Phylum Thermotogae Research Articles

Elucidation of microbial interactions, dynamics, and keystone microbes in high pressure anaerobic digestion

High-pressure anaerobic digestion (HPAD) is a promising technology for producing biogas enriched with high methane content in a single-step process. To enhance HPAD performance, a comprehensive understanding of microbial community dynamics and their interactions is essential. For this, mesophilic batch high-pressurized anaerobic reactors were operated under 3 bars (H3) and 6 bars (H6). The experimental results showed that the effect of high-pressure (up to 6 bar) on acidification was negligible while methanogenesis was significantly delayed. Microbial analysis showed the predominance of Defluviitoga affiliated with the phylum Thermotogae and the reduction of Thiopseudomonas under high-pressure conditions. In addition, the microbial cluster pattern in H3 and H6 was significantly different compared to the CR, indicating a clear shift in microbial community structure. Moreover, Methanobacterium, Methanomicrobiaceae, Alkaliphilus, and Petrimonas were strongly correlated in network analysis, and they could be identified as keystone microbes in the HPAD reactor.

Sulfidogenic Microbial Communities of the Uzen High-Temperature Oil Field in Kazakhstan.

Application of seawater for secondary oil recovery stimulates the development of sulfidogenic bacteria in the oil field leading to microbially influenced corrosion of steel equipment, oil souring, and environmental issues. The aim of this work was to investigate potential sulfide producers in the high-temperature Uzen oil field (Republic of Kazakhstan) exploited with seawater flooding and the possibility of suppressing growth of sulfidogens in both planktonic and biofilm forms. Approaches used in the study included 16S rRNA and dsrAB gene sequencing, scanning electron microscopy, and culture-based techniques. Thermophilic hydrogenotrophic methanogens of the genus Methanothermococcus (phylum Euryarchaeota) predominated in water from the zone not affected by seawater flooding. Methanogens were accompanied by fermentative bacteria of the genera Thermovirga, Defliviitoga, Geotoga, and Thermosipho (phylum Thermotogae), which are potential thiosulfate- or/and sulfur-reducers. In the sulfate- and sulfide-rich formation water, the share of Desulfonauticus sulfate-reducing bacteria (SRB) increased. Thermodesulforhabdus, Thermodesulfobacterium, Desulfotomaculum, Desulfovibrio, and Desulfoglaeba were also detected. Mesophilic denitrifying bacteria of the genera Marinobacter, Halomonas, and Pelobacter inhabited the near-bottom zone of injection wells. Nitrate did not suppress sulfidogenesis in mesophilic enrichments because denitrifiers reduced nitrate to dinitrogen; however, thermophilic denitrifiers produced nitrite, an inhibitor of SRB. Enrichments and a pure culture Desulfovibrio alaskensis Kaz19 formed biofilms highly resistant to biocides. Our results suggest that seawater injection and temperature of the environment determine the composition and functional activity of prokaryotes in the Uzen oil field.

Effects of plastics on reactor performance and microbial communities during acidogenic fermentation of food waste for production of volatile fatty acids

The aim of this work was to study the effects of plastics (high-density polyethylene (HDPE), polystyrene (PS), polypropylene (PP), and polyethylene terephthalate (PET)) on reactor performance and microbial communities during acidogenic fermentation of food waste for the production of volatile fatty acids (VFA). The addition of HDPE and PS increased total VFA yields by 28% and 47%, respectively, whereas the addition of PP and PET decreased total VFA yields by 6% and 2%, respectively. The highest enhancing performance of PS could be ascribed to its highly porous structure that could provide immobilization effects for microbial growth. Degradation of various plastics was confirmed by FESEM results, but the degrees were limited (i.e., 3.9–8.7%). Bacterial analysis showed that the addition of various plastics altered the community diversity. Phylum Thermotogae and genus Defluviitoga dominated all the reactors. Potential HDPE- and PS-degrading microbes could belong to genus Clostridium_sensu_stricto_8, while Tepidanaerobacter_syntrophicus could be PET-degrading microbes.

