Scholarly Work - Chemical & Biological Engineering

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    Rapamycin does not alter bone microarchitecture or material properties quality in young-adult and aged female C57BL/6 mice
    (Wiley, 2024-01) Devine, Connor C.; Brown, Kenna C.; Paton, Kat O.; Heveran, Chelsea M.; Martin, Stephen A.
    Advancing age is the strongest risk factor for osteoporosis and skeletal fragility. Rapamycin is an FDA-approved immunosuppressant that inhibits the mechanistic target of rapamycin (mTOR) complex, extends lifespan, and protects against aging-related diseases in multiple species; however, the impact of rapamycin on skeletal tissue is incompletely understood. We evaluated the effects of a short-term, low-dosage, interval rapamycin treatment on bone microarchitecture and strength in young-adult (3 mo old) and aged female (20 mo old) C57BL/6 mice. Rapamycin (2 mg/kg body mass) was administered via intraperitoneal injection 1×/5 d for a duration of 8 wk; this treatment regimen has been shown to induce geroprotective effects while minimizing the side effects associated with higher rapamycin dosages and/or more frequent or prolonged delivery schedules. Aged femurs exhibited lower cancellous bone mineral density, volume, trabecular connectivity density and number, higher trabecular thickness and spacing, and lower cortical thickness compared to young-adult mice. Rapamycin had no impact on assessed microCT parameters. Flexural testing of the femur revealed that both yield strength and ultimate strength were lower in aged mice compared to young-adult mice. There were no effects of rapamycin on these or other measures of bone biomechanics. Age, but not rapamycin, altered local and global measures of bone turnover. These data demonstrate that short-term, low-dosage interval rapamycin treatment does not negatively or positively impact the skeleton of young-adult and aged mice.
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    Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels
    (Wiley, 2023-09) Fredrikson, Jacob P.; Brahmachary, Priyanka P.; June, Ronald K.; Cox, Lewis M.; Chang, Connie B.
    One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50 µm diameter alginate microgels using drop-based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten-fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0 and 38.0 kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.
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    Simulation of catalase-dependent tolerance of microbial biofilm to hydrogen peroxide with a biofilm computer model
    (Springer Science and Business Media LLC, 2023-08) Stewart, Philip S.; Owkes, Mark
    Hydrogen peroxide (HP) is a common disinfectant and antiseptic. When applied to a biofilm, it may be expected that the top layer of the biofilm would be killed by HP, the HP would penetrate further, and eventually eradicate the entire biofilm. However, using the Biofilm.jl computer model, we demonstrate a mechanism by which the biofilm can persist, and even become thicker, in the indefinite treatment with an HP solution at concentrations that are lethal to planktonic microorganisms. This surprising result is found to be dependent on the neutralization of HP by dead biomass, which provides protection for living biomass deeper within the biofilm. Practically, to control a biofilm, this result leads to the concept of treating with an HP dose exceeding a critical threshold concentration rather than a sustained, lower-concentration treatment.
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    Composition analysis of canola and intermediate wheatgrass biomass and the effects of extraction
    (BioResources, 2023-01) Johnsrude, Lauren M.; Scheffel, Aidan J.; Allen, Brett L.; Wettstein, Stephanie G.
    Knowing the composition of biomass is critical for determining accurate yields of renewable chemicals and fuels; however, nonstructural components can affect the results of standard composition procedures, leading to inaccurate reactant amounts. To remove these nonstructural components, solvent extractions can be done, but the impact on composition values has not been well-reported. For this study, compositional analysis was performed on as-received canola (Brassica napus) and intermediate wheatgrass (Thinopyrum intermedium), as well as ethanol, water, and water/ethanol extracted biomasses. Water/ethanol extraction of the intermediate wheatgrass resulted in significantly lower xylose and both acid soluble and insoluble lignin amounts when compared to the as-received analysis. Since sugar was removed during the extractions, it is recommended to use the as-received composition values for glucuronoarabinoxylans; however, the extractives may interfere with the lignin analysis and therefore, the extracted lignin values are likely more reflective of the composition.
