Benign Fractionation of Lignin with CO₂‑Expanded Solvents of Acetic Acid + Water

Kraft lignin was fractionated by molecular weight (MW), using CO₂-expanded solutions of acetic acid/water in a 90/10 wt/wt ratio. In particular, as CO₂ pressures were increased from 7 bar to 48 bar, expanding the liquid-solvent phase and reducing its dielectric strength, lignin fractions decreasing in MW from 15 000 to 1250, with polydispersity index (PDI) values decreasing from 3.7 to 1.6, were obtained. The recovered lignin fractions were similar, in terms of chemical functionality. With the use of gas-expanded liquids (GXL), only one solvent composition is required, and recovery and reuse of the biorenewable CO₂ + acetic acid–water solution is facilitated through pressure release and recompression. The process was demonstrated for the recovery of seven lignin fractions to demonstrate its versatility and effectiveness, but simpler operation with recovery of just a low MW lignin fraction and a high MW lignin fraction is more consistent with anticipated applications

Metabolism of Multiple Aromatic Compounds in Corn Stover Hydrolysate by <i>Rhodopseudomonas palustris</i>

Lignocellulosic biomass hydrolysates hold great potential as a feedstock for microbial biofuel production, due to their high concentration of fermentable sugars. Present at lower concentrations are a suite of aromatic compounds that can inhibit fermentation by biofuel-producing microbes. We have developed a microbial-mediated strategy for removing these aromatic compounds, using the purple nonsulfur bacterium Rhodopseudomonas palustris. When grown photoheterotrophically in an anaerobic environment, R. palustris removes most of the aromatics from ammonia fiber expansion (AFEX) treated corn stover hydrolysate (ACSH), while leaving the sugars mostly intact. We show that R. palustris can metabolize a host of aromatic substrates in ACSH that have either been previously described as unable to support growth, such as methoxylated aromatics, and those that have not yet been tested, such as aromatic amides. Removing the aromatics from ACSH with R. palustris, allowed growth of a second microbe that could not grow in the untreated ACSH. By using defined mutants, we show that most of these aromatic compounds are metabolized by the benzoyl-CoA pathway. We also show that loss of enzymes in the benzoyl-CoA pathway prevents total degradation of the aromatics in the hydrolysate, and instead allows for biological transformation of this suite of aromatics into selected aromatic compounds potentially recoverable as an additional bioproduct

Engineered Lignin in Poplar Biomass Facilitates Cu-Catalyzed Alkaline-Oxidative Pretreatment

Both untransformed poplar and genetically modified “zip-lignin” poplar, in which additional ester bonds were introduced into the lignin backbone, were subjected to mild alkaline and copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment. Our hypothesis was that the lignin in zip-lignin poplar would be removed more easily than lignin in untransformed poplar during this alkaline pretreatment, resulting in higher sugar yields following enzymatic hydrolysis. We observed improved glucose and xylose hydrolysis yields for zip-lignin poplar compared to untransformed poplar following both alkaline-only pretreatment (56% glucose yield for untransformed poplar compared to 67% for zip-lignin poplar) and Cu-AHP pretreatment (77% glucose yield for untransformed poplar compared to 85% for zip-lignin poplar). Compositional analysis, glycome profiling, and microscopy all supported the notion that the ester linkages increase delignification and improve sugar yields. Essentially no differences were noted in the molecular weight distributions of solubilized lignins between the zip-lignin poplar and the control line. Significantly, when zip-lignin poplar was utilized as the feedstock, hydrogen peroxide, catalyst, and enzyme loadings could all be substantially reduced while maintaining high sugar yields

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant <i>Saccharomyces cerevisiae</i> Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

<div><p>The inability of the yeast <i>Saccharomyces cerevisiae</i> to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of <i>S. cerevisiae</i> to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting <i>S. cerevisiae</i> strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent <i>S. cerevisiae</i> strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in <i>GRE3</i>, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust <i>S. cerevisiae</i> strain with the ability to ferment xylose anaerobically from ACSH.</p></div

The GLBRCY127 strain developed by directed engineering with xylose isomerase coupled with batch evolution can rapidly consume xylose aerobically.

<p>Average sugar consumption and cell growth of unevolved GLBRCY22-3 strain engineered with <i>ScTAL1</i>, <i>CpxylA</i> and <i>SsXYL3</i> cultured in bioreactors containing YPDX media and sparged with air from biological duplicates is shown (<b>A</b>). Indicated components were quantified from media samples at times from initial inoculation. In (<b>B</b>), the average percentage of xylose consumed and change in cell density per day are plotted for each transfer during the adaption of the Y22-3 strain in YP media containing 0.1% glucose and 2% xylose. The pattern of lower % of xylose consumed and change in cell density per day during every third transfer is due to reaching saturated growth prior to transfer. Average extracellular xylose concentrations and cell density measurements from parental Y22-3 and evolved Y127 strains grown aerobically in culture tubes with YPX media from three independent biological replicates are plotted in (<b>C</b>). In (<b>D</b>), evolved isolate Y127 was cultured in the same conditions as in (<b>A</b>), and samples measurements taken in an identical manner.</p

Fermentation kinetic profiles for engineered and evolved <i>S. cerevisiae</i> strains.

