Effect of pH on the activity of ice-binding protein from Marinomonas primoryensis.

The ability of an ice-binding protein (IBP) from Marinomonas primoryensis (MpIBP) to influence ice crystal growth and structure in nonphysiological pH environments was investigated in this work. The ability for MpIBP to retain ice interactivity under stressed environmental conditions was determined via (1) a modified splat assay to determine ice recrystallization inhibition (IRI) of polycrystalline ice and (2) nanoliter osmometry to evaluate the ability of MpIBP to dynamically shape the morphology of a single ice crystal. Circular dichroism (CD) was used to relate the IRI and DIS activity of MpIBP to secondary structure. The results illustrate that MpIBP secondary structure was stable between pH 6 and pH 10. It was found that MpIBP did not interact with ice at pH ≤ 4 or pH ≥ 13. At 6 ≤ pH ≥ 12 MpIBP exhibited a reduction in grain size of ice crystals as compared to control solutions and demonstrated dynamic ice shaping at 6 ≤ pH ≥ 10. The results substantiate that MpIBP retains some secondary structure and function in non-neutral pH environments; thereby, enabling its potential utility in nonphysiological materials science and engineering applications.

A New Estimate of Building Floor Space in North America.

Floor space is a key variable used to understand the energy and material demands of buildings. Using recent data sets of building footprints, we employ a random forest regression model to estimate the total floor space (conditioned and unconditioned) of the North American building stock. Our estimate for total floor space in 2016 is 88033 (+15907/-21861) million m2, which is 2.9 times higher than current estimates from national statistics offices. We also show how floor space per capita (m2 cap-1) is not constant across the North American region, highlighting the heterogeneous nature of building stocks. As a critical variable in integrated assessment models to project energy and material demands, this result suggests that there is much more unconditioned floor space than previously realized. Furthermore, when estimating material stocks, flows, and associated embodied carbon emissions, total floor space per-capita estimates, such as those presented in this study, offer a more comprehensive approach in comparison to national statistics that do not capture unconditioned floor space. This result also calls for an investigation as to why there is such a vast difference between estimates of conditioned and total floor space.

Genome engineering of E. coli for improved styrene production.

Microbial production of exogenous organic compounds is challenging as biosynthetic pathways are often complex and produce metabolites that are toxic to the hosts. Biogenic styrene is an example of this problem, which if addressed could result in a more sustainable supply of this important component of the plastics industry. In this study, we engineered Escherichia coli for the production of styrene. We systematically optimized the production capability by first screening different pathway expression levels in E. coli strains. We then further designed and constructed a transcription regulator library targeting 54 genes with 85,420 mutations, and tested this library for increased styrene resistance and production. A series of tolerant mutants not only exhibited improved styrene tolerance but also produced higher styrene concentrations compared to the parent strain. The best producing mutant, ST05 LexA_E45I, produced a 3.45-fold increase in styrene compared to the parent strain. The produced styrene was extracted via gas stripping into dodecane and used in a direct free radical synthesis of polystyrene.

On-Demand Microwave-Assisted Fabrication of Gelatin Foams.

Ultraporous gelatin foams (porosity >94%, ρ ≈ 0.039⁻0.056 g/cm³) have been fabricated via microwave radiation. The resulting foam structures are unique with regard to pore morphology (i.e., closed-cell) and exhibit 100% macroporosity (pore size 332 to 1700 µm), presence of an external skin, and densities similar to aerogels. Results indicate that the primary foaming mechanism is governed by the vaporization of water that is tightly bound in secondary structures (i.e., helices, ß-turns, ß-sheets) that are present in dehydrated gelatin films but not present in the foams after microwave radiation (700 Watts).

Algal Biochar-Metal Nanocomposite Particles Tailor the Hydration Kinetics and Compressive Strength of Portland Cement Paste.

Biochar can improve the mechanical properties of portland cement paste and concrete. In this work, we produced algal biochar-zinc (biochar-Zn) and algal biochar-calcium (biochar-Ca) nanocomposite particles and studied their effect on the hydration kinetics and compressive strength of cement paste. Results show that 3 wt % biochar-Zn delayed peak heat evolution during cement hydration from 8.3 to 10.0 h, while 3 wt % addition of biochar-Ca induced a minor acceleration of peak heat from 8.3 to 8.2 h. Both biochar-Zn and biochar-Ca nanocomposite particles increased the compressive strength of cement paste at 28 days by 22.6 and 17.0%, respectively. Data substantiate that retardation or minor acceleration of the reaction kinetics was due exclusively to the presence of Zn and Ca phases, respectively, while the enhanced strength was attributed to a nucleation effect induced by such phases and the internal curing effect of biochar.