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    Capacity at All-Way Stop Control Intersections: Case Study
    (SAGE Publications, 2023-03) Al-Kaisy, Ahmed; Doruk, Dorukhan
    This paper presents an empirical investigation into the capacity of all-way stop-controlled (AWSC) intersections. Video data was collected over four days at an AWSC intersection site in Bozeman, Montana. The site is characterized by single-lane approaches and high level of vehicular and pedestrian traffic. Using strict protocols, video records were processed at the individual vehicle level and several information metrics were extracted for each vehicle in the data set on all approaches. Study results indicate that the total intersection capacity at the study site varied between 400 and 1,400 vehicles per hour. The study suggests that the wide range of capacity observations is largely associated with the pedestrian crossing activity at the study site. Statistical tests confirmed that both pedestrian crossing activity and the level of conflict have significant effects on intersection capacity at the 95% confidence level. For movement type, the right-turn movement was not found to have a significant effect on intersection capacity while left-turn movement was found to negatively affect the intersection capacity. The results presented in this paper offer valuable information on AWSC intersection capacity, given the limited amount of information in the literature and the dated nature of those empirical observations.
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    Sex-specific effects of calving season on joint health and biomarkers in Montana ranchers
    (Springer Science and Business Media LLC, 2023-01) Thompson, Matthew A.; Martin, Stephen A.; Hislop, Brady D.; Younkin, Roubie; Andrews, Tara M.; Miller, Kaleena; June, Ronald K.; Adams, Erik S.
    Background. Agricultural workers have a higher incidence of osteoarthritis (OA), but the etiology behind this phenomenon is unclear. Calving season, which occurs in mid- to late-winter for ranchers, includes physical conditions that may elevate OA risk. Our primary aim was to determine whether OA biomarkers are elevated at the peak of calving season compared to pre-season, and to compare these data with joint health survey information from the subjects. Our secondary aim was to detect biomarker differences between male and female ranchers. Methods. During collection periods before and during calving season, male (n = 28) and female (n = 10) ranchers completed joint health surveys and provided samples of blood, urine, and saliva for biomarker analysis. Statistical analyses examined associations between mean biomarker levels and survey predictors. Ensemble cluster analysis identified groups having unique biomarker profiles. Results. The number of calvings performed by each rancher positively correlated with plasma IL-6, serum hyaluronic acid (HA) and urinary CTX-I. Thiobarbituric acid reactive substances (TBARS), a marker of oxidative stress, was significantly higher during calving season than pre-season and was also correlated with ranchers having more months per year of joint pain. We found evidence of sexual dimorphism in the biomarkers among the ranchers, with leptin being elevated and matrix metalloproteinase-3 diminished in female ranchers. The opposite was detected in males. WOMAC score was positively associated with multiple biomarkers: IL-6, IL-2, HA, leptin, C2C, asymmetric dimethylarginine, and CTX-I. These biomarkers represent enzymatic degradation, inflammation, products of joint destruction, and OA severity. Conclusions. The positive association between number of calvings performed by each rancher (workload) and both inflammatory and joint tissue catabolism biomarkers establishes that calving season is a risk factor for OA in Montana ranchers. Consistent with the literature, we found important sex differences in OA biomarkers, with female ranchers showing elevated leptin, whereas males showed elevated MMP-3.
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    Effect of Dilute Acid Pretreatment and Lignin Extraction Conditions on Lignin Properties and Suitability as a Phenol Replacement in Phenol-Formaldehyde Wood Adhesives
    (American Chemical Society, 2022-12) Saulnier, Brian K.; Siahkamari, Mohsen; Singh, Sandip K.; Nejad, Mojgan; Hodge, David B.
