The first sequenced Sphaerotilus natans bacteriophage- characterization and potential to control its filamentous bacterium host.

Bacteriophages (phages) are ubiquitous entities present in every conceivable habitat as a result of their bacterial parasitism. Their prevalence and impact in the ecology of bacterial communities and their ability to control pathogens make their characterization essential, particularly of new phages, improving knowledge and potential application. The isolation and characterization of a new lytic phage against Sphaerotilus natans strain DSM 6575, named vB_SnaP-R1 (SnaR1), is here described. Besides being the first sequenced genome of a Sphaerotilus natans infecting phage, 99% of its 41507 bp genome lacks homology with any other sequenced phage, revealing its uniqueness and previous lack of knowledge. Moreover, SnaR1 is the first Podoviridae phage described infecting this bacterium. Sphaerotilus natans is an important filamentous bacterium due to its deleterious effect on wastewater treatment plants (WWTP) and thus, phages may play a role as novel biotechnological tools against filamentous overgrowth in WWTP. The lytic spectrum of SnaR1 was restricted to its host strain, infecting only one out of three S. natans strains and infection assays revealed its ability to reduce bacterial loads. Results suggest SnaR1 as the prototype of a new phage genus and demonstrates its potential as a non-chemical alternative to reduce S. natans DSM 6575 cells.

Reclassification of Sphaerotilus natans subsp. sulfidivorans Gridneva et al. 2011 as Sphaerotilus sulfidivorans sp. nov. and comparative genome analysis of the genus Sphaerotilus.

Filamentous iron oxides accumulating bacteria Sphaerotilus natans subsp. natans and S. natans subsp. sulfidivorans were described as subspecies based on 99.7% identity of their 16S rRNA sequences, in spite of important physiological difference. The ANI between their genomes was 94.7%, which indicate their assignment to different species. S. natans subsp. sulfidivorans and S. montanus possess genes for a complete SOX system, while S. natans subsp. natans encode only SoxYZ. There are genes for the Calvin cycle in the genomes of S. hippei DSM 566T, S. natans subsp. sulfidivorans D-501T, and S. montanus HST. Lithoautotrophy on reduced sulfur compounds is probably possible for S. natans subsp. sulfidivorans and S. montanus, but not for S. natans subsp. natans. Considering significant differences in the genome characteristics and metabolic potential of S. natans subsp. sulfidivorans and S. natans subsp. natans, we propose their classification as different species, S. natans and S. sulfidivorans sp. nov.

Advanced biofilm analysis in streams receiving organic deicer runoff.

Prolific heterotrophic biofilm growth is a common occurrence in airport receiving streams containing deicers and anti-icers, which are composed of low-molecular weight organic compounds. This study investigated biofilm spatiotemporal patterns and responses to concurrent and antecedent (i.e., preceding biofilm sampling) environmental conditions at stream sites upstream and downstream from Milwaukee Mitchell International Airport in Milwaukee, Wisconsin, during two deicing seasons (2009-2010; 2010-2011). Biofilm abundance and community composition were investigated along spatial and temporal gradients using field surveys and microarray analyses, respectively. Given the recognized role of Sphaerotilus in organically enriched environments, additional analyses were pursued to specifically characterize its abundance: a consensus sthA sequence was determined via comparison of whole metagenome sequences with a previously identified sthA sequence, the primers developed for this gene were used to characterize relative Sphaerotilus abundance using quantitative real-time PCR, and a Sphaerotilus strain was isolated to validate the determined sthA sequence. Results indicated that biofilm abundance was stimulated by elevated antecedent chemical oxygen demand concentrations, a surrogate for deicer concentrations, with minimal biofilm volumes observed when antecedent chemical oxygen demand concentrations remained below 48 mg/L. Biofilms were composed of diverse communities (including sheathed bacterium Thiothrix) whose composition appeared to shift in relation to antecedent temperature and chemical oxygen demand. The relative abundance of sthA correlated most strongly with heterotrophic biofilm volume (positive) and dissolved oxygen (negative), indicating that Sphaerotilus was likely a consistent biofilm member and thrived under low oxygen conditions. Additional investigations identified the isolate as a new strain of Sphaerotilus montanus (strain KMKE) able to use deicer components as carbon sources and found that stream dissolved oxygen concentrations related inversely to biofilm volume as well as to antecedent temperature and chemical oxygen demand. The airport setting provides insight into potential consequences of widescale adoption of organic deicers for roadway deicing.

