Effects of the Wastewater Flow Rate on Interactions between the Genus Nitrosomonas and Diverse Populations in an Activated Sludge Microbiome.

The present study characterized the interactions of microbial populations in activated sludge systems during the operational period after an increase in the wastewater flow rate and consequential ammonia accumulation using a 16S rRNA gene sequencing-based network analysis. Two hundred microbial populations accounting for 81.8% of the total microbiome were identified. Based on a co-occurrence analysis, Nitrosomonas-type ammonia oxidizers had one of the largest number of interactions with diverse bacteria, including a bulking-associated Thiothrix organism. These results suggest that an increased flow rate has an impact on constituents by changing ammonia concentrations and also that Nitrosomonas- and Thiothrix-centric responses are critical for ammonia removal and microbial community recovery.

Respirometric characterization of aerobic sulfide, thiosulfate and elemental sulfur oxidation by S-oxidizing biomass.

Respirometry was used to reveal the mechanisms involved in aerobic biological sulfide oxidation and to characterize the kinetics and stoichiometry of a microbial culture obtained from a desulfurizing biotrickling filter. Physical-chemical processes such as stripping and chemical oxidation of hydrogen sulfide were characterized since they contributed significantly to the conversions observed in respirometric tests. Mass transfer coefficient for hydrogen sulfide and the kinetic parameters for chemical oxidation of sulfide with oxygen were estimated. The stoichiometry of the process was determined and the different steps in the sulfide oxidation process were identified. The conversion scheme proposed includes intermediate production of elemental sulfur and thiosulfate and the subsequent oxidation of both compounds to sulfate. A kinetic model describing each of the reactions observed during sulfide oxidation was calibrated and validated. The product selectivity was found to be independent of the dissolved oxygen to hydrogen sulfide concentration ratio in the medium at sulfide concentrations ranging from 3 to 30 mg S L(-1). Sulfide was preferentially consumed (SOURmax = 49.2 mg DO g(-1) VSS min(-1)) and oxidized to elemental sulfur at dissolved oxygen concentrations above 0.8 mg DO L(-1). Substrate inhibition of sulfide oxidation was observed (K(i,S(2-))= 42.4 mg S L(-1)). Intracellular sulfur accumulation also affected negatively the sulfide oxidation rate. The maximum fraction of elemental sulfur accumulated inside cells was estimated (25.6% w/w) and a shrinking particle equation was included in the kinetic model to describe elemental sulfur oxidation. The microbial diversity obtained through pyrosequencing analysis revealed that Thiothrix sp. was the main species present in the culture (>95%).

Identification and quantification of Thiothrix eikelboomii using qPCR for early detection of bulking incidents in a full-scale water reclamation plant.

This study found that the ratio of Thiothrix eikelboomii to total bacterial concentrations (TH/TB) (%) was a better indicator of bulking incidents affecting effluent quality compared to absolute T. eikelboomii abundance alone. This was determined using a genus-specific Thiothrix quantitative PCR primer and probe set, which was developed in this study to monitor specific Thiothrix populations over a 1-year period. T. eikelboomii was identified as the source of bulking incidents based on sequencing of the 16S rRNA gene at a nitrifying-denitrifying wastewater treatment plant. Peak T. eikelboomii concentrations observed in March, April, and July 2009 were 2.32 × 10(10), 2.64 × 10(10), and 1.84 × 10(10) cells/l, respectively. The highest fraction of T. eikelboomii to total bacterial population was measured at 0.24% in March, and a ratio >0.19% caused increases of suspended solids and biochemical oxygen demand in the secondary effluent. Additionally, food/mass ratios, dissolved oxygen concentrations in the anoxic selector, and ammonium ion concentrations in the primary effluent were three parameters displaying statistically significant correlations (r = 0.40, r = 0.50, and r = 0.32, respectively) to Thiothrix spp. abundance in an aeration tank. No bulking events caused by T. eikelboomii occurred when the dissolved oxygen concentrations in the anoxic selector was maintained at lower than 0.12 mg/l and the TH/TB ratios were <0.10%.

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.

Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov., gammaproteobacteria isolated from sulfide springs.