    Corn stover was subjected to dilute sulfuric acid pretreatment to assess the impact of pretreatment conditions on lignin extractability, properties, and utility as a phenol replacement in wood phenol-formaldehyde (PF) adhesives. It was identified that both formic acid and NaOH could extract and recover 60–70% of the lignin remaining after pretreatment and enzymatic hydrolysis under the mildest pretreatment conditions while simultaneously achieving reasonable enzymatic hydrolysis yields (>60%). The availability of reaction sites for the incorporation of lignins into the PF polymer matrix (i.e., unsubstituted phenolic hydroxyl groups) was shown to be strongly impacted by the pretreatment time and the recovery. Finally, a lignin-based wood adhesive was formulated by replacing 100% of the phenol with formic-acid-extracted lignin, which exhibited a dry shear strength exceeding a conventional PF adhesive. These findings suggest that both pretreatment and lignin extraction conditions can be tailored to yield lignins with properties targeted for this co-product application.
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    Particle classification by image analysis improves understanding of corn stover degradation mechanisms during deconstruction
    (Elsevier BV, 2023-03) Cousins, Dylan S.; Pedersen, Kristian P.; Otto, William G.; Rony, Asif Hasan; Lacey, Jeffrey A.; Aston, John E.; Hodge, David B.
    iomass feedstock heterogeneity is a principal roadblock to implementation of the biorefinery concept. Even within an identical cultivar of corn stover, different bales contain not only varying abundance moisture, ash, glucan, and other chemical compounds, but also varying abundance of tissue anatomies (e.g., leaf, husk, cob, or stalk). These different anatomical components not only differ in their response to pretreatment and enzymatic hydrolysis to glucose, but also vary in their mechanical and conveyance properties. Although this heterogeneous nature of corn stover feedstock has been identified as a challenge, a fundamental knowledge gap of how these tissues behave during biorefining processing remains. In this work, we demonstrate the use of a commercial fiber image analyzer typically used for wood fiber characterization to monitor the particle size and shapes of non-woody feedstock during milling, pretreatment, and hydrolysis. Additionally, we present novel use of Gaussian process classification to distinguish bundle, parenchyma, and fiber particles to an accuracy of 96.4%. Quantitative probability distribution plots for characteristics such as length and roundness allow elucidation of particle morphology as pretreatment and enzymatic hydrolysis progress. In both stalk pith and stalk rind, particles peel into individual cells whose walls are subsequently fragmented during enzymatic hydrolysis.
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    Environment Constrains Fitness Advantages of Division of Labor in Microbial Consortia Engineered for Metabolite Push or Pull Interactions
    (American Society for Microbiology, 2022-08) Beck, Ashely E.; Pintar, Kathryn; Schepens, Diana; Schrammeck, Ashely; Johnson, Timothy; Bleem, Alissa; Du, Martina; Harcombe, William R.; Bernstein, Hans C.; Heys, Jeffrey J.; Gedeon, Tomas; Carlson, Ross P.
    Fitness benefits from division of labor are well documented in microbial consortia, but the dependency of the benefits on environmental context is poorly understood. Two synthetic Escherichia coli consortia were built to test the relationships between exchanged organic acid, local environment, and opportunity costs of different metabolic strategies. Opportunity costs quantify benefits not realized due to selecting one phenotype over another. The consortia catabolized glucose and exchanged either acetic or lactic acid to create producer-consumer food webs. The organic acids had different inhibitory properties and different opportunity costs associated with their positions in central metabolism. The exchanged metabolites modulated different consortial dynamics. The acetic acid-exchanging (AAE) consortium had a “push” interaction motif where acetic acid was secreted faster by the producer than the consumer imported it, while the lactic acid-exchanging (LAE) consortium had a “pull” interaction motif where the consumer imported lactic acid at a comparable rate to its production. The LAE consortium outperformed wild-type (WT) batch cultures under the environmental context of weakly buffered conditions, achieving a 55% increase in biomass titer, a 51% increase in biomass per proton yield, an 86% increase in substrate conversion, and the complete elimination of by-product accumulation all relative to the WT. However, the LAE consortium had the trade-off of a 42% lower specific growth rate. The AAE consortium did not outperform the WT in any considered performance metric. Performance advantages of the LAE consortium were sensitive to environment; increasing the medium buffering capacity negated the performance advantages compared to WT.