Quantifying in situ growth rate of a filamentous bacterial species in activated sludge using rRNA:rDNA ratio.

If the in situ growth rate of filamentous bacteria in activated sludge can be quantified, researchers can more accurately assess the effect of operating conditions on the growth of filaments and improve the mathematical modeling of filamentous bulking. We developed a method to quantify the in situ specific growth rate of Sphaerotilus natans (a model filament) in activated sludge using the species-specific 16S rRNA:rDNA ratio. Primers targeting the 16S rRNA of S. natans were designed, and real-time PCR and RT-PCR were used to quantify DNA and RNA levels of S. natans, respectively. A positive linear relationship was found between the rRNA:rDNA ratio (from 440 to 4500) and the specific growth rate of S. natans (from 0.036 to 0.172 h-1) using chemostat experiments. The in situ growth rates of S. natans in activated sludge samples from three water reclamation facilities were quantified, illustrating how the approach can be applied in a complex environment such as activated sludge.

Moderate oxygen depletion as a factor favouring the filamentous growth of Sphaerotilus natans.

Sphaerotilus natans is a neutrophilic iron-related sheath-forming filamentous microorganism that presents dual morphotype: single cells and ensheathed cells forming filaments. As S. natans has been proposed as a sorbent for inorganic pollutants and it is occasionally involved in bulking episodes, elucidating factors affecting its filamentous growth is of crucial interest. The purpose of this work was to evaluate the effect of dissolved oxygen (DO) as a factor affecting S. natans filamentation from single cells. A method to quantify S. natans in its filamentous and single-cell morphotypes, based on a differential filtration procedure coupled with quantitative real-time PCR, was developed here. Scanning Electron Microscopy was used to validate the filtration step. Under actively aerated conditions (DO maintained at 7.6 ± 0.1 mg l(-1)), S. natans grew mainly as single cells throughout the experiment, while a depletion in DO concentration (to ~3 mg l(-1)) induced its filamentous growth. Indeed, when oxygen was reduced the proportion of single cells diminished from 83.3 ± 5.9 to 14.3 ± 3.4% while the filaments increased from 16.7 ± 5.9 to 85.7 ± 3.4%. Our results suggest that oxygen plays a key role in S. natans filamentation and contribute to better understanding of the filamentous proliferation of this bacterium. In addition, the proposed method will be helpful to evaluate other factors favouring filamentous growth.

Isolation of Sphaerotilus-Leptothrix strains from iron bacteria communities in Tierra del Fuego wetlands.

Sheath-forming iron- and manganese-depositing bacteria belonging to the Sphaerotilus-Leptothrix group (SLG) are widespread in natural and artificial water systems. Known requirements for their growth include the presence of organic substrates and molecular oxygen. High concentrations of reduced iron or manganese, although not necessary for most species, make their growth a noticeable phenomenon. Such microbial communities have been studied mostly in the Northern Hemisphere. Here, we present descriptions of diverse ochre-depositing microbial communities in Tierra del Fuego, Argentina, using a combined approach of microscopical examination, clone library construction and cultivation focused on SLG bacteria. To date, only few SLG type strains are available. The present work increases the number and diversity of cultivated SLG bacteria by obtaining isolates from biofilms and sediment samples of wetlands in Tierra del Fuego. Thirty isolates were selected based on morphological features such as sheath formation and iron/manganese deposition. Five operational taxonomic units (OTUs) were deduced. Sequencing of 16S rRNA genes showed that one OTU is identical to the Leptothrix mobilis Feox-1(T) -sequence while the four remaining OTUs show similarity values related to previously described type strains. Similarity values ranged from 96.5% to 98.8%, indicating possible new species and subspecies.

Biofilm formation and microbial community analysis of the simulated river bioreactor for contaminated source water remediation.

