Leveraging the versatile properties of bacterial spores in materials.

Inspired by biological functions of living systems, researchers have engineered cells as independent functional materials or integrated them within a natural or synthetic matrix to create engineered living materials (ELMs). However, the 'livingness' of cells in such materials poses serious drawbacks, such as a short lifespan and the need for cold-chain logistics. Bacterial spores have emerged as a game changer to bypass these shortcomings as a result of their intrinsic dormancy and resistance against harsh conditions. Emerging synthetic biology tools tailored for engineering spores and better understanding of their physical properties have led to novel applications of spore-based materials. Here, we review recent advances in such materials and discuss future challenges for the development of time- and cost-efficient spore-based materials with high performance.

Growth, Distribution, and Photosynthesis of Chlamydomonas Reinhardtii in 3D Hydrogels.

Engineered living materials (ELMs) are a novel class of functional materials that typically feature spatial confinement of living components within an inert polymer matrix to recreate biological functions. Understanding the growth and spatial configuration of cellular populations within a matrix is crucial to predicting and improving their responsive potential and functionality. Here, this work investigates the growth, spatial distribution, and photosynthetic productivity of eukaryotic microalga Chlamydomonas reinhardtii (C. reinhardtii) in three-dimensionally shaped hydrogels in dependence of geometry and size. The embedded C. reinhardtii cells photosynthesize and form confined cell clusters, which grow faster when located close to the ELM periphery due to favorable gas exchange and light conditions. Taking advantage of location-specific growth patterns, this work successfully designs and prints photosynthetic ELMs with increased CO2 capturing rate, featuring high surface to volume ratio. This strategy to control cell growth for higher productivity of ELMs resembles the already established adaptations found in multicellular plant leaves.

Automated quantification of lipid contents of Lipomyces starkeyi using deep-learning-based image segmentation.

Intracellular lipid droplets (LDs), subcellular organelles playing a role in long-term carbon storage, have immense potential in biofuel and dietary lipid production. Monitoring the state of LDs in living cells is of utmost importance for quick biomass harvest and screening promising isolates. Here, a deep-learning-based segmentation model was developed for automatic detection and segmentation of LDs using the model yeast species Lipomyces starkeyi, leading to fast and accurate quantification of lipid contents in liquid cultures. The trained model detected the yeast's cell and LDs in light microscopic images with an accuracy of 98% and 92%, respectively. Lipid content prediction using pixel numbers counted in segmented LDs showed high similarity to lipid quantification results obtained with gas chromatography-mass spectrometry. This automated quantification can highly reduce cost and time in real-time monitoring of lipid production, thereby providing an efficient tool in bio-fermentation.

A strategic approach to apply bacterial substances for increasing metabolite productions of Euglena gracilis in the bioreactor.

Bacterial extracellular polymeric substances (EPS) are promising materials that have a role in enhancing growth, metabolite production, and harvesting efficiency. However, the validity of the EPS effectiveness in scale-up cultivation of microalgae is still unknown. Therefore, in order to verify whether the bacterial metabolites work in the scale-up fermentation of microalgae, we conducted a bioreactor fermentation following the addition of bacterial EPS derived from the marine bacterium, Pseudoalteromonas sp., to Euglena gracilis. Various culture strategies (i.e., batch, glucose fed-batch, and glucose and EPS fed-batch) were conducted to maximize metabolite production of E. gracilis in scale-up cultivation. Consequently, biomass and paramylon concentrations in the continuous glucose and EPS-treated culture were enhanced by 3.0-fold and 4.2-fold (36.1 ± 1.4 g L-1 and 25.6 ± 0.1 g L-1), respectively, compared to the non-treated control (12.0 ± 0.3 g L-1 and 6.1 ± 0.1 g L-1). Also, the supplementation led to the enhanced concentrations of α-tocopherols and total fatty acids by 3.7-fold and 2.8-fold, respectively. The harvesting efficiency was enhanced in EPS-supplemented cultivation due to the flocculation of E. gracilis. To the best of our knowledge, this is the first study that verifies the effect of bacterial EPS in scale-up cultivation of microalgae. Also, our results showed the highest paramylon productivity than any other previous reports. The results obtained in this study showed that the scale-up cultivation of E. gracilis using bacterial EPS has the potential to be used as a platform to guide further increases in scale and in the industrial environment. KEY POINTS: Effect of EPS on Euglena gracilis fermentation was tested in bioreactor scale. EPS supplement was effective for the paramylon, α-tocopherol, and lipid production. EPS supplement induced the flocculation of E. gracilis.