Effects of granular activated carbon and temperature on the viscosity and methane yield of anaerobically digested of corn straw with different dry matter concentrations

Anaerobic digestion (AD) systems with high substrate concentrations are characterized by high viscosity, which affects material and energy transfer efficiencies, thereby influencing methane production efficiency. In this study, adding granular activated carbon (GAC) and increasing the temperature decreased the viscosity by 4.56–10.19% and 27.13–28.85%, respectively, and improved AD efficiency. Adding GAC and increasing the temperature enhanced the methane yields by 34.37–38.15% and 25.60–28.31%, respectively. Distance-based redundancy analysis showed that the viscosity, temperature, and GAC had the greatest effects on the composition of the microbial community. The dominant bacteria in the medium-temperature AD system at the phylum level belonged to Firmicutes, Bacteroidetes, and Euryarchaeota. In addition to the dominant bacteria in the medium-temperature AD system, the thermophilic phylum Thermotogae was abundant in the high-temperature AD system. Moreover, the relative abundance of Euryarchaeota, which contained most of the methanogens, was higher in the high-temperature AD system than in the medium-temperature AD system.

Effect of Cultivation Parameters on Fermentation and Hydrogen Production in the Phylum Thermotogae.

The phylum Thermotogae is composed of a single class (Thermotogae), 4 orders (Thermotogales, Kosmotogales, Petrotogales, Mesoaciditogales), 5 families (Thermatogaceae, Fervidobacteriaceae, Kosmotogaceae, Petrotogaceae, Mesoaciditogaceae), and 13 genera. They have been isolated from extremely hot environments whose characteristics are reflected in the metabolic and phenotypic properties of the Thermotogae species. The metabolic versatility of Thermotogae members leads to a pool of high value-added products with application potentials in many industry fields. The low risk of contamination associated with their extreme culture conditions has made most species of the phylum attractive candidates in biotechnological processes. Almost all members of the phylum, especially those in the order Thermotogales, can produce bio-hydrogen from a variety of simple and complex sugars with yields close to the theoretical Thauer limit of 4 mol H2/mol consumed glucose. Acetate, lactate, and L-alanine are the major organic end products. Thermotagae fermentation processes are influenced by various factors, such as hydrogen partial pressure, agitation, gas sparging, culture/headspace ratio, inoculum, pH, temperature, nitrogen sources, sulfur sources, inorganic compounds, metal ions, etc. Optimization of these parameters will help to fully unleash the biotechnological potentials of Thermotogae and promote their applications in industry. This article gives an overview of how these operational parameters could impact Thermotogae fermentation in terms of sugar consumption, hydrogen yields, and organic acids production.

Insights on comparative bacterial diversity between different arid zones of Cholistan Desert, Pakistan.

The present study was conducted to analyze bacterial diversity profile of Cholistan desert located in Pakistan. The study investigates the influence of physicochemical parameters of soil on distribution of different bacteria at all taxonomic levels and also study the distribution pattern between different desert environments, particularly rhizospheric and bulk desert sands. Species richness showed phyla Proteobacteria and Chloroflexi as the dominant OTUs in all the samples. Besides the two phyla, the rhizospheric soils with root remnants were dominated by Firmicutes, Deinococcus-Thermus, Actinobacteria and Acidobacteri, while phylum Thermotogae was present in significant quantity in rhizosheaths devoid of roots. In non-rhizospheric desert soils, a considerable number of OTUs belonged to phyla Proteobacteria, Chloroflexi, Bacteroidetes and Acidobacteria. An important finding from this study is that a bulk portion of the OTUs were assigned to unclassified taxa, indicating a large repertoire of unexplored taxa in the desert ecology of Pakistan. Distribution of taxonomic groups among various regions of the desert was collaborating well with the physicochemical parameters of the sites. The findings of this study establish the fundamental relationships between desert ecosystem, specific native plant and the total bacterial flora. This is the first study of microbial community analysis of any desert in Pakistan and thus, will serve as a future platform to explore further on desert ecosystem functioning by employing the ever-changing biotechnological tools.

Complete Genome Sequence of Athalassotoga saccharophila Strain NAS-01, a Deep-Branching Thermophilic Lineage in the Phylum Thermotogae.

