Integrative Conjugative Elements (ICEs) of the SXT/R391 family drive adaptation and evolution in γ-Proteobacteria.

Integrative Conjugative Elements (ICEs) are mosaics containing functional modules allowing maintenance by site-specific integration and excision into and from the host genome and conjugative transfer to a specific host range. Many ICEs encode a range of adaptive functions that aid bacterial survival and evolution in a range of niches. ICEs from the SXT/R391 family are found in γ-Proteobacteria. Over 100 members have undergone epidemiological and molecular characterization allowing insight into their diversity and function. Comparative analysis of SXT/R391 elements from a wide geographic distribution has revealed conservation of key functions, and the accumulation and evolution of adaptive genes. This evolution is associated with gene acquisition in conserved hotspots and variable regions within the SXT/R391 ICEs catalysed via element-encoded recombinases. The elements can carry IS elements and transposons, and a mutagenic DNA polymerase, PolV, which are associated with their evolution. SXT/R391 ICEs isolated from different niches appear to have retained adaptive functions related to that specific niche; phage resistance determinants in ICEs carried by wastewater bacteria, antibiotic resistance determinants in clinical isolates and metal resistance determinants in bacteria recovered from polluted environments/ocean sediments. Many genes found in the element hotspots are undetermined and have few homologs in the nucleotide databases.

Novel Corrugated Long Period Grating Surface Balloon-Shaped Heterocore-Structured Plastic Optical Fibre Sensor for Microalgal Bioethanol Production.

A novel long period grating (LPG) inscribed balloon-shaped heterocore-structured plastic optical fibre (POF) sensor is described and experimentally demonstrated for real-time measurement of the ultra-low concentrations of ethanol in microalgal bioethanol production applications. The heterocore structure is established by coupling a 250 µm core diameter POF between two 1000 µm diameter POFs, thus representing a large core-small core-large core configuration. Before coupling as a heterocore structure, the sensing region or small core fibre (SCF; i.e., 250 µm POF) is modified by polishing, LPG inscription, and macro bending into a balloon shape to enhance the sensitivity of the sensor. The sensor was characterized for ethanol-water solutions in the ethanol concentration ranges of 20 to 80 %v/v, 1 to 10 %v/v, 0.1 to 1 %v/v, and 0.00633 to 0.0633 %v/v demonstrating a maximum sensitivity of 3 × 106 %/RIU, a resolution of 7.9 × 10-6 RIU, and a limit of detection (LOD) of 9.7 × 10-6 RIU. The experimental results are included for the intended application of bioethanol production using microalgae. The characterization was performed in the ultra-low-level ethanol concentration range, i.e., 0.00633 to 0.03165 %v/v, that is present in real culturing and production conditions, e.g., ethanol-producing blue-green microalgae mixtures. The sensor demonstrated a maximum sensitivity of 210,632.8 %T/%v/v (or 5 × 106 %/RIU as referenced from the RI values of ethanol-water solutions), resolution of 2 × 10-4%v/v (or 9.4 × 10-6 RIU), and LOD of 4.9 × 10-4%v/v (or 2.3 × 10-5 RIU). Additionally, the response and recovery times of the sensor were investigated in the case of measurement in the air and the ethanol-microalgae mixtures. The experimentally verified, extremely high sensitivity and resolution and very low LOD corresponding to the initial rate of bioethanol production using microalgae of this sensor design, combined with ease of fabrication, low cost, and wide measurement range, makes it a promising candidate to be incorporated into the bioethanol production industry as a real-time sensing solution as well as in other ethanol sensing and/or RI sensing applications.

A Review of Optical Fibre Ethanol Sensors: Current State and Future Prospects.

A range of optical fibre-based sensors for the measurement of ethanol, primarily in aqueous solution, have been developed and are reviewed here. The sensing approaches can be classified into four groups according to the measurement techniques used, namely absorption (or absorbance), external interferometric, internal fibre grating and plasmonic sensing. The sensors within these groupings can be compared in terms of their characteristic performance indicators, which include sensitivity, resolution and measurement range. Here, particular attention is paid to the potential application areas of these sensors as ethanol production is globally viewed as an important industrial activity. Potential industrial applications are highlighted in the context of the emergence of the internet of things (IoT), which is driving widespread utilization of these sensors in the commercially significant industrial and medical sectors. The review concludes with a summary of the current status and future prospects of optical fibre ethanol sensors for industrial use.

