Glycerol-driven adaptive evolution for the production of low-molecular-weight Welan gum: Characterization and activity evaluation.

Through adaptive laboratory evolution (ALE) of Sphingomonas sp. ATCC 31555, fermentation for production of low-molecular-weight welan gum (LMW-WG) was performed using glycerol as sole carbon source. During ALE, GPC-MALS analysis revealed a gradual decrease in WG molecular weight with the increase of adaptation cycles, accompanied by changes in solution conformation. LMW-WG was purified and structurally analyzed using GPC-MALS, monosaccharide composition analysis, infrared spectroscopy, NMR analysis, atomic force microscopy, and scanning electron microscopy. Subsequently, LMW-WG obtains hydration, transparency, antioxidant activity, and rheological properties. Finally, an in vitro simulation colon reactor was used to evaluate potential prebiotic properties of LMW-WG as dietary fiber. Compared with WG produced using sucrose as substrate, LMW-WG exhibited a fourfold reduction in molecular weight while maintaining moderate viscosity. Structurally, L-Rha nearly completely replaced L-Man. Furthermore, LMW-WG demonstrated excellent hydration, antioxidant activity, and high transparency. It also exhibited resistance to saliva and gastrointestinal digestion, showcasing a favorable colonization effect on Bifidobacterium, making it a promising symbiotic agent.

Regulation of dye-decolorizing peroxidase gene expression in Pleurotus ostreatus grown on glycerol as the carbon source.

Dye-decolorizing peroxidases (DyPs) (E.C. 1.11.1.19) are heme peroxidases that catalyze oxygen transfer reactions similarly to oxygenases. DyPs utilize hydrogen peroxide (H2O2) both as an electron acceptor co-substrate and as an electron donor when oxidized to their respective radicals. The production of both DyPs and lignin-modifying enzymes (LMEs) is regulated by the carbon source, although less readily metabolizable carbon sources do improve LME production. The present study analyzed the effect of glycerol on Pleurotus ostreatus growth, total DyP activity, and the expression of three Pleos-dyp genes (Pleos-dyp1, Pleos-dyp2 and Pleos-dyp4), via real-time RT-qPCR, monitoring the time course of P. ostreatus cultures supplemented with either glycerol or glucose and Acetyl Yellow G (AYG) dye. The results obtained indicate that glycerol negatively affects P. ostreatus growth, giving a biomass production of 5.31 and 5.62 g/L with respective growth rates (micra; m) of 0.027 and 0.023 h-1 for fermentations in the absence and presence of AYG dye. In contrast, respective biomass production levels of 7.09 and 7.20 g/L and growth rates (µ) of 0.033 and 0.047 h-1 were observed in equivalent control fermentations conducted with glucose in the absence and presence of AYG dye. Higher DyP activity levels, 4,043 and 4,902 IU/L, were obtained for fermentations conducted on glycerol, equivalent to 2.6-fold and 3.16-fold higher than the activity observed when glucose is used as the carbon source. The differential regulation of the DyP-encoding genes in P. ostreatus were explored, evaluating the carbon source, the growth phase, and the influence of the dye. The global analysis of the expression patterns throughout the fermentation showed the up- and down- regulation of the three Pleos-dyp genes evaluated. The highest induction observed for the control media was that found for the Pleos-dyp1 gene, which is equivalent to an 11.1-fold increase in relative expression (log2) during the stationary phase of the culture (360 h), and for the glucose/AYG media was Pleos-dyp-4 with 8.28-fold increase after 168 h. In addition, glycerol preferentially induced the Pleos-dyp1 and Pleos-dyp2 genes, leading to respective 11.61 and 4.28-fold increases after 144 h. After 360 and 504 h of culture, 12.86 and 4.02-fold increases were observed in the induction levels presented by Pleos-dyp1 and Pleos-dyp2, respectively, in the presence of AYG. When transcription levels were referred to those found in the control media, adding AYG led to up-regulation of the three dyp genes throughout the fermentation. Contrary to the fermentation with glycerol, where up- and down-regulation was observed. The present study is the first report describing the effect of a less-metabolizable carbon source, such as glycerol, on the differential expression of DyP-encoding genes and their corresponding activity.

