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.

Wastewater Treatment with Bacterial Representatives of the Thiothrix Morphotype.

Bacteria of the Thiothrix morphotype, comprising the genera Thiothrix, Thiolinea and Thiofilum, are frequently encountered in domestic and industrial wastewater treatment systems, but they are usually not clearly differentiated due to the marked similarity in their morphologies. Methods ranging from light microscopy, FISH and PCR to modern high-throughput sequencing are used to identify them. The development of these bacteria in wastewater treatment systems has both advantages and disadvantages. On the one hand, the explosive growth of these bacteria can lead to activated sludge bulking or clogging of the treatment system's membranes, with a consequent decrease in the water treatment efficiency. On the other hand, members of the Thiothrix morphotype can improve the quality of granular sludge and increase the water treatment efficiency. This may be due to their capacity for sulfide oxidation, denitrification combined with the oxidation of reduced sulfur compounds, enhanced biological phosphate removal and possibly denitrifying phosphate removal. The recently obtained pangenome of the genus Thiothrix allows the explanation, at the genomic level, of the experimental results of various studies. Moreover, this review summarizes the data on the factors affecting the proliferation of representatives of the Thiothrix morphotype.

Metagenomics Revealed a New Genus 'Candidatus Thiocaldithrix dubininis' gen. nov., sp. nov. and a New Species 'Candidatus Thiothrix putei' sp. nov. in the Family Thiotrichaceae, Some Members of Which Have Traits of Both Na+- and H+-Motive Energetics.

Two metagenome-assembled genomes (MAGs), GKL-01 and GKL-02, related to the family Thiotrichaceae have been assembled from the metagenome of bacterial mat obtained from a sulfide-rich thermal spring in the North Caucasus. Based on average amino acid identity (AAI) values and genome-based phylogeny, MAG GKL-01 represented a new genus within the Thiotrichaceae family. The GC content of the GKL-01 DNA (44%) differed significantly from that of other known members of the genus Thiothrix (50.1-55.6%). We proposed to assign GKL-01 to a new species and genus 'Candidatus Thiocaldithrix dubininis' gen. nov., sp. nov. GKL-01. The phylogenetic analysis and estimated distances between MAG GKL-02 and the genomes of the previously described species of the genus Thiothrix allowed assigning GKL-02 to a new species with the proposed name 'Candidatus Thiothrix putei' sp. nov. GKL-02 within the genus Thiothrix. Genome data first revealed the presence of both Na+-ATPases and H+-ATPases in several Thiothrix species. According to genomic analysis, bacteria GKL-01 and GKL-02 are metabolically versatile facultative aerobes capable of growing either chemolithoautotrophically or chemolithoheterotrophically in the presence of hydrogen sulfide and/or thiosulfate or chemoorganoheterotrophically.

Effective adsorption of perrhenate ions on the filamentous sheath-forming bacteria, Sphaerotilus montanus, Sphaerotilus natans and Thiothrix fructosivorans.

AIMS: This study aimed to unveil perrhenate sorption properties of the filamentous sheaths formed by Sphaerotilus montanus, Sphaerotilus natans and Thiothrix fructosivorans. METHODS AND RESULTS: The adsorptions of perrhenate on lyophilizates of the above-mentioned filamentous sheaths were analysed by ICP, IR, XPS and EDX. The capacity reached 82 mg per g-adsorbent, when using S. natans. The Langmuir coefficient of this adsorbent was found to be the largest of the three. The adsorption capacity was discussed with respect to the amount of nitrogen and phosphorus in the adsorbents. The occurrence of anion exchange was implied by the IR spectrum changes before and after adsorption. The adsorption data fitted well with a pseudo-second-order equation, suggesting that the rate is determined by the chemical bond formation. CONCLUSIONS: A significant amount of perrhenate was adsorbed on the sheaths formed by S. montanus, S. natans and T. fructosivorans. The adsorption was correlated with the elemental compositions. A strong chemical bond formation was suggested from the results of the Langmuir adsorption isotherm and kinetic analysis. SIGNIFICANCE AND IMPACT OF STUDY: The capacity obtained for S. natans is one of the largest adsorptions amongst the similar biomaterials, implying the possibility of providing economical adsorbents of rare metal oxyanions.

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.

Identification and characterization of the S-layer formed on the sheath of Thiothrix nivea.