BACKGROUND, AIM, AND SCOPE: The start-up pattern of biofilm remediation system affects the biofilm characteristics and operating performances. The objective of this study was to evaluate the performances of the contaminated source water remediation systems with different start-up patterns in view of the pollutants removal performances and microbial community succession. METHODS: The operating performances of four lab-scale simulated river biofilm reactors were examined which employed different start-up methods (natural enrichment and artificial enhancement via discharging sediment with influent velocity gradient increase) and different bio-fillers (Elastic filler and AquaMats® ecobase). At the same time, the microbial communities of the bioreactors in different phases were analyzed by polymerase chain reaction, denaturing gradient gel electrophoresis, and sequencing. RESULTS AND DISCUSSION: The pollutants removal performances became stable in the four reactors after 2 months' operation, with ammonia nitrogen and permanganate index (COD(Mn)) removal efficiencies of 84.41-94.21% and 69.66-76.60%, respectively. The biomass of mature biofilm was higher in the bioreactors by artificial enhancement than that by natural enrichment. Microbial community analysis indicated that elastic filler could enrich mature biofilm faster than AquaMats®. The heterotrophic bacteria diversity of biofilm decreased by artificial enhancement, which favored the ammonia-oxidizing bacteria (AOB) developing on the bio-fillers. Furthermore, Nitrosomonas- and Nitrosospira-like AOB coexisted in the biofilm, and Pseudomonas sp., Sphaerotilus sp., Janthinobacterium sp., Corynebacterium aurimucosum were dominant in the oligotrophic niche. CONCLUSION: Artificial enhancement via the combination of sediment discharging and influent velocity gradient increasing could enhance the biofilm formation and autotrophic AOB enrichment in oligotrophic niche.

Taxonomic investigation of representatives of the genus Sphaerotilus: descriptions of Sphaerotilus montanus sp. nov., Sphaerotilus hippei sp. nov., Sphaerotilus natans subsp. natans subsp. nov. and Sphaerotilus natans subsp. sulfidivorans subsp. nov., and an emended description of the genus Sphaerotilus.

Seven strains of the genus Sphaerotilus were obtained from natural thermal sulfide (strains D-501(T), D-502, D-504, D-505 and D-507) and low-temperature ferrous (strain HS(T)) springs and from an activated sludge system (strain D-380). These Sphaerotilus isolates and strains of Sphaerotilus natans obtained from the DSMZ (S. natans DSM 6575(T), DSM 565 and DSM 566) were studied using a polyphasic taxonomic approach. All strains had Q-8 as the major quinone and C(16 : 1)ω7, C(16 : 0) and C(18 : 1)ω7 as the major fatty acids. The DNA-DNA hybridization results and 16S rRNA, hsp60 and gyrB gene sequencing experiments showed that isolates D-501(T), D-502, D-504, D-505, D-507 and D-380 were closely related to the type strain of S. natans DSM 6575(T). However, strains D-501(T), D-502, D-504, D-505 and D-507 significantly differed from the heterotrophic strain S. natans DSM 6575(T) by their capability for lithotrophic growth with reduced sulfur compounds as an electron donor for energy conservation and some other phenotypic features. For this reason, strains D-501(T), D-502, D-504, D-505 and D-507 merit a separate taxonomic classification at the subspecies level. The name Sphaerotilus natans subsp. sulfidivorans subsp. nov. (type strain D-501(T) = DSM 22545(T) = VKM B-2573(T)) is proposed. The subspecies Sphaerotilus natans subsp. natans subsp. nov. is automatically created as a result of this proposal. Strain D-380 was phenotypically closely related to S. natans DSM 6575(T). Strains D-380 and S. natans DSM 6575(T) were assigned to the subspecies Sphaerotilus natans subsp. natans subsp. nov. (type strain DSM 6575(T) = ATCC 13338(T)). The 16S rRNA, hsp60 and gyrB gene sequences obtained for strains HS(T) and DSM 565 showed very low sequence similarity values of 97.3 %, 89.7 % and 88.4 %, respectively, with S. natans DSM 6575(T). Strain HS(T) shared 99 % DNA-DNA relatedness with strain.

Biodiversity and monitoring of colorless filamentous bacteria in sulfide aquatic systems of North Caucasus region.

Bacterial mats in sulfide aquatic systems of North Caucasus are basically composed by the species of genera Thiothrix and Sphaerotilus. Additionally, several non-filamentous sulfur-oxidizing bacteria were isolated from the mats and several minor 16S rRNA phylotypes were found in clone libraries from these mats. The minor components were affiliated with Proteobacteria, Chlorobia, Cyanobacteria and Firmicutes. Even in an individual mat population heterogeneity of Thiothrix spp. was revealed by analysis of 16S rRNA gene and RAPD-PCR. Five Thiothrix isolates were described as new species Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov. In the Thiothrix-Sphaerotilus type of bacterial mat the proportion of dominant organisms might be influenced by sulfide concentration in the spring water. The higher sulfide concentration (more than 10 mg/l) in the spring water is more favorable for the development of bacterial mats with dominant Thiothrix organisms than for Thiothrix-Sphaerotilus type of sulfur mat.

