Industrializing methanotrophs and other methylotrophic bacteria: from bioengineering to product recovery.

Microbes that use the single-carbon substrates methanol and methane offer great promise to bioindustry along with substantial environmental benefits. Methanotrophs and other methylotrophs can be engineered and optimized to produce a wide range of products, from biopolymers to biofuels and beyond. While significant limitations remain, including delivery of single-carbon feedstock to bioreactors, efficient growth, and scale-up, these challenges are being addressed and notable improvements have been rapid. Development of expression chassis, use of genome-scale and regulatory models based on omics data, improvements in bioreactor design and operation, and development of green product recovery schemes are enabling the rapid development of single-carbon bioconversion in the industrial space.

Effects of different cryopreservation parameters on the differences between trypan blue and fluorescent SYTO 13/GelRed assays.

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell-cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.

Targeted spectroscopy in the eye fundus.

Significance: The assessment of biomarkers in the eye is rapidly gaining traction for the screening, diagnosis, and monitoring of ocular and neurological diseases. Targeted ocular spectroscopy is a technology that enables concurrent imaging of the eye fundus and analysis of high-quality spectra from a targeted region within the imaged area. This provides structural, compositional, and functional information of specific regions of the eye fundus from a non-invasive approach to ocular biomarker detection. Aim: The aim of our study was to demonstrate the multimodal functionality and validation of targeted ocular spectroscopy. This was done in vitro, using a reference target and a model eye, and in vivo. Approach: Images and spectra from different regions of a reference target and a model eye were acquired and analyzed to validate the system. Targeted ocular fluorescence spectroscopy was also demonstrated with the same model. Subsequently, in vivo imaging and diffuse reflectance spectra were acquired to assess blood oxygen saturation in the optic nerve head and the parafovea of healthy subjects. Results: Tests conducted with the reference target showed accurate spectral analysis within specific areas of the imaging space. In the model eye, distinct spectral signatures were observed for the optic disc, blood vessels, the retina, and the macula, consistent with the variations in tissue composition and functions between these regions. An ocular oximetry algorithm was applied to in vivo spectra from the optic nerve head and parafovea of healthy patients, showing significant differences in blood oxygen saturation. Finally, targeted fluorescence spectral analysis was performed in vitro. Conclusions: Diffuse reflectance and fluorescence spectroscopy in specific regions of the eye fundus open the door to a whole new range of monitoring and diagnostic capabilities, from assessment of oxygenation in glaucoma and diabetic retinopathy to photo-oxidation and photodegradation in age-related macular degeneration.

Accelerating the Biodegradation of Poly(lactic acid) through the Inclusion of Plant Fibers: A Review of Recent Advances.

As the global demand for plastics continues to grow, plastic waste is accumulating at an alarming rate with negative effects on the natural environment. The industrially compostable biopolymer poly(lactic acid) (PLA) is therefore being adopted for use in many applications, but the degradation of this material is slow under many end-of-life conditions. This Perspective explores the feasibility of accelerating the degradation of PLA through the formation of PLA-plant fiber composites. Topics include: (a) key properties of PLA, plant-based fibers, and biocomposites; (b) mechanisms of both hydrolytic degradation and biodegradation of PLA-fiber composites; (c) end-of-life degradation of PLA and PLA-plant fiber composites in aerobic and anaerobic conditions, relevant to compost, soil and seawater (aerobic), and landfills (anaerobic); and (d) sustainability and environmental impact of PLA and PLA-plant fiber composites, as evaluated using life cycle assessment. Additional degradation modes, including thermal and photodegradation, which are relevant during processing and use, have been omitted for clarity, as have other types of PLA biocomposites. Multiple studies have shown that the addition of some types of plant fibers to PLA (to form PLA biocomposites) accelerates both water transport in the material and hydrolysis, presenting a possible avenue for improving the end-of-life degradation of these materials. To facilitate the continued development of materials with enhanced biodegradability, we identify a need to implement testing protocols that can distinguish between different degradation mechanisms.

Sensitivity of visible range multi-wavelength algorithms for retinal tissue oximetry to acquisition parameters.

This study examined the sensitivity of broadband spectroscopy algorithms for retinal tissue oximetry to spectral acquisition parameters. Monte Carlo simulations were conducted on a 4-layer retinal model to assess the impact of various parameters. The optimal spectral range for accurate measurements was determined to be 530 nm to 585 nm. Decreased spectral resolution below 4 nm significantly reduced accuracy. Using an acquisition area larger than the blood vessel resulted in an underestimation of oxygen saturation, especially for high values. A threshold was observed where increased light intensity had no significant impact on measurement variability. The study highlights the importance of informed parameter selection for accurately assessing retinal microcapillary oxygenation and studying local hemodynamics.

Adapted feeding strategies in fed-batch fermentation improve sugar delivery and ethanol productivity.

