Cas9-mediated mutagenesis of potato starch-branching enzymes generates a range of tuber starch phenotypes.

We investigated whether Cas9-mediated mutagenesis of starch-branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium-mediated transformation or by PEG-mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low-SBE potato tubers. HPLC-SEC and 1 H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild-type starch. Mutants with strong reductions in SBE2 protein alone had near-normal amylopectin chain-length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 µm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9-mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.

High Molecular Weight Mixed-Linkage Glucan as a Mechanical and Hydration Modulator of Bacterial Cellulose: Characterization by Advanced NMR Spectroscopy.

Bacterial cellulose (BC) consists of a complex three-dimensional organization of ultrafine fibers which provide unique material properties such as softness, biocompatibility, and water-retention ability, of key importance for biomedical applications. However, there is a poor understanding of the molecular features modulating the macroscopic properties of BC gels. We have examined chemically pure BC hydrogels and composites with arabinoxylan (BC-AX), xyloglucan (BC-XG), and high molecular weight mixed-linkage glucan (BC-MLG). Atomic force microscopy showed that MLG greatly reduced the mechanical stiffness of BC gels, while XG and AX did not exert a significant effect. A combination of advanced solid-state NMR methods allowed us to characterize the structure of BC ribbons at ultra-high resolution and to monitor local mobility and water interactions. This has enabled us to unravel the effect of AX, XG, and MLG on the short-range order, mobility, and hydration of BC fibers. Results show that BC-XG hydrogels present BC fibrils of increased surface area, which allows BC-XG gels to hold higher amounts of bound water. We report for the first time that the presence of high molecular weight MLG reduces the density of clusters of BC fibrils and dramatically increases water interactions with BC. Our data supports two key molecular features determining the reduced stiffness of BC-MLG hydrogels, that is, (i) the adsorption of MLG on the surface of BC fibrils precluding the formation of a dense network and (ii) the preorganization of bound water by MLG. Hence, we have produced and fully characterized BC-MLG hydrogels with novel properties which could be potentially employed as renewable materials for applications requiring high water retention capacity (e.g. personal hygiene products).

Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase.

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

Hybrid metagenome assemblies link carbohydrate structure with function in the human gut microbiome.

Complex carbohydrates that escape small intestinal digestion, are broken down in the large intestine by enzymes encoded by the gut microbiome. This is a symbiotic relationship between microbes and host, resulting in metabolic products that influence host health and are exploited by other microbes. However, the role of carbohydrate structure in directing microbiota community composition and the succession of carbohydrate-degrading microbes, is not fully understood. In this study we evaluate species-level compositional variation within a single microbiome in response to six structurally distinct carbohydrates in a controlled model gut using hybrid metagenome assemblies. We identified 509 high-quality metagenome-assembled genomes (MAGs) belonging to ten bacterial classes and 28 bacterial families. Bacterial species identified as carrying genes encoding starch binding modules increased in abundance in response to starches. The use of hybrid metagenomics has allowed identification of several uncultured species with the functional potential to degrade starch substrates for future study.

Vortex fluidic mediated direct transesterification of wet microalgae biomass to biodiesel.

A bottleneck in the production of biodiesel from microalgae is the dewatering and lipid extraction process which is the dominant energy penalty and cost. A novel biodiesel production platform based on vortex fluidic device (VFD)-assisted direct transesterification (DT) of wet microalgal biomass of Chloroparva pannonica was developed and evaluated. Fatty acid extraction and fatty acid to FAME conversion efficiencies were used at different parameter settings to evaluate performance of the processing technology in confined and continuous mode. A response surface method based on Box-Behnken experimental design was used to determine the effects of water content, the ratio of biomass to methanol and residence time in the VFD. Average extraction efficiencies were 41% and conversion efficiencies >90% with the processing technology showing a broad tolerance to parameter settings. The findings suggest that VFD-assisted DT is a simple and effective way to produce biodiesel directly from wet microalgae biomass at room temperature.

Lobster processing by-products as valuable bioresource of marine functional ingredients, nutraceuticals, and pharmaceuticals.

