The complex polyploid genome architecture of sugarcane.

Sugarcane, the world's most harvested crop by tonnage, has shaped global history, trade and geopolitics, and is currently responsible for 80% of sugar production worldwide1. While traditional sugarcane breeding methods have effectively generated cultivars adapted to new environments and pathogens, sugar yield improvements have recently plateaued2. The cessation of yield gains may be due to limited genetic diversity within breeding populations, long breeding cycles and the complexity of its genome, the latter preventing breeders from taking advantage of the recent explosion of whole-genome sequencing that has benefited many other crops. Thus, modern sugarcane hybrids are the last remaining major crop without a reference-quality genome. Here we take a major step towards advancing sugarcane biotechnology by generating a polyploid reference genome for R570, a typical modern cultivar derived from interspecific hybridization between the domesticated species (Saccharum officinarum) and the wild species (Saccharum spontaneum). In contrast to the existing single haplotype ('monoploid') representation of R570, our 8.7 billion base assembly contains a complete representation of unique DNA sequences across the approximately 12 chromosome copies in this polyploid genome. Using this highly contiguous genome assembly, we filled a previously unsized gap within an R570 physical genetic map to describe the likely causal genes underlying the single-copy Bru1 brown rust resistance locus. This polyploid genome assembly with fine-grain descriptions of genome architecture and molecular targets for biotechnology will help accelerate molecular and transgenic breeding and adaptation of sugarcane to future environmental conditions.

Association of gene expression with syringyl to guaiacyl ratio in sugarcane lignin.

KEY MESSAGE: A transcriptome analysis reveals the transcripts and alleles differentially expressed in sugarcane genotypes with contrasting lignin composition. Sugarcane bagasse is a highly abundant resource that may be used as a feedstock for the production of biofuels and bioproducts in order to meet increasing demands for renewable replacements for fossil carbon. However, lignin imparts rigidity to the cell wall that impedes the efficient breakdown of the biomass into fermentable sugars. Altering the ratio of the lignin units, syringyl (S) and guaiacyl (G), which comprise the native lignin polymer in sugarcane, may facilitate the processing of bagasse. This study aimed to identify genes and markers associated with S/G ratio in order to accelerate the development of sugarcane bioenergy varieties with modified lignin composition. The transcriptome sequences of 12 sugarcane genotypes that contrasted for S/G ratio were compared and there were 2019 transcripts identified as differentially expressed (DE) between the high and low S/G ratio groups. These included transcripts encoding possible monolignol biosynthetic pathway enzymes, transporters, dirigent proteins and transcriptional and post-translational regulators. Furthermore, the frequencies of single nucleotide polymorphisms (SNPs) were compared between the low and high S/G ratio groups to identify specific alleles expressed with the phenotype. There were 2063 SNP loci across 787 unique transcripts that showed group-specific expression. Overall, the DE transcripts and SNP alleles identified in this study may be valuable for breeding sugarcane varieties with altered S/G ratio that may provide desirable bioenergy traits.

Comparison of different pretreatments for the production of bioethanol and biomethane from corn stover and switchgrass.

In this study the efficiency of mild ionic liquid (IL) pretreatment and pressurized hot water (PHW) is evaluated and compared in terms of bioethanol and biomethane yields, with corn stover (CS) and switchgrass (SG) as model bioenergy crops. Both feedstocks pretreated with the IL 1-ethyl-3-methylimidazolium acetate [C2C1Im][OAc] at 100°C for 3h exhibited lower glucose yield that those treated with harsher pretreatment conditions previously used. Compared to PHW, IL pretreatment demonstrated higher bioethanol yields; moreover IL pretreatment enhanced biomethane production. Taking into consideration both bioethanol and biomethane productions, results indicated that when using IL pretreatment, the total energy produced per kg of total solids was higher compared to untreated biomasses. Specifically energy produced from CS and SG was +18.6% and +34.5% respectively, as compared to those obtained by hot water treatment, i.e. +2.3% and +23.4% for CS and SG, respectively.

Effect of inoculum source on the enrichment of microbial communities on two lignocellulosic bioenergy crops under thermophilic and high-solids conditions.

