Practical deployment of automation to expedite aqueous two-phase extraction.

The feasibility of bioprocess development relies heavily on the successful application of primary recovery and purification techniques. Aqueous two-phase extraction (ATPE) disrupts the definition of "unit operation" by serving as an integrative and intensive technique that combines different objectives such as the removal of biomass and integrated recovery and purification of the product of interest. The relative simplicity of processing large samples renders this technique an attractive alternative for industrial bioprocessing applications. However, process development is hindered by the lack of easily predictable partition behaviours, the elucidation of which necessitates a large number of experiments to be conducted. Liquid handling devices can assist to address this problem; however, they are configured to operate using low viscosity fluids such as water and water-based solutions as opposed to highly viscous polymeric solutions, which are typically required in ATPE. In this work, an automated high throughput ATPE process development framework is presented by constructing phase diagrams and identifying the binodal curves for PEG6000, PEG3000, and PEG2000. Models were built to determine viscosity- and volume-independent transfer parameters. The framework provided an appropriate strategy to develop a very precise and accurate operation by exploiting the relationship between different liquid transfer parameters and process error. Process accuracy, measured by mean absolute error, and device precision, evaluated by the coefficient of variation, were both shown to be affected by the mechanical properties, particularly viscosity, of the fluids employed. For PEG6000, the mean absolute error improved by six-fold (from 4.82% to 0.75%) and the coefficient of variation improved by three-fold (from 0.027 to 0.008) upon optimisation of the liquid transfer parameters accounting for the viscosity effect on the PEG-salt buffer utilising ATPE operations. As demonstrated here, automated liquid handling devices can serve to streamline process development for APTE enabling wide adoption of this technique in large scale bioprocess applications.

Spray Drying Is a Viable Technology for the Preservation of Recombinant Proteins in Microalgae.

Microalgae are promising host organisms for the production of encapsulated recombinant proteins such as vaccines. However, bottlenecks in bioprocess development, such as the drying stage, need to be addressed to ensure feasibility at scale. In this study, we investigated the potential of spray drying to produce a recombinant vaccine in microalgae. A transformant line of Chlamydomonas reinhardtii carrying a subunit vaccine against salmonid alphavirus was created via chloroplast engineering. The integrity of the recombinant protein after spray drying and its stability after 27 months storage at -80 °C, +4 °C and room temperature were assessed by immunoblotting. The protein withstood spray drying without significant losses. Long-term storage at +4 °C and room temperature resulted in 50% and 92% degradation, respectively. Optimizing spray drying and storage conditions should minimize degradation and favour short-term storage at positive temperatures. Using data on yield and productivity, the economics of spray drying- and freeze drying-based bioprocesses were compared. The drying stage corresponded to 41% of the total production cost. Process optimization, genetic engineering and new market strategies were identified as potential targets for cost reduction. Overall, this study successfully demonstrates the suitability of spray drying as a process option for recombinant protein production in microalgae at the industrial scale.

Iron-dependent mutualism between Chlorella sorokiniana and Ralstonia pickettii forms the basis for a sustainable bioremediation system.

Phototrophic communities of autotrophic microalgae and heterotrophic bacteria perform complex tasks of nutrient acquisition and tackling environmental stress but remain underexplored as a basis for the bioremediation of emerging pollutants. In industrial monoculture designs, poor iron uptake by microalgae limits their productivity and biotechnological efficacy. Iron supplementation is expensive and ineffective because iron remains insoluble in an aqueous medium and is biologically unavailable. However, microalgae develop complex interkingdom associations with siderophore-producing bacteria that help solubilize iron and increase its bioavailability. Using dye degradation as a model, we combined environmental isolations and synthetic ecology as a workflow to design a simplified microbial community based on iron and carbon exchange. We established a mutualism between the previously non-associated alga Chlorella sorokiniana and siderophore-producing bacterium Ralstonia pickettii. Siderophore-mediated increase in iron bioavailability alleviated Fe stress for algae and increased the reductive iron uptake mechanism and bioremediation potential. In exchange, C. sorokiniana produced galactose, glucose, and mannose as major extracellular monosaccharides, supporting bacterial growth. We propose that extracellular iron reduction by ferrireductase is crucial for azoreductase-mediated dye degradation in microalgae. These results demonstrate that iron bioavailability, often overlooked in cultivation, governs microalgal growth, enzymatic processes, and bioremediation potential. Our results suggest that phototrophic communities with an active association for iron and carbon exchange have the potential to overcome challenges associated with micronutrient availability, while scaling up bioremediation designs.

