Selective enrichment, identification, and isolation of diclofenac, ibuprofen, and carbamazepine degrading bacteria from a groundwater biofilm.

Diclofenac, ibuprofen, and carbamazepine are three of the most widely detected and most concerning pharmaceutical residues in aquatic ecosystems. The aim of this study was to identify bacteria that may be involved in their degradation from a bacterial biofilm. Selective enrichment cultures in mineral salt solution containing pharmaceutical compounds as sole source of carbon and energy were set up, and population dynamics were monitored using shotgun metagenome sequencing. Bacterial genomes were reconstructed using genome-resolved metagenomics. Thirty bacterial isolates were obtained, identified at species level, and tested regarding pharmaceutical biodegradation at an initial concentration of 1.5 mg l-1. The results indicated that most probably diclofenac biodegrading cultures consisted of members of genera Ferrovibrio, Hydrocarboniphaga, Zavarzinia, and Sphingopyxis, while in ibuprofen biodegradation Nocardioides and Starkeya, and in carbamazepine biodegradation Nocardioides, Pseudonocardia, and Sphingopyxis might be involved. During the enrichments, compared to the initial state the percentage relative abundance of these genera increased up to three orders of magnitude. Except Starkeya, the genomes of these bacteria were reconstructed and annotated. Metabolic analyses of the annotated genomes indicated that these bacteria harbored genes associated with pharmaceutical biodegradation. Stenotrophomonas humi DIC_5 and Rhizobium daejeonense IBU_18 isolates eliminated diclofenac and ibuprofen during the tests in the presence of either glucose (3 g l-1) or in R2A broth. Higher than 90% concentration reduction was observed in the case of both compounds.

Nocardioides carbamazepini sp. nov., an ibuprofen degrader isolated from a biofilm bacterial community enriched on carbamazepine.

From the metagenome of a carbamazepine amended selective enrichment culture the genome of a new to science bacterial species affiliating with the genus Nocardioides was reconstructed. From the same enrichment an aerobic actinobacterium, strain CBZ_1T, sharing 99.4% whole-genome sequence similarity with the reconstructed Nocardioides sp. bin genome was isolated. On the basis of 16S rRNA gene sequence similarity the novel isolate affiliated to the genus Nocardioides, with the closest relatives Nocardioides kongjuensis DSM19082T (98.4%), Nocardioides daeguensis JCM17460T (98.4%) and Nocardioides nitrophenolicus DSM15529T (98.2%). Using a polyphasic approach it was confirmed that the isolate CBZ_1T represents a new phyletic lineage within the genus Nocardioides. According to metagenomic, metatranscriptomic studies and metabolic analyses strain CZB_1T was abundant in both carbamazepine and ibuprofen enrichments, and harbors biodegradative genes involved in the biodegradation of pharmaceutical compounds. Biodegradation studies supported that the new species was capable of ibuprofen biodegradation. After 7 weeks of incubation, in mineral salts solution supplemented with glucose (3 g l-1) as co-substrate, 70% of ibuprofen was eliminated by strain CBZ_1T at an initial conc. of 1.5 mg l-1. The phylogenetic, phenotypic and chemotaxonomic data supported the classification of strain CBZ_1T to the genus Nocardioides, for which the name Nocardioides carbamazepini sp. nov. (CBZ_1T = NCAIM B.0.2663 = LMG 32395) is proposed. To the best of our knowledge, this is the first study that reports simultaneous genome reconstruction of a new to science bacterial species using metagenome binning and at the same time the isolation of the same novel bacterial species.

Pinisolibacter aquiterrae sp. nov., a novel aromatic hydrocarbon-degrading bacterium isolated from benzene-, and xylene-degrading enrichment cultures, and emended description of the genus Pinisolibacter.

Two Gram-reaction-negative strains, designated as B13T and MA2-2, were isolated from two different aromatic hydrocarbon-degrading enrichment cultures and characterized using a polyphasic approach to determine their taxonomic position. The two strains had identical 16S rRNA gene sequences and were most closely related to Pinisolibacter ravus E9T (97.36 %) and Siculibacillus lacustris SA-279T (96.33 %). Cells were facultatively aerobic rods and motile with a single polar flagellum. The strains were able to degrade ethylbenzene as sole source of carbon and energy. The assembled genome of strain B13T had a total length of 4.91 Mb and the DNA G+C content was 68.8 mol%. The predominant fatty acids (>5 % of the total) of strains B13T and MA2-2 were C18 : 1 ω7c/C18 : 1 ω6c, C16 : 1 ω7c/C16 : 1 ω6c and C16 : 0. The major ubiquinone of strain B13T was Q10, while the major polar lipids were phosphatidyl-N-methylethanolamine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and a phospholipid. Based on phenotypic characteristics and phylogenetic data, it is concluded that strains B13T and MA2-2 are members of the genus Pinisolibacter and represent a novel species for which the name Pinisolibacter aquiterrae sp. nov. is proposed. The type strain of the species is strain B13T (=LMG 32346T=NCAIM B.02665T).