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    Quantitative analysis of macroscopic solute transport in the murine brain
    (Springer Nature, 2021-12) Ray, Lori A.; Pike, Martin; Simon, Matthew; Iliff, Jeffrey J.; Heys, Jeffrey J.
    Background. Understanding molecular transport in the brain is critical to care and prevention of neurological disease and injury. A key question is whether transport occurs primarily by diffusion, or also by convection or dispersion. Dynamic contrast-enhanced (DCE-MRI) experiments have long reported solute transport in the brain that appears to be faster than diffusion alone, but this transport rate has not been quantified to a physically relevant value that can be compared to known diffusive rates of tracers. Methods. In this work, DCE-MRI experimental data is analyzed using subject-specific finite-element models to quantify transport in different anatomical regions across the whole mouse brain. The set of regional effective diffusivities (Deff), a transport parameter combining all mechanisms of transport, that best represent the experimental data are determined and compared to apparent diffusivity (Dapp), the known rate of diffusion through brain tissue, to draw conclusions about dominant transport mechanisms in each region. Results. In the perivascular regions of major arteries, Deff for gadoteridol (550 Da) was over 10,000 times greater than Dapp. In the brain tissue, constituting interstitial space and the perivascular space of smaller blood vessels, Deff was 10–25 times greater than Dapp. Conclusions. The analysis concludes that convection is present throughout the brain. Convection is dominant in the perivascular space of major surface and branching arteries (Pe > 1000) and significant to large molecules (> 1 kDa) in the combined interstitial space and perivascular space of smaller vessels (not resolved by DCE-MRI). Importantly, this work supports perivascular convection along penetrating blood vessels.
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    An emerging view of the diversity, ecology and function of Archaea in alkaline hydrothermal environments
    (Oxford University Press, 2020-12) Mueller, Rebecca C; Peach, Jesse T; Skorupa, Dana J; Copie, Valerie; Bothner, Brian; Peyton, Brent M
    The described diversity within the domain Archaea has recently expanded due to advances in sequencing technologies, but many habitats that likely harbor novel lineages of archaea remain understudied. Knowledge of archaea within natural and engineered hydrothermal systems, such as hot springs and engineered subsurface habitats, has been steadily increasing, but the majority of the work has focused on archaea living in acidic or circumneutral environments. The environmental pressures exerted by the combination of high temperatures and high pH likely select for divergent communities and distinct metabolic pathways from those observed in acidic or circumneutral systems. In this review, we examine what is currently known about the archaea found in thermoalkaline environments, focusing on the detection of novel lineages and knowledge of the ecology, metabolic pathways and functions of these populations and communities. We also discuss the potential of emerging multi–omics approaches, including proteomics and metabolomics, to enhance our understanding of archaea within extreme thermoalkaline systems.
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    Lignin properties and cell wall response to deconstruction by alkaline pretreatment and enzymatic hydrolysis in brown midrib sorghums
    (Elsevier BV, 2022-04) Singh, Sandip K.; Saulnier, Brian K.; Hodge, David B.
    Lignin has an adverse impact on the deconstruction of plant cell wall biopolymers in biorefining processes and its reduction and/or alteration during biosynthesis is one target for decreasing plant cell wall recalcitrance. In this work, the impact of two brown midrib mutations (bmr6 and bmr12) in two sorghum background lines (the commercial hybrid Atlas and near-isogenic BTx623) on lignin properties and the plants’ response to cell wall deconstruction to monomeric sugars via alkaline pretreatment and enzymatic hydrolysis is investigated with the goal of assessing how differences in lignin content and properties impact the plant’s response to pretreatment. We identify that both bmr sorghum lines show significantly lower abundance of water-extractable sugars (glucose, sucrose, and fructose) and alkali-saponifiable p-coumarate. Furthermore, both these properties exhibited identical trends across both background lines. Next, both untreated and mild alkali-pretreated bmr sorghums were shown to exhibit higher glucose hydrolysis yields following enzymatic hydrolysis than the control lines. Following pretreatment, the Atlas bmr sorghums exhibited more lignin solubilization and the solubilized lignin was of lower molar mass than the background control line suggesting that differences in the lignin response to pretreatment resulted these differences. Finally, significant differences were observed in the lignin content, lignin monomer distribution, and inter-unit linkages in the Atlas bmr line relative to the control line with key differences including lower syringyl monomer content in both bmr lines, higher relative abundance of β-O-4 linkages in the bmr6 line, and the presence of 5-hydroxy guaiacyl monomers and benzodioxane (α-O-5/β-O-4) linkages in the bmr12 line.