Exploring the impact of magnetic fields on biomass production efficiency under aerobic and anaerobic batch fermentation of Saccharomyces cerevisiae.

In this work, the effect of moderate electromagnetic fields (2.5, 10, and 15 mT) was studied using an immersed coil inserted directly into a bioreactor on batch cultivation of yeast under both aerobic and anaerobic conditions. Throughout the cultivation, parameters, including CO2 levels, O2 saturation, nitrogen consumption, glucose uptake, ethanol production, and yeast growth (using OD 600 measurements at 1-h intervals), were analysed. The results showed that 10 and 15 mT magnetic fields not only statistically significantly boosted and sped up biomass production (by 38-70%), but also accelerated overall metabolism, accelerating glucose, oxygen, and nitrogen consumption, by 1-2 h. The carbon balance analysis revealed an acceleration in ethanol and glycerol production, albeit with final concentrations by 22-28% lower, with a more pronounced effect in aerobic cultivation. These findings suggest that magnetic fields shift the metabolic balance toward biomass formation rather than ethanol production, showcasing their potential to modulate yeast metabolism. Considering coil heating, opting for the 10 mT magnetic field is preferable due to its lower heat generation. In these terms, we propose that magnetic field can be used as novel tool to increase biomass yield and accelerate yeast metabolism.

Bioproduction of yeast single cell oil with acute oral toxicity study intended for edible oil application.

Human nutrition and health rely on edible oils. Global demand for edible oils is expanding, necessitating the discovery of new natural oil sources subjected to adequate quality and safety evaluation. However, in contrast to other agricultural products, India's edible oil supply is surprisingly dependent on imports. The microbial oil is generated by fermentation of oleaginous yeast Rhodotorula mucilaginosa IIPL32 MTCC 25056 using biodiesel plant byproduct crude glycerol as a fermentable carbon source. Enriched with monounsaturated fatty acid, nutritional indices mapping based on the fatty acid composition of the yeast SCO, suggested its plausible use as an edible oil blend. In the present study, acute toxicity evaluation of the yeast SCO in C57BL/6 mice has been performed by randomly dividing the animals into 5 groups with 50, 300, 2000, and 5000 mg/Kg yeast SCO dosage, respectively, and predicted the median lethal dose (LD50). Detailed blood biochemistry and kidney and liver histopathology analyses were also reported. The functions of the liver enzymes were also evaluated to check and confirm the anticipated toxicity. To determine cell viability and in vitro biocompatibility, the 3T3-L1 cell line and haemolysis tests were performed. The results suggested the plausible use of yeast SCO as an edible oil blend due to its non-toxic nature in mice models.

Improving the production of recombinant L-Asparaginase-II in Escherichia coli by co-expressing catabolite repressor activator (cra) gene.

Identification of a single genetic target for microbial strain improvement is difficult due to the complexity of the genetic regulatory network. Hence, a more practical approach is to identify bottlenecks in the regulatory networks that control critical metabolic pathways. The present work focuses on enhancing cellular physiology by increasing the metabolic flux through the central carbon metabolic pathway. Global regulator cra (catabolite repressor activator), a DNA-binding transcriptional dual regulator was selected for the study as it controls the expression of a large number of operons that modulate central carbon metabolism. To upregulate the activity of central carbon metabolism, the cra gene was co-expressed using a plasmid-based system. Co-expression of cra led to a 17% increase in the production of model recombinant protein L-Asparaginase-II. A pulse addition of 0.36% of glycerol every two hours post-induction, further increased the production of L-Asparaginase-II by 35% as compared to the control strain expressing only recombinant protein. This work exemplifies that upregulating the activity of central carbon metabolism by tuning the expression of regulatory genes like cra can relieve the host from cellular stress and thereby promote the growth as well as expression of recombinant hosts.