Development of a bacterial consortium from Variovorax paradoxus and Pseudomonas veronii isolates applicable in the removal of BTEX.

In this study, we report on the development of a novel bacterial consortium, consisting of Variovorax paradoxus and Pseudomonas veronii isolates, applicable in the biodegradation of all six BTEX compounds (benzene, toluene, ethylbenzene, o-, m- and p-xylene) and the bioremediation of contaminated sites. The co-cultivability of the selected bacterial isolates was determined in nutrient-rich medium, as well as in BTEX amended mineral salts solution using Terminal Restriction Fragment Length Polymorphism (T-RFLP) and CFU determinations. BTEX biodegradation capacity of the two-strain consortium was assessed in mineral salts solution, where a series of BTEX depletions and supplementations occurred, as well as in a real, BTEX polluted environmental sample (contaminated groundwater) in the presence of the autochthonous bacterial community. The obtained results indicated that the developed bacterial consortium is very efficient in BTEX biodegradation. Under laboratory conditions, the acclimatized bacterial consortium completely degraded the BTEX mixture with a concentration as high as 20 mg l-1 in a mineral salt medium within a short span of 6 h. Close to in situ groundwater conditions (incubated at 15 °C under static conditions in the absence of light), groundwater microcosms containing the autochthonous bacterial community inoculated with the developed bacterial consortium showed more efficient toluene, o-, m-and p-xylene biodegradation capacity than microcosms containing solely the native microbial population originally found in the groundwater. In the inoculated microcosms, after 115 h of incubation the concentration (~ 1.7 mg l-1 each) of o-, m- and p-xylene decreased to zero, whereas in the non-inoculated microcosms the concentration of xylene isomers was still 0.2, 0.3 and 0.3 mg l-1, respectively. The allochthonous bioaugmentation of the contaminated groundwater with the obtained inoculant was successful and manifested in a better BTEX degradation rate. Our results suggest that the obtained bacterial consortium can be a new, stable and efficient bioremediation agent applicable in the synergistic elimination of BTEX compounds from contaminated sites.

Potential of Variovorax paradoxus isolate BFB1_13 for bioremediation of BTEX contaminated sites.

Here, we report and discuss the applicability of Variovorax paradoxus strain BFB1_13 in the bioremediation of BTEX contaminated sites. Strain BFB1_13 was capable of degrading all the six BTEX-compounds under both aerobic (O2 conc. 8 mg l-1) and micro-aerobic/oxygen-limited (O2 conc. 0.5 mg l-1) conditions using either individual (8 mg‧l-1) or a mixture of compounds (~ 1.3 mg‧l-1 of each BTEX compound). The BTEX biodegradation capability of SBP-encapsulated cultures (SBP-Small Bioreactor Platform) was also assessed. The fastest degradation rate was observed in the case of aerobic benzene biodegradation (8 mg l-1 per 90 h). Complete biodegradation of other BTEX occurred after at least 168 h of incubation, irrespective of the oxygenation and encapsulation. No statistically significant difference was observed between aerobic and microaerobic BTEX biodegradation. Genes involved in BTEX biodegradation were annotated and degradation pathways were predicted based on whole-genome shotgun sequencing and metabolic analysis. We conclude that V. paradoxus strain BFB1_13 could be used for the development of reactive biobarriers for the containment and in situ decontamination of BTEX contaminated groundwater plumes. Our results suggest that V. paradoxus strain BFB1_13-alone or in co-culture with other BTEX degrading bacterial isolates-can be a new and efficient commercial bioremediation agent for BTEX contaminated sites.

Effect of oxygen limitation on the enrichment of bacteria degrading either benzene or toluene and the identification of Malikia spinosa (Comamonadaceae) as prominent aerobic benzene-, toluene-, and ethylbenzene-degrading bacterium: enrichment, isolation and whole-genome analysis.

