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Proteome-wide systems analysis of a cellulosic biofuel-producing microbe
Tolonen, Andrew C
Haas, Wilhelm
Chilaka, Amanda C
Aach, John
Gygi, Steven P
Church, George M
Molecular Systems Biology
We apply mass spectrometry-based ReDi proteomics to quantify the Clostridium phytofermentans proteome during fermentation of cellulosic substrates. ReDi proteomics gives accurate, low-cost quantification of an extra and intracellular microbial proteome. When combined with physiological measurements, these methods form a general systems biology strategy to evaluate the efficiency of cellulosic bioconversion and to identify enzyme targets to engineer for improving this process.C. phytofermentans expressed more than 100 carbohydrate-active enzymes, of which distinct subsets were upregulated on cellulose and hemicellulose. Numerous extracellular enzymes cleave insoluble plant polysaccharides into oligosaccharides, which are transported into the cell to be further degraded by intracellular carbohydratases. Sugars are catabolized by EMP glycolysis incorporating alternative glycolytic enzymes to maximize the ATP yield of anaerobic metabolism.During cellulosic fermentation, cells adhered to the substrate and altered metabolic processes such as upregulation of tryptophan and nicotinamide synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. These diverse metabolic changes highlight how a systems approach can identify novel ways to optimize cellulosic fermentation.
Cellulose is the world's most abundant renewable, biological energy source (Leschine, 1995). Microbial fermentation of cellulosic biomass could sustainably provide enough ethanol for 65% of US ground transportation fuel at current levels (Somerville, 2006). However, cellulose in plant biomass is packaged into a crystalline matrix, making biomass deconstruction a key roadblock to using it as a feedstock (Houghton et al, 2006). A promising strategy to overcome biomass recalcitrance is consolidated bioprocessing (Lynd et al, 2002), which uses microbes such as Clostridium phytofermentans to both secrete enzymes to depolymerize biomass and then ferment the resulting hexose and pentose sugars to a biofuel such as ethanol. The C. phytofermentans genome encodes 161 carbohydrate-active enzymes (CAZy) including 108 glycoside hydrolases spread across 39 families (Cantarel et al, 2009), highlighting the elaborate set of enzymes needed to breakdown different cellulosic polysaccharides. Faced with the complexity of metabolizing biomass, systems biology strategies are needed to comprehensively identify which cellulolytic and metabolic enzymes are used to ferment different cellulosic substrates.
This study presents a systems-level analysis of how C. phytofermentans ferments different cellulosic substrates that incorporates quantitative mass spectrometry-based proteomics of over 2500 proteins. Protein concentrations within each carbon source treatment were calculated by machine learning-supported spectral counting (Absolute Protein EXpression, APEX) (Lu et al, 2007). Protein levels on hemicellulose and cellulose relative to glucose were determined using reductive methylation (Hsu et al, 2003; Boersema et al, 2009), here called ReDi labeling, to chemically incorporate hydrogen or deuterium isotopes at lysines and N-terminal amines of tryptic peptides. We show that ReDi proteomics gives accurate, low-cost quantification of a microbial proteome and can be used to discern extracellular proteins. Further, we combine these quantitative proteomics with detailed measurements of growth, biomass consumption, fermentation product analyses, and electron microscopy. Together, these methods form a general strategy to evaluate the efficiency of cellulosic bioconversion and to identify enzyme targets to engineer for improving this process (Figure 1).
We found that fermentation of cellulosic substrates by C. phytofermentans involves secretion of numerous CAZy as well as proteins for binding of extracellular solutes, proteolysis, and motility. The most highly expressed protein in the proteome is a secreted protein that appears to compose a surface layer to support the cell and anchor cell surface proteins, including some enzymes for plant degradation. Once the secreted CAZy cleave insoluble plant polysaccharides into oligosaccharides, they are taken into the cell to be further degraded by intracellular CAZy, enabling more efficient sugar transport, conserving energy by phosphorolytic cleavage, and ensuring the sugar monomers were not available to competing microbes. Sugars are catabolized by EMP glycolysis incorporating reversible, PPi-dependent glycolytic enzymes, and pyruvate ferredoxin oxidoreductase. The genome encodes seven alcohol dehydrogenases, among which two iron-dependent enzymes are highly expressed and likely facilitate the high ethanol yields. Growth on cellulose also resulted in indirect changes such as increased tryptophan and nicotinamide synthesis and repression of fatty acid synthesis. We distilled the data into a model showing the highly expressed enzymes enabling efficient cellulosic fermentation by C. phytofermentans (Figure 7). Collectively, these data help understand how bacteria recycle plant biomass works towards enabling the use of plant biomass as a low-cost chemical feedstock.
Fermentation of plant biomass by microbes like Clostridium phytofermentans recycles carbon globally and can make biofuels from inedible feedstocks. We analyzed C. phytofermentans fermenting cellulosic substrates by integrating quantitative mass spectrometry of more than 2500 proteins with measurements of growth, enzyme activities, fermentation products, and electron microscopy. Absolute protein concentrations were estimated using Absolute Protein EXpression (APEX); relative changes between treatments were quantified with chemical stable isotope labeling by reductive dimethylation (ReDi). We identified the different combinations of carbohydratases used to degrade cellulose and hemicellulose, many of which were secreted based on quantification of supernatant proteins, as well as the repertoires of glycolytic enzymes and alcohol dehydrogenases (ADHs) enabling ethanol production at near maximal yields. Growth on cellulose also resulted in diverse changes such as increased expression of tryptophan synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. This study gives a systems-level understanding of how this microbe ferments biomass and provides a rational, empirical basis to identify engineering targets for industrial cellulosic fermentation.
bioenergy; clostridium; proteomics
Related Articles
Evaluating the composition and processing potential of novel sources of Brazilian biomass for sustainable biorenewables production
Lima, Marisa A
Gomez, Leonardo D
Steele-King, Clare G
Simister, Rachael
Bernardinelli, Oigres D
Carvalho, Marcelo A
Rezende, Camila A
Labate, Carlos A
deAzevedo, Eduardo R
McQueen-Mason, Simon J
Polikarpov, Igor
Biotechnology for Biofuels
The search for promising and renewable sources of carbohydrates for the production of biofuels and other biorenewables has been stimulated by an increase in global energy demand in the face of growing concern over greenhouse gas emissions and fuel security. In particular, interest has focused on non-food lignocellulosic biomass as a potential source of abundant and sustainable feedstock for biorefineries. Here we investigate the potential of three Brazilian grasses (Panicum maximum, Pennisetum purpureum and Brachiaria brizantha), as well as bark residues from the harvesting of two commercial Eucalyptus clones (E. grandis and E. grandis x urophylla) for biofuel production, and compare these to sugarcane bagasse. The effects of hot water, acid, alkaline and sulfite pretreatments (at increasing temperatures) on the chemical composition, morphology and saccharification yields of these different biomass types were evaluated.
The average yield (per hectare), availability and general composition of all five biomasses were compared. Compositional analyses indicate a high level of hemicellulose and lignin removal in all grass varieties (including sugarcane bagasse) after acid and alkaline pretreatment with increasing temperatures, whilst the biomasses pretreated with hot water or sulfite showed little variation from the control. For all biomasses, higher cellulose enrichment resulted from treatment with sodium hydroxide at 130°C. At 180°C, a decrease in cellulose content was observed, which is associated with high amorphous cellulose removal and 5-hydroxymethyl-furaldehyde production. Morphological analysis showed the effects of different pretreatments on the biomass surface, revealing a high production of microfibrillated cellulose on grass surfaces, after treatment with 1% sodium hydroxide at 130°C for 30 minutes. This may explain the higher hydrolysis yields resulting from these pretreatments, since these cellulosic nanoparticles can be easily accessed and cleaved by cellulases.
Our results show the potential of three Brazilian grasses with high productivity yields as valuable sources of carbohydrates for ethanol production and other biomaterials. Sodium hydroxide at 130°C was found to be the most effective pretreatment for enhanced saccharification yields. It was also efficient in the production of microfibrillated cellulose on grass surfaces, thereby revealing their potential as a source of natural fillers used for bionanocomposites production.
Bioethanol; Brachiaria brizantha; Brazilian grasses; Chemical composition; Enzymatic saccharification; Eucalyptus barks; Panicum maximum; Pennisetum purpureum; Pretreatments; Scanning electron microscopy; Sugarcane bagasse
Related Articles
Next-generation cellulosic ethanol technologies and their contribution to a sustainable Africa
van Zyl, W. H.
Chimphango, A. F. A.
den Haan, R.
Görgens, J. F.
Chirwa, P. W. C.
