Genomic and morphological characterization of a new Thiothrix species from a sulfide hot spring of the Zmeinaya bay (Northern Baikal, Russia).

A survey for bacteria of the genus Thiothrix indicated that they inhabited the area where the water of the Zmeiny geothermal spring (northern basin of Lake Baikal, Russia) mixed with the lake water. In the coastal zone of the lake oxygen (8.25 g/L) and hydrogen sulfide (up to 1 mg/L) were simultaneously present at sites of massive growth of these particular Thiothrix bacteria. Based on the analysis of the morphological characteristics and sequence of individual genes (16S rRNA, rpoB and tilS), we could not attribute the Thiothrix from Lake Baikal to any of the known species of this genus. To determine metabolic capabilities and phylogenetic position of the Thiothrix sp. from Lake Baikal, we analyzed their whole genome. Like all members of this genus, the bacteria from Lake Baikal were capable of organo-heterotrophic, chemolithoheterotrophic, and chemolithoautotrophic growth and differed from its closest relatives in the spectrum of nitrogen and sulfur cycle genes as well as in the indices of average nucleotide identity (ANI < 75-94%), amino acid identity (AAI < 94%) and in silico DNA-DNA hybridization (dDDH < 17-57%), which were below the boundary of interspecies differences, allowing us to identify them as novel candidate species.

History of the Study of the Genus Thiothrix: From the First Enrichment Cultures to Pangenomic Analysis.

Representatives of the genus Thiothrix are filamentous, sulfur-oxidizing bacteria found in flowing waters with counter-oriented sulfide and oxygen gradients. They were first described at the end of the 19th century, but the first pure cultures of this species only became available 100 years later. An increase in the number of described Thiothrix species at the beginning of the 21st century shows that the classical phylogenetic marker, 16S rRNA gene, is not informative for species differentiation, which is possible based on genome analysis. Pangenome analysis of the genus Thiothrix showed that the core genome includes genes for dissimilatory sulfur metabolism and central metabolic pathways, namely the Krebs cycle, Embden-Meyerhof-Parnas pathway, glyoxylate cycle, Calvin-Benson-Bassham cycle, and genes for phosphorus metabolism and amination. The shell part of the pangenome includes genes for dissimilatory nitrogen metabolism and nitrogen fixation, for respiration with thiosulfate. The dispensable genome comprises genes predicted to encode mainly hypothetical proteins, transporters, transcription regulators, methyltransferases, transposases, and toxin-antitoxin systems.

Aggregability of ß(1→4)-linked glucosaminoglucan originating from a sulfur-oxidizing bacterium Thiothrix nivea.

ß-1,4-glucosaminoglucan (GG) was prepared from the sheath of a sulfur-oxidizing bacterium Thiothrix nivea. Recently, GG was found to be adsorbed by cellulose (paper) and is therefore potentially applicable as an aminating agent for cellulose. We attempted to increase the yield of GG using a fed-batch cultivation method. Furthermore, the behavior of GG molecules in water was theoretically and experimentally investigated. NMR analysis in combination with molecular dynamics calculation suggested that GG molecules tend to form soluble aggregates in water. It was experimentally revealed that the self-aggregation is enhanced by the addition of NaCl and reduced temperature. Adsorption of GG onto cellulose via hydrogen bonding was confirmed by molecular dynamics simulation. Adsorption was also promoted in the presence of NaCl but was inhibited by a reduction in temperature. Only 11% of the amino groups in the GG-treated paper was reactive, suggesting that GG molecules adsorbed by the paper were forming aggregates.

Low frequency Raman Spectroscopy for micron-scale and in vivo characterization of elemental sulfur in microbial samples.