Athalassotoga saccharophila strain NAS-01 (=JCM 19762T) is an anaerobic thermoacidophilic bacterium which is one of the deepest branching lineages of the phylum Thermotogae Here, we report the complete genome sequence (1.96 Mbp) and two plasmid sequences (43.7 kbp and 43.5 kbp) of this strain.

Biochar enhanced thermophilic anaerobic digestion of food waste: Focusing on biochar particle size, microbial community analysis and pilot-scale application

Effectiveness of biochar addition to enhance thermophilic semi-continuous anaerobic digestion (AD) of food waste for methane production was investigated with a focus on dosage and particle size of biochar, pilot-scale application and elucidation of methanogenic pathways. Optimal dosage range of biochar was determined as 7.5 to 15 g per L working volume based on lab-scale batch AD. Effects of biochar with different particle sizes at a model dosage of 15 g/L were evaluated in a semi-continuous AD experiment, results of which showed that all the examined biochars with different particle sizes (<50 μm to 3 cm) substantially enhanced the average methane yields (0.465–0.543 L/gVS) compared to control digesters which failed due to overloading (≥3.04 gVS/L/d). No significant difference in methane yields, however, was observed among digesters with different particle sizes of biochars, except for 1–3 cm. The core reason for this phenomenon was that the biochars with different particle sizes had similar properties (e.g. density, surface area and pore size) and that the floating of large particle size (1–3 cm) of biochar with a density of 847 kg/m3 was not conducive to microbial growth. Metagenomic analysis was performed to determine the predominant microbial species and to explain the main methanogenic pathways in biochar-amended digesters using 16S rRNA sequencing. In the biochar-amended digester, bacterial phylum Thermotogae containing a major genus of Defluviitoga was selectively enriched with gradual increase of organic loadings, while simultaneously enriched methanogen genera Methanothermobacter and Methanosarcina, which showed a synergy of hydrogenotrophic and acetoclastic methanogenic pathways, jointly enhanced the methane productivity. Both technical feasibility and economic feasibility of adding biochar with simple pretreatment (e.g. smash) were validated in the pilot-scale thermophilic semi-continuous AD operations.

Vestiges of Adaptation to the Mesophilic Environment in the Genome of Tepiditoga spiralis gen. nov., sp. nov.

A novel anaerobic heterotrophic strain, designated strain sy52T, was isolated from a hydrothermal chimney at Suiyo Seamount in the Pacific Ocean. A 16S rRNA gene analysis revealed that the strain belonged to the family Petrotogaceae in the phylum Thermotogae. The strain was mesophilic with optimum growth at 48°C and the phylum primarily comprised hyperthermophiles and thermophiles. Strain sy52T possessed unique genomic characteristics, such as an extremely low G+C content and 6 copies of rRNA operons. Genomic analyses of strain sy52T revealed that amino acid usage in the predicted proteins resulted from adjustments to mesophilic environments. Genomic features also indicated independent adaptions to the mesophilic environment of strain sy52T and Mesotoga species, which belong to the mesophilic lineage in the phylum Thermotogae. Based on phenotypic and phylogenetic evidence, strain sy52T is considered to represent a novel genus and species in the family Petrotogaceae with the proposed name Tepiditoga spiralis gen. nov., sp. nov.

Towards a congruent reclassification and nomenclature of the thermophilic species of the genus Pseudothermotoga within the order Thermotogales

The phylum Thermotogae gathers thermophilic, hyperthermophic, mesophilic, and thermo-acidophilic anaerobic bacteria that are mostly originated from geothermally heated environments. The metabolic and phenotypic properties harbored by the Thermotogae species questions the evolutionary events driving the emergence of this early branch of the universal tree of life. Recent reshaping of the Thermotogae taxonomy has led to the description of a new genus, Pseudothermotoga, a sister group of the genus Thermotoga within the order Thermotogales. Comparative genomics of both Pseudothermotoga and Thermotoga spp., including 16S-rRNA-based phylogenetic, pan-genomic analysis as well as signature indel conservation, provided evidence that Thermotoga caldifontis and Thermotoga profunda species should be reclassified within the genus Pseudothermotoga and renamed as Pseudothermotoga caldifontis comb. nov. (type strain=AZM44c09T) and Pseudothermotoga profunda comb. nov. (type strain=AZM34c06T), respectively. In addition, based upon whole-genome relatedness indices and DNA–DNA Hybridization results, the reclassification of Pseudothermotoga lettingae and Pseudothermotoga subterranea as latter heterotypic synonyms of Pseudothermotoga elfii is proposed. Finally, potential genetic elements resulting from the distinct evolutionary story of the Thermotoga and Pseudothermotoga clades are discussed.