Interface Focus
The world is currently heavily dependent on oil, especially in the transport sector. However, rising oil prices, concern about environmental impact and supply instability are among the factors that have led to greater interest in renewable fuel and green chemistry alternatives. Lignocellulose is the only foreseeable renewable feedstock for sustainable production of transport fuels. The main technological impediment to more widespread utilization of lignocellulose for production of fuels and chemicals in the past has been the lack of low-cost technologies to overcome the recalcitrance of its structure. Both biological and thermochemical second-generation conversion technologies are currently coming online for the commercial production of cellulosic ethanol concomitantly with heat and electricity production. The latest advances in biological conversion of lignocellulosics to ethanol with a focus on consolidated bioprocessing are highlighted. Furthermore, integration of cellulosic ethanol production into existing bio-based industries also using thermochemical processes to optimize energy balances is discussed. Biofuels have played a pivotal yet suboptimal role in supplementing Africa's energy requirements in the past. Capitalizing on sub-Saharan Africa's total biomass potential and using second-generation technologies merit a fresh look at the potential role of bioethanol production towards developing a sustainable Africa while addressing food security, human needs and local wealth creation.
cellulosic ethanol; consolidated bioprocessing; integrating bio-based industries; sustainable biofuels in Africa
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Energy Potential and Greenhouse Gas Emissions from Bioenergy Cropping Systems on Marginally Productive Cropland
Schmer, Marty R.
Vogel, Kenneth P.
Varvel, Gary E.
Follett, Ronald F.
Mitchell, Robert B.
Jin, Virginia L.
PLoS ONE
Low-carbon biofuel sources are being developed and evaluated in the United States and Europe to partially offset petroleum transport fuels. Current and potential biofuel production systems were evaluated from a long-term continuous no-tillage corn (Zea mays L.) and switchgrass (Panicum virgatum L.) field trial under differing harvest strategies and nitrogen (N) fertilizer intensities to determine overall environmental sustainability. Corn and switchgrass grown for bioenergy resulted in near-term net greenhouse gas (GHG) reductions of −29 to −396 grams of CO2 equivalent emissions per megajoule of ethanol per year as a result of direct soil carbon sequestration and from the adoption of integrated biofuel conversion pathways. Management practices in switchgrass and corn resulted in large variation in petroleum offset potential. Switchgrass, using best management practices produced 3919±117 liters of ethanol per hectare and had 74±2.2 gigajoules of petroleum offsets per hectare which was similar to intensified corn systems (grain and 50% residue harvest under optimal N rates). Co-locating and integrating cellulosic biorefineries with existing dry mill corn grain ethanol facilities improved net energy yields (GJ ha−1) of corn grain ethanol by >70%. A multi-feedstock, landscape approach coupled with an integrated biorefinery would be a viable option to meet growing renewable transportation fuel demands while improving the energy efficiency of first generation biofuels.
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Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin
Foster, Cliff E.
Martin, Tina M.
Pauly, Markus
Journal of Visualized Experiments : JoVE
The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.
Related Articles
Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates
Foster, Cliff E.
Martin, Tina M.
Pauly, Markus
Journal of Visualized Experiments : JoVE
The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.
Related Articles
Bioconversion of Sugarcane Biomass into Ethanol: An Overview about Composition, Pretreatment Methods, Detoxification of Hydrolysates, Enzymatic Saccharification, and Ethanol Fermentation
Canilha, Larissa
Chandel, Anuj Kumar
Suzane dos Santos Milessi, Thais
Antunes, Felipe Antônio Fernandes
Luiz da Costa Freitas, Wagner
das Graças Almeida Felipe, Maria
da Silva, Silvio Silvério
Journal of Biomedicine and Biotechnology
Depleted supplies of fossil fuel, regular price hikes of gasoline, and environmental damage have necessitated the search for economic and eco-benign alternative of gasoline. Ethanol is produced from food/feed-based substrates (grains, sugars, and molasses), and its application as an energy source does not seem fit for long term due to the increasing fuel, food, feed, and other needs. These concerns have enforced to explore the alternative means of cost competitive and sustainable supply of biofuel. Sugarcane residues, sugarcane bagasse (SB), and straw (SS) could be the ideal feedstock for the second-generation (2G) ethanol production. These raw materials are rich in carbohydrates and renewable and do not compete with food/feed demands. However, the efficient bioconversion of SB/SS (efficient pretreatment technology, depolymerization of cellulose, and fermentation of released sugars) remains challenging to commercialize the cellulosic ethanol. Among the technological challenges, robust pretreatment and development of efficient bioconversion process (implicating suitable ethanol producing strains converting pentose and hexose sugars) have a key role to play. This paper aims to review the compositional profile of SB and SS, pretreatment methods of cane biomass, detoxification methods for the purification of hydrolysates, enzymatic hydrolysis, and the fermentation of released sugars for ethanol production.
Related Articles
Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production
Shen, Hui
Poovaiah, Charleson R
Ziebell, Angela
Tschaplinski, Timothy J
Pattathil, Sivakumar
Gjersing, Erica
Engle, Nancy L
Katahira, Rui
Pu, Yunqiao
Sykes, Robert
Chen, Fang
Ragauskas, Arthur J
Mielenz, Jonathan R
Hahn, Michael G
Davis, Mark
Stewart, C Neal
Dixon, Richard A
Biotechnology for Biofuels
Lignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, "absent technological breakthroughs", it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022. We here describe the properties of switchgrass (Panicum virgatum) biomass that has been genetically engineered to increase the cellulosic ethanol yield by more than 2-fold.
We have increased the cellulosic ethanol yield from switchgrass by 2.6-fold through overexpression of the transcription factor PvMYB4. This strategy reduces carbon deposition into lignin and phenolic fermentation inhibitors while maintaining the availability of potentially fermentable soluble sugars and pectic polysaccharides. Detailed biomass characterization analyses revealed that the levels and nature of phenolic acids embedded in the cell-wall, the lignin content and polymer size, lignin internal linkage levels, linkages between lignin and xylans/pectins, and levels of wall-bound fucose are all altered in PvMYB4-OX lines. Genetically engineered PvMYB4-OX switchgrass therefore provides a novel system for further understanding cell wall recalcitrance.
Our results have demonstrated that overexpression of PvMYB4, a general transcriptional repressor of the phenylpropanoid/lignin biosynthesis pathway, can lead to very high yield ethanol production through dramatic reduction of recalcitrance. MYB4-OX switchgrass is an excellent model system for understanding recalcitrance, and provides new germplasm for developing switchgrass cultivars as biomass feedstocks for biofuel production.
Switchgrass; Bioenergy; Biofuel; Feedstock; Cellulosic ethanol; PvMYB4; Transcription factor; Cell wall; Recalcitrance; Lignin; Hemicellulose; Pectin
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Impact of pretreatment and downstream processing technologies on economics and energy in cellulosic ethanol production
Kumar, Deepak
Murthy, Ganti S
Biotechnology for Biofuels
While advantages of biofuel have been widely reported, studies also highlight the challenges in large scale production of biofuel. Cost of ethanol and process energy use in cellulosic ethanol plants are dependent on technologies used for conversion of feedstock. Process modeling can aid in identifying techno-economic bottlenecks in a production process. A comprehensive techno-economic analysis was performed for conversion of cellulosic feedstock to ethanol using some of the common pretreatment technologies: dilute acid, dilute alkali, hot water and steam explosion. Detailed process models incorporating feedstock handling, pretreatment, simultaneous saccharification and co-fermentation, ethanol recovery and downstream processing were developed using SuperPro Designer. Tall Fescue (Festuca arundinacea Schreb) was used as a model feedstock.
Projected ethanol yields were 252.62, 255.80, 255.27 and 230.23 L/dry metric ton biomass for conversion process using dilute acid, dilute alkali, hot water and steam explosion pretreatment technologies respectively. Price of feedstock and cellulose enzymes were assumed as $50/metric ton and 0.517/kg broth (10% protein in broth, 600 FPU/g protein) respectively. Capital cost of ethanol plants processing 250,000 metric tons of feedstock/year was $1.92, $1.73, $1.72 and $1.70/L ethanol for process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Ethanol production cost of $0.83, $0.88, $0.81 and $0.85/L ethanol was estimated for production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Water use in the production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment was estimated 5.96, 6.07, 5.84 and 4.36 kg/L ethanol respectively.
Ethanol price and energy use were highly dependent on process conditions used in the ethanol production plant. Potential for significant ethanol cost reductions exist in increasing pentose fermentation efficiency and reducing biomass and enzyme costs. The results demonstrated the importance of addressing the tradeoffs in capital costs, pretreatment and downstream processing technologies.
Grass straw; cellulosic ethanol; pretreatment; process model; process economics.
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Agave proves to be a low recalcitrant lignocellulosic feedstock for biofuels production on semi-arid lands
Li, Hongjia
Pattathil, Sivakumar
Foston, Marcus B
Ding, Shi-You
Kumar, Rajeev
Gao, Xiadi
Mittal, Ashutosh
Yarbrough, John M
Himmel, Michael E
Ragauskas, Arthur J
Hahn, Michael G
Wyman, Charles E
Biotechnology for Biofuels
Agave, which is well known for tequila and other liquor production in Mexico, has recently gained attention because of its attractive potential to launch sustainable bioenergy feedstock solutions for semi-arid and arid lands. It was previously found that agave cell walls contain low lignin and relatively diverse non-cellulosic polysaccharides, suggesting unique recalcitrant features when compared to conventional C4 and C3 plants.