Elemental sulfur (S(0)) is an important intermediate of the sulfur cycle and is generated by chemical and biological sulfide oxidation. Raman spectromicroscopy can be applied to environmental samples for the detection of S(0), as a practical non-destructive micron-scale method for use on wet material and living cells. Technical advances in filter materials enable the acquisition of ultra-low frequency (ULF) Raman measurements in the 10-100 cm-1 range using a single-stage spectrometer. Here we demonstrate the potency of ULF Raman spectromicroscopy to harness the external vibrational modes of previously unrecognized S(0) structures present in environmental samples. We investigate the chemical and structural nature of intracellular S(0) granules stored within environmental mats of sulfur-oxidizing γ-Proteobacteria (Thiothrix). In vivo intracellular ULF scans indicate the presence of amorphous cyclooctasulfur (S8), clarifying enduring uncertainties regarding the content of microbial sulfur storage globules. Raman scattering of extracellular sulfur clusters in Thiothrix mats furthermore reveals an unexpected abundance of metastable ß-S8 and γ-S8, in addition to the stable α-S8 allotrope. We propose ULF Raman spectroscopy as a powerful method for the micron-scale determination of S(0) structure in natural and laboratory systems, with a promising potential to shine new light on environmental microbial and chemical sulfur cycling mechanisms.

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.

Enzymatic degradation of ß-1,4-linked N-acetylglucosaminoglucan prepared from Thiothrix nivea.

Thiothrix nivea is a filamentous sulfur-oxidizing bacterium commonly found in activated sludge. The filament of this bacterium is covered with a sheath. The sheath is an assemblage of macromolecular glucosaminoglucan (GG), [4)-ß-d-GlcN-(1 → 4)-ß-d-Glc-(1 → ]n, modified with an unidentified deoxy-sugar at position 3 of Glc. GG was obtained by dialysis after the partial hydrolysis of the sheath. The GG hydrogel was prepared by drying a GG solution. Then, the hydrogel was N-acetylated to prepare a stable hydrogel of N-acetylglucosaminoglucan (NGG), [4)-ß-d-GlcNAc-(1 → 4)-ß-d-Glc-(1 → ]n. The NGG hydrogel was stable in phosphate buffer but was disrupted by lysozyme addition, suggesting that NGG is susceptible to lysozyme degradation and has potential for medical use. The GG solution was N-acetylated to prepare a NGG suspension to confirm enzymatic degradation. The turbidity of the NGG suspension was decreased by lysozyme addition. Sugars released in the reaction mixture were derivatized with 4-aminobenzoic acid ethyl ester (ABEE) followed by HPLC analysis. Two major derivatives were detected, and their concentration was increased in reverse proportion to the turbidity of the reaction mixture. The derivatives were identified as GlcNAc-Glc-GlcNAc-Glc-ABEE and GlcNAc-Glc-ABEE by mass spectrometry. Consequently, NGG was found to be degraded by lysozyme via a mechanism similar to that of chitin degradation.

Filamentous bulking caused by Thiothrix species is efficiently controlled in full-scale wastewater treatment plants by implementing a sludge densification strategy.

Filamentous bulking caused by Thiothrix species is responsible for sludge washout and loss of performance in dairy wastewater treatment plants. A long-term study was conducted over 1.5 years to test three different mitigation strategies in a full-scale plant composed of two parallel sequential batch reactors (SBR1 and 2). Strategies based on polyaluminium chloride addition and volatile fatty acids reduction were ineffective to permanently solve the problem. On the contrary, modification of the reactor cycle based on the implementation of a periodic starvation proved efficient to solve the biomass wash-out and drastically reduce the sludge volume index in both reactors. Bacterial diversity analysis using 16S amplicon sequencing and quantitative PCR indicated a reduction of Thiothrix abundance from 51.9 to 1.0% in SBR1 and from 71.8 to 0.6% in SBR2. Simultaneously, the abundance of the glycogen-accumulating bacterium Candidatus Competibacter increased in both reactors. Microscopy analysis confirmed the transition between a bulking sludge towards a granular-like sludge. This study confirms the applicability of a periodic starvation to (1) solve recurring Thiothrix bulking, (2) convert loose aggregates into dense and compact granular-like structures and (3) considerably reduce energy demand for aeration.

Effect of culture residence time on substrate uptake and storage by a pure culture of Thiothrix (CT3 strain) under continuous or batch feeding.