Studying intramuscular fat deposition and muscle regeneration: insights from a comparative analysis of mouse strains, injury models, and sex differences.

Intramuscular fat (IMAT) infiltration, pathological adipose tissue that accumulates between muscle fibers, is a shared hallmark in a diverse set of diseases including muscular dystrophies and diabetes, spinal cord and rotator cuff injuries, as well as sarcopenia. While the mouse has been an invaluable preclinical model to study skeletal muscle diseases, they are also resistant to IMAT formation. To better understand this pathological feature, an adequate pre-clinical model that recapitulates human disease is necessary. To address this gap, we conducted a comprehensive in-depth comparison between three widely used mouse strains: C57BL/6J, 129S1/SvlmJ and CD1. We evaluated the impact of strain, sex and injury type on IMAT formation, myofiber regeneration and fibrosis. We confirm and extend previous findings that a Glycerol (GLY) injury causes significantly more IMAT and fibrosis compared to Cardiotoxin (CTX). Additionally, females form more IMAT than males after a GLY injury, independent of strain. Of all strains, C57BL/6J mice, both females and males, are the most resistant to IMAT formation. In regard to injury-induced fibrosis, we found that the 129S strain formed the least amount of scar tissue. Surprisingly, C57BL/6J of both sexes demonstrated complete myofiber regeneration, while both CD1 and 129S1/SvlmJ strains still displayed smaller myofibers 21 days post injury. In addition, our data indicate that myofiber regeneration is negatively correlated with IMAT and fibrosis. Combined, our results demonstrate that careful consideration and exploration are needed to determine which injury type, mouse model/strain and sex to utilize as preclinical model especially for modeling IMAT formation.

Influence of fumarate on interspecies electron transfer and the metabolic shift induced in Clostridium pasteurianum by Geobacter sulfurreducens.

AIMS: In previous studies, it was demonstrated that co-culturing Clostridium pasteurianum and Geobacter sulfurreducens triggers a metabolic shift in the former during glycerol fermentation. This shift, attributed to interspecies electron transfer and the exchange of other molecules, enhances the production of 1,3-propanediol at the expense of the butanol pathway. The aim of this investigation is to examine the impact of fumarate, a soluble compound usually used as an electron acceptor for G. sulfurreducens, in the metabolic shift previously described in C. pasteurianum. METHODS AND RESULTS: Experiments were conducted by adding along with glycerol, acetate, and different quantities of fumarate in co-cultures of G. sulfurreducens and C. pasteurianum. A metabolic shift was exhibited in all the co-culture conditions. This shift was more pronounced at higher fumarate concentrations. Additionally, we observed G. sulfurreducens growing even in the absence of fumarate and utilizing small amounts of this compound as an electron donor rather than an electron acceptor in the co-cultures with high fumarate addition. CONCLUSIONS: This study provided evidence that interspecies electron transfer continues to occur in the presence of a soluble electron acceptor, and the metabolic shift can be enhanced by promoting the growth of G. sulfurreducens.

Large enrichments in fatty acid 2H/1H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae.

Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2εfattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2εfattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550‰ 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200‰. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2εfattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2εfattyacid/water variations (<30‰) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.

Effects of salinity on glycerol conversion and biological phosphorus removal by aerobic granular sludge.