Ice-binding proteins and bioinspired synthetic mimics in non-physiological environments.

Ice-binding proteins (IBPs) are produced by a variety of organisms to prevent internal damage caused by ice crystal growth. Synthetic analogs are being designed to mimic beneficial properties of IBPs while mitigating drawbacks related to the use of biological proteins. While a multitude of engineering applications could benefit from the inhibition and control of ice formation and growth, synthetic analogs tend to be less potent than biological IBPs, and both IBPs and synthetic analogs tend to exhibit lower performance in non-physiological (i.e., non-biological) solutions. This review examines the ice interaction properties and performance of IBPs and their synthetic analogs in non-physiological environments. Common methods to measure ice interactions are discussed (i.e., thermal hysteresis, ice recrystallization inhibition, ice growth rate, and ice nucleation). A quantitative meta-analysis of material performance in non-physiological environments is presented, along with a discussion of future research directions. The findings presented herein can inform IBP and synthetic mimic selection to control ice interactions in a wide variety of materials science and engineering applications, including cell, tissue, and organ cryopreservation, food storage and transport, freeze-thaw damage of cementitious materials, and anti-icing surfaces for aerospace vehicles, solar panels, and wind turbines.

Bioinspired Threonine-Based Polymers with Potent Ice Recrystallization Inhibition Activity.

Ice growth mitigation is a pervasive challenge for multiple industries. In nature, ice-binding proteins (IBPs) demonstrate potent ice growth prevention through ice recrystallization inhibition (IRI). However, IBPs are expensive, difficult to produce in large quantities, and exhibit minimal resilience to nonphysiological environmental stressors, such as pH. For these reasons, researchers have turned to bioinspired polymeric materials that mimic IBP behavior. To date, however, no synthetic polymer has rivaled the ability of native IBPs to display IRI activity at ultralow nanomolar concentrations. In this work, we study the IRI activity of peptides and polypeptides inspired by common ice-binding residues of IBPs to inform the synthesis and characterization of a potent bioinspired polymer that mimics IBP behavior. We show first that the threonine polypeptide (pThr) displays the best IRI activity in phosphate-buffered saline (PBS). Second, we use pThr as a molecular model to synthesize and test a bioinspired polymer, poly(2-hydroxypropyl methacrylamide) (pHPMA). We show that pHPMA exhibits potent IRI activity in neutral PBS at ultralow concentrations (0.01 mg/mL). pHPMA demonstrates potent IRI activity at low molecular weights (2.3 kDa), with improved activity at higher molecular weights (32.8 kDa). These results substantiate that pHPMA is a robust molecule that mitigates ice crystal growth at concentrations similar to native IBPs.

Engineered Living Materials: Taxonomies and Emerging Trends.

At the intersection of synthetic biology and materials science, the field of engineered living materials (ELMs) has evolved into a new, standalone discipline. The fusion of bioengineering's design-build-test-learn approaches with classical materials science has yielded breakthrough innovations in the synthesis of complex, biologically active materials for functional applications in therapeutics, electronics, construction, and beyond. However, the transdisciplinary nature of the ELM field - and its rapid growth - has made holistic comprehension of achievements related to the tools, techniques, and applications of ELMs difficult across disciplines. To this end, this review proposes an emergent taxonomy of ELM research and uses the categorization to discuss current trends and state-of-the-art advancements, significant opportunities, and imminent challenges for scientists and engineers in the field.

Engineering living building materials for enhanced bacterial viability and mechanical properties.

Living building materials (LBMs) utilize microorganisms to produce construction materials that exhibit mechanical and biological properties. A hydrogel-based LBM containing bacteria capable of microbially induced calcium carbonate precipitation (MICP) was recently developed. Here, LBM design factors, i.e., gel/sand ratio, inclusion of trehalose, and MICP pathways, are evaluated. The results show that non-saturated LBM (gel/sand = 0.13) and gel-saturated LBM (gel/sand = 0.30) underwent distinct failure modes. The inclusion of trehalose maintains bacterial viability under ambient conditions with low relative humidity, without affecting mechanical properties of the LBM. Comparison of biotic and abiotic LBM shows that MICP efficiency in this material is subject to the pathway selected: the LBM with heterotrophic ureolytic Escherichia coli demonstrated the most mechanical enhancement from the abiotic controls, compared with either ureolytic or CO2-concentrating mechanisms from Synechococcus. The study shows that tailoring of LBM properties can be accomplished in a manner that considers both LBM microstructure and MICP pathways.

