Dimethyl Sulfoxide Assisted Ionic Liquid Pretreatment of Switchgrass for Isoprenol Production

The production cost and viscosity of certain ionic liquids (ILs) are among the major factors preventing the establishment of economically viable IL-based biomass pretreatment technologies. Recently, mixtures of an IL with an organic solvent have been proposed for cellulose processing and biomass pretreatment. Dimethyl sulfoxide (DMSO) is an inexpensive organic solvent that is industrially produced from lignin, a byproduct of the pulping process. We carry out a mechanistic study of DMSO-assisted IL pretreatment of switchgrass. The physical structures of biomass samples are studied by X-ray diffraction (XRD), N₂ adsorption analysis, and small angle neutron scattering (SANS). Both dry and aqueous suspensions of biomass samples are measured by SANS which provides unique information on biomass pretreatment. A mixture of 42 wt % [C₂C₁Im]­[OAc] and 58 wt % DMSO is proposed as the optimal pretreatment solution, and the recycling and reuse of the mixture of solvents are also studied. The fermentability of the hydrolysates generated after pretreatment is evaluated using an E. coli strain engineered to produce isoprenol. This study suggests an avenue for developing more efficient and cost-effective IL-based processes for the production of lignocellulosic biofuels and bioproducts

Metal Affinity Enrichment Increases the Range and Depth of Proteome Identification for Extracellular Microbial Proteins

Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added ∼20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as “hypothetical” were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO

Image_1_Microbial Community Structure and Functional Potential Along a Hypersaline Gradient.TIF

<p>Salinity is one of the strongest environmental drivers of microbial evolution and community composition. Here we aimed to determine the impact of salt concentrations (2.5, 7.5, and 33.2%) on the microbial community structure of reclaimed saltern ponds near San Francisco, California, and to discover prospective enzymes with potential biotechnological applications. Community compositions were determined by 16S rRNA amplicon sequencing revealing both higher richness and evenness in the pond sediments compared to the water columns. Co-occurrence network analysis additionally uncovered the presence of microbial seed bank communities, potentially primed to respond to rapid changes in salinity. In addition, functional annotation of shotgun metagenomic DNA showed different capabilities if the microbial communities at different salinities for methanogenesis, amino acid metabolism, and carbohydrate-active enzymes. There was an overall shift with increasing salinity in the functional potential for starch degradation, and a decrease in degradation of cellulose and other oligosaccharides. Further, many carbohydrate-active enzymes identified have acidic isoelectric points that have potential biotechnological applications, including deconstruction of biofuel feedstocks under high ionic conditions. Metagenome-assembled genomes (MAGs) of individual halotolerant and halophilic microbes were binned revealing a variety of carbohydrate-degrading potential of individual pond inhabitants.</p

Metal Affinity Enrichment Increases the Range and Depth of Proteome Identification for Extracellular Microbial Proteins

Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added ∼20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as “hypothetical” were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO

Metal Affinity Enrichment Increases the Range and Depth of Proteome Identification for Extracellular Microbial Proteins

Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added ∼20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as “hypothetical” were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO

Pylum composition of microbial communities from mesophilic and thermophilic enrichments on rice straw.

<p>Pylum composition of microbial communities from mesophilic and thermophilic enrichments on rice straw.</p

Contig cluster properties for selected clusters (Figure 3) with high abundance or large contigs in thermophilic and mesophilic communities.

*<p>T, thermophilic community; M, mesophilic community.</p

Rarefaction curves from pyrotag data for enriched mesophilic and thermophilic microbial communities.

<p>Dashed lines indicate ±1 standard error.</p

Ecological measures for microbial communities from mesophilic and thermophilic enrichments on rice straw.

<p>Ecological measures for microbial communities from mesophilic and thermophilic enrichments on rice straw.</p

Scatterplots of contig properties for select genus bins in thermophilic and mesophilic communities.

<p>Plotted contigs correspond to (A) <i>Micromonospora</i> (<i>Actinobacteria</i>) in thermophilic community, (B) <i>Mycobacterium</i> (<i>Actinobacteria</i>) in thermophilic community, (C) <i>Pseudoxanthomonas</i> (<i>Proteobacteria</i>) in thermophilic community, (D) <i>Pseudoxanthomonas</i> (<i>Proteobacteria</i>) in mesophilic community, (E) <i>Chryseobacterium</i> (<i>Bacteroidetes</i>) in mesophilic community, (F) <i>Niabella</i> (<i>Bacteroidetes</i>) in thermophilic community, (G) <i>Niastella</i> (<i>Bacteroidetes</i>) in mesophilic community, and (H) <i>Chelativorans</i> (<i>Proteobacteria</i>) in thermophilic community. Genera presented in A–E account for >50% of total dissimilarity between thermophilic and mesophilic communities. Notable clusters with high abundance or large contigs are labeled for reference in subsequent analyses.</p