A pure culture of the filamentous bacterium Thiothrix, strain CT3, was aerobically cultured in a chemostat under continuous acetate feeding at three different culture residence times (RT 6, 12 or 22 d) and the same volumetric organic load rate (OLR 0.12gCOD/L/d). Cells cultured at decreasing RT in the chemostat had an increasing transient response to acetate spikes in batch tests. The maximum specific acetate removal rate increased from 25 to 185mgCOD/gCOD/h, corresponding to a 1.8 to 8.1 fold higher respective steady-state rate in the chemostat. The transient response was mainly due to acetate storage in the form of poly(3-hydroxybutyrate) (PHB), whereas no growth response was observed at any RT. Interestingly, even though the storage rate also decreased as the RT increased, the storage yield increased from 0.41 to 0.50 COD/COD. This finding does not support the traditional view that storage plays a more important role as the transient response increases. The transient response of the steady-state cells was much lower than in cells cultured under periodic feeding (at 6 d RT, from 82 to 247mgCOD/gCOD/h), with the latter cells showing both storage and growth responses. On the other hand, even though steady-state cells had no growth response and their storage rate was also less, steady-state cells showed a higher storage yield than cells cultured under dynamic feeding. This suggests that in Thiothrix strain CT3, the growth response is triggered by periodic feeding, whereas the storage response is a constitutive mechanism, independent from previous acclimation to transient conditions.

Presence of N-L-lactyl-D-perosamine residue in the sheath-forming polysaccharide of Thiothrix fructosivorans.

Thiothrix fructosivorans forms a microtube (sheath) that encloses a line of cells. This sheath is an assemblage of [→4)-GlcN-(1→4)-Glc-(1→]n with side chains of Rha4N-(1→3)-Fuc(1→ at position 3 of Glc. The sheath-forming polysaccharide (SFP) may have some substitutions but this is not yet confirmed. To investigate the possible substitutions, the sheath was prepared by mild treatments. Solid-state NMR analysis suggested the presence of N-substitution. The sheath was hydrolyzed with concentrated HCl at 0°C, followed by derivatization with 4-aminobenzoic acid ethyl ester (ABEE). The presence of N-lactyl-Rha4N-Fuc-ABEE was suggested by NMR spectroscopy. Lactic acid was determined to be the l-isomer by chiral HPLC analysis. To estimate the N-lactylation degree, the sheath was N-acetylated. N-Acetyl-Rha4N-Fuc-ABEE and N-lactyl-Rha4N-Fuc-ABEE were then collectively recovered, and their abundance ratio was determined to be 1:4 by NMR analysis. When hydrolysis was performed at 40°C, GlcNAc-ABEE was obtained. For estimation of the N-acetylation degree, the sheath was N-acetylated with deuterated acetic anhydride and then N-acetyl-GlcN-ABEE was prepared. The content of deuterated N-acetyl-GlcN-ABEE was determined to be 50% based on the relative intensity of the acetyl proton signal in the 1D-(1)H NMR spectrum. It was concluded that Rha4N is mostly N-l-lactylated and GlcN is substoichiometrically N-acetylated.

Multiple sulphur and oxygen isotopes reveal microbial sulphur cycling in spring waters in the Lower Engadin, Switzerland.

Highly mineralized springs in the Scuol-Tarasp area of the Lower Engadin and in the Albula Valley near Alvaneu, Switzerland, display distinct differences with respect to the source and fate of their dissolved sulphur species. High sulphate concentrations and positive sulphur (δ(34)S) and oxygen (δ(18)O) isotopic compositions argue for the subsurface dissolution of Mesozoic evaporitic sulphate. In contrast, low sulphate concentrations and less positive or even negative δ(34)S and δ(18)O values indicate a substantial contribution of sulphate sulphur from the oxidation of sulphides in the crystalline basement rocks or the Jurassic sedimentary cover rocks. Furthermore, multiple sulphur (δ(34)S, Δ(33)S) isotopes support the identification of microbial sulphate reduction and sulphide oxidation in the subsurface, the latter is also evident through the presence of thick aggregates of sulphide-oxidizing Thiothrix bacteria.

