Ecophysiology of abundant denitrifying bacteria in activated sludge

The abundance of potential denitrifiers in full-scale wastewater treatment plants with biological nitrogen and phosphorus removal was investigated by FISH and various oligonucleotide probes. The potential denitrifiers were characterized as probe-defined populations that were able to consume radiolabelled substrate with oxygen, nitrate and nitrite as electron acceptor as determined by microautoradiography. The most abundant potential denitrifiers were related to the genera Aquaspirillum, Azoarcus, Thauera and Rhodocyclus, all within the Betaproteobacteria. They made up 20-49% of all bacteria in most of the 17 nitrogen removal plants investigated and were hardly present in four plants without denitrification. The ecophysiology of Aquaspirillum, Azoarcus and Thauera-related bacteria was consistent within each probe-defined group in the plants investigated. These three groups showed distinct physiological differences, with the Aquaspirillum-related bacteria appearing as the most specialized one, consuming only amino acids among the substrates tested, and Thauera as the most versatile consuming some volatile fatty acids, ethanol and amino acids. The coexistence of Aquaspirillum, Azoarcus and Thauera-related bacteria in a range of treatment plants with differences in wastewater, design and operation suggest that the populations ensure a functional stability of the plants by occupying different ecological niches related to the carbon transformation.     

The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium,strain EbN1

Recent research on microbial degradation of aromatic and other refractory compounds in anoxic waters and soils has revealed that nitrate-reducing bacteria belonging to the Betaproteobacteria contribute substantially to this process. Here we present the first complete genome of a metabolically versatile representative, strain EbN1, which metabolizes various aromatic compounds, including hydrocarbons. A circular chromosome (4.3 Mb) and two plasmids (0.21 and 0.22 Mb) encode 4603 predicted proteins. Ten anaerobic and four aerobic aromatic degradation pathways were recognized, with the encoding genes mostly forming clusters. The presence of paralogous gene clusters (e.g., for anaerobic phenylacetate oxidation), high sequence similarities to orthologs from other strains (e.g., for anaerobic phenol metabolism) and frequent mobile genetic elements (e.g., more than 200 genes for transposases) suggest high genome plasticity and extensive lateral gene transfer during metabolic evolution of strain EbN1. Metabolic versatility is also reflected by the presence of multiple respiratory complexes. A large number of regulators, including more than 30 two-component and several FNR-type regulators, indicate a finely tuned regulatory network able to respond to the fluctuating availability of organic substrates and electron acceptors in the environment. The absence of genes required for nitrogen fixation and specific interaction with plants separates strain EbN1 ecophysiologically from the closely related nitrogen-fixing plant symbionts of the Azoarcus cluster. Supplementary material on sequence and annotation are provided at the Web page .Electronic Supplementary Material Supplementary material is available for this article at Dedicated to Prof. Dr. h.c. Gerhard Gottschalk on the occasion of his 70th birthday.     

Cupriavidus necator isolates are able to fix nitrogen in symbiosis with different legume species

The aim of the present study was to identify a collection of 35 Cupriavidus isolates at the species level and to examine their capacity to nodulate and fix N(2). These isolates were previously obtained from the root nodules of two promiscuous trap species, Phaseolus vulgaris and Leucaena leucocephala, inoculated with soil samples collected near Sesbania virgata plants growing in Minas Gerais (Brazil) pastures. Phenotypic and genotypic methods applied for this study were SDS-PAGE of whole-cell proteins, and 16S rRNA and gyrB gene sequencing. To confirm the ability to nodulate and fix N(2), the presence of the nodC and nifH genes was also determined, and an experiment was carried out with two representative isolates in order to authenticate them as legume nodule symbionts. All 35 isolates belonged to the betaproteobacterium Cupriavidus necator, they possessed the nodC and nifH genes, and two representative isolates were able to nodulate five different promiscuous legume species: Mimosa caesalpiniaefolia, L. leucocephala, Macroptilium atropurpureum, P. vulgaris and Vigna unguiculata. This is the first study to demonstrate that C. necator can nodulate legume species.     