On the bacteriostatic activity of hyaluronic acid composite films.

Biofilm-related infections and contamination of biomaterials are major problems in the clinic. These contaminations are frequently caused by Staphylococcus aureus and are a pressing issue for implantable devices, catheters, contact lenses, prostheses, and wound dressings. Strategies to decrease contamination and biofilm related infections are vital for the success of implantable biomaterials. In this context, hyaluronic acid (HA), a naturally derived carbohydrate polymer, known to be biocompatible, degradable, and immunomodulatory, has shown some antimicrobial activity effects. Due to its poor structural stability, crosslinking strategies, and the incorporation of reinforcing fibres in HA gels is required to produce tailored gels for varying applications. Whilst carbon-based reinforcing materials, such as carbon nanofibers (CNF), present some intrinsic antimicrobial activity related to their high surface area, herein, a crosslinking strategy to enhance the mechanical properties and regulate the rate of degradation of HA is presented. We utilise bis-(ß-isocyanatoethyl) disulphide (BIED) as the crosslinker with the gel reinforced using 0.25 wt% CNF. The effects of CNF and BIED on the structural, mechanical, thermal, and swelling behaviour are examined. These new HA derivatives exhibit excellent mechanical properties and are capable of withstanding physiological stresses in vivo. Antimicrobial activity of the HA derivatives were tested against Staphylococcus aureus and the results reveal antibacterial effect. These carbohydrate based materials have potential application on surfaces within clinical settings where staphylococcal contamination is currently an issue.

The Genus Ochrobactrum as Major Opportunistic Pathogens.

Ochrobactrum species are non-enteric, Gram-negative organisms that are closely related to the genus Brucella. Since the designation of the genus in 1988, several distinct species have now been characterised and implicated as opportunistic pathogens in multiple outbreaks. Here, we examine the genus, its members, diagnostic tools used for identification, data from recent Ochrobactrum whole genome sequencing and the pathogenicity associated with reported Ochrobactrum infections. This review identified 128 instances of Ochrobactrum spp. infections that have been discussed in the literature. These findings indicate that infection review programs should consider investigation of possible Ochrobactrum spp. outbreaks if these bacteria are clinically isolated in more than one patient and that Ochrobactrum spp. are more important pathogens than previously thought.

Zymobacter palmae Pyruvate Decarboxylase is Less Effective Than That of Zymomonas mobilis for Ethanol Production in Metabolically Engineered Synechocystis sp. PCC6803.

To produce bioethanol from model cyanobacteria such as Synechocystis, a two gene cassette consisting of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are required to transform pyruvate first to acetaldehyde and then to ethanol. However the partition of pyruvate to ethanol comes at a cost, a reduction in biomass and pyruvate availability for other metabolic processes. Hence strategies to divert flux to ethanol as a biofuel in Synechocystis are of interest. PDC from Zymobacter palmae (ZpPDC) has been reported to have a lower Km then the Zymomonas mobilis PDC (ZmPDC), which has traditionally been used in metabolic engineering constructs. The Zppdc gene was combined with the native slr1192 alcohol dehydrogenase gene (adhA) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 over constructs created with the traditional Zmpdc. Native (Zppdc) and codon optimized (ZpOpdc) versions of the ZpPDC were cloned into a construct where pdc expression was controlled via the psbA2 light inducible promoter from Synechocystis sp. PCC 6803. These constructs were transformed into wildtype Synechocystis sp. PCC 6803 for expression and ethanol production. Ethanol levels were then compared with identical constructs containing the Zmpdc. While strains with the Zppdc (UL071) and ZpOpdc (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL070) expressing the pdc from Zymomonas mobilis. All constructs demonstrated lower biomass productivity illustrating that the flux from pyruvate to ethanol has a major effect on biomass and ultimately overall biofuel productivity. Thus the utilization of a PDC with a lower Km from Zymobacter palmae unusually did not result in enhanced ethanol production in Synechocystis sp. PCC 6803.

Autothermal Thermophilic Aerobic Digestion (ATAD) for Heat, Gas, and Production of a Class A Biosolids with Fertilizer Potential.