AIMS: Culturing compost-derived microbial communities on biofuel feedstocks under industrial conditions is a technique to enrich for organisms and lignocellulolytic enzymes for bioenergy feedstock deconstruction. In this study, microbial communities from green waste compost (GWC) and grape pomace compost (GPC) were cultured on switchgrass and eucalyptus to observe the impact of inoculation on feedstock decomposition and microbial community structure. METHODS AND RESULTS: Respiration was monitored as a measure of microbial activity, and 16S ribosomal RNA gene sequencing was used to characterize microbial community structure. The enriched community structure and respiration were influenced by the choice of feedstock, compost type, and application of thermophilic, high-solids conditions. However, the effect of compost source was significantly less than the effects of the other culture variables. CONCLUSIONS: Although there are subtle differences in potentially lignocellulolytic taxa between GPC- and GWC-derived communities, these differences do not affect the decomposition rates for these communities on switchgrass or eucalyptus. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are useful for designing future experiments to discover lignocellulolytic micro-organisms from compost. They suggest that such work may be better served by deemphasizing screening of compost sources and instead focusing on how compost-derived communities adapt to the feedstocks and process conditions relevant to biofuel production.

New experimental density data and soft-SAFT models of alkylimidazolium ([C(n)C1im]⁺) chloride (Cl⁻), methylsulfate ([MeSO4]⁻), and dimethylphosphate ([Me2PO4]⁻) based ionic liquids.

Ionic liquids have been shown to have application in several areas of importance in the context of sustainable industrial activity. One application of particular interest is the ability of certain ionic liquids to dissolve biomass. This clearly marks them as useful materials with application within biorefineries. In this contribution, we present new coarse-grained soft-SAFT models and experimental density data of chloride (Cl(-)), methylsulfate ([MeSO4](-)), and dimethylphosphate ([Me2PO4](-)) based ionic liquids which are relevant for biomass deconstruction processes. Model parameters were obtained by fitting to pure component temperature density data, and the models were subsequently tested by assessing their ability to accurately calculate viscosity and interfacial surface tension. We also developed models of mixtures of the ionic liquids with water and short-chain linear alcohols. We decomposed the contributions to the excess Gibbs energy of mixing to chemical and structural contributions, and used this to provide some insight into the driving forces for solubility of molecular species in these ionic liquids.

Thermophilic enrichment of microbial communities in the presence of the ionic liquid 1-ethyl-3-methylimidazolium acetate.

AIMS: The aim of the study was to develop an approach to enrich ionic liquid tolerant micro-organisms that efficiently decompose lignocellulose in a thermophilic and high-solids environment. METHODS AND RESULTS: High-solids incubations were conducted, using compost as an inoculum source, to enrich for thermophilic communities that decompose switchgrass in the presence of the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]). Ionic liquid levels were increased from 0 to 6% on a total weight basis incrementally. Successful enrichment of a community that decomposed lignocellulose at 55°C in the presence of 6% [C2mim][OAc] was achieved, when the [C2mim][OAc] level was increased stepwise from 2% to 4% to 5% to 6%. Pyrosequencing results revealed a shift in the community and a sharp decrease in richness, when thermophilic conditions were applied. CONCLUSIONS: A community tolerant to a thermophilic, high-solids environment containing 6% [C2mim][OAc] was enriched from compost. Gradually increasing [C2mim][OAc] concentrations allowed the community to adapt to [C2mim][OAc]. SIGNIFICANCE AND IMPACT OF THE STUDY: A successful approach to enrich communities that decompose lignocellulose under thermophilic high-solids conditions in the presence of elevated levels of [C2mim][OAc] has been developed. Communities yielded from this approach will provide resources for the discovery of enzymes and metabolic pathways relevant to biomass pretreatment and fuel production.

Exploring the effect of different plant lignin content and composition on ionic liquid pretreatment efficiency and enzymatic saccharification of Eucalyptus globulus L. mutants.

There are several approaches being investigated to improve the efficiency of biomass conversion into fermentable sugars, including those that engineer the feedstocks to enhance digestibility. In this study it was evaluated the impact of genotype modifications of three mutants of Eucalyptus globulus L., and of the corresponding wild type on cellulose hydrolyzability before and after ionic liquid (IL) pretreatment. Both untreated and IL-treated samples were chemically characterized and tested for cellulose hydrolizability. Results obtained indicate that genetic modifications altered wood lignin-S/G ratio. This alteration resulted in a different hydrolyzability of cellulose for untreated samples, i.e. high lignin-S/G ratio produced low glucose yield (r=-0.97; P<0.03; n=4), but did not affect glucose yield after IL pretreatment. IL pretreated samples had increased glucose yields compared to that of untreated samples due to the modification of microcrystalline cellulose I to mixtures of more hydrolysable cellulose II and amorphous cellulose, and to the partial removal of the steric impediment, or removal of the lignin "sheath" protecting cellulose, to enzymes. The efficiency of the IL pretreatment used in this study does not appear to be affected by the S/G content of the E. globulus.