Here, we report sugar release data from fungal enzymatic hydrolysis of non-pretreated and hydrothermally pretreated biomass that shows agave to be much less recalcitrant to deconstruction than poplar or switchgrass. In fact, non-pretreated agave has a sugar release five to eight times greater than that of poplar wood and switchgrass . Meanwhile, state of the art techniques including glycome profiling, nuclear magnetic resonance (NMR), Simon's Stain, confocal laser scanning microscopy and so forth, were applied to measure interactions of non-cellulosic wall components, cell wall hydrophilicity, and enzyme accessibility to identify key structural features that make agave cell walls less resistant to biological deconstruction when compared to poplar and switchgrass.
This study systematically evaluated the recalcitrant features of agave plants towards biofuels applications. The results show that not only does agave present great promise for feeding biorefineries on semi-arid and arid lands, but also show the value of studying agave's low recalcitrance for developments in improving cellulosic energy crops.
Agave; Biofuels; Feedstock; Low recalcitrance; Semi-arid land
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Genomic Analysis of the Hydrocarbon-Producing, Cellulolytic, Endophytic Fungus Ascocoryne sarcoides
Gianoulis, Tara A.
Griffin, Meghan A.
Spakowicz, Daniel J.
Dunican, Brian F.
Alpha, Cambria J.
Sboner, Andrea
Sismour, A. Michael
Kodira, Chinnappa
Egholm, Michael
Church, George M.
Gerstein, Mark B.
Strobel, Scott A.
PLoS Genetics
The microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Endophytes represent a promising group of organisms, as they are a mostly untapped reservoir of metabolic diversity. They are often able to degrade cellulose, and they can produce an extraordinary diversity of metabolites. The filamentous fungal endophyte Ascocoryne sarcoides was shown to produce potential-biofuel metabolites when grown on a cellulose-based medium; however, the genetic pathways needed for this production are unknown and the lack of genetic tools makes traditional reverse genetics difficult. We present the genomic characterization of A. sarcoides and use transcriptomic and metabolomic data to describe the genes involved in cellulose degradation and to provide hypotheses for the biofuel production pathways. In total, almost 80 biosynthetic clusters were identified, including several previously found only in plants. Additionally, many transcriptionally active regions outside of genes showed condition-specific expression, offering more evidence for the role of long non-coding RNA in gene regulation. This is one of the highest quality fungal genomes and, to our knowledge, the only thoroughly annotated and transcriptionally profiled fungal endophyte genome currently available. The analyses and datasets contribute to the study of cellulose degradation and biofuel production and provide the genomic foundation for the study of a model endophyte system.
Author Summary
A renewable source of energy is a pressing global need. The biological conversion of lignocellulose to biofuels by microorganisms presents a promising avenue, but few organisms have been studied thoroughly enough to develop the genetic tools necessary for rigorous experimentation. The filamentous-fungal endophyte A. sarcoides produces metabolites when grown on a cellulose-based medium that include eight-carbon volatile organic compounds, which are potential biofuel targets. Here we use broadly applicable methods including genomics, transcriptomics, and metabolomics to explore the biofuel production of A. sarcoides. These data were used to assemble the genome into 16 scaffolds, to thoroughly annotate the cellulose-degradation machinery, and to make predictions for the production pathway for the eight-carbon volatiles. Extremely high expression of the gene swollenin when grown on cellulose highlights the importance of accessory proteins in addition to the enzymes that catalyze the breakdown of the polymers. Correlation of the production of the eight-carbon biofuel-like metabolites with the expression of lipoxygenase pathway genes suggests the catabolism of linoleic acid as the mechanism of eight-carbon compound production. This is the first fungal genome to be sequenced in the family Helotiaceae, and A. sarcoides was isolated as an endophyte, making this work also potentially useful in fungal systematics and the study of plant–fungus relationships.
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Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality
Lee, Scott J
Warnick, Thomas A
Pattathil, Sivakumar
Alvelo-Maurosa, Jesús G
Serapiglia, Michelle J
McCormick, Heather
Brown, Virginia
Young, Naomi F
Schnell, Danny J
Smart, Lawrence B
Hahn, Michael G
Pedersen, Jeffrey F
Leschine, Susan B
Hazen, Samuel P
Biotechnology for Biofuels
There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency.
We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.).
Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality.
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Farm systems assessment of bioenergy feedstock production: Integrating bio-economic models and life cycle analysis approaches
Glithero, N.J.
Ramsden, S.J.
Wilson, P.
Agricultural Systems
► A modelling framework for managing energy and emissions trade-offs in agriculture. ► Combination of bio-economic process modelling and life cycle assessment. ► Captures financial, energy and emissions trade-offs and cereal straw use. ► The gross margin-energy trade-off is £36 GJ−1. ► All cereal straw is baled for sale when gross margin or net energy is maximised.
Climate change and energy security concerns have driven the development of policies that encourage bioenergy production. Meeting EU targets for the consumption of transport fuels from bioenergy by 2020 will require a large increase in the production of bioenergy feedstock. Initially an increase in 'first generation' biofuels was observed, however 'food competition' concerns have generated interest in second generation biofuels (SGBs). These SGBs can be produced from co-products (e.g. cereal straw) or energy crops (e.g. miscanthus), with the former largely negating food competition concerns. In order to assess the sustainability of feedstock supply for SGBs, the financial, environmental and energy costs and benefits of the farm system must be quantified. Previous research has captured financial costs and benefits through linear programming (LP) approaches, whilst environmental and energy metrics have been largely been undertaken within life cycle analysis (LCA) frameworks. Assessing aspects of the financial, environmental and energy sustainability of supplying co-product second generation biofuel (CPSGB) feedstocks at the farm level requires a framework that permits the trade-offs between these objectives to be quantified and understood. The development of a modelling framework for Managing Energy and Emissions Trade-Offs in Agriculture (MEETA Model) that combines bio-economic process modelling and LCA is presented together with input data parameters obtained from literature and industry sources. The MEETA model quantifies arable farm inputs and outputs in terms of financial, energy and emissions results. The model explicitly captures fertiliser: crop-yield relationships, plus the incorporation of straw or removal for sale, with associated nutrient impacts of incorporation/removal on the following crop in the rotation. Key results of crop-mix, machinery use, greenhouse gas (GHG) emissions per kg of crop product and energy use per hectare are in line with previous research and industry survey findings. Results show that the gross margin – energy trade-off is £36 GJ−1, representing the gross margin forgone by maximising net farm energy cf. maximising farm gross margin. The gross margin–GHG emission trade-off is £0.15 kg−1 CO2 eq, representing the gross margin forgone per kg of CO2 eq reduced when GHG emissions are minimised cf. maximising farm gross margin. The energy–GHG emission trade-off is 0.03 GJ kg−1 CO2 eq quantifying the reduction in net energy from the farm system per kg of CO2 eq reduced when minimising GHG emissions cf. maximising net farm energy. When both farm gross margin and net farm energy are maximised all the cereal straw is baled for sale. Sensitivity analysis of the model in relation to different prices of cereal straw shows that it becomes financially optimal to incorporate wheat straw at price of £11 t−1 for this co-product. Local market conditions for straw and farmer attitudes towards incorporation or sale of straw will impact on the straw price at which farmers will supply this potential bioenergy feedstock and represent important areas for future research.
Bioenergy; Cereal straw; Greenhouse gas emissions; Modelling; Farm systems
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Scale-up and evaluation of high solid ionic liquid pretreatment and enzymatic hydrolysis of switchgrass
Li, Chenlin
Tanjore, Deepti
He, Wei
Wong, Jessica
Gardner, James L
Sale, Kenneth L
Simmons, Blake A
Singh, Seema
Biotechnology for Biofuels
Ionic liquid (IL) pretreatment is receiving significant attention as a potential process that enables fractionation of lignocellulosic biomass and produces high yields of fermentable sugars suitable for the production of renewable fuels. However, successful optimization and scale up of IL pretreatment involves challenges, such as high solids loading, biomass handling and transfer, washing of pretreated solids and formation of inhibitors, which are not addressed during the development stages at the small scale in a laboratory environment. As a first in the research community, the Joint BioEnergy Institute, in collaboration with the Advanced Biofuels Process Demonstration Unit, a Department of Energy funded facility that supports academic and industrial entities in scaling their novel biofuels enabling technologies, have performed benchmark studies to identify key challenges associated with IL pretreatment using 1-ethyl-3-methylimidazolium acetate and subsequent enzymatic saccharification beyond bench scale.
Using switchgrass as the model feedstock, we have successfully executed 600-fold, relative to the bench scale (6 L vs 0.01 L), scale-up of IL pretreatment at 15% (w/w) biomass loading. Results show that IL pretreatment at 15% biomass generates a product containing 87.5% of glucan, 42.6% of xylan and only 22.8% of lignin relative to the starting material. The pretreated biomass is efficiently converted into monosaccharides during subsequent enzymatic hydrolysis at 10% loading over a 150-fold scale of operations (1.5 L vs 0.01 L) with 99.8% fermentable sugar conversion. The yield of glucose and xylose in the liquid streams were 94.8% and 62.2%, respectively, and the hydrolysate generated contains high titers of fermentable sugars (62.1 g/L of glucose and 5.4 g/L cellobiose). The overall glucan and xylan balance from pretreatment and saccharification were 95.0% and 77.1%, respectively. Enzymatic inhibition by [C2mim][OAc] at high solids loadings requires further process optimization to obtain higher yields of fermentable sugars.
