Scholarly Work - Chemical & Biological Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/8718
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Item 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.Item 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, MarkHydrogen 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.Item 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.Item 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.Item 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.Item 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.Item 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.Item 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.Item 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 MThe 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.Item 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.