Industrial wastewater often has high levels of salt, either due to seawater or e.g. sodium chloride (NaCl) usage in the processing. Previous work indicated that aerobic granular sludge (AGS) is differently affected by seawater or saline water at similar osmotic strength. Here we investigate in more detail the impact of NaCl concentrations and seawater on the granulation and conversion processes for AGS wastewater treatment. Glycerol was used as the carbon source since it is regularly present in industrial wastewaters, and to allow the evaluation of microbial interactions that better reflect real conditions. Long-term experiments were performed to evaluate and compare the effect of salinity on granulation, anaerobic conversions, phosphate removal, and the microbial community. Smooth and stable granules as well as enhanced biological phosphorus removal (EBPR) were achieved up to 20 g/L NaCl or when using seawater. However, at NaCl levels comparable to seawater strength (30 g/L) incomplete anaerobic glycerol uptake and aerobic phosphate uptake were observed, the effluent turbidity increased, and filamentous granules began to appear. The latter is likely due to the direct aerobic growth on the leftover substrate after the anaerobic feeding period. In all reactor conditions, except the reactor with 30 g/L NaCl, Ca. Accumulibacter was the dominant microorganism. In the reactor with 30 g/L NaCl, the relative abundance of Ca. Accumulibacter decreased to ≤1 % and an increase in the genus Zoogloea was observed. Throughout all reactor conditions, Tessaracoccus and Micropruina, both actinobacteria, were present which were likely responsible for the anaerobic conversion of glycerol into volatile fatty acids. None of the glycerol metabolizing proteins were detected in Ca. Accumulibacter which supports previous findings that glycerol can not be directly utilized by Ca. Accumulibacter. The proteome profile of the dominant taxa was analysed and the results are further discussed. The exposure of salt-adapted biomass to hypo-osmotic conditions led to significant trehalose and PO43--P release which can be related to the osmoregulation of the cells. Overall, this study provides insights into the effect of salt on the operation and stability of the EBPR and AGS processes. The findings suggest that maintaining a balanced cation ratio is likely to be more important for the operational stability of EBPR and AGS systems than absolute salt concentrations.

Up-regulation of Retrograde Response in yeast increases glycerol and reduces ethanol during wine fermentation.

Nutrient signaling pathways play a pivotal role in regulating the balance among metabolism, growth and stress response depending on the available food supply. They are key factors for the biotechnological success of the yeast Saccharomyces cerevisiae during food-producing fermentations. One such pathway is Retrograde Response, which controls the alpha-ketoglutarate supply required for the synthesis of amino acids like glutamate and lysine. Repressor MKS1 is linked with the TORC1 complex and negatively regulates this pathway. Deleting MKS1 from a variety of industrial strains causes glycerol to increase during winemaking, brewing and baking. This increase is accompanied by a reduction in ethanol production during grape juice fermentation in four commercial wine strains. Interestingly, this does not lead volatile acidity to increase because acetic acid levels actually lower. Aeration during winemaking usually increases acetic acid levels, but this effect reduces in the MKS1 mutant. Despite the improvement in the metabolites of oenological interest, it comes at a cost given that the mutant shows slower fermentation kinetics when grown in grape juice, malt and laboratory media and using glucose, sucrose and maltose as carbon sources. The deletion of RTG2, an activator of Retrograde Response that acts as an antagonist of MKS1, also results in a defect in wine fermentation speed. These findings suggest that the deregulation of this pathway causes a fitness defect. Therefore, manipulating repressor MKS1 is a promising approach to modulate yeast metabolism and to produce low-ethanol drinks.

Mixotrophic culture enhances fucoxanthin production in the haptophyte Pavlova gyrans.