Obligate sugar oxidation in Mesotoga spp., phylum Thermotogae, in the presence of either elemental sulfur or hydrogenotrophic sulfate-reducers as electron acceptor.

Mesotoga prima strain PhosAc3 is a mesophilic representative of the phylum Thermotogae comprising only fermentative bacteria so far. We show that while unable to ferment glucose, this bacterium is able to couple its oxidation to reduction of elemental sulfur. We demonstrate furthermore that M. prima strain PhosAc3 as well as M. prima strain MesG1 and Mesotoga infera are able to grow in syntrophic association with sulfate-reducing bacteria (SRB) acting as hydrogen scavengers through interspecies hydrogen transfer. Hydrogen production was higher in M. prima strain PhosAc3 cells co-cultured with SRB than in cells cultured alone in the presence of elemental sulfur. We propose that the efficient sugar-oxidizing metabolism by M. prima strain PhosAc3 in syntrophic association with a hydrogenotrophic sulfate-reducing bacterium can be extrapolated to all members of the Mesotoga genus. Genome comparison of Thermotogae members suggests that the metabolic difference between Mesotoga and Thermotoga species (sugar oxidation versus fermentation) is mainly due to the absence of the bifurcating [FeFe]-hydrogenase in the former. Such an obligate oxidative process for using sugars, unusual within prokaryotes, is the first reported within the Thermotogae. It is hypothesized to be of primary ecological importance for growth of Mesotoga spp. in the environments that they inhabit.

Evidence of horizontal gene transfer by transposase gene analyses in Fervidobacterium species.

Horizontal Gene Transfer (HGT) plays an important role in the physiology and evolution of microorganisms above all thermophilic prokaryotes. Some members of the Phylum Thermotogae (i.e., Thermotoga spp.) have been reported to present genomes constituted by a mosaic of genes from a variety of origins. This study presents a novel approach to search on the potential plasticity of Fervidobacterium genomes using putative transposase-encoding genes as the target of analysis. Transposases are key proteins involved in genomic DNA rearrangements. A comprehensive comparative analysis, including phylogeny, non-metric multidimensional scaling analysis of tetranucleotide frequencies, repetitive flanking sequences and divergence estimates, was performed on the transposase genes detected in four Fervidobacterium genomes: F. nodosum, F. pennivorans, F. islandicum and a new isolate (Fervidobacterium sp. FC2004). Transposase sequences were classified in different groups by their degree of similarity. The different methods used in this study pointed that over half of the transposase genes represented putative HGT events with closest relative sequences within the phylum Firmicutes, being Caldicellulosiruptor the genus showing highest gene sequence proximity. These results confirmed a direct evolutionary relationship through HGT between specific Fervidobacterium species and thermophilic Firmicutes leading to potential gene sequence and functionality sharing to thrive under similar environmental conditions. Transposase-encoding genes represent suitable targets to approach the plasticity and potential mosaicism of bacterial genomes.

Diversity and Distribution of Thermophilic Bacteria in Hot Springs of Pakistan.

Chilas and Hunza areas, located in the Main Mantle Thrust and Main Karakoram Thrust of the Himalayas, host a range of geochemically diverse hot springs. This Himalayan geothermal region encompassed hot springs ranging in temperature from 60 to 95°C, in pH from 6.2 to 9.4, and in mineralogy from bicarbonates (Tato Field), sulfates (Tatta Pani) to mixed type (Murtazaabad). Microbial community structures in these geothermal springs remained largely unexplored to date. In this study, we report a comprehensive, culture-independent survey of microbial communities in nine samples from these geothermal fields by employing a bar-coded pyrosequencing technique. The bacterial phyla Proteobacteria and Chloroflexi were dominant in all samples from Tato Field, Tatta Pani, and Murtazaabad. The community structures however depended on temperature, pH, and physicochemical parameters of the geothermal sites. The Murtazaabad hot springs with relatively higher temperature (90-95°C) favored the growth of phylum Thermotogae, whereas the Tatta Pani thermal spring site TP-H3-b (60°C) favored the phylum Proteobacteria. At sites with low silica and high temperature, OTUs belonging to phylum Chloroflexi were dominant. Deep water areas of the Murtazaabad hot springs favored the sulfur-reducing bacteria. About 40% of the total OTUs obtained from these samples were unclassified or uncharacterized, suggesting the presence of many undiscovered and unexplored microbiota. This study has provided novel insights into the nature of ecological interactions among important taxa in these communities, which in turn will help in determining future study courses in these sites.