Microbiome-Inspired Green Infrastructure: A Toolkit for Multidisciplinary Landscape Design.

Incorporating recent advances in environmental microbiome research and policy is a major challenge for urban design. We set out a framework for managing construction projects so that multidisciplinary teams of researchers and practitioners can explicitly consider environmental microbiota in design and construction contexts, thereby increasing ecosystem functionality and public health.

Robotic Extrusion of Algae-Laden Hydrogels for Large-Scale Applications.

A bioprinting technique for large-scale, custom-printed immobilization of microalgae is developed for potential applications within architecture and the built environment. Alginate-based hydrogels with various rheology modifying polymers and varying water percentages are characterized to establish a window of operation suitable for layer-by-layer deposition on a large scale. Hydrogels formulated with methylcellulose and carrageenan, with water percentages ranging from 80% to 92.5%, demonstrate a dominant viscoelastic solid-like property with G' > G″ and a low phase angle, making them the most suitable for extrusion-based printing. A custom multimaterial pneumatic extrusion system is developed to be attached on the end effector of an industrial multiaxis robot arm, allowing precision-based numerically controlled layered deposition of the viscous hydrogel. The relationship between the various printing parameters, namely air pressure, material viscosity, viscoelasticity, feed rate, printing distance, nozzle diameter, and the speed of printing, are characterized to achieve the desired resolution of the component. Printed prototypes are postcured in CaCl2 via crosslinking. Biocompatibility tests show that cells can survive for 21 days after printing the constructs. To demonstrate the methodology for scale-up, a 1000 × 500 mm fibrous hydrogel panel is additively deposited with 3 different hydrogels with varying water percentages.

Downstream Processing of Chlamydomonas reinhardtii TN72 for Recombinant Protein Recovery.

The green microalga Chlamydomonas reinhardtii is under development as a production host for recombinant proteins and whole-cell therapeutics. In particular, the cell wall-reduced strain TN72 is used as a model organism for protein expression and algal synthetic biology. However, the bioprocessing characteristics of TN72 and other C. reinhardtii strains have yet to be examined. Here we use a TN72 strain expressing a protein-based antibiotic (Pal) to study the scale-up of cell harvest and product recovery. Cell harvest was examined with 100L cultures in two intermittent-discharge continuous-flow disc-stack centrifuges at flow rates of 150-250 L.h-1, as well as with an ultra scale-down (USD) mimic of the centrifuges. Solids recovery exceeded 99.5% and the loss of product to the supernatant was below 2-3%. TN72 is intact following the high shear conditions of the feed zone, however discharge from both disc-stack centrifuges resulted in full cell breakage and in the case of Pal, partial degradation in the subsequent hours. We demonstrated that shake flask cultivation and the USD centrifuge technique can be used to predict the pilot-scale clarification efficiency and product release at the centrifuge inlet for TN72, but not the cell breakage on discharge. This study outlines a number of challenges for scale-up of recombinant protein production in the microalgal host in particular for whole cell therapeutics, but also opportunities for the bioprocessing of intracellular products from TN72.

Electrochemical Characterisation of Bio-Bottle-Voltaic (BBV) Systems Operated with Algae and Built with Recycled Materials.