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    Near-Infrared Spectroscopy can Predict Anatomical Abundance in Corn Stover
    (Frontiers Media SA, 2022-02) Cousins, Dylan S.; Otto, William G.; Rony, Asif Hasan; Pedersen, Kristian P.; Aston, John E.; Hodge, David B.
    Feedstock heterogeneity is a key challenge impacting the deconstruction and conversion of herbaceous lignocellulosic biomass to biobased fuels, chemicals, and materials. Upstream processing to homogenize biomass feedstock streams into their anatomical components via air classification allows for a more tailored approach to subsequent mechanical and chemical processing. Here, we show that differing corn stover anatomical tissues respond differently to pretreatment and enzymatic hydrolysis and therefore, a one-size-fits-all approach to chemical processing biomass is inappropriate. To inform on-line downstream processing, a robust and high-throughput analytical technique is needed to quantitatively characterize the separated biomass. Predictive correlation of near-infrared spectra to biomass chemical composition is such a technique. Here, we demonstrate the capability of models developed using an “off-the-shelf,” industrially relevant spectrometer with limited spectral range to make strong predictions of both cell wall chemical composition and the relative abundance of anatomical components of the corn stover, the latter for the first time ever. Gaussian process regression (GPR) yields stronger correlations (average R2v = 88% for chemical composition and 95% for anatomical relative abundance) than the more commonly used partial least squares (PLS) regression (average R2v = 84% for chemical composition and 92% for anatomical relative abundance). In nearly all cases, both GPR and PLS outperform models generated using neural networks. These results highlight the potential for coupling NIRS with predictive models based on GPR due to the potential to yield more robust correlations.
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    Metabolomic Profiling and Mechanotransduction of Single Chondrocytes Encapsulated in Alginate Microgels
    (MDPI AG, 2022-03) Fredrikson, Jacob P.; Brahmachary, Priyanka P.; Erdoğan, Ayten E.; Archambault, Zachary K.; Wilking, James N.; June, Ronald K.; Chang, Connie B.
    Articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened, resulting in increased chondrocyte stress. As chondrocytes are responsible for matrix synthesis and maintenance, it is important to understand how mechanical loads affect the cellular responses of chondrocytes. Many studies have examined chondrocyte responses to in vitro mechanical loading by embedding chondrocytes in 3-D hydrogels. However, these experiments are mostly performed in the absence of PCM, which may obscure important responses to mechanotransduction. Here, drop-based microfluidics is used to culture single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes formed PCM over 10 days in these single-cell 3-D microenvironments. Mechanotransduction studies were performed, in which single-cell microgels mimicking the cartilage PCM were embedded in high-stiffness agarose. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.