The worldwide annual production of lobster was 165,367 tons valued over $3.32 billion in 2004, but this figure rose up to 304,000 tons in 2012. Over half the volume of the worldwide lobster production has been processed to meet the rising global demand in diversified lobster products. Lobster processing generates a large amount of by-products (heads, shells, livers, and eggs) which account for 50-70% of the starting material. Continued production of these lobster processing by-products (LPBs) without corresponding process development for efficient utilization has led to disposal issues associated with costs and pollutions. This review presents the promising opportunities to maximize the utilization of LPBs by economic recovery of their valuable components to produce high value-added products. More than 50,000 tons of LPBs are globally generated, which costs lobster processing companies upward of about $7.5 million/year for disposal. This not only presents financial and environmental burdens to the lobster processors but also wastes a valuable bioresource. LPBs are rich in a range of high-value compounds such as proteins, chitin, lipids, minerals, and pigments. Extracts recovered from LPBs have been demonstrated to possess several functionalities and bioactivities, which are useful for numerous applications in water treatment, agriculture, food, nutraceutical, pharmaceutical products, and biomedicine. Although LPBs have been studied for recovery of valuable components, utilization of these materials for the large-scale production is still very limited. Extraction of lobster components using microwave, ultrasonic, and supercritical fluid extraction were found to be promising techniques that could be used for large-scale production. LPBs are rich in high-value compounds that are currently being underutilized. These compounds can be extracted for being used as functional ingredients, nutraceuticals, and pharmaceuticals in a wide range of commercial applications. The efficient utilization of LPBs would not only generate significant economic benefits but also reduce the problems of waste management associated with the lobster industry. This comprehensive review highlights the availability of the global LPBs, the key components in LPBs and their current applications, the limitations to the extraction techniques used, and the suggested emerging techniques which may be promising on an industrial scale for the maximized utilization of LPBs. Graphical abstractLobster processing by-product as bioresource of several functional and bioactive compounds used in various value-added products.

Emerging Technologies for the Production of Renewable Liquid Transport Fuels from Biomass Sources Enriched in Plant Cell Walls.

Plant cell walls are composed predominantly of cellulose, a range of non-cellulosic polysaccharides and lignin. The walls account for a large proportion not only of crop residues such as wheat straw and sugarcane bagasse, but also of residues of the timber industry and specialist grasses and other plants being grown specifically for biofuel production. The polysaccharide components of plant cell walls have long been recognized as an extraordinarily large source of fermentable sugars that might be used for the production of bioethanol and other renewable liquid transport fuels. Estimates place annual plant cellulose production from captured light energy in the order of hundreds of billions of tons. Lignin is synthesized in the same order of magnitude and, as a very large polymer of phenylpropanoid residues, lignin is also an abundant, high energy macromolecule. However, one of the major functions of these cell wall constituents in plants is to provide the extreme tensile and compressive strengths that enable plants to resist the forces of gravity and a broad range of other mechanical forces. Over millions of years these wall constituents have evolved under natural selection to generate extremely tough and resilient biomaterials. The rapid degradation of these tough cell wall composites to fermentable sugars is therefore a difficult task and has significantly slowed the development of a viable lignocellulose-based biofuels industry. However, good progress has been made in overcoming this so-called recalcitrance of lignocellulosic feedstocks for the biofuels industry, through modifications to the lignocellulose itself, innovative pre-treatments of the biomass, improved enzymes and the development of superior yeasts and other microorganisms for the fermentation process. Nevertheless, it has been argued that bioethanol might not be the best or only biofuel that can be generated from lignocellulosic biomass sources and that hydrocarbons with intrinsically higher energy densities might be produced using emerging and continuous flow systems that are capable of converting a broad range of plant and other biomasses to bio-oils through so-called 'agnostic' technologies such as hydrothermal liquefaction. Continued attention to regulatory frameworks and ongoing government support will be required for the next phase of development of internationally viable biofuels industries.

Prospecting for Energy-Rich Renewable Raw Materials: Sorghum Stem Case Study.

