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.

tilS and rpoB: New Molecular Markers for Phylogenetic and Biodiversity Studies of the Genus Thiothrix.

Currently, the phylogeny of the genus Thiothrix is based on comparative whole genome analysis because of the high homology of the 16S ribosomal RNA gene sequences within the genus. We analyzed the possibility of using various conservative genes as phylogenetic markers for the genus Thiothrix. We found that the levels of similarity of the nucleotide sequences of the tRNA(Ile)-lysidine synthase (tilS) and the ß subunit of RNA polymerase (rpoB) genes are in good agreement with the average nucleotide identity (ANI) values between the genomes of various representatives of the genus Thiothrix. The genomes of Thiothrix strains MK1, WS, DNT52, DNT53, and H33 were sequenced. Taxonomic analysis using both whole genomes and the tilS gene consistently showed that MK1 and WS belong to Thiothrix lacustris, while DNT52, DNT53, and H33 belong to Thiothrix subterranea. The tilS gene fragments were subjected to high-throughput sequencing to profile the Thiothrix mat of a sulfidic spring, which revealed the presence of known species of Thiothrix and new species-level phylotypes. Thus, the use of tilS and rpoB as phylogenetic markers will allow for rapid analyses of pure cultures and natural communities for the purpose of phylogenetic identification of representatives of the genus Thiothrix.

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.

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.

Recurrent disease outbreak in a warm temperate marginal coral community.

Coral diseases contribute to the rapid degradation of coral reefs on a global scale. Although widespread in tropical and subtropical reefs, disease outbreaks have not been described in warm temperate areas. Here, we report the outbreak of a new coral disease in a warm temperate marginal coral community in Japan. Outbreaks of the disease have been observed during the summer and autumn months since 2014. It affects the coral species Porites heronensis and was tentatively named "White Mat Syndrome" (WMS) as it consists of a white microbial mat dominated by Thiothrix sp., a sulfide oxidizing bacteria. Outbreaks followed high seasonal temperatures and were associated with the macroalga Gelidium elegans, which acts as a pathogen reservoir. With ocean warming and the anticipated increase in novel coral-algae interactions as some coral species shift poleward, WMS and emerging diseases could hinder the role of temperate areas as a future coral refuge.

Two New Species of Filamentous Sulfur Bacteria of the Genus Thiothrix, Thiothrix winogradskyi sp. nov. and 'Candidatus Thiothrix sulfatifontis' sp. nov.

The metagenome of foulings from sulfidic spring "Serovodorodny" (Tatarstan, Russia), where members of the genus Thiothrix was observed, was sequenced. Representatives of the phyla Gammaproteobacteria, Cyanobacteria and Campilobacteriota dominated in the microbial community. The complete genome of Thiothrix sp. KT was assembled from the metagenome. It displayed 93.93-99.72% 16S rRNA gene sequence identity to other Thiothrix species. The average nucleotide identity (ANI) и digital DNA-DNA hybridization (dDDH) showed that the genome designated KT represents a new species within the genus Thiothrix, 'Candidatus Thiothrix sulfatifontis' sp. nov. KT. The taxonomic status has been determined of the strain Thiothrix sp. CT3, isolated about 30 years ago and not assigned to any of Thiothrix species due to high 16S rRNA gene sequence identity with related species (i.e., 98.8-99.4%). The complete genome sequence of strain CT3 was determined. The ANI between CT3 and other Thiothrix species was below 82%, and the dDDH values were less than 40%, indicating that strain CT3 belongs to a novel species, Thiothrix winogradskyi sp. nov. A genome analysis showed that both strains are chemo-organoheterotrophs, chemolithotrophs (in the presence of hydrogen sulfide and thiosulfate) and chemoautotrophs. For the first time, representatives of Thiothrix showed anaerobic growth in the presence of thiosulfate.

Comparative Genome Analysis of the Genus Thiothrix Involving Three Novel Species, Thiothrix subterranea sp. nov. Ku-5, Thiothrix litoralis sp. nov. AS and "Candidatus Thiothrix anitrata" sp. nov. A52, Revealed the Conservation of the Pathways of Dissimilatory Sulfur Metabolism and Variations in the Genetic Inventory for Nitrogen Metabolism and Autotrophic Carbon Fixation.

Two strains of filamentous, colorless sulfur bacteria were isolated from bacterial fouling in the outflow of hydrogen sulfide-containing waters from a coal mine (Thiothrix sp. Ku-5) and on the seashore of the White Sea (Thiothrix sp. AS). Metagenome-assembled genome (MAG) A52 was obtained from a sulfidic spring in the Volgograd region, Russia. Phylogenetic analysis based on the 16S rRNA gene sequences showed that all genomes represented the genus Thiothrix. Based on their average nucleotide identity and digital DNA-DNA hybridization data these new isolates and the MAG represent three species within the genus Thiothrix with the proposed names Thiothrix subterranea sp. nov. Ku-5T, Thiothrix litoralis sp. nov. AST, and "Candidatus Thiothrix anitrata" sp. nov. A52. The complete genome sequences of Thiothrix fructosivorans QT and Thiothrix unzii A1T were determined. Complete genomes of seven Thiothrix isolates, as well as two MAGs, were used for pangenome analysis. The Thiothrix core genome consisted of 1,355 genes, including ones for the glycolysis, the tricarboxylic acid cycle, the aerobic respiratory chain, and the Calvin cycle of carbon fixation. Genes for dissimilatory oxidation of reduced sulfur compounds, namely the branched SOX system (SoxAXBYZ), direct (soeABC) and indirect (aprAB, sat) pathways of sulfite oxidation, sulfur oxidation complex Dsr (dsrABEFHCEMKLJONR), sulfide oxidation systems SQR (sqrA, sqrF), and FCSD (fccAB) were found in the core genome. Genomes differ in the set of genes for dissimilatory reduction of nitrogen compounds, nitrogen fixation, and the presence of various types of RuBisCO.