Autothermal thermophilic aerobic digestion (ATAD) is a microbial fermentation process characterized as a tertiary treatment of waste material carried out in jacketed reactors. The process can be carried out on a variety of waste sludge ranging from human, animal, food, or pharmaceutical waste where the addition of air initiates aerobic digestion of the secondary treated sludge material. Digestion of the sludge substrates generates heat, which is retained within the reactor resulting in elevation of the reactor temperature to 70-75 °C. During the process, deamination of proteinaceous materials also occurs resulting in liberation of ammonia and elevation of pH to typically pH 8.4. These conditions result in a unique microbial consortium, which undergoes considerable dynamic change during the heat-up and holding phases. The change in pH and substrate as digestion occurs also contributes to this dynamic change. Because the large reactors are not optimized for aeration, and because low oxygen solubility at elevated temperatures occurs, there are considerable numbers of anaerobes recovered which also contributes to the overall digestion. As the reactors are operated in a semi-continuous mode, the reactors are rarely washed, resulting in considerable biofilm formation. Equally, because of the fibrous nature of the sludge, fiber adhering organisms are frequently found which play a major role in the overall digestion process. Here, we review molecular tools needed to examine the ATAD sludge consortia, what has been determined through phylogenetic analysis of the consortia and the nature of the dynamics occurring within this unique fermentation environment.

A Novel Arsenate-Resistant Determinant Associated with ICEpMERPH, a Member of the SXT/R391 Group of Mobile Genetic Elements.

ICEpMERPH, the first integrative conjugative element (ICE) of the SXT/R391 family isolated in the United Kingdom and Europe, was analyzed to determine the nature of its adaptive functions, its genetic structure, and its homology to related elements normally found in pathogenic Vibrio or Proteus species. Whole genome sequencing of Escherichia coli (E. coli) isolate K802 (which contains the ICEpMERPH) was carried out using Illumina sequencing technology. ICEpMERPH has a size of 110 Kb and 112 putative open reading frames (ORFs). The "hotspot regions" of the element were found to contain putative restriction digestion systems, insertion sequences, and heavy metal resistance genes that encoded resistance to mercury, as previously reported, but also surprisingly to arsenate. A novel arsenate resistance system was identified in hotspot 4 of the element, unrelated to other SXT/R391 elements. This arsenate resistance system was potentially linked to two genes: orf69, encoding an organoarsenical efflux major facilitator superfamily (MFS) transporter-like protein related to ArsJ, and orf70, encoding nicotinamide adenine dinucleotide (NAD)-dependent glyceraldehyde-3-phosphate dehydrogenase. Phenotypic analysis using isogenic strains of Escherichia coli strain AB1157 with and without the ICEpMERPH revealed resistance to low levels of arsenate in the range of 1-5 mM. This novel, low-level resistance may have an important adaptive function in polluted environments, which often contain low levels of arsenate contamination. A bioinformatic analysis on the novel determinant and the phylogeny of ICEpMERPH was presented.

Evaluation of the ethanol tolerance for wild and mutant Synechocystis strains by flow cytometry.

Flow cytometry was used to evaluate the effect of initial ethanol concentrations on cyanobacterial strains of Synechocystis PCC 6803 [wild-type (WT), and ethanol producing recombinants (UL 004 and UL 030)] in batch cultures. Ethanol recombinants, containing one or two metabolically engineered cassettes, were designed towards the development of an economically competitive process for the direct production of bioethanol from microalgae through an exclusive autotrophic route. It can be concluded that the recombinant Synechocystis UL 030 containing two copies of the genes per genome was the most tolerant to ethanol. Nevertheless, to implement a production process using recombinant strains, the bioethanol produced will be required to be continuously extracted from the culture media via a membrane-based technological process for example to prevent detrimental effects on the biomass. The results presented here are of significance in defining the maximum threshold for bulk ethanol concentration in production media.

Brevundimonas spp: Emerging global opportunistic pathogens.

Non-fermenting Gram-negative bacteria are problematic in clinical locations, being one of the most prevalent causes of nosocomial infections. Many of these non-fermenting Gram-negative bacteria are opportunistic pathogens that affect patients that are suffering with underlying medical conditions and diseases. Brevundimonas spp., in particular Brevundimonas diminuta and Brevundimonas vesicularis, are a genus of non-fermenting Gram-negative bacteria considered of minor clinical importance. Forty-nine separate instances of infection relating to Brevundimonas spp were found in the scientific literature along with two pseudo-infections. The majority of these instances were infection with Brevundimonas vesicularis (thirty-five cases - 71%). The major condition associated with Brevundimonas spp infection was bacteraemia with seventeen individual cases/outbreaks (35%). This review identified forty-nine examples of Brevundimonas spp. infections have been discussed in the literature. These findings indicate that infection review programs should consider investigation of possible Brevundimonas spp outbreaks if these bacteria are clinically isolated in more than one patient.