Thiothrix nivea is a filamentous sulfur-oxidizing bacterium common in activated sludge and its filament is covered with a polysaccharide layer called sheath. In this study, we found that T. nivea aggregates under acidic conditions. A hexagonal lattice pattern, a typical morphological feature of proteinaceous S-layers, was newly observed on the surface of the sheath by transmission electron microscopy. The pattern and the acid-dependent aggregation were not observed in T. fructosivorans, a relative sheath-forming bacterium of T. nivea. The putative S-layer of T. nivea was detached by washing with unbuffered tris(hydroxymethyl)aminomethane base (Tris) solution and a protein of 160 kDa was detected by electrophoresis. Based on partial amino acid sequences of the protein, its structural gene was identified. The gene encodes an acidic protein which has a putative secretion signal and a Ca2+-binding domain. The protein was solubilized with urea followed by dialysis in the presence of calcium. A hexagonal lattice pattern was observed in the aggregates formed during dialysis, revealing that the protein is responsible for S-layer formation. Biosorption ability of copper, zinc, and cadmium onto the T. nivea filament decreased upon pretreatment with Tris, demonstrating that the S-layer was involved in metal adsorption. Moreover, aggregation of Escherichia coli was promoted by acidification in the presence of the S-layer protein, suggesting that the protein is potentially applicable as an acid-driven flocculant for other bacteria.

Evaluation of the genus Thiothrix Winogradsky 1888 (Approved Lists 1980) emend. Aruga et al. 2002: reclassification of Thiothrix disciformis to Thiolinea disciformis gen. nov., comb. nov., and of Thiothrix flexilis to Thiofilum flexile gen. nov., comb nov., with emended description of Thiothrix.

Thiothrix is the type genus of the Thiotrichaceae in the Thiotrichales of the Gammaproteobacteria, comprising nine species of sulfur-oxidising filamentous bacteria, which are variously autotrophic, heterotrophic or have mixed metabolic modes. Within the genus, four species show 16S rRNA gene identities lower the Yarza threshold for the rank of genus (94.5 %) - Thiothrix disciformis, Thiothrix flexilis, Thiothrix defluvii and Thiothrix eikelboomii - as they show no affiliation to extant genera, a polyphasic study was undertaken including biochemical, physiological and genomic properties and phylogeny based on the 16S rRNA gene (rrs), recombination protein A (RecA), polynucleotide nucleotidyltransferase (Pnp), translation initiation factor IF-2 (InfB), glyceraldehyde-3-phosphate dehydrogenase (GapA), glutaminyl-tRNA synthetase (GlnS), elongation factor EF-G (FusA) and concatamers of 53 ribosomal proteins encoded by rps, rpl and rpm operons, all of which support the reclassification of these species. We thus propose Thiolinea gen. nov. and Thiofilum gen. nov. for which the type species are Thiolinea disciformis gen. nov., comb. nov. and Thiofilum flexile gen. nov., comb. nov. We also propose that these genera are each circumscribed into novel families Thiolinaceae fam. nov. and Thiofilaceae fam. nov., and that Leucothrix and Cocleimonas are circumscribed into Leucotrichaceaefam. nov. and provide emended descriptions of Thiothrix and Thiotrichaceae.

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%.

Niphargus-Thiothrix associations may be widespread in sulphidic groundwater ecosystems: evidence from southeastern Romania.

Niphargus is a speciose amphipod genus found in groundwater habitats across Europe. Three Niphargus species living in the sulphidic Frasassi caves in Italy harbour sulphur-oxidizing Thiothrix bacterial ectosymbionts. These three species are distantly related, implying that the ability to form ectosymbioses with Thiothrix may be common among Niphargus. Therefore, Niphargus-Thiothrix associations may also be found in sulphidic aquifers other than Frasassi. In this study, we examined this possibility by analysing niphargids of the genera Niphargus and Pontoniphargus collected from the partly sulphidic aquifers of the Southern Dobrogea region of Romania, which are accessible through springs, wells and Movile Cave. Molecular and morphological analyses revealed seven niphargid species in this region. Five of these species occurred occasionally or exclusively in sulphidic locations, whereas the remaining two were restricted to nonsulphidic areas. Thiothrix were detected by PCR on all seven Dobrogean niphargid species and observed using microscopy to be predominantly attached to their hosts' appendages. 16S rRNA gene sequences of the Thiothrix epibionts fell into two main clades, one of which (herein named T4) occurred solely on niphargids collected in sulphidic locations. The other Thiothrix clade was present on niphargids from both sulphidic and nonsulphidic areas and indistinguishable from the T3 ectosymbiont clade previously identified on Frasassi-dwelling Niphargus. Although niphargids from Frasassi and Southern Dobrogea are not closely related, the patterns of their association with Thiothrix are remarkably alike. The finding of similar Niphargus-Thiothrix associations in aquifers located 1200 km apart suggests that they may be widespread in European groundwater ecosystems.

New insights into mycelial pellets for aerobic sludge granulation in membrane bioreactor: Bio-functional interactions among metazoans, microbial communities and protein expression.