Photobioelectrochemical systems are an emerging possibility for renewable energy. By exploiting photosynthesis, they transform the energy of light into electricity. This study evaluates a simple, scalable bioelectrochemical system built from recycled plastic bottles, equipped with an anode made from recycled aluminum, and operated with the green alga Chlorella sorokiniana. We tested whether such a system, referred to as a bio-bottle-voltaic (BBV) device, could operate outdoors for a prolonged time period of 35 days. Electrochemical characterisation was conducted by measuring the drop in potential between the anode and the cathode, and this value was used to calculate the rate of charge accumulation. The BBV systems were initially able to deliver ~500 mC·bottle−1·day−1, which increased throughout the experimental run to a maximum of ~2000 mC·bottle−1·day−1. The electrical output was consistently and significantly higher than that of the abiotic BBV system operated without algal cells (~100 mC·bottle−1·day−1). The analysis of the rate of algal biomass accumulation supported the hypothesis that harvesting a proportion of electrons from the algal cells does not significantly perturb the rate of algal growth. Our finding demonstrates that bioelectrochemical systems can be built using recycled components. Prototypes of these systems have been displayed in public events; they could serve as educational toolkits in schools and could also offer a solution for powering low-energy devices off-grid.

Ambulatory cardiovascular functioning in healthy postmenopausal women with victimization histories.

Criminal victimization is a prevalent stressor among women, with potentially long-lasting emotional consequences. The present study examined associations among severity of lifetime victimization, current posttraumatic stress disorder (PTSD) symptoms, and ambulatory blood pressure (ABP) and heart rate (AHR) levels and variability. Thirty-nine healthy post-menopausal women who endorsed a history of criminal victimization completed a measure of PTSD symptom severity and participated in an average of 18 h of ambulatory cardiovascular monitoring. PTSD symptom clusters were associated with AHR. Symptom clusters and lifetime victimization severity jointly predicted ABP, with ABP the highest among severely victimized women with high levels of current intrusions. Given the prevalence of criminal victimization, biopsychosocial research on women's cardiovascular disease risk may benefit from increased attention to this stressor and its psychological sequelae.

Treatment of Phaeodactylum tricornutum cells with papain facilitates lipid extraction.

Triacylglycerols (TAGs) from microalgae have the potential to be used for biodiesel, but several technical and economic hurdles have to be overcome. A major challenge is efficient extraction of intracellular TAGs from algae. Here we investigate the use of enzymes to deconstruct algal cell walls/membranes. We describe a rapid and simple assay that can assess the efficacy of different enzyme treatments on TAG-containing algae. By this means crude papain and bromelain were found to be effective in releasing TAGs from the diatom Phaeodactylum tricornutum, most likely because of their cysteine protease activity. Pre-treating algal biomass with crude papain enabled complete extraction of TAGs using heptane/isopropyl alcohol. Heptane as a single solvent was also effective, although complete recovery of TAG was not obtained. Economic implications of these findings are discussed, with the aim to reduce the complexity of, and energy needed in, TAG extraction.

The continued lived experience of the unexpected death of a child.

The purpose of this qualitative study was to further examine the lived experiences of families that faced the sudden loss of a child. Six mothers that had previously participated approximately 9 years ago in the study entitled "The Lived Experience of the Unexpected Death of a Child" were interviewed. All of the mothers were Caucasian and had achieved a high school or greater level of education. Taped interviews and field notes were transcribed for data display. Together, two raters completed data reduction and coding for theme identification and categorization. Six themes were revealed that described helpful coping behaviors used by the mothers: positive beliefs, faith beliefs, everlasting love, pleasant remembrances, social engagement, and staying connected. Two themes, avoidance and rumination, described the behaviors that did not help the mothers cope with their loss. Finally, answers to the questions regarding acceptance and healing time were also revealed.

Directed evolution of a thermostable l-aminoacylase biocatalyst.

Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution strategies to identify improved variants. Mutants P237S and F251Y were 2.4-fold more active towards N-benzoyl valine relative to the wild type at 65°C. F251 mutations to basic residues resulted in 4.5-11-fold shifts in the substrate preference towards N-benzoyl phenylalanine relative to N-benzoyl valine. The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of k(cat) values for N-benzoyl valine and N-benzoyl phenylalanine. Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance, whereas those improving activity towards N-benzoyl-valine occurred in the hinge region loops linking the dimerization and zinc-binding domains in each monomer. These observations support a previously proposed substrate induced conformational transition between open and closed forms of aminoacylases.