Results from this initial scale up evaluation indicate that the IL-based conversion technology can be effectively scaled to larger operations and the current study establishes the first scaling parameters for this conversion pathway but several issues must be addressed before a commercially viable technology can be realized, most notably reduction in water consumption and efficient IL recycle.
Scale-up; Pretreatment; Saccharification; Ionic liquid; High solid loading; Viscosity; Inhibition
Related Articles
Co-production of ethanol and squalene using a Saccharomyces cerevisiae ERG1 (squalene epoxidase) mutant and agro-industrial feedstock
Hull, Claire M
Loveridge, E Joel
Rolley, Nicola J
Donnison, Iain S
Kelly, Steven L
Kelly, Diane E
Biotechnology for Biofuels
Genetically customised Saccharomyces cerevisiae that can produce ethanol and additional bio-based chemicals from sustainable agro-industrial feedstocks (for example, residual plant biomass) are of major interest to the biofuel industry. We investigated the microbial biorefinery concept of ethanol and squalene co-production using S. cerevisiae (strain YUG37-ERG1) wherein ERG1 (squalene epoxidase) transcription is under the control of a doxycycline-repressible tet07-CYC1 promoter. The production of ethanol and squalene by YUG37-ERG1 grown using agriculturally sourced grass juice supplemented with doxycycline was assessed.
Use of the tet07-CYC1 promoter permitted regulation of ERG1 expression and squalene accumulation in YUG37-ERG1, allowing us to circumvent the lethal growth phenotype seen when ERG1 is disrupted completely. In experiments using grass juice feedstock supplemented with 0 to 50 μg doxycycline mL−1, YUG37-ERG1 fermented ethanol (22.5 [±0.5] mg mL−1) and accumulated the highest squalene content (7.89 ± 0.25 mg g−1 dry biomass) and yield (18.0 ± 4.18 mg squalene L−1) with supplements of 5.0 and 0.025 μg doxycycline mL−1, respectively. Grass juice was found to be rich in water-soluble carbohydrates (61.1 [±3.6] mg sugars mL−1) and provided excellent feedstock for growth and fermentation studies using YUG37-ERG1.
Residual plant biomass components from crop production and rotation systems represent possible substrates for microbial fermentation of biofuels and bio-based compounds. This study is the first to utilise S. cerevisiae for the co-production of ethanol and squalene from grass juice. Our findings underscore the value of the biorefinery approach and demonstrate the potential to integrate microbial bioprocess engineering with existing agriculture.
Bio-based products; ERG1; Ethanol; Sterol; Squalene; Squalene epoxidase
Related Articles
Life-cycle energy use and greenhouse gas emissions of production of bioethanol from sorghum in the United States
Cai, Hao
Dunn, Jennifer B
Wang, Zhichao
Han, Jeongwoo
Wang, Michael Q
Biotechnology for Biofuels
The availability of feedstock options is a key to meeting the volumetric requirement of 136.3 billion liters of renewable fuels per year beginning in 2022, as required in the US 2007 Energy Independence and Security Act. Life-cycle greenhouse gas (GHG) emissions of sorghum-based ethanol need to be assessed for sorghum to play a role in meeting that requirement.
Multiple sorghum-based ethanol production pathways show diverse well-to-wheels (WTW) energy use and GHG emissions due to differences in energy use and fertilizer use intensity associated with sorghum growth and differences in the ethanol conversion processes. All sorghum-based ethanol pathways can achieve significant fossil energy savings. Relative to GHG emissions from conventional gasoline, grain sorghum-based ethanol can reduce WTW GHG emissions by 35% or 23%, respectively, when wet or dried distillers grains with solubles (DGS) is the co-product and fossil natural gas (FNG) is consumed as the process fuel. The reduction increased to 56% or 55%, respectively, for wet or dried DGS co-production when renewable natural gas (RNG) from anaerobic digestion of animal waste is used as the process fuel. These results do not include land-use change (LUC) GHG emissions, which we take as negligible. If LUC GHG emissions for grain sorghum ethanol as estimated by the US Environmental Protection Agency (EPA) are included (26 g CO2e/MJ), these reductions when wet DGS is co-produced decrease to 7% or 29% when FNG or RNG is used as the process fuel. Sweet sorghum-based ethanol can reduce GHG emissions by 71% or 72% without or with use of co-produced vinasse as farm fertilizer, respectively, in ethanol plants using only sugar juice to produce ethanol. If both sugar and cellulosic bagasse were used in the future for ethanol production, an ethanol plant with a combined heat and power (CHP) system that supplies all process energy can achieve a GHG emission reduction of 70% or 72%, respectively, without or with vinasse fertigation. Forage sorghum-based ethanol can achieve a 49% WTW GHG emission reduction when ethanol plants meet process energy demands with CHP. In the case of forage sorghum and an integrated sweet sorghum pathway, the use of a portion of feedstock to fuel CHP systems significantly reduces fossil fuel consumption and GHG emissions.
This study provides new insight into life-cycle energy use and GHG emissions of multiple sorghum-based ethanol production pathways in the US. Our results show that adding sorghum feedstocks to the existing options for ethanol production could help in meeting the requirements for volumes of renewable, advanced and cellulosic bioethanol production in the US required by the EPA's Renewable Fuel Standard program.
Grain sorghum; Sweet sorghum; Forage sorghum; Ethanol; Life-cycle analysis; Greenhouse gas emissions
Related Articles
Development of microorganisms for cellulose-biofuel consolidated bioprocessings: metabolic engineers' tricks
Mazzoli, Roberto
Computational and Structural Biotechnology Journal
Cellulose waste biomass is the most abundant and attractive substrate for "biorefinery strategies" that are aimed to produce high-value products (e.g. solvents, fuels, building blocks) by economically and environmentally sustainable fermentation processes. However, cellulose is highly recalcitrant to biodegradation and its conversion by biotechnological strategies currently requires economically inefficient multistep industrial processes. The need for dedicated cellulase production continues to be a major constraint to cost-effective processing of cellulosic biomass.
Research efforts have been aimed at developing recombinant microorganisms with suitable characteristics for single step biomass fermentation (consolidated bioprocessing, CBP). Two paradigms have been applied for such, so far unsuccessful, attempts: a) "native cellulolytic strategies", aimed at conferring high-value product properties to natural cellulolytic microorganisms; b) "recombinant cellulolytic strategies", aimed to confer cellulolytic ability to microorganisms exhibiting high product yields and titers.
By starting from the description of natural enzyme systems for plant biomass degradation and natural metabolic pathways for some of the most valuable product (i.e. butanol, ethanol, and hydrogen) biosynthesis, this review describes state-of-the-art bottlenecks and solutions for the development of recombinant microbial strains for cellulosic biofuel CBP by metabolic engineering. Complexed cellulases (i.e. cellulosomes) benefit from stronger proximity effects and show enhanced synergy on insoluble substrates (i.e. crystalline cellulose) with respect to free enzymes. For this reason, special attention was held on strategies involving cellulosome/designer cellulosome-bearing recombinant microorganisms.
metabolic engineering; butanol; ethanol; hydrogen; cellulosome; cellulase
Related Articles
Efficient conversion of biomass into lipids by using the simultaneous saccharification and enhanced lipid production process
Gong, Zhiwei
Shen, Hongwei
Wang, Qian
Yang, Xiaobing
Xie, Haibo
Zhao, Zongbao K
Biotechnology for Biofuels
Microbial lipid production by using lignocellulosic biomass as the feedstock holds a great promise for biodiesel production and biorefinery. This usually involves hydrolysis of biomass into sugar-rich hydrolysates, which are then used by oleaginous microorganisms as the carbon and energy sources to produce lipids. However, the costs of microbial lipids remain prohibitively high for commercialization. More efficient and integrated processes are pivotal for better techno-economics of microbial lipid technology.
Here we describe the simultaneous saccharification and enhanced lipid production (SSELP) process that is highly advantageous in terms of converting cellulosic materials into lipids, as it integrates cellulose biomass hydrolysis and lipid biosynthesis. Specifically, Cryptococcus curvatus cells prepared in a nutrient-rich medium were inoculated at high dosage for lipid production in biomass suspension in the presence of hydrolytic enzymes without auxiliary nutrients. When cellulose was loaded at 32.3 g/L, cellulose conversion, cell mass, lipid content and lipid coefficient reached 98.5%, 12.4 g/L, 59.9% and 204 mg/g, respectively. Lipid yields of the SSELP process were higher than those obtained by using the conventional process where cellulose was hydrolyzed separately. When ionic liquid pretreated corn stover was used, both cellulose and hemicellulose were consumed simultaneously. No xylose was accumulated over time, indicating that glucose effect was circumvented. The lipid yield reached 112 mg/g regenerated corn stover. This process could be performed without sterilization because of the absence of auxiliary nutrients for bacterial contamination.