Inhella inkyongensis gen. nov., sp. nov., a new freshwater bacterium in the order Burkholderiales.

A freshwater bacterium, designated IMCC1713(T), was isolated from a highly eutrophic artificial pond. Cells of the strain were Gram-negative, chemoheterotrophic, polybeat and obligately aerobic short rods that were motile with a single polar flagellum. The 16S rRNA gene sequence similarity analysis showed that the novel strain was most closely related to the species Roseateles depolymerans (96.3%), Mitsuaria chitosanitabida (96.2%), Ideonella dechloratans (96.2%), and Pelomonas saccharophila (96.1%) in the Sphaerotilus-Leptothrix group within the order Burkholderiales. Phylogenetic trees based on 16S rRNA gene sequences indicated that the isolate formed an independent monophyletic clade within the order Burkholderiales. The relatively low DNA G+C content (57.4 mol%), together with several phenotypic characteristics, differentiated the novel strain from other members of the Sphaerotilus-Leptothrix group. From the taxonomic data, therefore, the strain should be classified as a novel genus and species, for which the name Inhella inkyongensis gen. nov., sp. nov. is proposed. The type strain of the proposed species is strain IMCC1713(T) (=KCTC 12791(T)=NBRC 103252(T)=CCUG 54308(T)).

[Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide stream in the North Caucasus].

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide stream (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Spahaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism's population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or "threads" of natural sulfide springs.

Methylibium petroleiphilum gen. nov., sp. nov., a novel methyl tert-butyl ether-degrading methylotroph of the Betaproteobacteria.

A Gram-negative, rod-shaped, motile, non-pigmented, facultative aerobe that grew optimally at pH 6.5 and 30 degrees C (strain PM1T) was isolated for its ability to completely degrade the gasoline additive methyl tert-butyl ether. Analysis of the 16S rRNA gene sequence indicated that this bacterium was a member of the class Betaproteobacteria in the Sphaerotilus-Leptothrix group. The 16S rRNA gene sequence identity to other genera in this group, Leptothrix, Aquabacterium, Roseateles, Sphaerotilus, Ideonella and Rubrivivax, ranged from 93 to 96 %. The chemotaxonomic data including Q-8 as the major quinone, C16 : 1omega7c and C16 : 0 as the major fatty acids and a DNA G+C content of 69 mol%, support the inclusion of strain PM1T in the class Betaproteobacteria. It differed from other members of the Sphaerotilus-Leptothrix group by being a facultative methylotroph that used methanol as a sole carbon source, and by also being able to grow heterotrophically in defined media containing ethanol, toluene, benzene, ethylbenzene and dihydroxybenzoates as sole carbon sources. On the basis of the morphological, physiological, biochemical and genetic information, a new genus and species, Methylibium petroleiphilum gen. nov., sp. nov., is proposed, with PM1T (=ATCC BAA-1232T=LMG 22953T) as the type strain.

Novel approach to quantitative detection of specific rRNA in a microbial community, using catalytic DNA.

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.

Relationship of species-specific filament levels to filamentous bulking in activated sludge.

To examine the relationship between activated-sludge bulking and levels of specific filamentous bacteria, we developed a statistics-based quantification method for estimating the biomass levels of specific filaments using 16S rRNA-targeted fluorescent in situ hybridization (FISH) probes. The results of quantitative FISH for the filament Sphaerotilus natans were similar to the results of quantitative membrane hybridization in a sample from a full-scale wastewater treatment plant. Laboratory-scale reactors were operated under different flow conditions to develop bulking and nonbulking sludge and were bioaugmented with S. natans cells to stimulate bulking. Instead of S. natans, the filament Eikelboom type 1851 became dominant in the reactors. Levels of type 1851 filaments extending out of the flocs correlated strongly with the sludge volume index, and extended filament lengths of approximately 6 x 10(8) micro m ml(-1) resulted in bulking in laboratory-scale and full-scale activated-sludge samples. Quantitative FISH showed that high levels of filaments occurred inside the flocs in nonbulking sludge, supporting the "substrate diffusion limitation" hypothesis for bulking. The approach will allow the monitoring of incremental improvements in bulking control methods and the delineation of the operational conditions that lead to bulking due to specific filaments.