Enhancing stability of aerobic granules by microbial selection pressure using height-adjustable influent strategy.

Optimizing granules size distribution is critical for both reactor performance and stability. In this research, an optimal size range of 1800 to 3000 µm was proposed regarding mass transfer and granules stability based on granules developed at DO around 8.0 mg L-1 with the feed COD:N:P at 100:5:1. A height-adjustable influent strategy was applied to facilitate the nutrient storage of granules at optimum size range via microbial selective pressure. Results suggested insufficient hydraulic shear stress led to overgrowth of granules size. High abundance of filamentous bacteria (Thiothrix sp.) was observed in oversized granules, which detached and affected the remaining granules, resulting in severe sludge bulking. Strong hydraulic shear stress suppressed uncontrolled growth of granules. However, fewer abundance of simultaneous nitrification and denitrification (SND) bacterium was acquired, which led to unfavored SND effect and total nitrogen (TN) removal efficiency. The height-adjustable influent strategy facilitated the poly-ß-hydroxybutyrate (PHB) storage of granules at optimum size range, while limiting the overgrowth of granules size. Additionally, more than 87.51% of total granules situated in optimal sizes range, which led to higher abundance of SND bacterium and higher TN removal efficiency.

Metagenomic insights into the effect of sulfate on enhanced biological phosphorus removal.

Excess phosphorus in water supplies causes eutrophication, which degrades water quality. Hence, the efficient removal of phosphorus from wastewater represents a highly desirable process. Here, we evaluated the effect of sulfate concentration on enhanced biological phosphorus removal (EBPR), in which phosphorus is typically removed under anaerobic-oxic cycles, with sulfate reduction the predominant process in the anaerobic phase. Two sequencing batch EBPR reactors operated under high- (SBR-H) vs. low-sulfate (SBR-L) concentrations for 189 days and under three periods, i.e., start-up, sufficient acetate, and limited acetate. Under acetate-rich conditions, phosphorus removal efficiency was > 90% for both reactors; however, under acetate-limited conditions, only 34% and 91.3% of the phosphorus were removed for the SBR-L and the SBR-H, respectively. Metagenomic sequencing of the reactors showed that the relative abundance of the polyphosphate-accumulating and sulfur-reducing bacteria (SRB) was higher in the SBR-H, consistent with its higher phosphorus removal activity. Ten high-quality metagenome-assembled genomes, including one closely related to the genus Thiothrix disciformis (99.81% average amino acid identity), were recovered and predicted to simultaneously metabolize phosphorus and sulfur by the presence of phosphorus (ppk, ppx, pst, and pit) and sulfur (sul, sox, dsr, sqr, apr, cys, and sat) metabolism marker genes. The omics-based analysis provided a holistic view of the microbial ecosystem in the EBPR process and revealed that SRB and Thiothrix play key roles in the presence of high sulfate.Key points• We observed high phosphorus-removal efficiency in high-sulfate EBPR.• Metagenome-based analysis revealed sulfate-related metabolic mechanisms in EBPR.• SRB and PAOs showed interrelationships in the EBPR-sulfur systems.

Genomic and Metabolic Insights into Two Novel Thiothrix Species from Enhanced Biological Phosphorus Removal Systems.

Two metagenome-assembled genomes (MAGs), obtained from laboratory-scale enhanced biological phosphorus removal bioreactors, were analyzed. The values of 16S rRNA gene sequence identity, average nucleotide identity, and average amino acid identity indicated that these genomes, designated as RT and SSD2, represented two novel species within the genus Thiothrix, 'Candidatus Thiothrix moscowensis' and 'Candidatus Thiothrix singaporensis'. A complete set of genes for the tricarboxylic acid cycle and electron transport chain indicates a respiratory type of metabolism. A notable feature of RT and SSD2, as well as other Thiothrix species, is the presence of a flavin adenine dinucleotide (FAD)-dependent malate:quinone oxidoreductase instead of nicotinamide adenine dinucleotide (NAD)-dependent malate dehydrogenase. Both MAGs contained genes for CO2 assimilation through the Calvin-Benson-Bassam cycle; sulfide oxidation (sqr, fccAB), sulfur oxidation (rDsr complex), direct (soeABC) and indirect (aprBA, sat) sulfite oxidation, and the branched Sox pathway (SoxAXBYZ) of thiosulfate oxidation to sulfur and sulfate. All these features indicate a chemoorganoheterotrophic, chemolithoautotrophic, and chemolithoheterotrophic lifestyle. Both MAGs comprise genes for nitrate reductase and NO-reductase, while SSD2 also contains genes for nitrite reductase. The presence of polyphosphate kinase and exopolyphosphatase suggests that RT and SSD2 could accumulate and degrade polyhosphates during the oxic-anoxic growth cycle in the bioreactors, such as typical phosphate-accumulating microorganisms.