Analysis and comparative genomics of R997, the first SXT/R391 integrative and conjugative element (ICE) of the Indian Sub-Continent.

The aim of this study was to analyse R997, the first integrative and conjugative element (ICE) isolated from the Indian Sub-Continent, and to determine its relationship to the SXT/R391 family of ICEs. WGS of Escherichia coli isolate AB1157 (which contains R997) was performed using Illumina sequencing technology. R997 context was assessed by de novo assembly, gene prediction and annotation tools, and compared to other SXT/R391 ICEs. R997 has a size of 85 Kb and harbours 85 ORFs. Within one of the variable regions a HMS-1 ß-lactamase resistance gene is located. The Hotspot regions of the element contains restriction digestion systems and insertion sequences. R997 is very closely related to the SXT-like elements from widely dispersed geographic areas. The sequencing of R997 increases the knowledge of the earliest isolated SXT/R391 elements and may provide insight on the emergence of these elements on the Indian sub-continent.

Mutagenic activities of biochars from pyrolysis.

Biochar production, from pyrolysis of lignocellulosic feedstocks, agricultural residues, and animal and poultry manures are emerging globally as novel industrial and commercial products. It is important to develop and to validate a series of suitable protocols for the ecological monitoring of the qualities and properties of biochars. The highly sensitive Salmonella mutagenicity assays (the Ames test) are used widely by the toxicology community and, via the rat liver extract (S9), can reflect the potential for mammalian metabolic activation. We examined the Ames test for analyses of the mutagenic activities of dimethylsulphoxide (DMSO) extracts of biochars using two bacterial models (S. typhimurium strains TA98 and TA100) in the presence and in the absence of the metabolic activation with the S9-mix. Tester strain TA98 was most sensitive in detecting mutagenic biochar products, and the contribution of S9 was established. Temperature and times of pyrolysis are important. Biochar pyrolysed at 400°C for 10min, from a lignocellulose precursor was mutagenic, but not when formed at 800°C for 60min, or at 600°C for 30min. Biochars from poultry litter, and manures of calves fed on grass had low mutagenicities. Biochar from pig manure had high mutagenicity; biochars from manures of cows fed on a grass plus cereals, those of calves fed on mother's milk, and biochars from solid industrial waste had intermediate mutagenicities. The methods outlined can indicate the need for further studies for screening and detection of the mutagenic residuals in a variety of biochar products.

SXT/R391 Integrative and Conjugative Elements (ICEs) Encode a Novel 'Trap-Door' Strategy for Mobile Element Escape.

Integrative conjugative elements (ICEs) are a class of bacterial mobile elements that have the ability to mediate their own integration, excision, and transfer from one host genome to another by a mechanism of site-specific recombination, self-circularisation, and conjugative transfer. Members of the SXT/R391 ICE family of enterobacterial mobile genetic elements display an unusual UV-inducible sensitization function which results in stress induced killing of bacterial cells harboring the ICE. This sensitization has been shown to be associated with a stress induced overexpression of a mobile element encoded conjugative transfer gene, orf43, a traV homolog. This results in cell lysis and release of a circular form of the ICE. Induction of this novel system may allow transfer of an ICE, enhancing its survival potential under conditions not conducive to conjugative transfer.

Utilising the native plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC6803 as a cloning site for enhanced product production.