The SSELP process facilitates direct conversion of both cellulose and hemicellulose of lignocellulosic materials into microbial lipids. It greatly reduces time and capital costs while improves lipid coefficient. Optimization of the SSELP process at different levels should further improve the efficiency of microbial lipid technology, which in turn, promote the biotechnological production of fatty acid-derived products from lignocellulosic biomass.
Microbial lipids; Cryptococcus curvatus; Biodiesel; Corn stover; Simultaneous saccharification and enhanced lipid production
Related Articles
Energy Efficiency Analysis: Biomass-to-Wheel Efficiency Related with Biofuels Production, Fuel Distribution, and Powertrain Systems
Huang, Wei-Dong
Zhang, Y-H Percival
PLoS ONE
Energy efficiency analysis for different biomass-utilization scenarios would help make more informed decisions for developing future biomass-based transportation systems. Diverse biofuels produced from biomass include cellulosic ethanol, butanol, fatty acid ethyl esters, methane, hydrogen, methanol, dimethyether, Fischer-Tropsch diesel, and bioelectricity; the respective powertrain systems include internal combustion engine (ICE) vehicles, hybrid electric vehicles based on gasoline or diesel ICEs, hydrogen fuel cell vehicles, sugar fuel cell vehicles (SFCV), and battery electric vehicles (BEV).
Methodology/Principal Findings
We conducted a simple, straightforward, and transparent biomass-to-wheel (BTW) analysis including three separate conversion elements -- biomass-to-fuel conversion, fuel transport and distribution, and respective powertrain systems. BTW efficiency is a ratio of the kinetic energy of an automobile's wheels to the chemical energy of delivered biomass just before entering biorefineries. Up to 13 scenarios were analyzed and compared to a base line case – corn ethanol/ICE. This analysis suggests that BEV, whose electricity is generated from stationary fuel cells, and SFCV, based on a hydrogen fuel cell vehicle with an on-board sugar-to-hydrogen bioreformer, would have the highest BTW efficiencies, nearly four times that of ethanol-ICE.
In the long term, a small fraction of the annual US biomass (e.g., 7.1%, or 700 million tons of biomass) would be sufficient to meet 100% of light-duty passenger vehicle fuel needs (i.e., 150 billion gallons of gasoline/ethanol per year), through up to four-fold enhanced BTW efficiencies by using SFCV or BEV. SFCV would have several advantages over BEV: much higher energy storage densities, faster refilling rates, better safety, and less environmental burdens.
Related Articles
A comparative study of ethanol production using dilute acid, ionic liquid and AFEX™ pretreated corn stover
Uppugundla, Nirmal
da Costa Sousa, Leonardo
Chundawat, Shishir PS
Yu, Xiurong
Simmons, Blake
Singh, Seema
Gao, Xiadi
Kumar, Rajeev
Wyman, Charles E
Dale, Bruce E
Balan, Venkatesh
Biotechnology for Biofuels
In a biorefinery producing cellulosic biofuels, biomass pretreatment will significantly influence the efficacy of enzymatic hydrolysis and microbial fermentation. Comparison of different biomass pretreatment techniques by studying the impact of pretreatment on downstream operations at industrially relevant conditions and performing comprehensive mass balances will help focus attention on necessary process improvements, and thereby help reduce the cost of biofuel production.
An on-going collaboration between the three US Department of Energy (DOE) funded bioenergy research centers (Great Lakes Bioenergy Research Center (GLBRC), Joint BioEnergy Institute (JBEI) and BioEnergy Science Center (BESC)) has given us a unique opportunity to compare the performance of three pretreatment processes, notably dilute acid (DA), ionic liquid (IL) and ammonia fiber expansion (AFEXTM), using the same source of corn stover. Separate hydrolysis and fermentation (SHF) was carried out using various combinations of commercially available enzymes and engineered yeast (Saccharomyces cerevisiae 424A) strain. The optimal commercial enzyme combination (Ctec2: Htec2: Multifect Pectinase, percentage total protein loading basis) was evaluated for each pretreatment with a microplate-based assay using milled pretreated solids at 0.2% glucan loading and 15 mg total protein loading/g of glucan. The best enzyme combinations were 67:33:0 for DA, 39:33:28 for IL and 67:17:17 for AFEX. The amounts of sugar (kg) (glucose: xylose: total gluco- and xylo-oligomers) per 100 kg of untreated corn stover produced after 72 hours of 6% glucan loading enzymatic hydrolysis were: DA (25:2:2), IL (31:15:2) and AFEX (26:13:7). Additionally, the amounts of ethanol (kg) produced per 100 kg of untreated corn stover and the respective ethanol metabolic yield (%) achieved with exogenous nutrient supplemented fermentations were: DA (14.0, 92.0%), IL (21.2, 93.0%) and AFEX (20.5, 95.0%), respectively. The reason for lower ethanol yield for DA is because most of the xylose produced during the pretreatment was removed and not converted to ethanol during fermentation.
Compositional analysis of the pretreated biomass solids showed no significant change in composition for AFEX treated corn stover, while about 85% of hemicellulose was solubilized after DA pretreatment, and about 90% of lignin was removed after IL pretreatment. As expected, the optimal commercial enzyme combination was different for the solids prepared by different pretreatment technologies. Due to loss of nutrients during the pretreatment and washing steps, DA and IL pretreated hydrolysates required exogenous nutrient supplementation to ferment glucose and xylose efficiently, while AFEX pretreated hydrolysate did not require nutrient supplementation.
AFEX; Dilute acid; Ionic liquid; Pretreatment; Enzymatic hydrolysis; Cellulosic ethanol
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Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept
Hull, Claire M
Loveridge, E. Joel
Donnison, Iain S
Kelly, Diane E
Kelly, Steven L
AMB Express
Microbial biotechnology and biotransformations promise to diversify the scope of the biorefinery approach for the production of high-value products and biofuels from industrial, rural and municipal waste feedstocks. In addition to bio-based chemicals and metabolites, microbial biomass itself constitutes an obvious but overlooked by-product of existing biofermentation systems which warrants fuller attention. The probiotic yeast Saccharomyces boulardii is used to treat gastrointestinal disorders and marketed as a human health supplement. Despite its relatedness to S. cerevisiae that is employed widely in biotechnology, food and biofuel industries, the alternative applications of S. boulardii are not well studied. Using a biorefinery approach, we compared the bioethanol and biomass yields attainable from agriculturally-sourced grass juice using probiotic S. boulardii (strain MYA-769) and a commercial S. cerevisiae brewing strain (Turbo yeast). Maximum product yields for MYA-769 (39.18 [±2.42] mg ethanol mL−1 and 4.96 [±0.15] g dry weight L−1) compared closely to those of Turbo (37.43 [±1.99] mg mL−1 and 4.78 [±0.10] g L−1, respectively). Co-production, marketing and/or on-site utilisation of probiotic yeast biomass as a direct-fed microbial to improve livestock health represents a novel and viable prospect for rural biorefineries. Given emergent evidence to suggest that dietary yeast supplementations might also mitigate ruminant enteric methane emissions, the administration of probiotic yeast biomass could also offer an economically feasible way of reducing atmospheric CH4.
Bioethanol; Biomass; Biorefinery; Cholesterol; Probiotic; Saccharomyces boulardii
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Genotype, development and tissue-derived variation of cell-wall properties in the lignocellulosic energy crop Miscanthus
da Costa, Ricardo M. F.
Lee, Scott J.
Allison, Gordon G.
Hazen, Samuel P.
Winters, Ana
Bosch, Maurice
Annals of Botany
Background and Aims
Species and hybrids of the genus Miscanthus contain attributes that make them front-runners among current selections of dedicated bioenergy crops. A key trait for plant biomass conversion to biofuels and biomaterials is cell-wall quality; however, knowledge of cell-wall composition and biology in Miscanthus species is limited. This study presents data on cell-wall compositional changes as a function of development and tissue type across selected genotypes, and considers implications for the development of miscanthus as a sustainable and renewable bioenergy feedstock.
Cell-wall biomass was analysed for 25 genotypes, considering different developmental stages and stem vs. leaf compositional variability, by Fourier transform mid-infrared spectroscopy and lignin determination. In addition, a Clostridium phytofermentans bioassay was used to assess cell-wall digestibility and conversion to ethanol.
Key Results
Important cell-wall compositional differences between miscanthus stem and leaf samples were found to be predominantly associated with structural carbohydrates. Lignin content increased as plants matured and was higher in stem tissues. Although stem lignin concentration correlated inversely with ethanol production, no such correlation was observed for leaves. Leaf tissue contributed significantly to total above-ground biomass at all stages, although the extent of this contribution was genotype-dependent.
It is hypothesized that divergent carbohydrate compositions and modifications in stem and leaf tissues are major determinants for observed differences in cell-wall quality. The findings indicate that improvement of lignocellulosic feedstocks should encompass tissue-dependent variation as it affects amenability to biological conversion. For gene–trait associations relating to cell-wall quality, the data support the separate examination of leaf and stem composition, as tissue-specific traits may be masked by considering only total above-ground biomass samples, and sample variability could be mostly due to varying tissue contributions to total biomass.
