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Abstract Background Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. Results We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared. Conclusions The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.

Abstract Background Brachypodium distachyon constitutes an excellent model species for grasses. It is a small, easily propagated, temperate grass with a rapid life cycle and a small genome. It is a self-fertile plant that can be transformed with high efficiency using Agrobacteria and callus derived from immature embryos. In addition, considerable genetic and genomic resources are becoming available for this species in the form of mapping populations, large expressed sequence tag collections, T-DNA insertion lines and, in the near future, the complete genome sequence. The development of Brachypodium as a model species is of particular value in the areas of cell wall and biomass research, where differences between dicots and grasses are greatest. Here we explore the effect of mild conditions of pretreatment and hydrolysis in Brachypodium stem segments as a contribution for the establishment of sensitive screening of the saccharification properties in different genetic materials. Results The non-cellulosic monosaccharide composition of Brachypodium is closely related to grasses of agricultural importance and significantly different from the dicot model Arabidopsis thaliana. Diluted acid pretreatment of stem segments produced significant release of sugars and negatively affected the amount of sugars obtained by enzymatic hydrolysis. Monosaccharide and oligosaccharide analysis showed that the hemicellulose fraction is the main target of the enzymatic activity under the modest hydrolytic conditions used in our assays. Scanning electron microscopy analysis of the treated materials showed progressive exposure of fibrils in the stem segments. Conclusion Results presented here indicate that under mild conditions cellulose and hemicellulose are hydrolysed to differing extents, with hemicellulose hydrolysis predominating. We anticipate that the sub-optimal conditions for hydrolysis identified here will provide a sensitive assay to detect variations in saccharification among Brachypodium plants, providing a useful analytical tool for identifying plants with alterations in this trait.

Abstract Malaria is a major public health problem for countries in the Southern Africa Development Community (SADC). While the endemicity of malaria varies enormously across this region, many of the countries have districts that are prone to periodic epidemics, which can be regional in their extent, and to resurgent outbreaks that are much more localized. These epidemics are frequently triggered by climate anomalies and often follow periods of drought. Many parts of Southern Africa have suffered rainfall deficit over the past three years and countries expect to see increased levels of malaria when the rains return to more 'normal' levels. Problems with drug and insecticide resistance are documented widely and the region contains countries with the highest rates of HIV prevalence to be found anywhere in the world. Consequently, many communities are vulnerable to severe disease outcomes should epidemics occur. The SADC countries have adopted the Abuja targets for Roll Back Malaria in Africa, which include improved epidemic detection and response, i.e., that 60% of epidemics will be detected within two weeks of onset, and 60% of epidemics will be responded to within two weeks of detection. The SADC countries recognize that to achieve these targets they need improved information on where and when to look for epidemics. The WHO integrated framework for improved early warning and early detection of malaria epidemics has been recognized as a potentially useful tool for epidemic preparedness and response planning. Following evidence of successful adoption and implementation of this approach in Botswana, the SADC countries, the WHO Southern Africa Inter-Country Programme on Malaria Control, and the SADC Drought Monitoring Centre decided to organize a regional meeting where countries could gather to assess their current control status and community vulnerability, consider changes in epidemic risk, and develop a detailed plan of action for the forthcoming 2004–2005 season. The following is a report on the 1st Southern African Regional Epidemic Outlook Forum, which was held in Harare, Zimbabwe, 26th–29th September, 2004.

Little has been documented about the benefits and impacts of the recent growth in climate services, despite a growing call to justify their value and stimulate investment. Regional Climate Outlook Forums (RCOFs), an integral part of the public and private enterprise of climate services, have been implemented over the last 20 years with the objectives of producing and disseminating seasonal climate forecasts to inform improved climate risk management and adaptation. In proposing guidance on how to measure the success of RCOFs, we offer three broad evaluative categories that are based on the primary stated goals of the RCOFs: 1) quality of the climate information used and developed at RCOFs; 2) legitimacy of RCOF processes focused on consensus forecasts, broad user engagement, and capacity building; and 3) usability of the climate information produced at RCOFs. Evaluating the quality of information relies largely on quantitative measures and statistical techniques that are standardized and transferrable, but assessing the RCOF processes and perceived usability of RCOF products will necessitate a combination of quantitative and qualitative social science methods that are sensitive to highly variable regional contexts. As RCOFs have taken up different formats and procedures to adapt to diverse institutional and political settings and varied technical and scientific capacities, objective evaluation methods adopted should align with the goals and intent of the evaluation and be performed in a participatory, coproduction manner where producers and users of climate services together design the evaluation metrics and processes. To fully capture the potential benefits of the RCOFs, it may be necessary to adjust or recalibrate the goals of these forums to better fit the evolving landscape of climate services development, needs, and provision. [ABSTRACT FROM AUTHOR]