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    Varying perivascular astroglial endfoot dimensions along the vascular tree maintain perivascular‐interstitial flux through the cortical mantle
    (Wiley, 2020-10) Wang, Marie Xun; Ray, Lori; Tanaka, Kenji F.; Iliff, Jeffrey J.; Heys, Jeffrey
    The glymphatic system is a recently defined brain-wide network of perivascular spaces along which cerebrospinal fluid (CSF) and interstitial solutes exchange. Astrocyte endfeet encircling the perivascular space form a physical barrier in between these two compartments, and fluid and solutes that are not taken up by astrocytes move out of the perivascular space through the junctions in between astrocyte endfeet. However, little is known about the anatomical structure and the physiological roles of the astrocyte endfeet in regulating the local perivascular exchange. Here, visualizing astrocyte endfoot-endfoot junctions with immunofluorescent labeling against the protein megalencephalic leukoencephalopathy with subcortical cysts-1 (MLC1), we characterized endfoot dimensions along the mouse cerebrovascular tree. We observed marked heterogeneity in endfoot dimensions along vessels of different sizes, and of different types. Specifically, endfoot size was positively correlated with the vessel diameters, with large vessel segments surrounded by large endfeet and small vessel segments surrounded by small endfeet. This association was most pronounced along arterial, rather than venous segments. Computational modeling simulating vascular trees with uniform or varying endfeet dimensions demonstrates that varying endfoot dimensions maintain near constant perivascularinterstitial flux despite correspondingly declining perivascular pressures along the cerebrovascular tree through the cortical depth. These results describe a novel anatomical feature of perivascular astroglial endfeet and suggest that endfoot heterogeneity may be an evolutionary adaptation to maintain perivascular CSF-interstitial fluid exchange through deep brain structures.
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    A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions
    (2021-11) Ozay, Burcu; McCalla, Stephanie E.
    Isothermal nucleic acid amplification techniques are used to detect a variety of molecules such as RNAs, DNAs and proteins. When compared to the gold standard polymerase chain reaction, isothermal methods are rapid and simple but often have lower specificity and sensitivity. In this review, we provide a summary of reaction additives and techniques that enhance the sensitivity, specificity, stability, robustness, or speed of isothermal nucleic acid amplification reactions used for sensing applications. We also provide the mechanism and purpose of each enhancement technique, which can provide guidance when selecting reaction additives to optimize novel isothermal amplification systems and assays.
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    Effect of catalyst and reaction conditions on aromatic monomer yields, product distribution, and sugar yields during lignin hydrogenolysis of silver birch wood
    (2020-11) Phongpreecha, Thanaphong; Christy, Kendall F.; Singh, Sandip K.; Hao, Pengchao; Hodge, David B.
    The impact of catalyst choice and reaction conditions during catalytic hydrogenolysis of silver birch biomass are assessed for their effect on aromatic monomer yields and selectivities, lignin removal, and sugar yields from enzymatic hydrolysis. At a reaction temperature of 220 °C with no supplemental H2, it was demonstrated that both Co/C and Ni/C exhibited aromatic monomer yields of >50%, which were close to the theoretical maximum expected for the lignin based on total β-O-4 content and exhibited high selectivities for 4-propylguaiacol and 4-propylsyringol. Pd/C exhibited a significantly different set of products, and using a model lignin dimer, showed a product profile that shifted upon inclusion of supplemental H2, suggesting that the generation of surface hydrogen is critical for this catalyst system. Lignin removal during hydrogenolysis could be correlated to glucose yields and inclusion of lignin depolymerizing catalysts significantly improves lignin removal and subsequent enzymatic hydrolysis yields.
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    Impact of dilute acid pretreatment conditions on p-coumarate removal in diverse maize lines
    (2020-10) Saulnier, Brian K.; Phongpreecha, Thanaphong; Singh, Sandip K.; Hodge, David B.
    Prior work has identified that lignins recovered from dilute acid-pretreated corn stover exhibit superior performance in phenol–formaldehyde resins used in wood adhesive applications when compared to diverse process-modified lignins derived from other sources. This improved performance is hypothesized to be due to the higher content of unsubstituted phenolic groups specifically p-coumarate lignin esters. In this work, a diverse set of corn stover samples are employed that exhibit diversity in p-coumarate content and total lignin content to explore the relationship between dilute acid pretreatment conditions, p-coumarate ester hydrolysis, xylan solubilization, and the resulting glucose enzymatic hydrolysis yields. The goal of this study is to identify pretreatment conditions that preserve a significant fraction of the p-coumarate esters while simultaneously achieving high enzymatic hydrolysis yields. Kinetic parameters for p-coumarate ester hydrolysis were quantified and pretreatment-biomass combinations were identified that result in glucose hydrolysis yields of more than 90% while retaining nearly 50 mg p-coumarate/g lignin.