Sorghum vegetative tissues are becoming increasingly important for biofuel production. The composition of sorghum stem tissues is influenced by genotype, environment and photoperiod sensitivity, and varies widely between varieties and also between different stem tissues (outer rind vs inner pith). Here, the amount of cellulose, (1,3;1,4)-ß-glucan, arabinose and xylose in the stems of twelve diverse sorghum varieties, including four photoperiod-sensitive varieties, was measured. At maturity, most photoperiod-insensitive lines had 1% w/w (1,3;1,4)-ß-glucan in stem pith tissue whilst photoperiod-sensitive varieties remained in a vegetative stage and accumulated up to 6% w/w (1,3;1,4)-ß-glucan in the same tissue. Three sorghum lines were chosen for further study: a cultivated grain variety (Sorghum bicolor BTx623), a sweet variety (S. bicolor Rio) and a photoperiod-sensitive wild line (S. bicolor ssp. verticilliflorum Arun). The Arun line accumulated 5.5% w/w (1,3;1,4)-ß-glucan and had higher SbCslF6 and SbCslH3 transcript levels in pith tissues than did photoperiod-insensitive varieties Rio and BTx623 (<1% w/w pith (1,3;1,4)-ß-glucan). To assess the digestibility of the three varieties, stem tissue was treated with either hydrolytic enzymes or dilute acid and the release of fermentable glucose was determined. Despite having the highest lignin content, Arun yielded significantly more glucose than the other varieties, and theoretical calculation of ethanol yields was 10 344 L ha-1 from this sorghum stem tissue. These data indicate that sorghum stem (1,3;1,4)-ß-glucan content may have a significant effect on digestibility and bioethanol yields. This information opens new avenues of research to generate sorghum lines optimised for biofuel production.

Grape marc as a source of carbohydrates for bioethanol: Chemical composition, pre-treatment and saccharification.

Global grape production could generate up to 13 Mt/yr of wasted biomass. The compositions of Cabernet Sauvignon (red marc) and Sauvignon Blanc (white marc) were analyzed with a view to using marc as raw material for biofuel production. On a dry weight basis, 31-54% w/w of the grape marc consisted of carbohydrate, of which 47-80% was soluble in aqueous media. Ethanol insoluble residues consisted mainly of polyphenols, pectic polysaccharides, heteroxylans and cellulose. Acid and thermal pre-treatments were investigated for their effects on subsequent cellulose saccharification. A 0.5M sulfuric acid pre-treatment yielded a 10% increase in the amount of liberated glucose after enzymatic saccharification. The theoretical amount of bioethanol that could be produced by fermentation of grape marc was up to 400 L/t. However, bioethanol from only soluble carbohydrates could yield 270 L/t, leaving a polyphenol enriched fraction that may be used in animal feed or as fertilizer.

Prospecting for Energy-Rich Renewable Raw Materials: Agave Leaf Case Study.

Plant biomass from different species is heterogeneous, and this diversity in composition can be mined to identify materials of value to fuel and chemical industries. Agave produces high yields of energy-rich biomass, and the sugar-rich stem tissue has traditionally been used to make alcoholic beverages. Here, the compositions of Agave americana and Agave tequilana leaves are determined, particularly in the context of bioethanol production. Agave leaf cell wall polysaccharide content was characterized by linkage analysis, non-cellulosic polysaccharides such as pectins were observed by immuno-microscopy, and leaf juice composition was determined by liquid chromatography. Agave leaves are fruit-like--rich in moisture, soluble sugars and pectin. The dry leaf fiber was composed of crystalline cellulose (47-50% w/w) and non-cellulosic polysaccharides (16-22% w/w), and whole leaves were low in lignin (9-13% w/w). Of the dry mass of whole Agave leaves, 85-95% consisted of soluble sugars, cellulose, non-cellulosic polysaccharides, lignin, acetate, protein and minerals. Juice pressed from the Agave leaves accounted for 69% of the fresh weight and was rich in glucose and fructose. Hydrolysis of the fructan oligosaccharides doubled the amount of fermentable fructose in A. tequilana leaf juice samples and the concentration of fermentable hexose sugars was 41-48 g/L. In agricultural production systems such as the tequila making, Agave leaves are discarded as waste. Theoretically, up to 4000 L/ha/yr of bioethanol could be produced from juice extracted from waste Agave leaves. Using standard Saccharomyces cerevisiae strains to ferment Agave juice, we observed ethanol yields that were 66% of the theoretical yields. These data indicate that Agave could rival currently used bioethanol feedstocks, particularly if the fermentation organisms and conditions were adapted to suit Agave leaf composition.