Identification and genome reconstruction of abundant distinct taxa in microbiomes from one thermophilic and three mesophilic production-scale biogas plants.

BackgroundBiofuel production from conversion of biomass is indispensable in the portfolio of renewable energies. Complex microbial communities are involved in the anaerobic digestion process of plant material, agricultural residual products and food wastes. Analysis of the genetic potential and microbiology of communities degrading biomass to biofuels is considered to be the key to develop process optimisation strategies. Hence, due to the still incomplete taxonomic and functional characterisation of corresponding communities, new and unknown species are of special interest.ResultsThree mesophilic and one thermophilic production-scale biogas plants (BGPs) were taxonomically profiled using high-throughput 16S rRNA gene amplicon sequencing. All BGPs shared a core microbiome with the thermophilic BGP featuring the lowest diversity. However, the phyla Cloacimonetes and Spirochaetes were unique to BGPs 2 and 3, Fusobacteria were only found in BGP3 and members of the phylum Thermotogae were present only in the thermophilic BGP4. Taxonomic analyses revealed that these distinctive taxa mostly represent so far unknown species. The only exception is the dominant Thermotogae OTU featuring 16S rRNA gene sequence identity to Defluviitoga tunisiensis L3, a sequenced and characterised strain. To further investigate the genetic potential of the biogas communities, corresponding metagenomes were sequenced in a deepness of 347.5 Gbp in total. A combined assembly comprised 80.3 % of all reads and resulted in the prediction of 1.59 million genes on assembled contigs. Genome binning yielded genome bins comprising the prevalent distinctive phyla Cloacimonetes, Spirochaetes, Fusobacteria and Thermotogae. Comparative genome analyses between the most dominant Thermotogae bin and the very closely related Defluviitogatunisiensis L3 genome originating from the same BGP revealed high genetic similarity. This finding confirmed applicability and reliability of the binning approach. The four highly covered genome bins of the other three distinct phyla showed low or very low genetic similarities to their closest phylogenetic relatives, and therefore indicated their novelty.ConclusionsIn this study, the 16S rRNA gene sequencing approach and a combined metagenome assembly and binning approach were used for the first time on different production-scale biogas plants and revealed insights into the genetic potential and functional role of so far unknown species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0565-3) contains supplementary material, which is available to authorized users.

Effect of increased load of high-strength food wastewater in thermophilic and mesophilic anaerobic co-digestion of waste activated sludge on bacterial community structure

In recent years, anaerobic co-digestion (AcoD) has been widely used to improve reactor performance, especially methane production. In this study, we applied two different operating temperatures (thermophilic and mesophilic) and gradually increased the load of food wastewater (FWW) to investigate the bacterial communities during the AcoD of waste activated sludge (WAS) and FWW. As the load of FWW was increased, methane production rate (MPR; L CH4/L d) and methane content (%) in both Thermophilic AcoD (TAcoD) and Mesophilic AcoD (MAcoD) increased significantly; the highest MPR and methane content in TAcoD (1.423 L CH4/L d and 68.24%) and MAcoD (1.233 L CH4/L d and 65.21%) were observed when the FWW mixing ratio was 75%. However, MPR and methane yield in both reactors decreased markedly and methane production in TAcoD ceased completely when only FWW was fed into the reactor, resulting from acidification of the reactor caused by accumulation of organic acids. Pyrosequencing analysis revealed a decrease in bacterial diversity in TAcoD and a markedly different composition of bacterial communities between TAcoD and MAcoD with an increase in FWW load. For example, Bacterial members belonging to two genera Petrotoga (assigned to phylum Thermotogae) and Petrimonas (assigned to phylum Bacteroidetes) became dominant in TAcoD and MAcoD with an increase in FWW load, respectively. In addition, quantitative real-time PCR (qPCR) results showed higher bacterial and archaeal populations (expressed as 16S rRNA gene concentration) in TAcoD than MAcoD with an increase in FWW load and showed maximum population when the FWW mixing ratio was 75% in both reactors. Collectively, this study demonstrated the dynamics of key bacterial communities in TAcoD and MAcoD, which were highly affected by the load of FWW.

