Preparation and analysis of environmental DNA: optimisation of techniques for phylogenetic analysis of ATAD sludge

peer-reviewedNon-culture based analysis of microbial populations via phylogenetic analysis is becoming an increasingly important tool in the determination of microbial communities and community flux in a large number of environmental niches. The results obtained are however only as good as the preparative techniques used to isolate the original templates and the PCR processing techniques used to amplify these templates. In thermal niches such as autothermal thermophilic aerobic digestion systems (ATAD) there are many features that limit the optimal recovery of microbial diversity. Such features include lysis and release of products from the microbial population such as nucleases and proteases which effect both template recovery and polymerase functionality, inhibitory substances from the environmental source and even the nature of the primers used to amplify the resultant templates. In optimisation of the extraction and amplification of environmental DNA from an ATAD system we have identified a number of issues that affect the recovery of diversity which may have widespread applicability to the recovery of diversity by molecular techniques from other environmental niches. Methods of optimisation and analysis of diversity recovery will be discussed.HE

Molecular analysis of bacterial community DNA in sludge undergoing autothermal thermophilic aerobic digestion (ATAD): pitfalls and improved methodology to enhance diversity recovery

peer-reviewedMolecular analysis of the bacterial community structure associated with sludge processed by autothermal thermophilic aerobic digestion (ATAD), was performed using a number of extraction and amplification procedures which differed in yield, integrity, ability to amplify extracted templates and specificity in recovering species present. Interference to PCR and qPCR amplification was observed due to chelation, nuclease activity and the presence of thermolabile components derived from the ATAD sludge. Addition of selected adjuvant restored the ability to amplify community DNA, derived from the thermophilic sludge, via a number of primer sets of ecological importance and various DNA polymerases. Resolution of community profiles by molecular techniques was also influenced by the ATAD sludge extraction procedure as demonstrated by PCR-DGGE profiling and comparison of taxonomic affiliations of the most predominant members within 16S rRNA gene libraries constructed from ATAD DNA extracted by different methods. Several modifications have been shown to be necessary to optimize the molecular analysis of the ATAD thermal niche which may have general applicability to diversity recovery from similar environments

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.peer-reviewe

Secreted Endothelial Cell Factors Immobilized on Collagen Scaffolds Enhance the Recipient Endothelial Cell Environment

Strategies to design novel vascular scaffolds are a continuing aim in tissue engineering and often such designs encompass the use of recombinant factors to enhance the performance of the scaffold. The established use of cell secretion utilized in feeder systems and conditioned media offer a source of paracrine factors, which has potential to be used in tissue-engineered (TE) scaffolds. Here we utilize this principle from endothelial cells (ECs), to create a novel TE scaffold by harnessing secreted factors and immobilizing these to collagen scaffolds. This research revealed increased cellular attachment and positive angiogenic gene upregulation responses in recipient ECs grown on these conditioned scaffolds. Also, the conditioning method did not affect the mechanical structural integrity of the scaffolds. These results may advocate the potential use of this system to improve vascular scaffolds' in vivo performance. In addition, this process may be a future method utilized to improve other tissue engineering scaffold therapies

13C-NMR Assessment of the Pattern of Organic Matter Transformation during Domestic Wastewater Treatment by Autothermal Aerobic Digestion (ATAD)

The pattern of biodegradation and the chemical changes occurring in the macromolecular fraction of domestic sludge during autothermal thermophilic aerobic digestion (ATAD) was monitored and characterised via solid-state 13C-NMR CP-MAS. Major indexes such as aromaticity, hydrophobicity and alkyl/O-alkyl ratios calculated for the ATAD processed biosolids were compared by means of these values to corresponding indexes reported for sludges of different origin such as manures, soil organic matter and certain types of compost. Given that this is the first time that these techniques have been applied to ATAD sludge, the data indicates that long-chain aliphatics are easily utilized by the microbial populations as substrates for metabolic activities at all stages of aerobic digestion and serve as a key substrate for the temperature increase, which in turn results in sludge sterilization. The ATAD biosolids following treatment had a prevalence of O-alkyl domains, a low aromaticity index (10.4%) and an alkyl/O-alkyl ratio of 0.48 while the hydrophobicity index of the sludge decreased from 1.12 to 0.62 during the treatment. These results have important implications for the evolution of new ATAD modalities particularly in relation to dewatering and the future use of ATAD processed biosolids as a fertilizer, particularly with respect to hydrological impacts on the soil behaviour

The Tissue-Engineered Vascular Graft-Past, Present, and Future

Cardiovascular disease is the leading cause of death worldwide, with this trend predicted to continue for the foreseeable future. Common disorders are associated with the stenosis or occlusion of blood vessels. The preferred treatment for the long-term revascularization of occluded vessels is surgery utilizing vascular grafts, such as coronary artery bypass grafting and peripheral artery bypass grafting. Currently, autologous vessels such as the saphenous vein and internal thoracic artery represent the gold standard grafts for small-diameter vessels (<6 mm), outperforming synthetic alternatives. However, these vessels are of limited availability, require invasive harvest, and are often unsuitable for use. To address this, the development of a tissue-engineered vascular graft (TEVG) has been rigorously pursued. This article reviews the current state of the art of TEVGs. The various approaches being explored to generate TEVGs are described, including scaffold-based methods (using synthetic and natural polymers), the use of decellularized natural matrices, and tissue self-assembly processes, with the results of various in vivo studies, including clinical trials, highlighted. A discussion of the key areas for further investigation, including graft cell source, mechanical properties, hemodynamics, integration, and assessment in animal models, is then presented

Molecular Analysis of Bacterial Community DNA in Sludge Undergoing Autothermal Thermophilic Aerobic Digestion (ATAD): Pitfalls and Improved Methodology to Enhance Diversity Recovery

Molecular analysis of the bacterial community structure associated with sludge processed by autothermal thermophilic aerobic digestion (ATAD), was performed using a number of extraction and amplification procedures which differed in yield, integrity, ability to amplify extracted templates and specificity in recovering species present. Interference to PCR and qPCR amplification was observed due to chelation, nuclease activity and the presence of thermolabile components derived from the ATAD sludge. Addition of selected adjuvant restored the ability to amplify community DNA, derived from the thermophilic sludge, via a number of primer sets of ecological importance and various DNA polymerases. Resolution of community profiles by molecular techniques was also influenced by the ATAD sludge extraction procedure as demonstrated by PCR-DGGE profiling and comparison of taxonomic affiliations of the most predominant members within 16S rRNA gene libraries constructed from ATAD DNA extracted by different methods. Several modifications have been shown to be necessary to optimize the molecular analysis of the ATAD thermal niche which may have general applicability to diversity recovery from similar environments