Fucoxanthin is a versatile substance in the food and pharmaceutical industries owing to its excellent antioxidant and anti-obesity properties. Several microalgae, including the haptophyte Pavlova spp., can produce fucoxanthin and are potential industrial fucoxanthin producers, as they lack rigid cell walls, which facilitates fucoxanthin extraction. However, the commercial application of Pavlova spp. is limited owing to insufficient biomass production. In this study, we aimed to develop a mixotrophic cultivation method to increase biomass and fucoxanthin production in Pavlova gyrans OPMS 30543X. The effects of culturing OPMS 30543X with different organic carbon sources, glycerol concentrations, mixed-nutrient conditions, and light intensities on the consumption of organic carbon sources, biomass production, and fucoxanthin accumulation were analyzed. Several organic carbon sources, such as glycerol, glucose, sucrose, and acetate, were examined, revealing that glycerol was well-consumed by the microalgae. Biomass and fucoxanthin production by OPMS 30543X increased in the presence of 10 mM glycerol compared to that observed without glycerol. Metabolomic analysis revealed higher levels of the metabolites related to the glycolytic, Calvin-Benson-Bassham, and tricarboxylic acid cycles under mixotrophic conditions than under autotrophic conditions. Cultures grown under mixotrophic conditions with a light intensity of 100 µmol photons m-2 s-1 produced more fucoxanthin than autotrophic cultures. Notably, the amount of fucoxanthin produced (18.9 mg/L) was the highest reported thus far for Pavlova species. In conclusion, the use of mixotrophic culture is a promising strategy for increasing fucoxanthin production in Pavlova species. KEY POINTS: • Glycerol enhances biomass and fucoxanthin production in Pavlova gyrans • Metabolite levels increase under mixotrophic conditions • Mixotrophic conditions and medium-light intensity are appropriate for P. gyrans.

Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered ß-fructofuranosidase enzyme in Pichia pastoris.

The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

Towards controlled and simple design of non-enzymatic amperometric sensor for glycerol determination in yeast fermentation medium.

Glycerol is a widely used signaling bioanalyte in biotechnology. Glycerol can serve as a substrate or product of many metabolic processes in cells. Therefore, quantification of glycerol in fermentation samples with inexpensive, reliable, and rapid sensing systems is of great importance. In this work, an amperometric assay based on one-step designed electroplated functional Pd layers with controlled design was proposed for a rapid and selective measurement of glycerol in yeast fermentation medium. A novel assay utilizing electroplated Pd-sensing layers allows the quantification of glycerol in yeast fermentation medium in the presence of interfering species with RSD below 3% and recoveries ranged from 99 to 103%. The assay requires minimal sample preparation, viz. adjusting of sample pH to 12. The time taken to complete the electrochemical analysis was 3 min. Remarkably, during investigations, it was revealed that sensitivity and selectivity of glycerol determination on Pd sensors were significantly affected by its adsorption and did not depend on the surface structure of sensing layers. This study is expected to contribute to both fundamental and practical research fields related to a preliminary choice of functional sensing layers for specific biotechnology and life science applications in the future.

Mitochondrial membrane transporters as attractive targets for the fermentative production of succinic acid from glycerol in Saccharomyces cerevisiae.

Previously, we reported an engineered Saccharomyces cerevisiae CEN.PK113-1A derivative able to produce succinic acid (SA) from glycerol with net CO2 fixation. Apart from an engineered glycerol utilization pathway that generates NADH, the strain was equipped with the NADH-dependent reductive branch of the TCA cycle (rTCA) and a heterologous SA exporter. However, the results indicated that a significant amount of carbon still entered the CO2-releasing oxidative TCA cycle. The current study aimed to tune down the flux through the oxidative TCA cycle by targeting the mitochondrial uptake of pyruvate and cytosolic intermediates of the rTCA pathway, as well as the succinate dehydrogenase complex. Thus, we tested the effects of deletions of MPC1, MPC3, OAC1, DIC1, SFC1, and SDH1 on SA production. The highest improvement was achieved by the combined deletion of MPC3 and SDH1. The respective strain produced up to 45.5 g/L of SA, reached a maximum SA yield of 0.66 gSA/gglycerol, and accumulated the lowest amounts of byproducts when cultivated in shake-flasks. Based on the obtained data, we consider a further reduction of mitochondrial import of pyruvate and rTCA intermediates highly attractive. Moreover, the approaches presented in the current study might also be valuable for improving SA production when sugars (instead of glycerol) are the source of carbon.

Insights into intraspecific diversity of central carbon metabolites in Saccharomyces cerevisiae during wine fermentation.