Five strains of filamentous, sulfur-oxidizing bacteria were isolated from sulfur mats of different sulfide springs from various regions of the Northern Caucasus, Russia. A phylogenetic analysis based on 16S rRNA gene sequence comparison showed that all of the isolates are affiliated with the filamentous, colourless, sulfur-oxidizing bacteria of the genus Thiothrix within the Gammaproteobacteria and are closely related to Thiothrix fructosivorans. All strains are capable of growing heterotrophically, lithoautotrophically with thiosulfate or sulfide as the sole energy source and mixotrophically. Strains G1(T), G2, P and K2 are able to fix molecular nitrogen, but strain BL(T) is not. Randomly amplified polymorphic DNA (RAPD)-PCR analysis was used to assess the level of genetic relationships among the Thiothrix isolates. The Nei and Li similarity index revealed high genetic similarity among strains G1(T), G2, P and K2 (above 75 %), indicating that they are closely related. In combination with physiological and morphological data, strains G1(T), G2, P and K2 can be considered as members of the same species. The lowest genetic similarity (approx. 20 %) was reached between strain BL(T) and the other isolated Thiothrix strains. Strains BL(T) and G1(T) shared 35 % DNA-DNA relatedness and showed 51 and 53 % relatedness, respectively, to Thiothrix fructosivorans ATCC 49749. On the basis of this polyphasic analysis, strains G1(T), G2, P and K2 represent a novel species within the genus Thiothrix, for which the name Thiothrix caldifontis sp. nov. is proposed, with strain G1(T) (=DSM 21228(T) =VKM B-2520(T)) as the type strain. In addition, strain BL(T) represents a second novel species, Thiothrix lacustris sp. nov., with strain BL(T) (=DSM 21227(T) =VKM B-2521(T)) as the type strain.

Application of image analysis in activated sludge to evaluate correlations between settleability and features of flocs and filamentous species.

Digital image analysis is a useful tool to estimate some morphological parameters of flocs and filamentous species in activated sludge wastewater treatment processes. In this work we found the correlation between some morphological parameters and sludge volume index (SVI). The sludge was taken from a pilot-scale activated sludge plant, owned by ENEA, located side stream to the Trebbo di Reno (Bologna, Italy) municipal WWTP and fed by domestic wastewater. In order to use image analysis, we developed a correct method to acquire digital microbiological observations and to obtain images altogether representative of the sludge properties. We identified and assessed the parameters needed to estimate the settleability of the sludge and evaluated the morphological filamentous features. It is known that several conditions (i.e. low F/M, nutrient deficiency, low dissolved oxygen) select specific filamentous species and their excessive growth decrease floc-forming/filaments ratio, correspond to the worse settleability properties; we found a relationship between the relative abundance of filamentous species and SVI. We also evaluated the fractal dimension parameter (FD) and determined a threshold value useful to distinguish between the "weak" and "firm" floc and we found a correlation between FD and SVI.

A novel symbiosis between chemoautotrophic bacteria and a freshwater cave amphipod.

Symbioses involving animals and chemoautotrophic bacteria form the foundation of entire ecosystems at deep-sea hydrothermal vents and cold seeps, but have so far not been reported in terrestrial or freshwater environments. A rare example of a terrestrial ecosystem sustained by chemoautotrophy is found within the sulfide-rich Frasassi limestone cave complex of central Italy. In this study, we report the discovery of abundant filamentous bacteria on the exoskeleton of Niphargus ictus, a macroinvertebrate endemic to Frasassi. Using 16S rDNA sequencing and fluorescence in situ hybridization (FISH), we show that N. ictus throughout the large cave complex are colonized by a single phylotype of bacteria in the sulfur-oxidizing clade Thiothrix. The epibiont phylotype is distinct from Thiothrix phylotypes that form conspicuous biofilms in the cave streams and pools inhabited by N. ictus. Using a combination of 13C labeling, FISH, and secondary ion mass spectrometry (SIMS), we show that the epibiotic Thiothrix are autotrophic, establishing the first known example of a non-marine chemoautotroph-animal symbiosis. Conditions supporting chemoautotrophy, and the N. ictus-Thiothrix association, likely commenced in the Frasassi cave complex between 350,000 and 1 million years ago. Therefore, the N. ictus-Thiothrix symbiosis is probably significantly younger than marine chemoautotrophic symbioses, many of which have been evolving for tens to hundreds of million years.

[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.