Genomic Analysis and Assessment of Melanin Synthesis in Amorphotheca resinae KUC3009.

This study reports the draft genome of Amorphotheca resinae KUC30009, a fungal isolate with promising industrial-scale melanin production potential. The mechanisms for melanin or melanin-related pigment formation of this strain were examined through bioinformatic and biochemical strategies. The 30.11 Mb genome of A. resinae contains 9638 predicted genes. Genomic-based discovery analyses identified 14 biosynthetic gene clusters (BGCs) associated with secondary metabolite production. Moreover, genes encoding a specific type 1 polyketide synthase and 4-hydroxynaphthalene reductase were identified and predicted to produce intermediate metabolites of dihydroxy naphthalene (DHN)-melanin biosynthesis pathway, but not to DHN-melanin. These findings were further supported by the detection of increased flaviolin concentrations in mycelia and almost unchanged morphologies of the culture grown with tricyclazole. Apart from this, the formation of melanin in the culture filtrate appeared to depend on the laccase-like activity of multi-copper oxidases. Simultaneously, concentrations of nitrogen-containing sources decreased when the melanin formed in the media. Interestingly, melanin formation in the culture fluid was proportional to laccase-like activity. Based on these findings, we proposed novel strategies for the enhancement of melanin production in culture filtrates. Therefore, our study established a theoretical and methodological basis for synthesizing pigments from fungal isolates using genomic- and biochemical-based approaches.

Utilization of extracellular fungal melanin as an eco-friendly biosorbent for treatment of metal-contaminated effluents.

Fungal melanins have been considered as potential biosorbents due to their metal-binding properties, stability, and scalability. Previous studies established scalable fungal melanin production methods with promising strains, however, their applicability for metal-contaminated effluents treatment has not been sufficiently reported. Herein, melanin pigment derived from Amorphotheca resinae was produced and characterized using microscopy and spectroscopy techniques. Adsorptive properties towards Cu(II), Pb(II), Cd(II), and Zn(II) were evaluated using batch tests. Melanin pigment was composed of aggregates of nanosized particles with indole-based constituents. Adsorption capacities increased with the pH of solution, especially at pH > 4.0. Maximum binding capacities of Cu(II), Pb(II), Cd(II), and Zn(II) on melanin were 69.18, 103.23, 24.31, and 13.57 mg/g, respectively. The competitive adsorption experiments elucidated affinity as Cu(II)>Pb(II)≫Cd(II)>Zn(II). Adsorption time generally required <2.5 h to reach equilibrium; the pseudo-second-order kinetic model well described the kinetics. Chelating ability of free radicals in pigment was considered as a possible mechanism for adsorption. Initial adsorption capacities remained almost intact even after 5 consecutive adsorption-desorption cycles. Complete removal of Cu(II), Pb(II), and Cd(II) from metal-contaminated effluent was confirmed. Consequently, melanin pigment derived from A. resinae can be used as a biosorbent suitable for the treatment of metal-contaminated aqueous solutions.

Application of electrical treatment on Euglena gracilis for increasing paramylon production.

Paramylon also called ß-1,3-glucan is a value-added product produced from Euglena gracilis. Recently, researchers have developed various strategies for the enhanced paramylon production, among which electrical treatment for microbial stimulation can be an alternative owing to the applicability to large-scale cultivation. In this study, we applied the electrical treatment for enhanced paramylon production and found the proper treatment conditions. Under the treatment with platinum electrodes at 10 mA, the paramylon production of treated cells was significantly increased about 2.5-fold, compared to those of the untreated cells, although the density of cells was maintained due to considerable stress. The size of treated cells became larger, possibly due to the increased level of paramylon production within the cells. Accordingly, the contents of glucose uptake, glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), and uridine diphosphoglucose (UDPG) were shifted to appropriate states for the process of paramylon synthesis under the treatment. The increased level of transcripts encoding glucan synthase-like 2 (EgGSL2) was also confirmed via droplet digital PCR (ddPCR) under the treatment. Overall, this study makes a major contribution to research on electrical stimulation and provides new insights into E. gracilis metabolism like paramylon synthesis. KEY POINTS: • Electrical treatment induced the paramylon production and morphological change of Euglena gracilis. • The glucose uptake of E. gracilis was increased during the electrical treatment, fueling the paramylon synthesis.

Selection and characterization of toxic Aspergillus spore-specific DNA aptamer using spore-SELEX.

