Understanding the Impacts of Biomass Blending on the Uncertainty of Hydrolyzed Sugar Yield from a Stochastic Perspective

Feedstock price and availability are key challenges for biorefinery development. Biomass blending has been suggested as a route to overcome these limitations. However, the impacts of feedstock blending on the uncertainty in hydrolyzed sugar yields remain unclear. This study quantifies the uncertainties in the sugar yields from hydrolysis of the blends of corn stover, switchgrass, and grass clippings by considering both feedstock compositional variation and model uncertainty. The results indicate that feedstock blending reduces the uncertainties in sugar yields and delivers feedstock of more uniform quality. A 60/35/5 blend of corn stover, switchgrass, and grass clippings on average achieves a glucose yield of 32.6 g/100 g of biomass, which is comparable to those of corn stover (33.3 g/100 g) and switchgrass (32.9 g/100 g), but drastically higher than that of grass clippings (21.7 g/100 g). This same blend also achieves the lowest variance in glucose yield (2.9 g/100 g) compared to corn stover (3.1 g/100 g), switchgrass (3.3 g/100 g), and grass clippings (5.6 g/100 g). A further investigation on the breakdown of the variability of the hydrolyzed sugar yields reveals that the reduction in the variability of sugar yields for blended feedstocks is achieved by reduced feedstock compositional variation. Based on these results, the optimization of blending ratios is performed with respect to three objectives: (1) to maximize the probability of meeting the sugar yields target, (2) to maximize the expected sugar yields, and (3) to maximize sugar yields per unit feedstock expense, while satisfying constraints of feedstock availability and price. The maximized probability of meeting the sugar yield target, expected sugar yield, and glucose yield per unit feedstock expense are 91.33%, 32.66 g of sugar/100 g of biomass, and 40.83 g/$, respectively. The optimization method developed in this study is readily applied to other combinations of feedstocks, biofuel production processes, and constraints

Understanding the Impacts of Biomass Blending on the Uncertainty of Hydrolyzed Sugar Yield from a Stochastic Perspective

Feedstock price and availability are key challenges for biorefinery development. Biomass blending has been suggested as a route to overcome these limitations. However, the impacts of feedstock blending on the uncertainty in hydrolyzed sugar yields remain unclear. This study quantifies the uncertainties in the sugar yields from hydrolysis of the blends of corn stover, switchgrass, and grass clippings by considering both feedstock compositional variation and model uncertainty. The results indicate that feedstock blending reduces the uncertainties in sugar yields and delivers feedstock of more uniform quality. A 60/35/5 blend of corn stover, switchgrass, and grass clippings on average achieves a glucose yield of 32.6 g/100 g of biomass, which is comparable to those of corn stover (33.3 g/100 g) and switchgrass (32.9 g/100 g), but drastically higher than that of grass clippings (21.7 g/100 g). This same blend also achieves the lowest variance in glucose yield (2.9 g/100 g) compared to corn stover (3.1 g/100 g), switchgrass (3.3 g/100 g), and grass clippings (5.6 g/100 g). A further investigation on the breakdown of the variability of the hydrolyzed sugar yields reveals that the reduction in the variability of sugar yields for blended feedstocks is achieved by reduced feedstock compositional variation. Based on these results, the optimization of blending ratios is performed with respect to three objectives: (1) to maximize the probability of meeting the sugar yields target, (2) to maximize the expected sugar yields, and (3) to maximize sugar yields per unit feedstock expense, while satisfying constraints of feedstock availability and price. The maximized probability of meeting the sugar yield target, expected sugar yield, and glucose yield per unit feedstock expense are 91.33%, 32.66 g of sugar/100 g of biomass, and 40.83 g/$, respectively. The optimization method developed in this study is readily applied to other combinations of feedstocks, biofuel production processes, and constraints

Nucleation and Assembly of Silica into Protein-Based Nanocomposites as Effective Anticancer Drug Carriers Using Self-Assembled Silk Protein Nanostructures as Biotemplates

<i>Bombyx mori</i> (<i>B.</i> <i>mori</i>) silk fibroin and sericin can act as a great candidate in delivering drugs or other bioactive substances. Silica also has a great application in the field of drug delivery. To the best of our knowledge, there has been no report on the design of a nanocomposite made of silk protein and silica for drug delivery. Here, for the first time, we used <i>B.</i> <i>mori</i> silk fibroin (SF) and sericin (SS), self-assembled into nanospheres and nanofibers in situ in the aqueous solution, respectively, as a biotemplate to regulate the nucleation and self-assembly of silica for designing anticancer drug delivery. SF and SS mediated the nucleation and assembly of silica into monodispersed nanospheres (termed Si/SF) and nanofibers (termed Si/SS), respectively. The size and topography of the silica assemblies were dependent on the concentration of SF or SS as well as reaction conditions. Both Si/SF nanospheres and Si/SS nanofibers showed a high loading capability and sustained release profile of an anticancer drug, doxorubicin (DOX), in vitro. Si/SF nanospheres were found to be efficiently internalized in human cervical carcinoma (HeLa) cells and accumulate around the cell nuclei. Si/SS nanofibers could only adhere to the surface of the cancer cells. This indicates that DOX-loaded Si/SF nanospheres and Si/SS nanofibers are more effective in cancer therapy than free DOX. Our results suggest that the self-assembled Si/SF spheres and Si/SS nanofibers are potential effective anticancer drug carriers

Upgrading of Postconsumer Absorbent Hygiene Products for Bioethanol Production

Postconsumer absorbent hygiene products (AHPs), one of the most intractable municipal solid waste (MSW) streams, are generally subjected to landfills, causing urban waste management difficulties and environmental pollution. This study investigated the viability of recycling the cellulose component from postconsumer AHPs, demonstrated the fermentability of obtained cellulosic sugars for bioethanol production, and evaluated the technical feasibility of the process through scale-up. A set of integrated unit operations was developed to effectively upcycle the recovered AHPs into bioproducts. The processes were further optimized and scaled up 50-fold. Up to 83.5% glucose and 46.0% xylose yields were achieved during enzymatic saccharification. The sugars were readily fermentable through separate hydrolysis and fermentation (SHF) as well as simultaneous saccharification and fermentation (SSF) with maximum 89.9% ethanol theoretical yield

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

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

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

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