BACKGROUND: The use of photosynthetic autotrophs and in particular the model organism Synechocystis PCC6803 is receiving much attention for the production of sustainable biofuels and other economically useful products through metabolic engineering. Optimisation of metabolic-engineered organisms for high-level sustained production of product is a key element in the manipulation of this organism. A limitation to the utilisation of metabolically-engineered Synechocystis PCC6803 is the availability of strong controllable promoters and stable gene dosage methods for maximising gene expression and subsequent product formation following genetic manipulation. RESULTS: A native Synechocystis PCC6803 small plasmid, pCA2.4, is consistently maintained at a copy level of up to 7 times that of the polyploid chromosome. As this plasmid is stable during cell division, it is potentially an ideal candidate for maximising gene dosage levels within the organism. Here, we describe the construction of a novel expression vector generated from the native plasmid, pCA2.4. To investigate the feasibility of this new expression system, a yellow fluorescent protein (YFP) encoding gene was cloned downstream of the strong Ptrc promoter and integrated into a predicted neutral site within the pCA2.4 plasmid. The stability of the integrated construct was monitored over time compared to a control strain containing an identical YFP-expressing construct integrated at a known neutral site in a chromosomal location. CONCLUSIONS: A significantly higher fluorescence level of the yellow fluorescent protein was observed when its encoded gene was integrated into the pCA2.4 native plasmid when compared to the isogenic chromosomally integrated control strain. On average, a minimum of 20-fold higher fluorescence level could be achieved from integration into the native plasmid. Fluorescence was also monitored as a function of culture time and demonstrated to be stable over multiple sub-cultures even after the removal of selective pressure. Therefore, the native small plasmid, pCA2.4 may be utilised to stably increase gene expression levels in Synechocystis PCC6803. With the complementary utilisation of an inducible promoter system, rapid generation of commodity-producing Synechocystis PCC6803 strains having high level, controlled expression may be more achievable.

Cation Diffusion Facilitator family: Structure and function.

The Cation Diffusion Facilitators (CDFs) form a family of membrane-bound proteins capable of transporting zinc and other heavy metal ions. Involved in metal tolerance/resistance by efflux of ions, CDF proteins share a two-modular architecture consisting of a transmembrane domain (TMD) and C-terminal domain (CTD) that protrudes into the cytoplasm. Discovery of a Zn²âº and Cd²âº CDF transporter from a marine bacterium Maricaulis maris that does not possess the CTD questions current perceptions regarding this family of proteins. This article describes a new, CTD-lacking subfamily of CDFs and our current knowledge about this family of proteins in the view of these findings.

Examination of the cell sensitizing gene orf43 of ICE R391 suggests a role in ICE transfer enhancement to recipient cells.

SXT/R391 family of ICEs have been found to express an unusual function that enhances bacterial cell death post-UV irradiation. Previous analysis of ICE R391 found four core SXT/R391 ICE genes to be involved­orf96, orf90, orf91 and orf43. These genes functioned as part of a UV-inducible pathway, where upon exposure to UV, the levels of the Orf43 protein, a TraV homolog which we propose naming TraV(R391), were upregulated, resulting in increased cell sensitization. Here, we examined the effect of orf43 overexpression and found it led to host cell permeabilization. The inducing agent for orf43, UV irradiation, is also known to increase the ICE R391 extrachromosomal form and apparent conjugative transfer rate. We demonstrated, via conjugative transfer deficient mutants, that orf43 overexpression alone restored a small level of ICE R391 transfer to recipient cells via an unknown mechanism other than conjugation. TraV homologs have been reported to function in conjugative transfer. However, TraV(R391) is the first homolog to cause UV-associated cell sensitization. TraV(R391) when overexpressed must contain a unique adaptation or function which results in cell lysis and decreased survival. A hypothesis for retaining such a detrimental effect may be in its role of enhancing ICE survival upon cell damage.

Control of expression of the ICE R391 encoded UV-inducible cell-sensitising function.

BACKGROUND: Many SXT/R391-like enterobacterial Integrative Conjugative Elements (ICEs) have been found to express an atypical, recA-dependent, UV-inducible, cell-sensitising phenotype observed as a reduction in post-irradiation cell survival rates in host cells. Characterisation of a complete deletion library of the prototype ICE R391 identified the involvement of three core ICE genes, orfs90/91 encoding a putative transcriptional enhancer complex, and orf43, encoding a putative type IV secretion system, outer membrane-associated, conjugative transfer protein. RESULTS: In this study, expression analysis of orf43 indicated that it was up-regulated as a result of UV irradiation in an orfs90/91-dependent manner. Induced expression was found to be controlled from a site preceding the gene which required functional orfs90/91. Expression of orfs90/91 was in turn found to be regulated by orf96, a λ cI-like regulator. Targeted construction of ICE R391 deletions, RT-PCR and qRT-PCR analysis confirmed a regulatory link between orfs90/91 and orf43 while site-directed mutagenesis of orf43 suggested an association with the cell membrane was a prerequisite for the cytotoxic effect. CONCLUSIONS: Because of the recA-dependence of the effect, we hypothesise that UV induction of RecA results in cleavage of the cI-like ICE-encoded repressor protein, the product of orf96. This in turn allows expression of the transcriptional enhancer complex encoded by orfs90/91, which we conclude stimulates transcription of orf43, whose product is directly responsible for the effect.