Saccharomyces cerevisiae is a major actor in winemaking that converts sugars from the grape must into ethanol and CO2 with outstanding efficiency. Primary metabolites produced during fermentation have a great importance in wine. While ethanol content contributes to the overall profile, other metabolites like glycerol, succinate, acetate or lactate also have significant impacts, even when present in lower concentrations. S. cerevisiae is known for its great genetic diversity that is related to its natural or technological environment. However, the variation range of metabolic diversity which can be exploited to enhance wine quality depends on the pathway considered. Our experiment assessed the diversity of primary metabolites production in a set of 51 S. cerevisiae strains from various genetic backgrounds. Results pointed out great yield differences depending on the metabolite considered, with ethanol having the lowest variation. A negative correlation between ethanol and glycerol was observed, confirming glycerol synthesis as a suitable lever to reduce ethanol yield. Genetic groups were linked to specific yields, such as the wine group and high α-ketoglutarate and low acetate yields. This research highlights the potential of using natural yeast diversity in winemaking. It also provides a detailed data set on production of well known (ethanol, glycerol, acetate) or little-known (lactate) primary metabolites.

Continuous catalytic production of 1,3-dihydroxyacetone: Sustainable approach combining perfusion cultures and immobilized cells.

Currently, the predominant method for the industrial production of 1,3-dihydroxyacetone (DHA) from glycerol involves fed-batch fermentation. However, previous research has revealed that in the biocatalytic synthesis of DHA from glycerol, when the DHA concentration exceeded 50 g·L-1, it significantly inhibited microbial growth and metabolism, posing a challenge in maintaining prolonged and efficient catalytic production of DHA. In this study, a new integrated continuous production and synchronous separation (ICSS) system was constructed using hollow fiber columns and perfusion culture technology. Additionally, a cell reactivation technique was implemented to extend the biocatalytic ability of cells. Compared with fed-batch fermentation, the ICSS system operated for 360 h, yielding a total DHA of 1237.8 ± 15.8 g. The glycerol conversion rate reached 97.7 %, with a productivity of 3.44 g·L-1·h-1, representing 485.0 % increase in DHA production. ICSS system exhibited strong operational characteristics and excellent performance, indicating significant potential for applications in industrial bioprocesses.

Unveiling the ecological resilience and industrial potential of Skeletonema marinoi through mixotrophic cultivation in Nordic winter condition.

Mixotrophy, the concurrent use of inorganic and organic carbon in the presence of light for microalgal growth, holds ecological and industrial significance. However, it is poorly explored in diatoms, especially in ecologically relevant species like Skeletonema marinoi. This study strategically employed mixotrophic metabolism to optimize the growth of a strain of Skeletonema marinoi (Sm142), which was found potentially important for biomass production on the west coast of Sweden in winter conditions. The aim of this study was to discern the most effective organic carbon sources by closely monitoring microalgal growth through the assessment of optical density, chlorophyll a fluorescence, and biomass concentration. The impact of various carbon sources on the physiology of Sm142 was investigated using photosynthetic and respiratory parameters. The findings revealed that glycerol exhibited the highest potential for enhancing the biomass concentration of Sm142 in a multi-cultivator under the specified experimental conditions, thanks to the increase in respiration activity. Furthermore, the stimulatory effect of glycerol was confirmed at a larger scale using environmental photobioreactors simulating the winter conditions on the west coast of Sweden; it was found comparable to the stimulation by CO2-enriched air versus normal air. These results were the first evidence of the ability of Skeletonema marinoi to perform mixotrophic metabolism during the winter and could explain the ecological success of this diatom on the Swedish west coast. These findings also highlight the importance of both organic and inorganic carbon sources for enhancing biomass productivity in harsh winter conditions.

Serial adaptive laboratory evolution enhances mixed carbon metabolic capacity of Escherichia coli.