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    Xylan Is Critical for Proper Bundling and Alignment of Cellulose Microfibrils in Plant Secondary Cell Walls
    (2021-09) Crowe, Jacob D.; Hao, Pengchao; Pattathil, Sivakumar; Pan, Henry; Ding, Shi-You; Hodge, David B.; Krüger Jensen, Jacob
    Plant biomass represents an abundant and increasingly important natural resource and it mainly consists of a number of cell types that have undergone extensive secondary cell wall (SCW) formation. These cell types are abundant in the stems of Arabidopsis, a well-studied model system for hardwood, the wood of eudicot plants. The main constituents of hardwood include cellulose, lignin, and xylan, the latter in the form of glucuronoxylan (GX). The binding of GX to cellulose in the eudicot SCW represents one of the best-understood molecular interactions within plant cell walls. The evenly spaced acetylation and 4-O-methyl glucuronic acid (MeGlcA) substitutions of the xylan polymer backbone facilitates binding in a linear two-fold screw conformation to the hydrophilic side of cellulose and signifies a high level of molecular specificity. However, the wider implications of GX–cellulose interactions for cellulose network formation and SCW architecture have remained less explored. In this study, we seek to expand our knowledge on this by characterizing the cellulose microfibril organization in three well-characterized GX mutants. The selected mutants display a range of GX deficiency from mild to severe, with findings indicating even the weakest mutant having significant perturbations of the cellulose network, as visualized by both scanning electron microscopy (SEM) and atomic force microscopy (AFM). We show by image analysis that microfibril width is increased by as much as three times in the severe mutants compared to the wild type and that the degree of directional dispersion of the fibrils is approximately doubled in all the three mutants. Further, we find that these changes correlate with both altered nanomechanical properties of the SCW, as observed by AFM, and with increases in enzymatic hydrolysis. Results from this study indicate the critical role that normal GX composition has on cellulose bundle formation and cellulose organization as a whole within the SCWs.
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    Artificial consortium demonstrates emergent properties of enhanced cellulosic-sugar degradation and biofuel synthesis
    (2020-12) Park, Heejoon; Patel, Ayushi; Hunt, Kristopher A.; Henson, Michael A.; Carlson, Ross P.
    Planktonic cultures, of a rationally designed consortium, demonstrated emergent properties that exceeded the sums of monoculture properties, including a >200% increase in cellobiose catabolism, a >100% increase in glycerol catabolism, a >800% increase in ethanol production, and a >120% increase in biomass productivity. The consortium was designed to have a primary and secondary-resource specialist that used crossfeeding with a positive feedback mechanism, division of labor, and nutrient and energy transfer via necromass catabolism. The primary resource specialist was Clostridium phytofermentans (a.k.a. Lachnoclostridium phytofermentans), a cellulolytic, obligate anaerobe. The secondary-resource specialist was Escherichia coli, a versatile, facultative anaerobe, which can ferment glycerol and byproducts of cellobiose catabolism. The consortium also demonstrated emergent properties of enhanced biomass accumulation when grown as biofilms, which created high cell density communities with gradients of species along the vertical axis. Consortium biofilms were robust to oxic perturbations with E. coli consuming O2, creating an anoxic environment for C. phytofermentans. Anoxic/oxic cycling further enhanced biomass productivity of the biofilm consortium, increasing biomass accumulation ~250% over the sum of the monoculture biofilms. Consortium emergent properties were credited to several synergistic mechanisms. E. coli consumed inhibitory byproducts from cellobiose catabolism, driving higher C. phytofermentans growth and higher cellulolytic enzyme production, which in turn provided more substrate for E. coli. E. coli necromass enhanced C. phytofermentans growth while C. phytofermentans necromass aided E. coli growth via the release of peptides and amino acids, respectively. In aggregate, temporal cycling of necromass constituents increased flux of cellulose-derived resources through the consortium. The study establishes a consortia-based, bioprocessing strategy built on naturally occurring interactions for improved conversion of cellulose-derived sugars into bioproducts.
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