The primary aims of this present study were to evaluate the effect of oxygen limitation on the bacterial community structure of enrichment cultures degrading either benzene or toluene and to clarify the role of Malikia-related bacteria in the aerobic degradation of BTEX compounds. Accordingly, parallel aerobic and microaerobic enrichment cultures were set up and the bacterial communities were investigated through cultivation and 16S rDNA Illumina amplicon sequencing. In the aerobic benzene-degrading enrichment cultures, the overwhelming dominance of Malikia spinosa was observed and it was abundant in the aerobic toluene-degrading enrichment cultures as well. Successful isolation of a Malikia spinosa strain shed light on the fact that this bacterium harbours a catechol 2,3-dioxygenase (C23O) gene encoding a subfamily I.2.C-type extradiol dioxygenase and it is able to degrade benzene, toluene and ethylbenzene under clear aerobic conditions. While quick degradation of the aromatic substrates was observable in the case of the aerobic enrichments, no significant benzene degradation, and the slow degradation of toluene was observed in the microaerobic enrichments. Despite harbouring a subfamily I.2.C-type C23O gene, Malikia spinosa was not found in the microaerobic enrichments; instead, members of the Pseudomonas veronii/extremaustralis lineage dominated these communities. Whole-genome analysis of M. spinosa strain AB6 revealed that the C23O gene was part of a phenol-degrading gene cluster, which was acquired by the strain through a horizontal gene transfer event. Results of the present study revealed that bacteria, which encode subfamily I.2.C-type extradiol dioxygenase enzyme, will not be automatically able to degrade monoaromatic hydrocarbons under microaerobic conditions.

Aerobic and oxygen-limited naphthalene-amended enrichments induced the dominance of Pseudomonas spp. from a groundwater bacterial biofilm.

In this study, we aimed at determining the impact of naphthalene and different oxygen levels on a biofilm bacterial community originated from a petroleum hydrocarbon-contaminated groundwater. By using cultivation-dependent and cultivation-independent approaches, the enrichment, identification, and isolation of aerobic and oxygen-limited naphthalene degraders was possible. Results indicated that, regardless of the oxygenation conditions, Pseudomonas spp. became the most dominant in the naphthalene-amended selective enrichment cultures. Under low-oxygen conditions, P. veronii/P. extremaustralis lineage affiliating bacteria, and under full aerobic conditions P. laurentiana-related isolates were most probably capable of naphthalene biodegradation. A molecular biological tool has been developed for the detection of naphthalene 1,2-dioxygenase-related 2Fe-2S reductase genes of Gram-negative bacteria. The newly developed COnsensus DEgenerate Hybrid Oligonucleotide Primers (CODEHOP-PCR) technique may be used in the monitoring of the natural attenuation capacity of PAH-contaminated sites. A bacterial strain collection with prolific biofilm-producing and effective naphthalene-degrading organisms was established. The obtained strain collection may be applicable in the future for the development of biofilm-based bioremediation systems for the elimination of PAHs from groundwater (e.g., biofilm-based biobarriers).

First isolation of carbapenem-resistant Acinetobacter beijerinckii from an environmental sample.

The emergence of opportunistic Acinetobacter spp. in healthcare settings poses a significant threat to public health. The major reasons for nosocomial spread of these species are their abilities to develop and transfer drug resistance against various classes of antibiotics. Considering that Acinetobacter spp. are ubiquitous in nature, can utilize several carbon sources, and reach humans via various pathways, our aim was to obtain information about the environmental strains of this genus. Our first step was to develop and test a multistep isolation procedure based on traditional scientific methods. Antibiotic resistance patterns of the isolated strains were determined, as susceptibility to 12 antibiotics of 7 classes was tested by MIC Test Strip method. Altogether 366 samples (groundwater, surface water, and soil) of 24 sites were investigated and a collection of 37 Acinetobacter isolates was obtained. Among others, clinically important human pathogen Acinetobacter spp., such as A. baumannii, A. johnsonii, and A. gyllenbergii were identified. Three environmental strains were determined as multidrug-resistant including a carbapenem-resistant, hemolytic Acinetobacter beijerinckii strain isolated from a hydrocarbon-contaminated groundwater sample. In summary, it has been found that the applied multistep isolation procedure is applicable to isolate various species of Acinetobacter genus. Based on the antibiotic resistance assay, we can conclude that environmental representatives of Acinetobacter spp. are able to develop multidrug resistance, but at a lower rate than their clinical counterparts.