Mechanical evaluation of 3D printed biomimetic non-Euclidean saddle geometries mimicking the mantis shrimp.

Engineering design has drawn inspiration from naturally occurring structures to advance manufacturing processes and products, termed biomimetics. For example, the mantis shrimp, orderStomatopoda, is capable of producing one of the fastest appendage strikes in the world with marginal musculoskeletal displacement. The extreme speed of the mantis shrimp's raptorial appendage is due to the non-Euclidean hyperbolic paraboloid (i.e. saddle) shape within the dorsal region of the merus, which allows substantial energy storage through compression in the sagittal plane. Here, investigation of 3D printed synthetic geometries inspired by the mantis shrimp saddle geometry has revealed insights for elastic energy storage (i.e. spring-like) applications. Saddles composed of either astiffor aflexibleresin were investigated for spring response to explore the geometric effects. By modulating the saddle geometry and testing the spring response, it was found that, for thestiffresin, the spring constant was improved as the curvature of the contact and orthogonal faces were maximized and minimized, respectively. For theflexibleresin, it was found that the spring constant increased by less than 250 N mm-1as the saddle geometry changed, substantiating that the flexible component of mantis saddles does not contribute to energy storage capabilities. The geometries of two saddles from the mantis shrimp speciesO. scyllaruswere estimated and exhibited similar trends to manufactured saddles, suggesting that modulating saddle geometry can be used for tailored energy storage moduli in spatially constrained engineering applications.

Zeolite Adsorption of Chloride from a Synthetic Alkali-Activated Cement Pore Solution.

This work presents experimental evidence that confirms the potential for two specific zeolites, namely chabazite and faujasite (with a cage size ~2-13 Å), to adsorb small amounts of chloride from a synthetic alkali-activated cement (AAC) pore solution. Four synthetic zeolites were first exposed to a chlorinated AAC pore solution, two faujasite zeolites (i.e., FAU, X-13), chabazite (i.e., SSZ-13), and sodium-stabilized mordenite (i.e., Na-Mordenite). The mineralogy and chemical composition were subsequently investigated via X-ray diffraction (XRD) and both energy- and wavelength-dispersive X-ray spectroscopy (WDS), respectively. Upon exposure to a chlorinated AAC pore solution, FAU and SSZ-13 displayed changes to their diffraction patterns (i.e., peak shifting and broadening), characteristic of ion entrapment within zeolitic aluminosilicate frameworks. Elemental mapping with WDS confirmed the presence of small amounts of elemental chlorine. Results indicate that the chloride-bearing capacity of zeolites is likely dependent on both microstructural features (e.g., cage sizes) and chemical composition.

Optical Properties and Mechanical Modeling of Acetylated Transparent Wood Composite Laminates.

Transparent wood composites (TWCs) are a new class of light-transmitting wood-based materials composed of a delignified wood template that is infiltrated with a refractive- index-matched polymer resin. Recent research has focused primarily on the fabrication and characterization of single-ply TWCs. However, multi-ply composite laminates are of interest due to the mechanical advantages they impart compared to the single ply. In this work, 1- and 2-ply [0°/90°] TWC laminates were fabricated using a delignified wood template (C) and an acetylated delignified wood template (AC). The optical and mechanical properties of resultant C and AC TWC laminates were determined using ultraviolet-visible spectroscopy (UV-Vis) and tensile testing (5× replicates), respectively. In addition, the ability of classical lamination plate theory and simple rule of mixtures to predict multi-ply tensile modulus and strength, respectively, from ply-level mechanical properties were investigated and are reported herein. Experimental results highlight tradeoffs that exist between the mechanical and optical responses of both unmodified and chemically modified TWCs. Template acetylation reduced the stiffness and strength in the 0° fiber direction by 2.4 GPa and 58.9 MPa, respectively, compared to the unmodified samples. At high wavelengths of light (>515 nm), AC samples exhibited higher transmittance than the C samples. Above 687 nm, the 2-ply AC sample exhibited a higher transmittance than the 1-ply C sample, indicating that thickness-dependent optical constraints can be overcome with improved interfacial interactions. Finally, both predictive models were successful in predicting the elastic modulus and tensile strength response for the 2-ply C and AC samples.

Ice-Binding Protein from Shewanella frigidimarinas Inhibits Ice Crystal Growth in Highly Alkaline Solutions.