Miscanthus; biofuels; plant cell wall; Fourier transform mid-infrared spectroscopy; FTIR; lignin; fermentation; bioenergy; recalcitrance; lignocellulose; biomass; development; carbohydrates
Related Articles
Ethanol Distribution, Dispensing, and Use: Analysis of a Portion of the Biomass-to-Biofuels Supply Chain Using System Dynamics
Vimmerstedt, Laura J.
Bush, Brian
Peterson, Steve
PLoS ONE
The Energy Independence and Security Act of 2007 targets use of 36 billion gallons of biofuels per year by 2022. Achieving this may require substantial changes to current transportation fuel systems for distribution, dispensing, and use in vehicles. The U.S. Department of Energy and the National Renewable Energy Laboratory designed a system dynamics approach to help focus government action by determining what supply chain changes would have the greatest potential to accelerate biofuels deployment. The National Renewable Energy Laboratory developed the Biomass Scenario Model, a system dynamics model which represents the primary system effects and dependencies in the biomass-to-biofuels supply chain. The model provides a framework for developing scenarios and conducting biofuels policy analysis. This paper focuses on the downstream portion of the supply chain–represented in the distribution logistics, dispensing station, and fuel utilization, and vehicle modules of the Biomass Scenario Model. This model initially focused on ethanol, but has since been expanded to include other biofuels. Some portions of this system are represented dynamically with major interactions and feedbacks, especially those related to a dispensing station owner's decision whether to offer ethanol fuel and a consumer's choice whether to purchase that fuel. Other portions of the system are modeled with little or no dynamics; the vehicle choices of consumers are represented as discrete scenarios. This paper explores conditions needed to sustain an ethanol fuel market and identifies implications of these findings for program and policy goals. A large, economically sustainable ethanol fuel market (or other biofuel market) requires low end-user fuel price relative to gasoline and sufficient producer payment, which are difficult to achieve simultaneously. Other requirements (different for ethanol vs. other biofuel markets) include the need for infrastructure for distribution and dispensing and widespread use of high ethanol blends in flexible-fuel vehicles.
Related Articles
Comparison of sugar content for ionic liquid pretreated Douglas-fir woodchips and forestry residues
Socha, Aaron M
Plummer, Samuel P
Stavila, Vitalie
Simmons, Blake A
Singh, Seema
Biotechnology for Biofuels
The development of affordable woody biomass feedstocks represents a significant opportunity in the development of cellulosic biofuels. Primary woodchips produced by forest mills are considered an ideal feedstock, but the prices they command on the market are currently too expensive for biorefineries. In comparison, forestry residues represent a potential low-cost input but are considered a more challenging feedstock for sugar production due to complexities in composition and potential contamination arising from soil that may be present. We compare the sugar yields, changes in composition in Douglas-fir woodchips and forestry residues after pretreatment using ionic liquids and enzymatic saccharification in order to determine if this approach can efficiently liberate fermentable sugars.
These samples were either mechanically milled through a 2 mm mesh or pretreated as received with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate [C2mim][OAc] at 120°C and 160°C. IL pretreatment of Douglas-fir woodchips and forestry residues resulted in approximately 71-92% glucose yields after enzymatic saccharification. X-ray diffraction (XRD) showed that the pretreated cellulose was less crystalline after IL pretreatment as compared to untreated control samples. Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) revealed changes in lignin and hemicellulose structure and composition as a function of pretreatment. Mass balances of sugar and lignin streams for both the Douglas-fir woodchips and forestry residues throughout the pretreatment and enzymatic saccharification processes are presented.
While the highest sugar yields were observed with the Douglas-fir woodchips, reasonably high sugar yields were obtained from forestry residues after ionic liquid pretreatment. Structural changes to lignin, cellulose and hemicellulose in the woodchips and forestry residues of Douglas-fir after [C2mim][OAc] pretreatment are analyzed by XRD and 2D-NMR, and indicate that significant changes occurred. Irrespective of the particle sizes used in this study, ionic liquid pretreatment successfully allowed high glucose yields after enzymatic saccharification. These results indicate that forestry residues may be a more viable feedstock than previously thought for the production of biofuels.
Lignocellulose; Biomass pretreatment; Ionic liquid pretreatment; Douglas-fir; Softwood; Woodchips; Forestry residues; 1-ethyl-3-methylimidazolium acetate
Related Articles
Pretreatment on Miscanthus lutarioriparious by liquid hot water for efficient ethanol production
Li, Hong-Qiang
Li, Cheng-Lan
Sang, Tao
Xu, Jian
Biotechnology for Biofuels
The C4 perennial grass Miscanthus giganteus has proved to be a promising bio-energy crop. However, the biomass recalcitrance is a major challenge in biofuel production. Effective pretreatment is necessary for achieving a high efficiency in converting the crop to fermentable sugars, and subsequently biofuels and other valued products.
Miscanthus lutarioriparious was pretreated with a liquid hot water (LHW) reactor. Between the pretreatment severity (PS) of 2.56-4.71, the solid recovery was reduced; cellulose recovery remained nearly unchanged; and the Klason lignin content was slightly increased which was mainly due to the dissolving of hemicellulose and the production of a small amount of pseudo-lignin. The result shows that a LHW PS of 4.71 could completely degrade the hemicellulose in Miscanthus. Hemicellulose removal dislodged the enzymatic barrier of cellulose, and the ethanol conversion of 98.27% was obtained.
Our study demonstrated that LHW served as an effective pretreatment in case that Miscanthus lutarioriparious was used for ethanol production by simultaneous saccharification and fermentation. The combination and the pretreatment method of Miscanthus feedstock holds a great potential for biofuel production.
Miscanthus lutarioriparious; Liquid hot water pretreatment; Simultaneous saccharification and fermentation; Ethanol
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text:
I don't make New Year's Resolutions. I don't think I ever really did, but the last decade or two would have been enough to stifle that impulse. I've just been too aware that I don't have that much control over what happens in my life.
Discuss the article on the Forums.
To the PR Community: can someone help me with some of my labs and spect scan
Discussion in 'Diagnostic Guidelines and Laboratory Testing' started by loayachil, Mar 18, 2013.

subset: commoncrawl


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Science news articles about 'sun storm'
NASA's Solar Shield to Protect Power Grids From Sun StormsNASA has a new tool in the battle against massive sun storms.
Anatomy of Sun Storms & Solar Flares (Infographic)See how solar flares, sun storms and huge eruptions from the sun work in this SPACE.com infographic.
Space Pictures This Week: Sun Storm, Mars Lander, MoreA star nursery shines, a sun storm erupts, a Mars orbiter spies its cousin, and more in the week's best space pictures.
NASA spacecraft show three dimensional anatomy of a solar storm... lines and causing wide-spread blackouts. These sun storms can interfere with communications between ground controllers ... near Earth's poles. Radio noise from the storm also can disrupt cell phone service. Space ...
SPACE PHOTOS THIS WEEK: Sun Storm, Galactic Core, MoreThe active sun extends loops of particles, chaos reigns in the Milky Way's middle, tropical storm Ida gets spotted from above, and more in the week's best space pictures.
New Space Telescope to Target Sun StormsNASA plans to launch a new spacecraft, the Solar Dynamics Observatory (SDO), to take the most detailed observations ever of the sun and its complex weather.
Video: Solar Storms May Interrupt Cell PhonesNASA says intense sun storms have the ability to knock out our power and cell phone signals. Whit Johnson reports.
Sun storms may bring northern lights farther southBy 2010-08-02T22:04:25Z WASHINGTON (AP) -- The sun may be about to put on a colorful light show....
Sun Unleashes Impressive Solar FlareThe sun unleashed a huge solar flare and eruption Sept. 8, but the sun storm is not aimed at Earth.
Solar Shocker: Sun Storms Change DirectionsWhen storms of charged particles rage on the sun, they don't always travel in a straight line, a new study finds.
Solar flares bring Northern Lights to skies over Britain | Ian Sample... aurora may be visible for several nights as Sun storm brewsStargazers in Britain may witness the extraordinary lightshow ... opportunity to see the celestial spectacle follows a storm on the Sun that has produced the most powerful solar flare ...
'The Aurora': Photographer Films Amazing Northern Lights ShowWhen powerful sun storms set off dazzling auroras in March, photographer Torje Sorgjerd was ready.
Solar Flare Sparks Biggest Eruption Ever Seen on SunA sun storm has shot perhaps the largest amount of particles into space ever recorded, scientists say.
Solstice Sun Storm May Spark Dazzling Northern Lights TodaySkywatchers at high northern latitudes may see some supercharged auroras, NASA says.
As Sun Storms Ramp Up, Electric Grid Braces for ImpactWith the sun nearing the high point in its 11-year activity cycle, grid operators are seeking to protect a vulnerable power-delivery system.
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"Everything must be on the table."
"There must be shared sacrifice."