Aerobic and oxygen-limited enrichment of BTEX-degrading biofilm bacteria: dominance of Malikia versus Acidovorax species.

Due to their high resistance against environmental challenges, bacterial biofilms are ubiquitous and are frequently associated with undesired phenomena in environmental industry (e. g. biofouling). However, because of the high phylogenetic and functional diversity, bacterial biofilms are important sources of biotechnologically relevant microorganisms, e.g. those showing bioremediation potential. In our previous work, the high phylogenetic and metabolic diversity of a clogging biofilm, developed in a simple aromatic hydrocarbon (BTEX)-contaminated groundwater well was uncovered. The determination of relationships between different groups of biofilm bacteria and certain metabolic traits has been omitted so far. Therefore, by setting up new biofilm-based enrichment microcosms, the research goal of the present study was to identify the aerobic/hypoxic BTEX-degrading and/or prolific biofilm-forming bacteria. The initial bacterial community composition as well as temporal dynamics due to the selective enrichment has been determined. The obtained results indicated that the concentration of dissolved oxygen may be a strong selective force on the evolution and final structure of microbial communities, developed in hydrocarbon-contaminated environments. Accordingly, members of the genus Malikia proved to be the most dominant community members of the aerobic BTEX-degrading enrichments. Acidovorax spp. dominated the oxygen-limited/hypoxic setup. During the study, a strain collection of 23 different bacterial species was obtained. Non-pathogenic members of this strain collection, with outstanding biodegradation (e.g. Pseudomonas, Variovorax isolates) and biofilm-forming potential (e.g. Rhizobium), may potentially be applied in the development of biofilm-based semipermeable reactive biobarriers.

Sphingobium aquiterrae sp. nov., a toluene, meta- and para-xylene-degrading bacterium isolated from petroleum hydrocarbon-contaminated groundwater.

A Gram-negative, aerobic, slightly yellow-pigmented bacterium, designated as SKLS-A10T, was isolated from groundwater sample of the 'Siklós' petroleum hydrocarbon contaminated site (Hungary). Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain SKLS-A10T formed a distinct phyletic lineage within the genus Sphingobium. It shared the highest 16S rRNA gene homology with Sphingobium abikonense DSM 23268T (97.29 %), followed by Sphingobium lactosutens DSM 23389T (97.23 %), Sphingobium phenoxybenzoativorans KCTC 42448T (97.16 %) and Sphingobium subterraneum NBRC 109814T (96.74 %). The predominant fatty acids (>5 % of the total) are C18 : 1ω7c, C14 : 0 2-OH, C16 : 1ω7c/iso C15 : 0 2-OH, C17 : 1ω6c and C16 : 0. The major ubiquinone is Q-10. The predominant polyamine is spermidine. The major polar lipids are sphingoglycolipid and diphosphatidylglycerol. The DNA G+C content of strain SKLS-A10T is 65.9 mol%. On the basis of evidence from this taxonomic study using a polyphasic approach, strain SKLS-A10T represents a novel species of the genus Sphingobium for which the name Sphingobiumaquiterrae sp. nov. is proposed. The type strain is SKLS-A10T (=DSM 106441T=NCAIM B. 02634T).

Stable isotope probing of hypoxic toluene degradation at the Siklós aquifer reveals prominent role of Rhodocyclaceae.

The availability of oxygen is often a limiting factor for the degradation of aromatic hydrocarbons in subsurface environments. However, while both aerobic and anaerobic degraders have been intensively studied, degradation betwixt, under micro- or hypoxic conditions has rarely been addressed. It is speculated that in environments with limited, but sustained oxygen supply, such as in the vicinity of groundwater monitoring wells, hypoxic degradation may take place. A large diversity of subfamily I.2.C extradiol dioxygenase genes has been previously detected in a BTEX-contaminated aquifer in Hungary. Older literature suggests that such catabolic potentials could be associated to hypoxic degradation. Bacterial communities dominated by members of the Rhodocyclaceae were found, but the majority of the detected C23O genotypes could not be affiliated to any known bacterial degrader lineages. To address this, a stable isotope probing (SIP) incubation of site sediments with 13C7-toluene was performed under microoxic conditions. A combination of 16S rRNA gene amplicon sequencing and T-RFLP fingerprinting of C23O genes from SIP gradient fractions revealed the central role of degraders within the Rhodocyclaceae in hypoxic toluene degradation. The main assimilators of 13C were identified as members of the genera Quatrionicoccus and Zoogloea, and a yet uncultured group of the Rhodocyclaceae.