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.

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.

Glutamate as sole carbon source for enhanced biological phosphorus removal.

Enhanced Biological Phosphorus Removal (EBPR) is based on the enrichment of sludge in polyphosphate accumulating organisms (PAO). Candidatus Accumulibacter is the bacterial community member most commonly identified as PAO in EBPR systems when volatile fatty acids (VFA) are the carbon source. However, it is necessary to understand the role of non-Accumulibacter PAO in the case of wastewater with low VFA content. This work shows the first successful long-term operation of an EBPR system with glutamate as sole carbon and nitrogen source, resulting in the enrichment of sludge in the genus Thiothrix (37%), the family Comamonadaceae (15.6%) and Accumulibacter (7.7%). The enrichment was performed in an anaerobic/anoxic/oxic (A2/O) continuous pilot plant, obtaining stable biological N and P removal. This microbial community performed anaerobic P-release with only 18-29% of the observed PHA storage in Accumulibacter-enriched sludge and with slight glycogen storage instead of consumption, indicating the involvement of other carbon storage routes not related to PHA and glycogen. Thiothrix could be clearly involved in P-removal because it is able of accumulating Poly-P, probably without PHA synthesis, but with glutamate involvement. On the other hand, Comamonadaceae could participate in degradation of glutamate and denitrification, but its involvement in P-uptake cannot be reliably concluded.

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.

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.

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.

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.

Remediation of Thiothrix spp. associated bulking problems by raw wastewater feeding: A full-scale experience.

An industrial wastewater treatment plant (WWTP) in Australia has long suffered from bulking problems associated with the proliferation of Thiothrix spp. The WWTP consists of a covered anaerobic lagoon (CAL) followed by a sequencing batch reactor (SBR). The CAL functions as both an anaerobic digester and surge lagoon for the irregular flow of wastewater generated from the production of seasonal products. Chemical analysis of the raw influent showed it was composed of a mixture of organic acids, phenols and alcohols. The CAL effluent was characterised by high acetic acid and phenolic concentrations. An attempt was made to manipulate the SBR microbial community to improve settling by direct feeding small volumes of raw influent into the SBR. After raw feeding, the plant ceased bulking as the settled sludge volume reduced from 930 to 200mLL-1. 16S rRNA gene profiling and biovolumes of SBR samples revealed major changes in the microbial community. The Thiothrix spp. population decreased from 36.8% to 0.2%, and Zoogloea spp. dominated all samples after raw feeding. Therefore, direct feeding is proposed as a control method for industrial plants with surge/anaerobic lagoons in order to manage the bulking problems caused by Thiothrix spp. in downstream SBRs.

Novel Large Sulfur Bacteria in the Metagenomes of Groundwater-Fed Chemosynthetic Microbial Mats in the Lake Huron Basin.

Little is known about large sulfur bacteria (LSB) that inhabit sulfidic groundwater seeps in large lakes. To examine how geochemically relevant microbial metabolisms are partitioned among community members, we conducted metagenomic analysis of a chemosynthetic microbial mat in the Isolated Sinkhole, which is in a deep, aphotic environment of Lake Huron. For comparison, we also analyzed a white mat in an artesian fountain that is fed by groundwater similar to Isolated Sinkhole, but that sits in shallow water and is exposed to sunlight. De novo assembly and binning of metagenomic data from these two communities yielded near complete genomes and revealed representatives of two families of LSB. The Isolated Sinkhole community was dominated by novel members of the Beggiatoaceae that are phylogenetically intermediate between known freshwater and marine groups. Several of these Beggiatoaceae had 16S rRNA genes that contained introns previously observed only in marine taxa. The Alpena fountain was dominated by populations closely related to Thiothrix lacustris and an SM1 euryarchaeon known to live symbiotically with Thiothrix spp. The SM1 genomic bin contained evidence of H2-based lithoautotrophy. Genomic bins of both the Thiothrix and Beggiatoaceae contained genes for sulfur oxidation via the rDsr pathway, H2 oxidation via Ni-Fe hydrogenases, and the use of O2 and nitrate as electron acceptors. Mats at both sites also contained Deltaproteobacteria with genes for dissimilatory sulfate reduction (sat, apr, and dsr) and hydrogen oxidation (Ni-Fe hydrogenases). Overall, the microbial mats at the two sites held low-diversity microbial communities, displayed evidence of coupled sulfur cycling, and did not differ largely in their metabolic potentials, despite the environmental differences. These results show that groundwater-fed communities in an artesian fountain and in submerged sinkholes of Lake Huron are a rich source of novel LSB, associated heterotrophic and sulfate-reducing bacteria, and archaea.