Background: Worldwide 3.4 billion tonnes of municipal solid waste (MSW) will be produced annually by 2050, however, current approaches to MSW management predominantly involve unsustainable practices like landfilling and incineration. The organic fraction of MSW (OMSW) typically comprises ~ 50% lignocellulose-rich material but is underexplored as a biomanufacturing feedstock due to its highly inconsistent and heterogeneous composition. This study sought to overcome the limitations associated with studying MSW-derived feedstocks by using OMSW produced from a realistic and reproducible MSW mixture on a commercial autoclave system. The resulting OMSW fibre was enzymatically hydrolysed and used to screen diverse microorganisms of biotechnological interest to identify robust species capable of fermenting this complex feedstock. Results: The autoclave pre-treated OMSW fibre contained a polysaccharide fraction comprising 38% cellulose and 4% hemicellulose. Enzymatic hydrolysate of OMSW fibre was high in d-glucose (5.5% w/v) and d-xylose (1.8%w/v) but deficient in nitrogen and phosphate. Although relatively low levels of levulinic acid (30 mM) and vanillin (2 mM) were detected and furfural and 5-hydroxymethylfurfural were absent, the hydrolysate contained an abundance of potentially toxic metals (0.6% w/v). Hydrolysate supplemented with 1% yeast extract to alleviate nutrient limitation was used in a substrate-oriented shake-flask screen with eight biotechnologically useful microorganisms (Clostridium saccharoperbutylacetonicum, Escherichia coli, Geobacillus thermoglucosidasius, Pseudomonas putida, Rhodococcus opacus, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Zymomonas mobilis). Each species' growth and productivity were characterised and three species were identified that robustly and efficiently fermented OMSW fibre hydrolysate without significant substrate inhibition: Z. mobilis, S. cerevisiae and R. opacus, respectively produced product to 69%, 70% and 72% of the maximum theoretical fermentation yield and could theoretically produce 136 kg and 139 kg of ethanol and 91 kg of triacylglycerol (TAG) per tonne of OMSW. Conclusions: Developing an integrated biorefinery around MSW has the potential to significantly alleviate the environmental burden of current waste management practices. Substrate-oriented screening of a representative and reproducible OMSW-derived fibre identified microorganisms intrinsically suited to growth on OMSW hydrolysates. These species are promising candidates for developing an MSW biorefining platform and provide a foundation for future studies aiming to valorise this underexplored feedstock. [ABSTRACT FROM AUTHOR]

Background: The fungus Aspergillus niger is an important industrial organism for citric acid fermentation; one of the most efficient biotechnological processes. Previously we introduced a dynamic model that captures this process in the industrially relevant batch fermentation setting, providing a more accurate predictive platform to guide targeted engineering. In this article we exploit this dynamic modelling framework, coupled with a robust genetic algorithm for the in silico evolution of A. niger organic acid production, to provide solutions to complex evolutionary goals involving a multiplicity of targets and beyond the reach of simple Boolean gene deletions. We base this work on the latest metabolic models of the parent citric acid producing strain ATCC1015 dedicated to organic acid production with the required exhaustive genomic coverage needed to perform exploratory in silico evolution. Results: With the use of our informed evolutionary framework, we demonstrate targeted changes that induce a complete switch of acid output from citric to numerous different commercially valuable target organic acids including succinic acid. We highlight the key changes in flux patterns that occur in each case, suggesting potentially valuable targets for engineering. We also show that optimum acid productivity is achieved through a balance of organic acid and biomass production, requiring finely tuned flux constraints that give a growth rate optimal for productivity. Conclusions: This study shows how a genome-scale metabolic model can be integrated with dynamic modelling and metaheuristic algorithms to provide solutions to complex metabolic engineering goals of industrial importance. This framework for in silico guided engineering, based on the dynamic batch growth relevant to industrial processes, offers considerable potential for future endeavours focused on the engineering of organisms to produce valuable products. [ABSTRACT FROM AUTHOR]

Biotechnologies that aim to produce renewable fuels, chemicals, and bioproducts from residual ligno(hemi)cellulosic biomass mostly rely on enzymatic depolymerization of plant cell walls (PCW). This process requires an arsenal of diverse enzymes, including xylanases, which synergistically act on the hemicellulose, reducing the long and complex xylan chains to oligomers and simple sugars. Thus, xylanases play a crucial role in PCW depolymerization. Until recently, the largest xylanase family, glycoside hydrolase family 11 (GH11) has been exclusively represented by endo-catalytic β-1,4- and β-1,3-xylanases. Analysis of a metatranscriptome library from a microbial lignocellulose community resulted in the identification of an unusual exo-acting GH11 β-1,4-xylanase (MetXyn11). Detailed characterization has been performed on recombinant MetXyn11 including determination of its low-resolution small-angle X-ray scattering (SAXS) molecular envelope in solution. Our results reveal that MetXyn11 is a monomeric globular enzyme that liberates xylobiose from heteroxylans as the only product. MetXyn11 has an optimal activity in a pH range from 6 to 9 and an optimal temperature of 50 °C. The enzyme maintained above 65% of its original activity in the pH range 5 to 6 after being incubated for 72 h at 50 °C. Addition of the enzyme to a commercial enzymatic cocktail (CelicCtec3) promoted a significant increase of enzymatic hydrolysis yields of hydrothermally pretreated sugarcane bagasse (16% after 24 h of hydrolysis). [ABSTRACT FROM AUTHOR]