Expansion of ability of denitrification within the filamentous colorless sulfur bacteria of the genus Thiothrix.

Filamentous sulfur bacteria of the genus Thiothrix are able to respire nitrate (NO3-→NO2-) under anaerobic growth. Here, Thiothrix caldifontis (G1(T), G3), Thiothrix unzii (A1(T), TN) and Thiothrix lacustris AS were shown to be capable of further reduction of nitrite and/or nitrous oxides (denitrification). In particular, in the genomes of these strains, excluding T. unzii TN, the nirS gene encoding periplasmic respiratory nitrite reductase was detected, and for T. lacustris AS the nirS expression was confirmed during anaerobic growth. The nirK gene, coding for an alternative nitrite reductase, and the nrfA gene, encoding nitrite reduction to ammonia, were not found in any investigated strains. All Thiothrix species capable of denitrification possess the cnorB gene encoding cytochrome c-dependent NO reductase but not the qnorB gene coding for quinol-dependent NO reductase. Denitrifying capacity ('full' or 'truncated') can vary between strains belonging to the same species and correlates with physical-chemical parameters of the environment such as nitrate, hydrogen sulfide and oxygen concentrations. Phylogenetic analysis revealed the absence of recent horizontal transfer events for narG and nirS; however, cnorB was subjected to gene transfer before the separation of modern species from a last common ancestor of the Thiothrix species.

Presence of alternating glucosaminoglucan in the sheath of Thiothrix nivea.

A sheath-forming sulfa oxidizer, Thiothrix nivea, was mixotrophically cultured in a medium supplemented with acetic acid and sodium disulfide. Its sheath, a microtube-like extracellular supermolecule, was prepared by selectively removing the cells with lysozyme, sodium dodecyl sulfate, and sodium hydroxide. The sheath was not visibly affected by hydrazine treatment, suggesting that it is not a proteinous supermolecule. From the acid hydrolysate of the sheath, glucose and glucosamine were detected in an approximate molar ratio of 1:1. Three other saccharic compounds were detected and recovered by HPLC as fluorescent derivatives prepared by reaction with 4-aminobenzoic acid ethyl ester. Nuclear magnetic resonance (NMR) analysis suggested that one of the derivatives was derived from an unidentified deoxypentose. NMR analysis for the other 2 derivatives showed that they were derived from ß-1,4-linked disaccharides and tetrasaccharides, which were composed of glucose and glucosamine. The sheath was readily broken down by weak HCl treatment, releasing an unidentified deoxypentose and polymer. Chemical analysis showed the presence of ß-1,4-linked D-Glcp and D-GlcNp in the polymer. NMR analysis revealed that the polymer had a repeating unit of →4)-D-Glcp-(ß1→4)-D-GlcNp-(ß1→. The solid-state 1D-(13)C NMR spectrum of the polymer in N-acetylated form supported this result. The molecular weight of the polymer was estimated to be 8.2×10(4) by size exclusion chromatography. Based on these results, the sheath of T. nivea is hypothesized to be assembled from alternately ß-1,4-linked glucosaminoglucan grafted with unidentified deoxypentose.

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.

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.

Microbial community change of sulfate reduction and sulfur oxidation bacteria in the activated sludge cultivated with acetate and peptone.

The growth of sulfate reducing bacteria (SRB) and filamentous sulfur bacteria was monitored on a laboratory scale in activated sludge reactors using acetate and peptone as the artificial wastewater. When the artificial wastewater contained acetate and peptone, filamentous bacteria increased in the sludge and the SVI values increased. There was a good correlation between sulfate reducing activity and sulfur oxidation activity in the produced sludge. The microbial community change of filamentous sulfur bacteria and sulfate reducing bacteria was analyzed using the fluorescent in situ hybridization (FISH) method. The tendency for the growth of filamentous sulfur bacteria Thiothrix eikelboomii following the growth of SRB was observed. The percentage of SRB385- hybridized cells and DNMA657-hybridized cells found in the total cell area increased from 2-3% to 7-10% when the filamentous bulking occurred.