Direct cultivation of aerobic granular sludge (AGS) in membrane bioreactor (MBR) has gained increasing attention. Mycelial pellets (MPs) has been shown capable of promoting rapid granulation of aerobic sludge in MBR, yet mechanisms remain unclear and in-depth insight into cross-scale interactions between MPs and indigenous microbiota as well as the corresponding protein expression functions is necessary. Herein, we found that the addition of MPs in MBR resulted in massive growth of metazoans with 40-400 /mL for rotifers, 20-140 /mL for nematodes and 2-420 /mL for oligochaetes in the initial phase of granulation. This facilitated the MPs to rapidly aggregate with bacteria to form defensive granules for physical protection from predation by metazoans, which inhibited the overgrowth of filamentous bacteria Thiothrix and promoted the reproduction of functional bacteria related to nitrogen removal (Nitrospira, Trichococcus and Acinetobacter). Proteomic analysis demonstrated that the upregulation of functional proteins was mainly ascribed to the decrease of Thiothrix and the increase of Nitrospira, resulting in the enhancement of metabolic pathways involved in glycolysis/gluconeogenesis, citrate (TCA) cycle, oxidative phosphorylation, pyruvate metabolism, nitrogen metabolism and biosynthesis of amino acids, which was responsible for MPs-induced AGS with denser structure, more abundant proteins and ß-polysaccharides, higher species diversity, significant nitrogen removal (33.12-42.33%) and lower membrane fouling potential. This study provided a novel and comprehensive insight into the enhanced granulation of aerobic sludge by MPs and the functional superiority of MPs-induced AGS in MBR system.

Dominance of epiphytic filamentous Thiothrix spp. on an aquatic macrophyte in a hydrothermal vent flume in Sedge Bay, Yellowstone Lake, Wyoming.

Sublacustrine hydrothermal vents, geysers, and fumaroles impart regions of Yellowstone Lake with distinctive chemical compositions that generate unique freshwater habitats and support diverse microbial life. Some microbial communities within Sedge Bay manifest themselves as accumulations of white-colored films on the surfaces of aquatic macrophytes located within the hydrothermal flow of vents. It was hypothesized that the white films were the product of microbial growth, particularly sulfur-oxidizing bacteria. An investigation of the relevant biological compounds in the vent waters was conducted. Microscopy, non-culture molecular techniques, and phylogenetic analysis were used to assay the bacterial diversity associated with the films. Microscopic analysis of the white films revealed the presence of long filaments (>200 µm) that contained sulfur granules. Filaments with these characteristics were not detected on the normal macrophyte samples. Nucleic acids were extracted from the surface of macrophyte coated with the white film (SB1, SB2) and from the surface of an uncoated macrophyte (SC). 16S ribosomal (rRNA) genes were amplified with the polymerase chain reaction (PCR) and cloned. Amplified ribosomal DNA restriction analysis (ARDRA) was used to examine 100 clones from each library and identify unique phylotypes. S(Chao1) and the Shannon Index, mathematical measures of richness and heterogeneity, were employed to assess the ARDRA pattern diversity of each sample. The SC community contained 50 unique phylotypes, predominantly cyanobacteria and proteobacteria, and was the most heterogeneous. SB1 and SB2 communities were less heterogeneous and dominated by Thiothrix. Dilution to extinction PCR conducted with specific primers indicated that the relative abundance of Thiothrix 16S rRNA gene copies in all three samples were similar. However, reduced sulfur compounds from the vent resulted in a more narrow habitat that supported the sulfur-oxidizing Thiothrix in the white film to the exclusion of cyanobacteria and other proteobacteria found on the normal macrophyte. The majority of 16S rRNA gene sequences obtained in this study displayed similarities ≤98% to any known sequence in public data bases which suggests an abundance of new bacterial species in Sedge Bay.

Stable granulation of seawater-adapted aerobic granular sludge with filamentous Thiothrix bacteria.

Many sources of wastewater contain sulfides, which can cause excessive growth of filamentous bacteria such as Thiothrix sp. resulting in bulking sludge in conventional activated sludge systems. Granular sludge systems could potentially also suffer from the growth of filamentous bacteria. Uptake of easily degradable COD by the relatively slow growing Ca. Accumulibacter phosphatis bacteria and the absence of strong diffusion gradients due to plug flow feeding through the settled granular sludge bed are assumed to be the dominant factors for successful granulation. Sulfides will remain after this anaerobic phase and cause growth of sulfide-consuming bacteria such as Thiothrix sp. Here we observed the impact of growth of Thiothrix sp bacteria in a laboratory aerobic granular sludge reactor by feeding a mixture of acetate and thiosulfate in the influent. Thiothrix sp, proliferated when 18% of the influent COD was due to thiosulfate, forming 51.4 ± 8.3% of the total granular biomass. Despite the strong presence of these filamentous bacteria a well settling sludge was maintained (SVI10 equal to 13.3 mL/g). These results confirm that sludge morphology is not necessarily a reflection of the cell morphology of the bacteria, but is highly influence by reactor operation. It also reiterates the fact that compact biofilms are formed when the substrate consumption rate is lower than the substrate transport rate.