Athalassotoga saccharophila gen. nov., sp. nov., isolated from an acidic terrestrial hot spring, and proposal of Mesoaciditogales ord. nov. and Mesoaciditogaceae fam. nov. in the phylum Thermotogae.

A novel moderately thermophilic, weakly acidophilic, heterotrophic, anaerobic, short-rod bacterium having an outer sheath-like structure (toga) was isolated from a low-salt acidic terrestrial hot spring in Oku-Shiobara, Tochigi, Japan. The strain, designated NAS-01T, grew between 30 and 60 °C (optimum 55 °C), and at pH 4.5 and 7.5 (optimum pH 5.5 to 6.0) and could not grow in media with ≥ 1 % NaCl (optimum 0 % NaCl). It utilized Fe(III), thiosulfate or l-cystine as electron acceptor for growth, and yeast extract, peptone or a variety of sugars as carbon and energy sources. The major cellular fatty acid was C16 : 0, and no lipoquinone was detected. Strain NAS-01T contained phospholipids and glycolipids, but not aminolipids. The DNA G+C content was 41.1 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain was included in the phylum Thermotogae, and was closely related to Mesoaciditoga lauensis. The 16S rRNA gene sequence similarity between them was 90 %, and they were deeply branched off the rest of the known Thermotogae species. On the basis of the phylogenetic and ecophysiological properties, strain NAS-01T represents a novel species of a new genus in the phylum Thermotogae, for which we propose the name Athalassotoga saccharophila gen. nov., sp. nov. The type strain of the type species is NAS-01T ( = JCM 19762T = DSM 28954T). In addition, we propose a new order and family, Mesoaciditogales ord. nov. and Mesoaciditogaceae fam. nov., respectively, to accommodate the novel genus and the closely related genus Mesoaciditoga.

Insights into thermoadaptation and the evolution of mesophily from the bacterial phylum Thermotogae.

Thermophiles are extremophiles that grow optimally at temperatures >45 °C. To survive and maintain function of their biological molecules, they have a suite of characteristics not found in organisms that grow at moderate temperature (mesophiles). At the cellular level, thermophiles have mechanisms for maintaining their membranes, nucleic acids, and other cellular structures. At the protein level, each of their proteins remains stable and retains activity at temperatures that would denature their mesophilic homologs. Conversely, cellular structures and proteins from thermophiles may not function optimally at moderate temperatures. These differences between thermophiles and mesophiles presumably present a barrier for evolutionary transitioning between the 2 lifestyles. Therefore, studying closely related thermophiles and mesophiles can help us determine how such lifestyle transitions may happen. The bacterial phylum Thermotogae contains hyperthermophiles, thermophiles, mesophiles, and organisms with temperature ranges wide enough to span both thermophilic and mesophilic temperatures. Genomic, proteomic, and physiological differences noted between other bacterial thermophiles and mesophiles are evident within the Thermotogae. We argue that the Thermotogae is an ideal group of organisms for understanding of the response to fluctuating temperature and of long-term evolutionary adaptation to a different growth temperature range.

Complete genome sequence of the strain Defluviitoga tunisiensis L3, isolated from a thermophilic, production-scale biogas plant

An anaerobic, thermophilic bacterium belonging to the phylum Thermotogae was isolated from a rural, thermophilic biogas plant (54°C) producing methane-rich biogas from maize silage, barley, cattle and pig manure. Here we report the first complete genome sequence of the Defluviitoga tunisiensis strain L3, an isolate from the family Thermotogaceae. The strain L3 encodes several genes predicted to be involved in utilization of a large diversity of complex carbohydrates including cellobiose and xylan for the production of acetate, hydrogen (H2) and carbon dioxide (CO2). The genome sequence of D. tunisiensis L3 provides the basis for biotechnological exploitation of genetic determinants playing an important role in thermophilic fermentation processes utilizing renewable primary products.