Generation and analysis of an ICE R391 deletion library identifies genes involved in the element encoded UV-inducible cell-sensitising function.

ICE R391, a prototype member of the SXT/R391 family of site-specific integrative conjugative elements (ICEs), frequently isolated from enterobacterial pathogens, exhibits an unusual, recA-dependent, UV-inducible, cell-sensitising function. This significantly decreases postirradiation cell survival rates in Escherichia coli host cells, a trait that would at first appear to be counterproductive in terms of adaptation to stress conditions. Construction and screening of a complete ICE R391 deletion library in E. coli identified three ICE R391 genes, orfs90/91, encoding a putative transcriptional enhancer, and orf43, encoding a putative type IV secretion system outer membrane-associated conjugative transfer protein, in the cell-sensitising function. Cloning and complementation of these genes confirmed their involvement in UV sensitising. Expression of both orfs90/91 and orf43 in wild-type E. coli indicated that orf43 encodes a cytotoxic gene product upon up-regulation. Deletion of the orf43 homologue in SXT, s050, also abolished its associated UV sensitisation. We hypothesise that ICE R391 and other members of the SXT/R391 family display decreased survival rates upon exposure to UV irradiation through the induction of orf43.

Use of PCR-DGGE Based Molecular Methods to Analyse Microbial Community Diversity and Stability during the Thermophilic Stages of an ATAD Wastewater Sludge Treatment Process as an Aid to Performance Monitoring.

PCR and PCR-DGGE techniques have been evaluated to monitor biodiversity indexes within an ATAD (autothermal thermophilic aerobic digestion) system treating domestic sludge for land spread, by examining microbial dynamics in response to elevated temperatures during treatment. The ATAD process utilises a thermophilic population to generate heat and operates at elevated pH due to degradation of sludge solids, thus allowing pasteurisation and stabilisation of the sludge. Genera-specific PCR revealed that Archaea, Eukarya and Fungi decline when the temperature reaches 59°C, while the bacterial lineage constitutes the dominant group at this stage. The bacterial community at the thermophilic stage, its similarity index to the feed material, and the species richness present were evaluated by PCR-DGGE. Parameters such as choice of molecular target (16S rDNA or rpoB genes), and electrophoresis condition, were optimised to maximise the resolution of the method for ATAD. Dynamic analysis of microbial communities was best observed utilising PCR-DGGE analysis of the V6-V8 region of 16S rDNA, while rpoB gene profiles were less informative. Unique thermophilic communities were shown to quickly adapt to process changes, and shown to be quite stable during the process. Such techniques may be used as a monitoring technique for process health and efficiency.

Phylogenetic analysis of the bacterial community in a full scale autothermal thermophilic aerobic digester (ATAD) treating mixed domestic wastewater sludge for land spread.

The bacterial community associated with a full scale autothermal thermophilic aerobic digester (ATAD) treating sludge, originating from domestic wastewater and destined for land spread, was analysed using a number of molecular approaches optimised specifically for this high temperature environment. 16S rDNA genes were amplified directly from sludge with universally conserved and Bacteria-specific rDNA gene primers and a clone library constructed that corresponded to the late thermophilic stage (t = 23 h) of the ATAD process. Sequence analyses revealed various 16S rDNA gene sequence types reflective of high bacterial community diversity. Members of the bacterial community included α- and ß-Proteobacteria, Actinobacteria with High G + C content and Gram-Positive bacteria with a prevalence of the Firmicutes (Low G + C) division (class Clostridia and Bacillus). Most of the ATAD clones showed affiliation with bacterial species previously isolated or detected in other elevated temperature environments, at alkaline pH, or in cellulose rich environments. Several phylotypes associated with Fe(III)- and Mn(IV)-reducing anaerobes were also detected. The presence of anaerobes was of interest in such large scale systems where sub-optimal aeration and mixing is often the norm while the presence of large amounts of capnophiles suggest the possibility of limited convection and entrapment of CO(2) within the sludge matrix during digestion. Comparative analysis with organism identified in other ATAD systems revealed significant differences based on optimised techniques. The abundance of thermophilic, alkalophilic and cellulose-degrading phylotypes suggests that these organisms are responsible for maintaining the elevated temperature at the later stages of the ATAD process.