A strictly anaerobic betaproteobacterium Georgfuchsia toluolica gen. nov., sp. nov. degrades aromatic compounds with Fe(III), Mn(IV) or nitrate as an electron acceptor

A bacterium (strain G5G6) that grows anaerobically with toluene was isolated from a polluted aquifer (Banisveld, the Netherlands). The bacterium uses Fe(III), Mn(IV) and nitrate as terminal electron acceptors for growth on aromatic compounds. The bacterium does not grow on sugars, lactate or acetate. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain G5G6 belonged to the Betaproteobacteria . Its closest, but only distantly related, cultured relative is Sterolibacterium denitrificans Chol-1S^T (94.6% similarity of the 16S rRNA genes), a cholesterol-oxidizing, denitrifying bacterium. Strain G5G6 possesses the benzylsuccinate synthase A ( bssA ) gene encoding the α-subunit of Bss, which catalyzes the first step in anaerobic toluene degradation. The deduced BssA amino acid sequence is closely related to those of Azoarcus and Thauera species, which also belong to the Betaproteobacteria . Strain G5G6 is the first toluene-degrading, iron-reducing bacterium that does not belong to the Geobacteraceae within the Deltaproteobacteria . Based on phylogenetic and physiological comparison, strain G5G6 could not be assigned to a described species. Therefore, strain G5G6 (DSMZ 19032^T=JCM 14632^T) is a novel taxon of the Betaproteobacteria . We propose the name Georgfuchsia toluolica gen. nov., sp. nov.     

Temporal and spatial variability in nearshore bacterioplankton communities of Lake Michigan

The spatial and temporal variability of bacterial communities were determined for the nearshore waters of Lake Michigan, an oligotrophic freshwater inland sea. A freshwater estuary and nearshore sites were compared six times during 2006 using denaturing gradient gel electrophoresis (DGGE). Bacterial composition clustered by individual site and date rather than by depth. Seven 16S rRNA gene clone libraries were constructed, yielding 2717 bacterial sequences. Spatial variability was detected among the DGGE banding patterns and supported by clone library composition. The clone libraries from deep waters and the estuary environment revealed highest overall bacterial diversity. Betaproteobacteria sequence types were the most dominant taxa, comprising 40.2–67.7% of the clone libraries. BAL 47 was the most abundant freshwater cluster of Betaproteobacteria , indicating widespread distribution of this cluster in the nearshore waters of Lake Michigan. Incertae sedis 5 and Oxalobacteraceae sequence types were prevalent in each clone library, displaying more diversity than previously described in other freshwater environments. Among the Oxalobacteraceae sequences, a globally distributed freshwater cluster was determined. The nearshore waters of Lake Michigan are a dynamic environment that experience forces similar to the coastal ocean environment and share common bacterial diversity with other freshwater habitats.     

The role of a groundwater bacterial community in the degradation of the herbicide terbuthylazine

A bacterial community in an aquifer contaminated by s- triazines was studied. Groundwater microcosms were treated with terbuthylazine at a concentration of 100 μg L⁻¹ and degradation of the herbicide was assessed. The bacterial community structure (abundance and phylogenetic composition) and function (carbon production and cell viability) were analysed. The bacterial community was able to degrade the terbuthylazine; in particular, Betaproteobacteria were involved in the herbicide biotransformation. Identification of some bacterial isolates by PCR amplification of the 16S rRNA gene revealed the presence of two Betaproteobacteria species able to degrade the herbicide: Advenella incenata and Janthinobacterium lividum . PCR detection of the genes encoding s -triazine-degrading enzymes indicated the presence of the atz A and atz B genes in A. incenata and the atz B and atz C genes in J. lividum . The nucleotide sequences of the PCR fragments of the atz genes from these strains were 100% identical to the homologous genes of the Pseudomonas sp. strain ADP. In conclusion, the results show the potential for the use of a natural attenuation strategy in the treatment of aquifers polluted with the terbuthylazine. The two bacteria isolated could facilitate the implementation of effective bioremediation protocols, especially in the case of the significant amounts of herbicide that can be found in groundwater as a result of accidental spills.     

Diazotrophic Burkholderia species isolated from the Amazon region exhibit phenotypical, functional and genetic diversity

Forty-eight Burkholderia isolates from different land use systems in the Amazon region were compared to type strains of Burkholderia species for phenotypic and functional characteristics that can be used to promote plant growth. Most of these isolates (n=46) were obtained by using siratro (Macroptilium atropurpureum - 44) and common bean (Phaseolus vulgaris - 2) as the trap plant species; two isolates were obtained from nodules collected in the field from Indigofera suffruticosa and Pithecellobium sp. The evaluated characteristics were the following: colony characterisation on "79" medium, assimilation of different carbon sources, enzymatic activities, solubilisation of phosphates, nitrogenase activity and antifungal activity against Fusarium oxysporium f. sp. phaseoli. Whole cell protein profiles, 16S rRNA, gyrB, and recA gene sequencing and multilocus sequence typing were used to identify the isolates. The isolates showed different cultural and biochemical characteristics depending on the legume species from which they were obtained. Except for one isolate from I. suffruticosa, all isolates were able to solubilise calcium phosphate and present nitrogenase activity under free-living conditions. Only one isolate from common beans, showed antifungal activity. The forty four isolates from siratro nodules were identified as B. fungorum; isolates UFLA02-27 and UFLA02-28, obtained from common bean plants, were identified as B. contaminans; isolate INPA89A, isolated from Indigofera suffruticosa, was a close relative of B. caribensis but could not be assigned to an established species; isolate INPA42B, isolated from Pithecellobium sp., was identified as B. lata. This is the first report of nitrogenase activity in B. fungorum, B. lata and B. contaminans.     