Enrichment, isolation and characterization of fungi tolerant to 1-ethyl-3-methylimidazolium acetate.

AIMS: This work aimed to characterize microbial tolerance to 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid that has emerged as a novel biomass pretreatment for lignocellulosic biomass. METHODS AND RESULTS: Enrichment experiments performed using inocula treated with [C2mim][OAc] under solid and liquid cultivation yielded fungal populations dominated by Aspergilli. Ionic liquid-tolerant Aspergillus isolates from these enrichments were capable of growing in a radial plate growth assay in the presence of 10% [C2mim][OAc]. When a [C2mim][OAc]-tolerant Aspergillus fumigatus strain was grown in the presence of switchgrass, endoglucanases and xylanases were secreted that retained residual enzymatic activity in the presence of 20% [C2mim][OAc]. CONCLUSIONS: The results of the study suggest that tolerance to ionic liquids is a general property of the Aspergilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Tolerance to an industrially important ionic liquid was discovered in a fungal genera that is widely used in biotechnology, including biomass deconstruction.

Study of enzymatic digestion of cellulose by small angle neutron scattering.

Small angle neutron scattering (SANS) was used to study the structure of Avicel (FD100) microcrystalline cellulose during enzymatic digestion. Digestions were performed in either of two modes: a static, quiescent mode or a dynamic mode using a stirred suspension recycled through a flow cell. The scattering pattern for as-received Avicel in D(2)O buffer is comprised of a low Q power law region resulting from the surface fractal character of the microcrystalline fibers and a high Q roll-off due to scattering from water-filled nanopores with radii approximately 20 A. For digestions in the dynamic mode the high Q roll-off decreased in magnitude within approximately 1 h after addition of enzymes, whereas in the static digestions no change was observed in the high Q roll-off, even after 60 h. These results indicate that only with significant agitation does enzyme digestion affect the structure of the nanopores.

Microstructure determination of AOT + phenol organogels utilizing small-angle X-ray scattering and atomic force microscopy.

Dry reverse micelles of the anionic twin-tailed surfactant bis(2-ethylhexyl) sulfosuccinate (AOT) dissolved in nonpolar solvents spontaneously form an organogel when p-chlorophenol is added in a 1:1 AOT:phenol molar ratio. The solvents used were benzene, toluene, m-xylene, 2,2,4-trimethylpentane (isooctane), decane, dodecane, tetradecane, hexadecane, and 2,6,10,14-tetramethylpentadecane (TMPD). The proposed microstructure of the gel is based on strands of stacked phenols linked to AOT through hydrogen bonding. Small-angle X-ray scattering (SAXS) spectra of the organogels suggest a characteristic length scale for these phenol-AOT strands that is independent of concentration but dependent on the chemical nature of the nonpolar solvent used. Correlation lengths determined from the SAXS spectra indicate that the strands self-assemble into fibers. Direct visualization of the gel in its native state is accomplished by using tapping mode atomic force microscopy (AFM). It is shown that these organogels consist of fiber bundle assemblies. The SAXS and AFM data reinforce the theory of a molecular architecture consisting of three length scales-AOT/phenolic strands (ca. 2 nm in diameter) that self-assemble into fibers (ca. 10 nm in diameter), which then aggregate into fiber bundles (ca. 20-100 nm in diameter) and form the organogel.

Women's retirement, work and life paths: changes, disruptions and discontinuities.

In this qualitative study we examine women's perceptions and interpretations of the impact of retirement on their life experience, whether retirement was encountered from unexpected workplace redundancies, through their own decisions, or those of their partner. In the first instance we examine traditional definitions and meanings of retirement and their relevance to women's experience. Any theoretical explanation that work is central to defining core identity and social integration is challenged. Secondly, this study provides some alternative ways to examine changes in work, disruptions, and discontinuities in the context of women's life paths. Whereas specific identities and roles may be important, being able to contribute to social life, whether through paid or unpaid work or through self-interests, may be more appropriate determinants of societal integration, than identities that are formed and maintained by a production-oriented society.

A community response to elder abuse.

The 1987 amendments to the Older Americans Act of 1965 required that State Offices on Aging identify agencies involved in identifying and treating abused, neglected, and exploited elders and determine the need for appropriate services for such individuals. This paper discusses how the Dallas, Texas Area Agency on Aging met this requirement and what the community did to increase services for those in need. The creation of an Elder Abuse Task Force had a radiating impact on state law, the statewide adult protective service system, and regional policymaking bodies.