<p>ND, Not Determined for aerobic conditions; ND*, Not Determined – no ethanol produced.</p>1<p>In g xylose consumed/L/h.</p>2<p>In g xylose consumed/g DCW/h.</p>3<p>Calculated from the maximum ethanol concentration produced divided by the consumed xylose concentration at that time.</p>4<p>Calculated from the ethanol concentration produced between two time points after glucose depletion.</p>5<p>Yield of g DCW/g glucose consumed calculated at or near the time of glucose depletion and prior to xylose consumption. No cell growth was observed during xylose consumption.</p>6<p>Yield of g glycerol/g glucose consumed calculated at or near the time of glucose depletion and prior to xylose consumption.</p><p>Fermentation kinetic profiles for engineered and evolved <i>S. cerevisiae</i> strains.</p

The xylose consumption phenotypes of the evolved Y127 and Y128 strains are dependent upon <i>CpxylA</i> and <i>ScTAL1.</i>

<p>Extracellular xylose concentrations (solid lines) and cell density (dashed lines) were measured by YSI instrument and OD₆₀₀ readings, respectively, from cultures containing KanMX marker rescued versions of (<b>A</b>) GLBRCY127 (Y132) and GLBRCY132 <i>xylAΔ</i> or (<b>B</b>) Y132 and Y132 <i>tal1Δ</i> strains inoculated in aerobic YPX media. In (<b>C</b>), extracellular xylose concentrations (solid lines) and cell density (dashed lines) were measured as in (<b>A</b>, <b>B</b>) for KanMX marker rescued GLBRCY128 (Y133) and two independent GLBRCY133 <i>xylAΔ</i> strains inoculated in anaerobic YPX media. These selection marker-rescued Y128 strains were cultured in YPD media and total RNA isolated from a single time point. Expression of <i>CpxylA</i> was then quantified and normalized to <i>ScERV25</i> RNA levels by qPCR. The bar graph in (<b>D</b>) displays the average values and standard deviations for <i>CpxylA</i> RNA from three independent biological replicates.</p

Phenotypic screening of wild and domesticated <i>S. cerevisiae</i> strains identifies NRRL YB-210 with tolerance to hydrolysates made from a variety of pretreated lignocellulose.

<p>In (<b>A</b>), 117 <i>S. cerevisiae</i> strains (including some in duplicate) were cultured in 96-well plates and monitored for changes cell density and growth rates calculated as described in Materials and Methods. All strains in each condition were then ranked from 1 (highest growth rate in yellow) to 117 (lowest growth rate, or no growth, in blue) and hierarchically clustered. Arrows indicate clustered rows for BY4741 (green), CEN.PK2 (black) in duplicate microtiter wells, and NRRL YB-210/GLBRCY0 (red). Representative growth data for the YB-210/GLBRCY0 strain in the indicated media from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107499#pone-0107499-g002" target="_blank">Fig. 2A</a> are plotted (<b>B–C</b>). CS, corn stover; SG, switchgrass; YP; Yeast Extract and Peptone supplementation, 6%; 6% glucan loading ACSH, 9%; 9% glucan loading ACSH, Dtx.; Detoxified.</p

Second stage anaerobic adaptation on xylose enabled rapid xylose fermentation by evolved GLBRCY128 isolate.

<p>Average fermentation kinetic profiles of the GLBRCY127 strain cultured in bioreactors containing YPDX media and sparged with nitrogen from biological duplicates are shown (<b>A</b>). Average concentrations with standard deviations of indicated compounds were quantified from media samples at times from initial inoculation. In (<b>B</b>), the percentage of xylose consumed and change in cell density per day is plotted for each transfer during the anaerobic adaptation of Y127 in YP media containing 0.1% glucose and 2% xylose. In the first two transfers (hatched bars), Tween-80 and ergosterol were added to the media. In (<b>C</b>), evolved isolate Y128 was cultured in biological duplicate under the same conditions as in (<b>A</b>), and samples measurements taken in an identical manner.</p

GLBRCY128 can anaerobically ferment xylose from ACSH.

<p>A diagram summarizing the engineering and evolution of the YB-210 strain into the evolved Y128 strain is provided in (<b>A</b>). Fermentation kinetic profiles of the Y127 (<b>B</b>) and Y128 (<b>C</b>) strains cultured in bioreactors containing ACSH and sparged with nitrogen from biological duplicates are shown. Average concentrations and standard deviations of indicated components were quantified from media samples at times from initial inoculation. Vertical colored bars indicate time points at which samples were taken for metabolomic analysis described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107499#pone-0107499-g007" target="_blank">Fig. 7A–D</a>.</p