I keep hearing such pronouncements from voices both inside and outside of government who characterize themselves as "fiscally conservative." So why are many of them criticizing school spending cuts enacted by Gov. Jennifer Granholm through either line-item veto or executive order?
Stay Engaged
If one just looks just at the effect of the Governor's actions and ignores the motivations (the administration has all but admitted using school spending as a "hostage" to its demands for tax hikes) Ms. Granholm looks like a fiscal conservative's hero right now.
So where's the love?
For that matter, where's the love for taxpayers from the 12 Republicans and 62 Democrats who voted yesterday to appropriate $159 million in federal "stimulus" money being reserved for next year's school budget, and another $59 million the House proposes getting from various tax hikes, to "undo" school spending reductions?
The tax hikes (part of a proposed "Michigan Future Fund") include a proposed $300 million doctors tax, a $55 million suspension of income tax personal exemption inflation indexing, raising certain business taxes (by repealing deductions), a $43 million tobacco tax hike, and more.
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Middle East
Islamist rebels fighting President Bashar al-Assad's forces in Syria have rejected overtures from the United States to sit down and talk, a senior US diplomat said.
The Syrian government said it was "reprehensible" that Washington was prepared to enter a dialogue with the Islamic Front, which comprises six major Islamist rebel groups and which Damascus considers a terrorist organisation.
The developments highlighted Washington's difficulties in engaging with the faction-ridden rebels in Syria's civil war.
With five weeks to go before UN peace talks convene in Geneva, it is unclear who will represent the opposition, and the clock is ticking towards a December 27 deadline set by UN mediator Lakhdar Brahimi for both sides to name their delegations.
"The Islamic Front has refused to sit with us, without giving any reason," US Syria envoy Robert Ford told Al Arabiya television, speaking in Arabic, a day after Secretary of State John Kerry said such talks might take place.
"We are ready to sit with them because we talk to all parties and political groups in Syria," Mr Ford said.
The Islamic Front has overshadowed the more moderate Free Syrian Army, which is formally led by the Supreme Military Council (SMC) and backed by Western and Arab powers.
The Front has rejected the authority of the SMC, the military arm of the main political opposition in exile, and last week seized control of SMC weapons depots in northern Syria.
In a video statement given to Reuters by the SMC on Wednesday, Mr Ford described the takeover as "an extremely negative development" and urged disparate rebel groups to work together.
"If there is not tight cooperation between the different armed groups that are fighting the regime, the regime is going to be successful in surviving," he said.
"It is extremely important that the Supreme Military Council be able to play its role of coordinating. If it cannot do that, I do not see how the armed opposition can be truly effective."
Mr Kerry said on Tuesday a US meeting with the Islamic Front was possible as part of efforts to broaden opposition representation at the Geneva peace talks, but the Front's commanders are wary of openly coordinating with Washington.
Any such public engagement with the United States could put the newly formed Islamic Front on a collision course with the powerful al Qaeda-linked Islamic State in Iraq and the Levant.
The Syrian Foreign Ministry said in a statement carried on the state news agency that dialogue with the Islamic Front contradicted US and international commitments to combat terrorism, as well as "international pledges that terrorist organisations would not be given the chance to participate in the Geneva conference".
The long-delayed talks in Switzerland are meant to discuss a political transition to lead Syria out of a 33-month-old conflict in which more than 100,000 people have been killed.
The Islamic Front "agrees in principle, strategy and goals with the Nusra Front", the Syrian foreign ministry said, referring to another al Qaeda-linked rebel group that the State Department last year listed as a terrorist organisation.
- Reuters
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We report the measurements of charge collection efficiency for nuclear particle detectors made from electronic-grade chemical vapor deposition (CVD) diamond samples with thicknesses of 20 and 150 μm fabricated at CEA/LETI. Although it is well established that the transport properties of CVD diamond, and more specifically the μτ product, tend to improve with the sample thickness, it was observed that excellent collection efficiencies can be achieved on the 20-μm-thick detector owing to the reduced interelectrode distance. Due to the polycrystalline nature of CVD diamond, the charge collection efficiency is inhomogeneous throughout the detector surface. However, a mean collection efficiency of 50% was measured over the whole detector surface, with regions close to 100%, corresponding to the highest values reported in the literature for CVD diamond. This excellent performance enables the detection of very low-energy heavy charged particles with a detection limit well below 200 keV for protons
A not-for-profit organization, IEEE is the world's largest technical professional organization dedicated to advancing technology for the benefit of humanity.© Copyright 2016 IEEE - All rights reserved. Use of this web site signifies your agreement to the terms and conditions.


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Compliance & Standards Briefing: GHS Tools, 10,000th LEED, RoHS 2
3E Company has release a suite of new software and online data management tools to help companies comply with the Occupational Safety and Health Administration's (OSHA) alignment of its HazComm Standard with the United Nations' Globally Harmonized System of classification and labeling of chemicals, the company said. The new OSHA standards will change the way companies produce Material Safety Data Sheets and other labels.
The United Kingdom has collected 47 percent of WEEE for recycling so far in 2011, the nation's highest rate ever, the country's Environment Agency announced. So far this year, 249,284 metric tonnes of WEEE have been collected against 521, 785 metric tons that have been placed on the market. That collection rate would exceed the 45 percent of annual weight requirement that the EU is considering imposing on its members.
The U.S. Green Building Council last week awarded its 10,000th LEED certification to a community center in Santa Cruz, Calif., according to worldarchitecturenews.com. The milestone came twelve years after the council first began certifying energy efficient buildings.
The EU's RoHS 2 legislation, which extends the RoHS Directive's ban on the use of certain hazardous chemicals to almost any product with "electronic function," will significantly increase costs for suppliers who will need more documentation to prove compliance, a manager at TR Fastenings told supplymanagement.com. The new RoHS 2 legislation becomes effective in 2013.
China has changed its proposed RoHS measure to cover hazardous chemicals in "electrical and electronic products," Electronics Weekly reported. The previous proposal only applied to "electronic information products." No standards have been published yet.
A class action lawsuit has been filed alleging that Uponor, Inc. and Weil-McLain falsely represented that the plastic in their Multicor and AlumiPex plumbing systems met NSF Standard 61, the law firm Larson King announced. The suit claims that the companies submitted compliant pipes for NSF testing, and then manufactured the consumer products with different plastic containing toxic materials.
PEP Stations LLC, a provider of premium electric vehicle charging stations, announced that its PEP Stations PS2000 received ETL certification to UL 2231-1, UL 2231-2 and UL Subject 2594 from the third-party tester Intertek. Some of Pep Stations' clients include Hershey's Chocolate World, The Smithsonian Institution, and The Mandalay Bay.
The ZigBee Alliance, a group of companies creating wireless sensors and networks for use in energy management, commercial and consumer applications, announced that it has so far certified a total of 341 ZigBee Certified products.
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Energy Manager News
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156 Results for kyle long
Posted Jun 17, 2016
With Father's Day on Sunday, we asked Bears guard Kyle Long and some of his teammates to describe how instrumental their dads have been in their lives.
Posted Apr 15, 2016
As expected, the Bears have exercised their fifth-year option on offensive lineman Kyle Long, keeping him under contract through the 2017 season.
Posted Mar 11, 2016
Friends with Bobby Massie since they played high school football against each other in Virginia, Kyle Long helped convince the free-agent tackle to leave the Cardinals and sign with the Bears.
Posted Jun 15, 2016
Offensive lineman Kyle Long addresses the media Tuesday at Halas Hall.
Posted Jun 6, 2016
Kyle Long, Pat O'Donnell and Zach Miller check in from the 21st annual Bears Care Gala.
Posted May 25, 2016
Offensive lineman Kyle Long talks to the media Wednesday at Halas Hall during OTAs.
Posted May 4, 2016
Take a look behind the scenes as Bears players posed for a recent photo and video shoot at Halas Hall.
Posted Jan 14, 2016
Kyle Long has been named to the 2016 Pro Bowl. View photos of the Bears RT from this past season.
Posted Dec 5, 2014
On Kyle Long's 26th birthday, ChicagoBears.com takes a look at photos from the guard's 2-year career in the NFL.
Posted Aug 16, 2015
Hosts Zach Zaidman and Tom Thayer are joined by offensive lineman Kyle Long on the Bears All Access radio show.
Posted Aug 26, 2014
Guard Kyle Long joins Jeff Joniak on a conference call with fans.
Posted Apr 25, 2013
Bears first round Draft pick, number 20 overall, OT Kyle Long answers questions from the Chicago media.


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As Hurricane Sandy nears landfall on the East Coast, photos capturing the monstrous waves are being circulated across the Internet. Check out a collection of 10 amazing wave pictures that have surfaced so far.
Welcome to your discussion forum: Click the login link below to sign in with or to set up a Disqus account or to access your social networking account. When you do, your comment should be posted immediately, provided it meets the guidelines. (What are the guidelines?.) Report abuse by flagging a comment (mouse over the comment). Comments made here are the sole responsibility of the person posting them; these comments do not reflect the opinion of The Berkshire Eagle. So keep it civil.