Bioethanol is a renewable fuel widely used in road transportation and is generally regarded as a clean energy source. Although fermentation is one of the major processes in bioethanol production, studies on improving its efficiency through operational design are limited, especially compared to other steps (pretreatment and hydrolysis/saccharification). In this study, two adapted feeding strategies, in which feed medium addition (sugar delivery) was adjusted to increase the supply of fermentable sugar, were developed to improve ethanol productivity in 5-L fed-batch fermentation by Saccharomyces cerevisiae. Specifically, a linear adapted feeding strategy was established based on changes in cell biomass, and an exponential adapted feeding strategy was developed based on cell biomass accumulation. By implementing these two feeding strategies, the overall ethanol productivity reached 0.88±0.04 and 0.87±0.06 g/L/h, respectively. This corresponded to ~20% increases in ethanol productivity compared to fixed pulsed feeding operations. Additionally, there was no residual glucose at the end of fermentation, and final ethanol content reached 95±3 g/L under the linear adapted operation and 104±3 g/L under the exponential adapted feeding strategy. No statistical difference was observed in the overall ethanol yield (ethanol-to-sugar ratio) between fixed and adapted feeding strategies (~91%). These results demonstrate that sugar delivery controlled by adapted feeding strategies was more efficient than fixed feeding operations, leading to higher ethanol productivity. Overall, this study provides novel adapted feeding strategies to improve sugar delivery and ethanol productivity. Integration into the current practices of the ethanol industry could improve productivity and reduce production costs of fermentation processes.

Methanol bioconversion in Methylotuvimicrobium buryatense 5GB1C through self-cycling fermentation.

Methanol is an abundant and low-cost next-generation carbon source. While many species of methanotrophic bacteria can convert methanol into valuable bioproducts in bioreactors, Methylotuvimicrobium buryatense 5GB1C stands out as one of the most promising strains for industrialization. It has a short doubling time compared to most methanotrophs, remarkable resilience against contamination, and a suite of tools enabling genetic engineering. When approaching industrial applications, growing M. buryatense 5GB1C on methanol using common batch reactor operation has important limitations; for example methanol toxicity leads to mediocre biomass productivity. Advanced bioreactor operation strategies, such as fed-batch and self-cycling fermentation, have the potential to greatly improve the industrial prospects of methanotrophs growing on methanol. Herein, implementation of fed-batch operation led to a 26-fold increase in biomass density, while two different self-cycling fermentation (SCF) strategies led to 3-fold and 10-fold increases in volumetric biomass productivity. Interestingly, while synchronization is a typical trait of microbial populations undergoing SCF, M. buryatense 5GB1C cultures growing under this mode of operation led to stable, reproducible cycles but no significant synchronization.

The influence of self-cycling fermentation long- and short-cycle schemes on Saccharomyces cerevisiae and Escherichia coli.

Self-cycling fermentation (SCF), a cyclic process in which cells, on average, divide once per cycle, has been shown to lead to whole-culture synchronization and improvements in productivity during bioconversion. Previous studies have shown that the completion of synchronized cell replication sometimes occurs simultaneously with depletion of the limiting nutrient. However, cases in which the end of cell doubling occurred before limiting nutrient exhaustion were also observed. In order to better understand the impact of these patterns on bioprocessing, we investigated the growth of Saccharomyces cerevisiae and Escherichia coli in long- and short-cycle SCF strategies. Three characteristic events were identified during SCF cycles: (1) an optimum in control parameters, (2) the time of completion of synchronized cell division, and (3) the depletion or plateau of the limiting nutrient. Results from this study and literature led to the identification of three potential trends in SCF cycles: (A) co-occurrence of the three key events, (B) cell replication ending prior to the co-occurrence of the other two events, and (C) depletion or plateau of the limiting nutrient occurring later than the co-occurrence of the other two events. Based on these observations, microbial physiological differences were analyzed and a novel definition for SCF is proposed.

Monte-Carlo simulation and tissue-phantom model for validation of ocular oximetry.

Ocular oximetry, in which blood oxygen saturation is evaluated in retinal tissues, is a promising technique for the prevention, diagnosis and management of many diseases and conditions. However, the development of new tools for evaluating oxygen saturation in the eye fundus has often been limited by the lack of reference tools or techniques for such measurements. In this study, we describe a two-step validation method. The impact of scattering, blood volume fraction and lens yellowing on the oximetry model is investigated using a tissue phantom, while a Monte Carlo model of the light propagation in the eye fundus is used to study the effect of the fundus layered-structure. With this method, we were able to assess the performance of an ocular oximetry technique in the presence of confounding factors and to quantify the impact of the choroidal circulation on the accuracy of the measurements. The presented strategy will be useful to anyone involved in studies based on the eye fundus diffuse reflectance.

Use of uncoated magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and real-time-PCR.

Uncoated tosyl-activated magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Centrifugation and uncoated magnetic beads recovered more than 99% and 93%, respectively, of 1.68 × 107 CFU/mL, 1.68 × 106 CFU/mL and 1.68 × 105 CFU/mL M. smegmatis cells resuspended in phosphate buffer saline. The use of magnetic beads was more efficient to concentrate cells from 1.68 × 104 CFU/mL of M. smegmatis than centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p < 0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of greater recovery. Magnetic separation of MAP cells from milk, urine, and feces specimens was demonstrated by detection of F57 and IS900 sequences. Beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 × 105 and 2 × 103 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of tuberculosis and Johne's disease.