Microbes have inherent capacities for utilizing various carbon sources, however they often exhibit sub-par fitness due to low metabolic efficiency. To test whether a bacterial strain can optimally utilize multiple carbon sources, Escherichia coli was serially evolved in L-lactate and glycerol. This yielded two end-point strains that evolved first in L-lactate then in glycerol, and vice versa. The end-point strains displayed a universal growth advantage on single and a mixture of adaptive carbon sources, enabled by a concerted action of carbon source-specialists and generalist mutants. The combination of just four variants of glpK, ppsA, ydcI, and rph-pyrE, accounted for more than 80% of end-point strain fitness. In addition, machine learning analysis revealed a coordinated activity of transcriptional regulators imparting condition-specific regulation of gene expression. The effectiveness of the serial adaptive laboratory evolution (ALE) scheme in bioproduction applications was assessed under single and mixed-carbon culture conditions, in which serial ALE strain exhibited superior productivity of acetoin compared to ancestral strains. Together, systems-level analysis elucidated the molecular basis of serial evolution, which hold potential utility in bioproduction applications.

Anaerobic co-metabolic biodegradation of pharmaceuticals and personal care products driven by glycerol fermentation.

Anaerobic digestion in two sequential phases, acidogenesis and methanogenesis, has been shown to be beneficial for enhancing the biomethane generation from wastewater. In this work, the application of glycerol (GOH) as a fermentation co-substrate during the wastewater treatment was evaluated on the biodegradation of different pharmaceuticals and personal care products (PPCPs). GOH co-digestion during acidogenesis led to a significant increase in the biodegradation of acetaminophen (from 78 to 89%), ciprofloxacin (from 25 to 46%), naproxen (from 73 to 86%), diclofenac (from 36 to 48%), ibuprofen (from 65 to 88%), metoprolol (from 45 to 59%), methylparaben (from 64 to 78%) and propylparaben (from 68 to 74%). The heterotrophic co-metabolism of PPCPs driven by glycerol was confirmed by the biodegradation kinetics, in which kbio (biodegradation kinetics constant) values increased from 0.18 to 2.11 to 0.27-3.60 L g-1-VSS d-1, for the operational phases without and with GOH, respectively. The assessment of metabolic pathways in each phase revealed that the prevalence of aromatic compounds degradation, metabolism of xenobiotics by cytochrome P450, and benzoate degradation routes during acidogenesis are key factors for the enzymatic mechanisms linked to the PPCPs co-metabolism. The phase separation of anaerobic digestion was effective in the PPCPs biodegradation, and the co-fermentation of glycerol provided an increase in the generation potential of biomethane in the system (energetic potential of 5.0 and 6.3 kJ g-1-CODremoved, without and with GOH, respectively). This study showed evidence that glycerol co-fermentation can exert a synergistic effect on the PPCPs removal during anaerobic digestion mediated by heterotrophic co-metabolism.

[Enhancing the glycerol utilization of engineered yeast increases its bisabolene production].

Bisabolene is a compound commonly found in essential oils of various plants. It has a broad application in sectors such as chemical, pharmaceutical, and health-care products. This study focuses on modifying the glycerol metabolism pathway to obtain a high bisabolene-producing strain of Saccharomyces cerevisiae. To achieve this, the glycerol transporter gene PtFPS2 from Pachysolen tannophilus and the glycerol dehydrogenase gene Opgdh from Ogataea parapolymorpha were overexpressed in engineered yeast YS036, which was equipped with a GAL promoters-enhanced mevalonic acid pathway. Additionally, the glucose-inhibiting transcription factor MIG1 was knocked out to reduce glucose inhibition. The results showed that the GAL promoter transcription levels of the recombinant yeast strains increased, and the co-utilization of sucrose and glycerol was further improved in MIG1-knockout strain. Moreover, the maximum yield of bisabolene in shaking flask fermentation increased to 866.7 mg/L, an 82.2% increase compared to that of the original strain. By modifying the metabolic pathway of carbon sources, the yield of bisabolene was considerably improved. This study offers an effective strategy for enhancing the yield of terpene compounds in engineered yeast.