As airborne spores of toxic Aspergillus species cause mild symptoms to invasive fungal infections, their indoor concentration should be controlled through real-time management. Aptamer-based biosensors could provide economical and simple solutions for point-of-care. In this study, we isolated aptamers binding to the spores of three representative toxic Aspergillus species (A. fumigatus, A. flavus, and A. niger) for the first time, using cell-SELEX (systematic evolution of ligands through exponential enrichment). Among the aptamer candidates, Asp-3 showed a broad and high binding affinity for the Aspergillus spores. Considering the low binding affinity with proteinase-treated spores, we speculated that the Asp-3 binding sites could be possibly associated with cell surface proteins. The high Asp-3 specificity was confirmed by comparing the binding affinity between the Aspergillus target species and other common indoor fungal species. Moreover, we also established quantitative linear relationships between Asp-3 and the spore concentration of each Aspergillus species. Therefore, the selected Asp-3 aptamer, conjugated with detection sensors, could be an effective biorecognition element for the spores of three toxic Aspergillus species.

Fungal melanin as a biocompatible broad-spectrum sunscreen with high antioxidant activity.

Melanin is considered a bio-inspired dermo-cosmetic component due to its high UV absorption and antioxidant activity. Among various melanin sources, fungal melanin is a promising candidate for sunscreen because of its sustainability and scalability; however, quantitative assessment of its function has not yet been sufficiently explored. In this study, melanin samples derived from Amorphotheca resinae were prepared, followed by the evaluation of their sunscreen performance, antioxidant activity, and cytotoxicity. Melanin-blended cream was prepared by blending a melanin suspension and a pure cream. The cream showed an in vitro sun protection factor value of 2.5 when the pigment content was 5%. The cream showed a critical wavelength of approximately 388 nm and a UVA/UVB ratio of more than 0.81, satisfying the broad-spectrum sunscreen requirement. Oxygen radical absorbance capacity assays indicated that fungal melanin had antioxidant activity similar to ascorbic acid but higher than reduced glutathione. Fungal melanin had no statistically significant cytotoxicity to human keratinocyte cell lines until 72 h of exposure, even at a concentration of 4 mg mL-1. Consequently, melanin pigment can be used as a biocompatible broad-spectrum sunscreen with high antioxidant activity and as a practical alternative in dermo-cosmetic formulations.

Enzymatic production of sugar from fungi and fungi-infected lignocellulosic biomass by a new cellulosomal enzyme harboring N-acetyl-ß-d-glucosaminidase activity.

Cellulosomes are scaffold proteins displaying enzymes on the cell wall to efficiently obtain nutrient sources. CcGlcNAcase is a novel cellulosomal component. Based on sequence analysis, CcGlcNAcase was predicted to be a chitinolytic enzyme based on high homology with the discoidin domain-containing protein and chitobiase/ ß-hexosaminidase C terminal domain. CcGlcNAcase expression was notably increased when chitin was present. CcGlcNAcase produced N-acetyl-d-glucosamine from various lengths of N-acetyl-d-glucosamine. CcGlcNAcase bound to chitin (89%) and fungi (54.10%), whereas CcGlcNAcase exhibited a low binding ability to cellulose and xylan. CcGlcNAcase hydrolyzed fungi, yielding maximum 3.90 g/L N-acetyl-d-glucosamine. CcGlcNAcase enhanced cellulase toward fungi-infected lignocellulosic biomass, yielding 18 mg/L glucose (1.32-fold) and 1.72-fold increased total reducing sugar levels, whereas cellulase alone produced 13 mg/L glucose. Taken together, CcGlcNAcase can be utilized to enhance the degradation of fungi-infected lignocellulosic biomass and exhibits potential applications in the wood and sugar industry.

Production and characterization of melanin pigments derived from Amorphotheca resinae.

As melanin has emerged as functional pigment with cosmetic, health and food applications, the demand for the pigments is expected to increase. However, the conventional sources (e.g. mushroom, hair, and wool) of melanin production entail pigments inside the substrates which requires the costly extraction procedures, leading to inappropriate scalable production. In this study, we screened 102 of fungal isolates for their ability to produce melanin in the supernatant and selected the only Amorphotheca resinae as a promising candidate. In the peptone yeast extract glucose broth, A. resinae produced the melanin rapidly during the autolysis phase of growth, reaching up 4.5 g/L within 14 days. Structural characterization of the purified melanin from A. resinae was carried out by using elemental analysis, electron paramagnetic resonance, 13C solid-state nuclear magnetic resonance spectroscopy, and pyrolysis-gas chromatography-mass spectrometry in comparison with the standard melanins. The results indicate that the structural properties of A. resinae melanin is similar to the eumelanin which has a wide range of industrial uses. For example, the purified melanin from A. resinae has the potent antioxidant activities as a result of free radical scavenging assays. Consequently, A. resinae KUC3009 can be a promising candidate for scalable production of industrially applicable melanin.