Anaerobic codigestion of sewage sludge and glycerol, focusing on process kinetics, microbial dynamics and sludge dewaterability

Anaerobic codigestion (AcoD) is a proven option to significantly boost biogas production while utilizing existing digesters and infrastructure. The aim of the present research was to conduct an exhaustive study regarding anaerobic codigestion of mixed sewage sludge and crude glycerol considering impacts on organic load, hydraulic load, process performance and microbial community. The methane potential of crude glycerol varied from 370 mL CH4·g−1 VS to 483 mL CH4·g−1 VS for different samples tested. The half maximal inhibitory concentration of crude glycerol was 1.01 g VS L−1, and the primary mechanism of inhibition was through overload from rapid fermentation rather than the presence of toxic compounds in the crude glycerol. In continuous operation over 200 days, feeding glycerol at up to 2% v/v, increased organic load by up to 70% and resulted in a 50% increase in methane production. Glycerol dosing resulted in no change in apparent dewaterability, with both codigestion and control reactors returning values of 22%–24%. Members of the phylum Thermotogae emerged as a niche population during AcoD of sewage sludge and glycerol; however there was no gross change in microbial community structure and only minimal changes in diversity. AcoD did not result in synergisms between sewage sludge and crude glycerol. Actually, at dose rate up to 2% v/v glycerol dosing is still an effective strategy to increase the organic loading rate of continuous anaerobic digesters with minimal impact of the hydraulic retention time. Nonetheless, the dose rate must be managed to: (i) prevent process inhibition and (ii) ensure sufficient degradation time to produce a stable biosolids product.

Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations

All species from the phylum Thermotogae, class Thermotogae, are currently part of a single family, Thermotogaceae. Using genomic data from 17 Thermotogae species, detailed phylogenetic and comparative genomic analyses were carried out to understand their evolutionary relationships and identify molecular markers that are indicative of species relationships within the phylum. In the 16S rRNA gene tree and phylogenetic trees based upon two different large sets of proteins, members of the phylum Thermotogae formed a number of well-resolved clades. Character compatibility analysis on the protein sequence data also recovered a single largest clique that exhibited similar topology to the protein trees and where all nodes were supported by multiple compatible characters. Comparative genomic analyses have identified 85 molecular markers, in the form of conserved signature indels (CSIs), which are specific for different observed clades of Thermotogae at multiple phylogenetic depths. Eleven of these CSIs were specific for the phylum Thermotogae whereas nine others supported a clade comprising of the genera Thermotoga, Thermosipho and Fervidobacterium. Ten other CSIs provided evidence that the genera Thermosipho and Fervidobacterium shared a common ancestor exclusive of the other Thermotogae and four and eight CSIs in other proteins were specific for the genera Thermosipho and Fervidobacterium, respectively. Two other deep branching clades, one consisting of the genera Kosmotoga and Mesotoga and the other comprising of the genera Petrotoga and Marinitoga, were also supported by multiple CSIs. Based upon the consistent branching of the Thermotogae species using different phylogenetic approaches, and numerous identified CSIs supporting the distinctness of different clades, it is proposed that the class Thermotogae should be divided into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.). Additionally, the results of our phylogenetic/compatibility studies along with the species distribution patterns of 22 identified CSIs, provide compelling evidence that the current genus Thermotoga is comprised of two evolutionary distinct groups and that it should be divided into two genera. It is proposed that the emended genus Thermotoga should retain only the species Thermotoga maritima, Tt. neapolitana, Tt. petrophila, Tt. naphthophila, Thermotoga sp. EMP, Thermotoga sp. A7A and Thermotoga sp. RQ2 while the other Thermotoga species (viz. Tt. lettingae, Tt. thermarum, Tt. elfii, Tt. subterranean and Tt. hypogea) be transferred to a new genus, Pseudothermotoga gen. nov.