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Brittany Murphy death house for sale for $18 million
If you have a spare $18 million, you can buy a Hollywood Hills mansion where Brittany Murphy and her husband died under mysterious circumstances within months of each other.


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Masters And Servants (Raging Stallion)
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After many readers' requests I've decided to do some research into the fine art of giving head to a girl. But seeing that I've only ever been on the receiving end, I thought my advice might be a little, ah, inappropriately personal.
So I rounded up my gorgeous lesbian and bisexual girlfriends for a few drinks and some frank advice. For the sake of the sisterhood, you know?
This is what they taught me…
Step 1
Hot things up before you start
Okay, I don't actually think this needs saying yet again, but guys, most women need to be – at least a little – hot for you before you dive for her clit.
She might like it if you tell her exactly what you're going to do to her. She might want some time to fantasize while you stroke her body. She might just want to make out a little. She might want to read something sexy first or watch Grey's Anatomy or Twilight or a really filthy porno. She might need a massage, some breast fondling, a bit of sweet talking or some sexy wrestling.
Heck, I don't what your lady wants, but you should. If you don't and she's not talking, try different things and read her reactions closely. As soon as she's feeling a little frisky, you can go South…
Step 2
Don't attack. And don't fall back.
So, you have your face between her legs. Now what? L says you should forget all that nonsense about butterfly kisses on her thighs and around her pubic mound. That type of "expectation creation" works fine for first time encounters, but if you've been with your woman for a while now, you'll only succeed in tickling her. So no beating about the bush people!
Of course, all women are different. There's a spot just a few inches up and to the left of my pubic bone… but I digress. Point is, if she's squirming this early, it's probably because you're tickling her.
Easy does it, Tiger. Meet soft and wet (your tongue) with soft and wet (her inner labia and clitoris). Then allow her to set the pace and pressure. I.e. Don't put your mouth on her like it's a cake fest and everyone's invited. Think of your tongue as something that provides constant warm, intelligent attention, listen to the her body's feedback, and allow her to ravish your mouth.
Step 3
Take your sweet time…
If you're down there, stay there for a while. The beautiful T says she normally spends from 30 minutes to an hour in her girlfriends Delta of Venus, driving said girlfriend to numerous orgasms.
We should all be so lucky.
This "time thing" ties up with the Step 1. In general, women don't reach orgasm as fast as men do. And she won't if she's nervous or under pressure. So if she knows she only has the four minutes you give her every time to "get there" she won't.
Then again, if she knows she can rely on your mouth to be there for a while, she can get lost and allow herself to drown in the throes of ecstasy.
Quick tips and techniques:
· Circles are your friends
· Use a firm, not hard or limp tongue
· Use teeth only if you really thinks she'll be into it.
· Don't suck on the clitoris for more than a second or two at a time and only do this when you know she's ready for it.
· Make eye contact.
· Praise her. Tell her how hot she is, how good she smells and tastes etc.
· Keep your fingers out of the pie. Until the very last…
Okay ladies and gentlemen. Go out and enjoy! If you have any more queries or tips, feel free to add them in the box below…
- Women24
Because really, you don't have to be a kinky contortionist with a model's body to be spectacular in bed.
If you judge other women's sexual potency by how old they are, it's time to stop limiting yourself and the women around you, says Dorothy Black.
Read Women24's Comments Policy
24.com publishes all comments posted on articles provided that they adhere to our Comments Policy. Should you wish to report a comment for editorial review, please do so by clicking the 'Report Comment' button to the right of each comment.
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SEC: Apple reveals Cook's 2014 pay, Drexler retires from Apple board
In a new filing with the SEC, Apple revealed on Thursday that CEO Tim Cook has been able to double his total compensation from that of last year, with the increase based largely on performance bonuses based on how well the company has done over the last year. In addition to $1.75 million in salary, Cook reaped $6.7 million in non-equity incentive compensation for his work. In addition, Apple disclosed in the filing that board member Millard "Mickey" Drexler will retire.
Apple now requires executives to hold 3x salary in stock [U]
[Updated with details, stock grants for BoD] Despite recommending (and obtaining) a "no" vote on a similar measure, Apple has updated its bylaws and now requires executive officers to hold at least three times their annual salary in AAPL stock, reports the Wall Street Journal. The rule adopted by Apple has significant differences from the proposal put forth at Apple's annual shareholder meeting, but embraces the same principle -- that executives (and in this case non-executive directors as well) need to be personally invested in the companies they manage.
Apple execs net total of $26.6m for 2007
Apple's executive officers have taken home a considerable sum during 2007, with salaries ranging from $500,000 to $700,000, and stock option grants equaling that of the individual�s base salary. Despite his infamous $1 yearly salary, CEO Steve Jobs still holds the majority of company stock with 5.5 million shares, 5 million of which was a gift from the company in March 2003. Jobs also received a $43.5 million Gulfstream V jet as a bonus for outstanding performance from 1997 to 1999.
Now AAPL Stock: 93.4 ( -2.7 )
WhatsApp now handles over 100M calls daily
WhatsApp is celebrating that it is being used for over 100 million calls every day. In a brief notice, the Facebook-owned messaging platform advises the voice-calling feature it rolled out to its users last year now deals with an average of over 1,100 calls initiated per second. Earlier this year, it increased the security of its calls and other messages, by introducing end-to-end encryption on all platforms. http://bit.ly/292HqCX
Adele's '25' album now streaming
Recording artist Adele has "pulled a Kanye" after saying that her current album "25" would not be available for streaming. The seven-month-old record, which has yielded a number of hit singles, is now available for streaming on all the major streaming services, such as Apple Music and Spotify, as of today in most major markets, with worldwide distribution to come. Reportedly, the singer had demanded streaming be limited to paid subscribers -- a condition that has hurt some streamers with artists, who aren't paid royalties for free or trial listens . Apple pays performers its normal royalty rates during its free trial, avoiding the issue -- and having repeated success in both signing up exclusives and placing those exclusives into the top of the charts. http://ti.me/28U7NOu
SanDisk iXpand case has battery, storage
A new iPhone 6 and iPhone 6s case from SanDisk appears to be the "holy grail" of accessories: a stylish and protective case that offers both extra storage as well as the option of extra battery power as well. The iXpand Memory case offers either 32GB, 64GB, or 128GB of additional storage incorporated into the case, and an optional add-on battery pack (sold separately) adds up to an extra day or more of charge. Through the associated iXpand app, camera photos and videos can be automatically stored on the extra storage, optionally password-protected, The cost for the case is (in order of storage capacity) $60, $100, and $130. The battery pack's release data has not yet been announced, but the add-on should retail for an additional $30. http://bit.ly/291epHu
Fifth Hong Kong store to open June 30
The 46th official Apple store in what the company calls the "Greater China" region will open at 10AM local time in Hong Kong's New Town Plaza in the Sha Tin district on Thursday, June 30, according to a new page on Apple's retail website. Despite slowing growth due to economic turmoil in China, the country is still Apple's fastest-growing market. The new story will be the fifth for Hong Kong, a lucrative market for Apple thanks to the high concentration of wealth there and a thriving "gray" market. Normal store hours for the new store will be 10AM to 10PM every day. http://apple.co/291diaT
Apple Music bug bites very short songs
There appears to be a reproducible bug in the current version of iTunes -- version 12.4.1, running on OS X 10.11.5, both the current non-beta versions of the respective software -- that causes a streamed song to become "stuck" in endless buffering if the streamed item just before this happened was 60 seconds or less in length. The problem appears to stem from code in iTunes that signals the next track to start downloading one minute before the currently-playing track is done, and thus songs shorter than one minute don't trigger the download. Locally-stored music is unaffected by the bug, and the problem does not appear to exist in either the macOS Sierra developer beta or versions of iTunes earlier than 12.4, and does not affect the iOS Music app at all. http://bit.ly/291cN0l
Walmart Pay expands to 15 states
Walmart, one of the key progenitors of the failed CurrentC mobile payment system that was distinguished from Apple Pay by its ability to collect and share customer financial and buying data among its participants, has expanded the soft rollout of its alternative solution "Walmart Pay" to an additional 15 US states following a pilot program in Arkansas and Texas last month. Walmart continues to resist adding Apple Pay. The system, built into the Walmart app for iOS and Android, works with a complicated system of the camera scanning a purchase code at the register, then generating a QR code which is then scanned by the register. The system is available now in Alabama, Georgia, Indiana, Iowa, Kentucky, Louisiana, Michigan, Mississippi, Missouri, Nebraska, North Carolina, North Dakota, South Carolina, South Dakota, Tennessee, Virginia, and Washington, DC. http://apple.co/28SqZfu
Amazon refreshes entry-level Kindle
Amazon has added a new Kindle to its ebook reader range, one that is thinner and lighter than the previous entry-level model. Offering a six-inch 167-dpi touchscreen display, a doubled 4GB of storage, and an option for a white casing, the new model also adds in the ability to export highlights and notes to an email account as a PDF. Two versions of the all-new Kindle are available, priced at $80 including "Special Offers" and $100 without. http://amzn.to/28Q4c3R