Rapid and Accurate Quantification of Paramylon Produced from Euglena gracilis Using an ssDNA Aptamer.

The functional ingredients of microalgal biomass are receiving substantial recognition as the global demands for health supplements produced from natural sources are on the rise. Paramylon, a conglomerate of ß-1,3-glucans, is one of the major valuable sources derived from Euglena gracilis having multiple applications, thus necessitating the development of an efficient quantification method. Here, we employed a DNA aptamer to quantify the amount of paramylon produced by E. gracilis. Paramylon-specific aptamers were isolated by the systematic evolution of ligands by exponential enrichment (SELEX) process. To evaluate the potential aptamers, the binding affinity between aptamer candidates and paramylon granules was confirmed by a confocal laser scanning microscope and the dissociation constants of the selected aptamers were determined by nonlinear regression analysis. The selected DNA aptamer was successfully used for the quantification of paramylon, and the results were compared to those obtained by the standard methods. The new approach was also used for quantification of paramylon from E. gracilis cells cultured to different cell stages and physiologies. It can be concluded that the aptamer-based protocol for the measurement of paramylon proposed in this study is highly accurate and comparatively less time-consuming.

Improvement of Euglena gracilis Paramylon Production through a Cocultivation Strategy with the Indole-3-Acetic Acid-Producing Bacterium Vibrio natriegens.

We investigated the putative effects on the growth and paramylon production of Euglena gracilis of cocultivation with Vibrio natriegensE. gracilis heterotrophically cocultivated with V. natriegens displayed significant increases in biomass productivity and paramylon content. In addition, the effects of the bacterial inoculum density and the timing of inoculation on the growth of E. gracilis were examined, to determine the optimal conditions for cocultivation. With the optimal deployment of V. natriegens, biomass productivity and paramylon content were increased by more than 20% and 35%, respectively, compared to those in axenic E. gracilis cultures. Interestingly, indole-3-acetic acid biosynthesized by V. natriegens was responsible for these enhancements of E. gracilis The morphology of cocultured E. gracilis cells was assessed. Paramylon granules extracted from the cocultivation were significantly larger than those from axenic culture. Our study showed that screening for appropriate bacteria and subsequent cocultivation with E. gracilis represented an effective way to enhance biomass and metabolite production.IMPORTANCEEuglena gracilis has attracted special interest due to its ability to excessively accumulate paramylon. Paramylon is a linear ß-1,3-glucan polysaccharide that is the principal polymer for energy storage in E. gracilis The polysaccharide features high bioactive functionality in the immune system. This study explored a new method to enhance the production of paramylon by E. gracilis, through cocultivation with the indole-3-acetic acid-producing bacterium Vibrio natriegens The enhanced production was achieved indirectly with the phytohormone-producing bacteria, instead of direct application of the hormone. The knowledge obtained in this study furthers the understanding of the effects of V. natriegens on the growth and physiology of E. gracilis.

Enhancement of growth and paramylon production of Euglena gracilis by co-cultivation with Pseudoalteromonas sp. MEBiC 03485.

This study investigated the putative effects of co-cultivation of Euglena gracilis with Pseudoalteromonas sp. MEBiC 03485 on the growth of E. gracilis and its paramylon production. The strain MEBiC 03485 had beneficial effects on the growth and paramylon contents of E. gracilis. To determine the optimal conditions for co-cultivation, the effects of algal to bacterial inoculum ratios and E. gracilis growth stages were examined. Under optimal conditions, the biomass productivity and paramylon production were increased by more than 23% and 34%, respectively. These effects were attributed to the extracellular polymeric substances (EPS) from the strain MEBiC 03485. GC-MS and HPAEC were carried out to analyze the composition of EPS. It was found that the EPS consisted of rhamnose, galactose, glucose, and mannose. These results suggest a novel approach for potentially enhancing the growth of E. gracilis as well as its paramylon production, via co-culturing with the symbiotic strain MEBiC 03485.