Thauera and Azoarcus as functionally important genera in a denitrifying quinoline-removal bioreactor as revealed by microbial community structure comparison

Structural shifts associated with functional dynamics in a bacterial community may provide clues for identifying the most valuable members in an ecosystem. A laboratory-scale denitrifying reactor was adapted from use of non-efficient seeding sludge and was utilized to degrade quinoline and remove the chemical oxygen demand. Stable removal efficiencies were achieved after an adaptation period of six weeks. Both denaturing gradient gel electrophoresis profiling of the 16S rRNA gene V3 region and comparison of the 16S rRNA gene sequence clone libraries (LIBSHUFF analysis) demonstrated that microbial communities in the denitrifying reactor and seeding sludge were significantly distinct. The percentage of the clones affiliated with the genera Thauera and Azoarcus was 74% in the denitrifying reactor and 4% in the seeding sludge. Real-time quantitative PCR also indicated that species of the genera Thauera and Azoarcus increased in abundance by about one order of magnitude during the period of adaptation. The greater abundance of Thauera and Azoarcus in association with higher efficiency after adaptation suggested that these phylotypes might play an important role for quinoline and chemical oxygen demand removal under denitrifying conditions.     

An invasive Mimosa in India does not adopt the symbionts of its native relatives

Background and Aims

The large monophyletic genus Mimosa comprises approx. 500 species, most of which are native to the New World, with Central Brazil being the main centre of radiation. All Brazilian Mimosa spp. so far examined are nodulated by rhizobia in the betaproteobacterial genus Burkholderia. Approximately 10 Mya, transoceanic dispersal resulted in the Indian subcontinent hosting up to six endemic Mimosa spp. The nodulation ability and rhizobial symbionts of two of these, M. hamata and M. himalayana, both from north-west India, are here examined, and compared with those of M. pudica, an invasive species.

Methods

Nodules were collected from several locations, and examined by light and electron microscopy. Rhizobia isolated from them were characterized in terms of their abilities to nodulate the three Mimosa hosts. The molecular phylogenetic relationships of the rhizobia were determined by analysis of 16S rRNA, nifH and nodA gene sequences.

Key Results

Both native Indian Mimosa spp. nodulated effectively in their respective rhizosphere soils. Based on 16S rRNA, nifH and nodA sequences, their symbionts were identified as belonging to the alphaproteobacterial genus Ensifer, and were closest to the ‘Old World’ Ensifer saheli, E. kostiensis and E. arboris. In contrast, the invasive M. pudica was predominantly nodulated by Betaproteobacteria in the genera Cupriavidus and Burkholderia. All rhizobial strains tested effectively nodulated their original hosts, but the symbionts of the native species could not nodulate M. pudica.

Conclusions

The native Mimosa spp. in India are not nodulated by the Burkholderia symbionts of their South American relatives, but by a unique group of alpha-rhizobial microsymbionts that are closely related to the ‘local’ Old World Ensifer symbionts of other mimosoid legumes in north-west India. They appear not to share symbionts with the invasive M. pudica, symbionts of which are mostly beta-rhizobial.     

Changes in assembly processes in soil bacterial communities following a wildfire disturbance

Although recent work has shown that both deterministic and stochastic processes are important in structuring microbial communities, the factors that affect the relative contributions of niche and neutral processes are poorly understood. The macrobiological literature indicates that ecological disturbances can influence assembly processes. Thus, we sampled bacterial communities at 4 and 16 weeks following a wildfire and used null deviation analysis to examine the role that time since disturbance has in community assembly. Fire dramatically altered bacterial community structure and diversity as well as soil chemistry for both time-points. Community structure shifted between 4 and 16 weeks for both burned and unburned communities. Community assembly in burned sites 4 weeks after fire was significantly more stochastic than in unburned sites. After 16 weeks, however, burned communities were significantly less stochastic than unburned communities. Thus, we propose a three-phase model featuring shifts in the relative importance of niche and neutral processes as a function of time since disturbance. Because neutral processes are characterized by a decoupling between environmental parameters and community structure, we hypothesize that a better understanding of community assembly may be important in determining where and when detailed studies of community composition are valuable for predicting ecosystem function.