various methane and oxygen tensions, culture MM1 revealed the capability of a stable association consisting of one obligate methanotroph with type II intracytoplasmic membranes as the dominant strain, and four or five heterotrophs of different morphological, physiological and metabolic characteristics. Coexistence of different populations and the stability of culture MM1 under various conditions suggested that complex relationships may exist between the community members. Most of these relationships seem to be beneficial for both the methanotroph and heterotrophs, making the community adaptable to a range of environmental conditions containing methane as the only carbon source. Furthermore, faster and more complete transformation of 2-[4-(sulphophenyl)]decane (2C10LAS) by the community than by any of the community members alone, illustrates the role and importance of methanotrophic-heterotrophic interactions in combined metabolic attack on complex linear alkylbenzenesulphonates molecules.

Biodegradation of linear alkylbenzenesulphonates (LAS) by mixed methanotrophic-heterotrophic cultures.

The biodegradation of undecylbenzenesulphonate (C11LAS) was studied in shake flasks at 21 degrees C using two mixed bacterial cultures. The first culture, MM1, contained a type II methanotroph and four heterotrophs, and was enriched from a groundwater aquifer. The second culture, MC, consisted of five heterotrophic strains, most of them belonging to the genus Pseudomonas, and was isolated from the wastewater of a detergent plant. Methane, carbon dioxide and oxygen concentrations were determined by gas chromatography. Concentrations of C11LAS and the aromatic intermediates were determined by reversed-phase HPLC. In spite of faster transformation of the alkyl side-chain by the culture MC, the culture MM1 containing type II methanotroph was capable of further degradation of C11LAS aromatic intermediates (sulphophenylalkanoates). The most probable mechanism for the degradation of the alkyl part of the C11LAS molecule by both cultures was omega-oxidation of the terminal methyl group followed by beta-oxidation. Studies of methane utilization demonstrated an approximately three times higher second-order rate coefficient for methane consumption (kmax/Ks) in the absence of C11LAS. This indicates a possible metabolic activity of methanotrophs in the transformation of the complex LAS molecule due to the methane monooxygenase enzyme system.

Biodegradation of undecylbenzenesulphonate by mixed methane-oxidizing culture.

Biodegradation of undecylbenzenesulphonate (C(11)LAS) was performed in shake flasks at 21 degrees C by using a mixed methanotrophic-heterotrophic culture containing type II methanotrophs. Concentrations of C(11)LAS and aromatic intermediates were determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Methane and carbon dioxide concentrations were measured in headspace samples by using gas chromatography. RP-HPLC analyses of aqueous samples show that the culture MM1 expresses the capability of C(11)LAS transformation in the presence or absence of methane. Simultaneous methane oxidation and C(11)LAS degradation, and the inhibition of both transformation processes by acetylene were observed. This suggests the possibility that C(11)LAS transformation is catalyzed by methane monooxygenase (MMO). Comparable affinity of culture MM1 for both methane and C(11)LAS ( [Formula: see text], respectively), and more than four times higher maximum transformation rate for methane than for C(11)LAS ( [Formula: see text] (dry weight) cells day(-1), respectively), were determined. This further supports the involvement of MMO enzyme system in transformation and suggests a pronounced competitive inhibition of C(11)LAS degradation by methane.