The ability of a natural ice-binding protein from Shewanella frigidimarina (SfIBP) to inhibit ice crystal growth in highly alkaline solutions with increasing pH and ionic strength was investigated in this work. The purity of isolated SfIBP was first confirmed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography with an ultraviolet detector (SEC-UV). Protein stability was evaluated in the alkaline solutions using circular dichroism spectroscopy, SEC-UV, and SDS-PAGE. SfIBP ice recrystallization inhibition (IRI) activity, a measure of ice crystal growth inhibition, was assessed using a modified splat assay. Statistical analysis of results substantiated that, despite partial denaturation and misfolding, SfIBP limited ice crystal growth in alkaline solutions (pH ≤ 12.7) with ionic strength I ≤ 0.05 mol/L, but did not exhibit IRI activity in alkaline solutions where pH ≥ 13.2 and I ≥ 0.16 mol/L. IRI activity of SfIBP in solutions with pH ≤ 12.7 and I ≤ 0.05 mol/L demonstrated up to ≈ 66% reduction in ice crystal size compared to neat solutions.

Engineered Ureolytic Microorganisms Can Tailor the Morphology and Nanomechanical Properties of Microbial-Precipitated Calcium Carbonate.

We demonstrate for the first time that the morphology and nanomechanical properties of calcium carbonate (CaCO3) can be tailored by modulating the precipitation kinetics of ureolytic microorganisms through genetic engineering. Many engineering applications employ microorganisms to produce CaCO3. However, control over bacterial calcite morphology and material properties has not been demonstrated. We hypothesized that microorganisms genetically engineered for low urease activity would achieve larger calcite crystals with higher moduli. We compared precipitation kinetics, morphology, and nanomechanical properties for biogenic CaCO3 produced by two Escherichia coli (E. coli) strains that were engineered to display either high or low urease activity and the native producer Sporosarcina pasteurii. While all three microorganisms produced calcite, lower urease activity was associated with both slower initial calcium depletion rate and increased average calcite crystal size. Both calcite crystal size and nanoindentation moduli were also significantly higher for the low-urease activity E. coli compared with the high-urease activity E. coli. The relative resistance to inelastic deformation, measured via the ratio of nanoindentation hardness to modulus, was similar across microorganisms. These findings may enable design of novel advanced engineering materials where modulus is tailored to the application while resistance to irreversible deformation is not compromised.

Rational Control of Calcium Carbonate Precipitation by Engineered Escherichia coli.

Ureolytic bacteria ( e.g., Sporosarcina pasteurii) can produce calcium carbonate (CaCO3). Tailoring the size and shape of biogenic CaCO3 may increase the range of useful applications for these crystals. However, wild type Sporosarcina pasteurii is difficult to genetically engineer, limiting control of the organism and its crystal precipitates. Therefore, we designed, constructed, and compared different urease operons and expression levels for CaCO3 production in engineered Escherichia coli strains. We quantified urease expression and calcium uptake and characterized CaCO3 crystal phase and morphology for 13 engineered strains. There was a weak relationship between urease expression and crystal size, suggesting that genes surrounding the urease gene cluster affect crystal size. However, when evaluating strains with a wider range of urease expression levels, there was a negative relationship between urease activity and polycrystal size (e.g., larger crystals with lower activity). The resulting range of crystal morphologies created by the rationally designed strains demonstrates the potential for controlling biogenic CaCO3 precipitation.

Evaporation-based method for preparing gelatin foams with aligned tubular pore structures.

Gelatin-based foams with aligned tubular pore structures were prepared via liquid-to-gas vaporization of tightly bound water in dehydrated gelatin hydrogels. This study elucidates the mechanism of the foaming process by investigating the secondary (i.e., helical) structure, molecular interactions, and water content of gelatin films before and after foaming using X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry and thermogravimetric analysis (TGA), respectively. Experimental data from gelatin samples prepared at various gelatin-to-water concentrations (5-30 wt.%) substantiate that resulting foam structures are similar in pore diameter (approximately 350 µm), shape, and density (0.05-0.22 g/cm(3)) to those fabricated using conventional methods (e.g., freeze-drying). Helical structures were identified in the films but were not evident in the foamed samples after vaporization (~150 °C), suggesting that the primary foaming mechanism is governed by the vaporization of water that is tightly bound in secondary structures (i.e., helices, ß-turns, ß-sheets) that are present in dehydrated gelatin films. FTIR and TGA data show that the foaming process leads to more disorder and reduced hydrogen bonding to hydroxyl groups in gelatin and that no thermal degradation of gelatin occurs before or after foaming.