Rhodococcus sovatensis sp. nov., an actinomycete isolated from the hypersaline and heliothermal Lake Ursu.

A Gram-stain-positive, strictly aerobic, mesophilic bacterium, designated H004T, was isolated from a water sample of the hypersaline and heliothermal Lake Ursu, Sovata, Romania. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain H004T formed a distinct phyletic lineage within the genus Rhodococcus. It shared the highest 16S rRNA gene sequence similarity with Rhodococcus yunnanensis YIM 70056T (98.80 %), followed by Rhodococcus fascians LMG 3623T (98.73 %), Rhodococcus cercidiphylli YIM 65003T (98.73 %), Rhodococcus cerastii C5T (98.58 %) and Rhodococcus kyotonensis DS472T (98.53 %). The alkB-based phylogenetic analysis further confirmed that this strain constitutes a highly unique lineage within the genus. Chemotaxonomic characteristics, including the predominant fatty acids acids C15 : 0, C18 : 1ω9c, C19 : 1ω11c/C19 : 1ω9c and C16 : 1ω7c/iso-C15 : 0 2-OH, the major quinone MK-8(H2), the presence of mycolic acids and cell-wall chemotype IV were also consistent with the properties of members of the genus Rhodococcus. The DNA G+C content of strain H004T was 65.4 mol%. The results of DNA-DNA hybridization analyses with the closest relatives, in combination with the alkB-based phylogenetic analysis, as well as the chemotaxonomic and physiological data, demonstrated that isolate H004T represents a novel species of the genus Rhodococcus, for which the name Rhodococcus sovatensissp. nov. is proposed. The type strain is H004T (=DSM 102881T=NCAIM B.02632T).

Enrichment of dissimilatory Fe(III)-reducing bacteria from groundwater of the Siklós BTEX-contaminated site (Hungary).

Dissimilatory iron-reducing bacteria are commonly found in microbial communities of aromatic hydrocarbon-contaminated subsurface environments where they often play key role in the degradation of the contaminants. The Siklós benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated area is one of the best characterized petroleum hydrocarbon-contaminated sites of Hungary. Continuous monitoring of the microbial community in the center of the contaminant plume indicated the presence of an emerging Geobacter population and a Rhodoferax phylotype highly associated with aromatic hydrocarbon-contaminated subsurface environments. The aim of the present study was to make an initial effort to enrich Rhodoferax-related and other dissimilatory iron-reducing bacteria from this environment. Accordingly, four slightly different freshwater media were used to enrich Fe(III) reducers, differing only in the form of nitrogen source (organic, inorganic nitrogen or gaseous headspace nitrogen). Although enrichment of the desired Rhodoferax phylotype was not succeeded, Geobacter-related bacteria were readily enriched. Moreover, the different nitrogen sources caused the enrichment of different Geobacter species. Investigation of the diversity of benzylsuccinate synthase gene both in the enrichments and in the initial groundwater sample indicated that the Geobacter population in the center of the contaminant plume may not play a significant role in the anaerobic degradation of toluene.

Taibaiella coffeisoli sp. nov., isolated from the soil of a coffee plantation.

A Gram-stain-negative, obligately aerobic, non-motile, non-sporulating, rod-shaped bacterium, designated TZCO2T, was isolated from the soil of an irrigated coffee plantation in Arusha, Tanzania, East Africa. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that the isolate is affiliated with the genus Taibaiella in the family Chitinophagaceae. Its closest relative is Taibaiella koreensis THG-DT86T (96.7%). The pH and temperature ranges for growth were pH 6.0-8.5 (optimum 7.0-7.5) and 10-35 °C (optimum 30 °C, respectively. The predominant fatty acids were iso-C15:0 (32.4%), iso-C15:1 G (22.6%), iso-C17:0 (15.1%) and iso-C17:0 3-OH (10.0%) The only isoprenoid quinone detected in strain TZCO2T was menaquinone-7 (MK-7); the major polar lipids were phosphoaminolipid, phosphatidylethanolamine, unidentified aminolipids and lipids. The DNA G+C content was 51.9 mol%. Physiological and chemotaxonomic data further confirmed that strain TZCO2T is distinct from other members of the genus Taibaiella. Thus, strain TZCO2T is considered to represent a novel species of the genus, for which the name Taibaiella coffeisoli sp. nov. is proposed. The type strain is TZCO2T (=NCAIM B 02601T=CCM 8601T).