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.

Thiothrix eikelboomii interferes oxygen transfer in activated sludge.

This study revealed that, Thiothrix eikelboomii, a well-known filamentous bacterium that causes sludge bulking, could also interfere oxygen transfer during wastewater treatment. The volumetric oxygen transfer coefficient (KLa) in filamentous-bulking sludge (FBS) was found to be 43% lower than that in floc-forming sludge (FFS) at similar biomass concentrations, partially because the filamentous bacteria had increased the sludge apparent viscosity. The KLa value for FBS, however, was still significantly lower than that for FFS even if both sludges had similar apparent viscosity. Numerous tiny and free-swimming filaments were observed to attach on the air bubble surface, presumably reducing the liquid film renewal and increasing the liquid film thickness. Moreover, the filaments were co-coated with extracellular polymeric substances of protein and polysaccharide, which could make them performing like "amphiphilic molecules" of surfactants to hinder oxygen transfer. Therefore, the particular surface property of filaments and their interaction with air bubbles could also impact oxygen transfer. Thiothrix eikelboomii was identified to be the responsible filamentous bacterium that lowered the KLa value, while other filamentous bacteria with short filaments did not interfere oxygen transfer. This study implies that controlling sludge bulking benefits not only sludge settling but also oxygen transfer.

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.

Adding an anaerobic step can rapidly inhibit sludge bulking in SBR reactor.

Activated sludge from wastewater treatment plants was seeded into a sequencing batch reactor (SBR) in which synthetic wastewater was used as the influent. The sludge was bulked by decreasing the concentration of dissolved oxygen (DO). By adding a 30 min step of anaerobic stirring after the water inflow, the sludge bulking was rapidly inhibited after 10 running cycles, and the sludge volume index (SVI) decreased from 222 to 74 mL·g-1. The results of high-throughput sequencing showed that the relative abundance of bacteria Thiothrix, bacteria norank_o_Sphingobacteriales and fungi Trichosporon was increased by 6.3, 4.3 and 81.2%, after initial SBR stages, but these bacteria were inhibited by the addition of an anaerobic step, as their relative abundances decreased by 0.7, 0.8 and 14.7%, respectively. The proliferation of Thiothrix, norank_o_Sphingobacteriales and Trichosporon was the primary reason for the observed sludge bulking in the reactor. After the anaerobic step was added, the sludge extracellular polymeric substances (EPS) concentration was increased from 84.4 to 104.0 mg·(gMLSS)-1 (grams of mixed liquor suspended solids). Thus, the addition of an anaerobic step can inhibit the growth of filamentous bacteria, increasing the sludge EPS concentration and promoting the precipitation of activated sludge.

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.

Effect of periodic feeding on substrate uptake and storage rates by a pure culture of Thiothrix (CT3 strain).

A pure culture of Thiothrix strain CT3 has been aerobically cultured under periodic acetate feeding in a Sequencing Batch Reactor (SBR) at volumetric organic load rate of 0.12gCODL(-1)d(-1). Two different culture residence times (12d or 20d) were adopted as well as two different feed frequencies (1 and 4d(-1), for each culture residence time), the volumetric organic load rate being the same under all conditions. The transient response of the microorganism to the periodic acetate feed was investigated through batch tests with biomass withdrawn from the SBR, as function of the different SBR operating conditions. In all tested conditions, a quick transient response to the acetate spike was observed with fast increase of acetate uptake rate (ranging from 71 to 247mgCODgCOD(-1)h(-1)). This transient response was mainly due to acetate storage in form of poly-hydroxybutyrate (ranging from 45% to 64% of the observed yield) whereas the growth response (i.e. increase of production rate of active biomass) generally played a minor role (ranging from 21% to 38% of the observed yield). Apart from this general trend, culture residence time as well as feed frequency had a strong impact on transient behaviour of cultured cells. The overall transient response (i.e. maximum specific substrate removal rate) increased as culture residence time decreased or as feed frequency increased. Moreover, the ratio of storage response and growth response increased as the overall transient response decreased, i.e. the storage response was preferentially maintained when cells presented a lower transient response. The ability of the cells to increase their growth rate with respect to SBR average value was the lowest under the most unfavourable conditions (residence time 20d, feed frequency 1d(-1)) and increased with the increase in maximum substrate uptake rate.