The recovery of genomes from metagenomic datasets is a critical step to defining the functional roles of the underlying uncultivated populations. We previously developed MaxBin, an automated binning ...approach for high-throughput recovery of microbial genomes from metagenomes. Here we present an expanded binning algorithm, MaxBin 2.0, which recovers genomes from co-assembly of a collection of metagenomic datasets. Tests on simulated datasets revealed that MaxBin 2.0 is highly accurate in recovering individual genomes, and the application of MaxBin 2.0 to several metagenomes from environmental samples demonstrated that it could achieve two complementary goals: recovering more bacterial genomes compared to binning a single sample as well as comparing the microbial community composition between different sampling environments.
MaxBin 2.0 is freely available at http://sourceforge.net/projects/maxbin/ under BSD license.
Cellulose is inherently resistant to breakdown, and the native crystalline structure (cellulose I) of cellulose is considered to be one of the major factors limiting its potential in terms of ...cost-competitive lignocellulosic biofuel production. Here we report the impact of ionic liquid pretreatment on the cellulose crystalline structure in different feedstocks, including microcrystalline cellulose (Avicel), switchgrass ( Panicum virgatum ), pine ( Pinus radiata ), and eucalyptus ( Eucalyptus globulus ), and its influence on cellulose hydrolysis kinetics of the resultant biomass. These feedstocks were pretreated using 1-ethyl-3-methyl imidazolium acetate (C2mimOAc) at 120 and 160 °C for 1, 3, 6, and 12 h. The influence of the pretreatment conditions on the cellulose crystalline structure was analyzed by X-ray diffraction (XRD). On a larger length scale, the impact of ionic liquid pretreatment on the surface roughness of the biomass was determined by small-angle neutron scattering (SANS). Pretreatment resulted in a loss of native cellulose crystalline structure. However, the transformation processes were distinctly different for Avicel and for the biomass samples. For Avicel, a transformation to cellulose II occurred for all processing conditions. For the biomass samples, the data suggest that pretreatment for most conditions resulted in an expanded cellulose I lattice. For switchgrass, first evidence of cellulose II only occurred after 12 h of pretreatment at 120 °C. For eucalyptus, first evidence of cellulose II required more intense pretreatment (3 h at 160 °C). For pine, no clear evidence of cellulose II content was detected for the most intense pretreatment conditions of this study (12 h at 160 °C). Interestingly, the rate of enzymatic hydrolysis of Avicel was slightly lower for pretreatment at 160 °C compared with pretreatment at 120 °C. For the biomass samples, the hydrolysis rate was much greater for pretreatment at 160 °C compared with pretreatment at 120 °C. The result for Avicel can be explained by more complete conversion to cellulose II upon precipitation after pretreatment at 160 °C. By comparison, the result for the biomass samples suggests that another factor, likely lignin−carbohydrate complexes, also impacts the rate of cellulose hydrolysis in addition to cellulose crystallinity.
The efficiency of two biomass pretreatment technologies, dilute acid hydrolysis and dissolution in an ionic liquid, are compared in terms of delignification, saccharification efficiency and ...saccharide yields with switchgrass serving as a model bioenergy crop. When subject to ionic liquid pretreatment (dissolution and precipitation of cellulose by anti-solvent) switchgrass exhibited reduced cellulose crystallinity, increased surface area, and decreased lignin content compared to dilute acid pretreatment. Pretreated material was characterized by powder X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy and chemistry methods. Ionic liquid pretreatment enabled a significant enhancement in the rate of enzyme hydrolysis of the cellulose component of switchgrass, with a rate increase of 16.7-fold, and a glucan yield of 96.0% obtained in 24
h. These results indicate that ionic liquid pretreatment may offer unique advantages when compared to the dilute acid pretreatment process for switchgrass. However, the cost of the ionic liquid process must also be taken into consideration.
High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C) and thermophilic (55°C) conditions. Thermophilic enrichments ...yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2) were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production.
Auto-fluorescent mapping of plant cell walls was used to visualize cellulose and lignin in pristine switchgrass (Panicum virgatum) stems to determine the mechanisms of biomass dissolution during ...ionic liquid pretreatment. The addition of ground switchgrass to the ionic liquid 1-n-ethyl-3-methylimidazolium acetate resulted in the disruption and solubilization of the plant cell wall at mild temperatures. Swelling of the plant cell wall, attributed to disruption of inter- and intramolecular hydrogen bonding between cellulose fibrils and lignin, followed by complete dissolution of biomass, was observed without using imaging techniques that require staining, embedding, and processing of biomass. Subsequent cellulose regeneration via the addition of an anti-solvent, such as water, was observed in situ and provided direct evidence of significant rejection of lignin from the recovered polysaccharides. This observation was confirmed by chemical analysis of the regenerated cellulose. In comparison to untreated biomass, ionic liquid pretreated biomass produces cellulose that is efficiently hydrolyzed with commercial cellulase cocktail with high sugar yields over a relatively short time interval.
Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one ...promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50°C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.
Ionic liquids (ILs), solvents composed entirely of paired ions, have been used in a variety of process chemistry and renewable energy applications. Imidazolium-based ILs effectively dissolve biomass ...and represent a remarkable platform for biomass pretreatment. Although efficient, imidazolium cations are expensive and thus limited in their large-scale industrial deployment. To replace imidazolium-based ILs with those derived from renewable sources, we synthesized a series of tertiary amine-based ILs from aromatic aldehydes derived from lignin and hemicellulose, the major by-products of lignocellulosic biofuel production. Compositional analysis of switchgrass pretreated with ILs derived from vanillin, p -anisaldehyde, and furfural confirmed their efficacy. Enzymatic hydrolysis of pretreated switchgrass allowed for direct comparison of sugar yields and lignin removal between biomass-derived ILs and 1-ethyl-3-methylimidazolium acetate. Although the rate of cellulose hydrolysis for switchgrass pretreated with biomass-derived ILs was slightly slower than that of 1-ethyl-3-methylimidazolium acetate, 90–95% glucose and 70–75% xylose yields were obtained for these samples after 72-h incubation. Molecular modeling was used to compare IL solvent parameters with experimentally obtained compositional analysis data. Effective pretreatment of lignocellulose was further investigated by powder X-ray diffraction and glycome profiling of switchgrass cell walls. These studies showed different cellulose structural changes and differences in hemicellulose epitopes between switchgrass pretreatments with the aforementioned ILs. Our concept of deriving ILs from lignocellulosic biomass shows significant potential for the realization of a “closed-loop” process for future lignocellulosic biorefineries and has far-reaching economic impacts for other IL-based process technology currently using ILs synthesized from petroleum sources.
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