Polyphasic analysis of an Azoarcus-Leptothrix-dominated bacterial biofilm developed on stainless steel surface in a gasoline-contaminated hypoxic groundwater.

Pump and treat systems are widely used for hydrocarbon-contaminated groundwater remediation. Although biofouling (formation of clogging biofilms on pump surfaces) is a common problem in these systems, scarce information is available regarding the phylogenetic and functional complexity of such biofilms. Extensive information about the taxa and species as well as metabolic potential of a bacterial biofilm developed on the stainless steel surface of a pump submerged in a gasoline-contaminated hypoxic groundwater is presented. Results shed light on a complex network of interconnected hydrocarbon-degrading chemoorganotrophic and chemolitotrophic bacteria. It was found that besides the well-known hydrocarbon-degrading aerobic/facultative anaerobic biofilm-forming organisms (e.g., Azoarcus, Leptothrix, Acidovorax, Thauera, Pseudomonas, etc.), representatives of Fe(2+)-and Mn(2+)-oxidizing (Thiobacillus, Sideroxydans, Gallionella, Rhodopseudomonas, etc.) as well as of Fe(3+)- and Mn(4+)-respiring (Rhodoferax, Geobacter, Magnetospirillum, Sulfurimonas, etc.) bacteria were present in the biofilm. The predominance of ß-Proteobacteria within the biofilm bacterial community in phylogenetic and functional point of view was revealed. Investigation of meta-cleavage dioxygenase and benzylsuccinate synthase (bssA) genes indicated that within the biofilm, Azoarcus, Leptothrix, Zoogloea, and Thauera species are most probably involved in intrinsic biodegradation of aromatic hydrocarbons. Polyphasic analysis of the biofilm shed light on the fact that subsurface microbial accretions might be reservoirs of novel putatively hydrocarbon-degrading bacterial species. Moreover, clogging biofilms besides their detrimental effects might supplement the efficiency of pump and treat systems.

Zoogloea oleivorans sp. nov., a floc-forming, petroleum hydrocarbon-degrading bacterium isolated from biofilm.

A floc-forming, Gram-stain-negative, petroleum hydrocarbon-degrading bacterial strain, designated Buc(T), was isolated from a petroleum hydrocarbon-contaminated site in Hungary. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Buc(T) formed a distinct phyletic lineage within the genus Zoogloea. Its closest relative was found to be Zoogloea caeni EMB43(T) (97.2% 16S rRNA gene sequence similarity) followed by Zoogloea oryzae A-7(T) (95.9%), Zoogloea ramigera ATCC 19544(T) (95.5%) and Zoogloea resiniphila DhA-35(T) (95.4%). The level of DNA-DNA relatedness between strain Buc(T) and Z. caeni EMB43(T) was 31.6%. Cells of strain Buc(T) are facultatively aerobic, rod-shaped, and motile by means of a polar flagellum. The strain grew at temperatures of 5-35 °C (optimum 25-28 °C), and at pH 6.0-9.0 (optimum 6.5-7.5). The predominant fatty acids were C16:0, C10 : 0 3-OH, C12:0 and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The major respiratory quinone was ubiquinone-8 (Q-8) and the predominant polar lipid was phosphatidylethanolamine. The genomic DNA G+C content was 63.2 mol%. On the basis of the chemotaxonomic, molecular and phenotypic data, isolate Buc(T) is considered to represent a novel species of the genus Zoogloea, for which the name Zoogloea oleivorans sp. nov. is proposed. The type strain is Buc(T) ( =DSM 28387(T) =NCAIM B 02570(T)).

The detection and phylogenetic analysis of the alkane 1-monooxygenase gene of members of the genus Rhodococcus.

Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus.

Analysis of biofilm bacterial communities responsible for carbon removal through a reactor cascade treating wastewater.

In this study molecular microbiological and multivariate statistical analyses were carried out to determine the structure and dynamics of bacterial communities through a biofilm based, pilot-scale wastewater treatment cascade system comprised of eight reactors. Results indicated a vertical as well as horizontal differentiation of biofilm bacterial communities within individual reactors and through the reactor series, respectively. The richness of biofilm samples taken from dissolved oxygen rich sections of reactors were relatively lower than of samples taken from less oxygenized sections (one-way ANOVA P = 0.07). The Euclidean distance based one-way ANOSIM pointed out that in bacteriological point of view: (1) no statistically significant difference could be observed among the first five reactors (P ≥ 0.1); (2) the first seven reactors differed significantly from the last reactor, (P ≤ 0.03); (3) reactors 1 and 2 differed significantly from reactors 6 and 7 (P ≈ 0.02) and (4) reactor 3 from reactor 7 (P ≈ 0.03). 16S rRNA gene cloning revealed that through the cascade system the initially dominant heterotrophic bacteria (Acinetobacter, Acidovorax, Parabacteroides, Thauera, Desulfobacterium and Desulfomicrobium) were gradually replaced or supplemented by autotrophic nitrifying bacteria (Nitrosomonas, 'Candidatus Nitrotoga' and Nitrospira). Our results indicate that the vertical alteration of bacterial community structure within a particular reactor was driven by the alteration of dissolved oxygen concentration, while the horizontal alteration of bacterial community structure through the cascade system was driven mainly by the gradually decreasing dissolved organic matter content and increasing dissolved oxygen concentration.

One-year monitoring of meta-cleavage dioxygenase gene expression and microbial community dynamics reveals the relevance of subfamily I.2.C extradiol dioxygenases in hypoxic, BTEX-contaminated groundwater.

Aromatic hydrocarbons including benzene, toluene, ethyl-benzene, and xylene (BTEX) are frequent contaminants of groundwater, the major drinking water resource. Bioremediation is the only sustainable process to clean up these environments. Microbial degradation of BTEX compounds occurs rapidly under aerobic conditions but, in subsurface environments, the availability of oxygen is commonly restricted. Even so, the microaerobic degradation of aromatic compounds is still poorly understood. Hence, the dynamics of a bacterial community and the expression of meta-cleavage dioxygenase genes, with particular emphasis on subfamily I.2.C extradiol dioxygenase genes, were assessed over a 13-month period in a hypoxic, aromatic hydrocarbon-contaminated shallow groundwater by using sequence-aided terminal-restriction fragment length polymorphism (T-RFLP) and single-nucleotide primer extension (SNuPE), respectively. The bacterial 16S rRNA fingerprinting revealed the predominance of members of Rhodoferax, Azoarcus, Pseudomonas, and unknown bacteria related to Rhodocyclaceae. It was observed that mRNA transcripts of subfamily I.2.C extradiol dioxygenase genes were detected constantly over the monitoring period, and the detected sequences clustered into six distinct clusters. In order to reveal changes in the expression of these clusters over the monitoring period a SNuPE assay was developed. This quasi fingerprinting of functional gene expression provided the opportunity to link the investigated function to specific microbial populations. The results obtained can improve our understanding of aromatic hydrocarbon degradation under oxygen limitation and may benefit bioremediation research by demonstrating the usefulness of SNuPE for the monitoring of microbial populations involved in degradation process.

Remarkable impact of PAHs and TPHs on the richness and diversity of bacterial species in surface soils exposed to long-term hydrocarbon pollution.

Nowadays, because of substantial use of petroleum-derived fuels the number and extension of hydrocarbon polluted terrestrial ecosystems is in growth worldwide. In remediation of aforementioned sites bioremediation still tends to be an innovative, environmentally attractive technology. Although huge amount of information is available concerning the hydrocarbon degradation potential of cultivable hydrocarbonoclastic bacteria little is known about the in situ long-term effects of petroleum derived compounds on the structure of soil microbiota. Therefore, in this study our aim was to determine the long-term impact of total petroleum hydrocarbons (TPHs), volatile petroleum hydrocarbons (VPHs), total alkyl benzenes (TABs) as well as of polycyclic aromatic hydrocarbons (PAHs) on the structure of bacterial communities of four different contaminated soil samples. Our results indicated that a very high amount of TPH affected positively the diversity of hydrocarbonoclastic bacteria. This finding was supported by the occurrence of representatives of the α-, ß-, γ-Proteobacteria, Actinobacteria, Flavobacteriia and Bacilli classes. High concentration of VPHs and TABs contributed to the predominance of actinobacterial isolates. In PAH impacted samples the concentration of PAHs negatively correlated with the diversity of bacterial species. Heavily PAH polluted soil samples were mainly inhabited by the representatives of the ß-, γ-Proteobacteria (overwhelming dominance of Pseudomonas sp.) and Actinobacteria.