Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures

Background: The E. coli lac operon and its components have been studied for decades, and lac-derived systems are widely used for recombinant protein production. However, lac operon dynamics and induction behavior remain the paradigm of gene regulation. Recently, an HPLC-MS-based method to quantify IPTG in the medium and inside the biomass has been established, and this tool may be useful to uncover the lack of knowledge and allow optimization of biotechnological processes. Results: The results obtained from the study of IPTG distribution profiles in fed-batch, high cell density cultures allowed discrimination between two different depletion patterns of an inducer from the medium to the biomass in E. coli-expressing rhamnulose-1-phosphate aldolase (RhuA). Moreover, we could demonstrate that active transport mediates the uptake of this gratuitous inducer. Additionally, we could study the induction behaviors of this expression system by taking into account the biomass concentration at the induction time. Conclusions: In the bistable range, partial induction occurred, which led to intermediate levels of RhuA activity. There was a direct relationship between the initial inducer concentrations and the initial inducer transport rate together with the specific activity. A majority of the inducer remains in the medium to reach equilibrium with the intracellular level. The intracellular inducer accumulation was a further evidence of bistability of the lac operon

Comparison between two reactors using Trametes versicolor for agricultural wastewater treatment under non-sterile condition in sequencing batch mode

Altres ajuts: Acord transformatiu CRUE-CSICAgricultural wastewater is a major source of herbicides, which pose environmental and health concerns owing to their substantial use and poor elimination rate in conventional wastewater treatment plants. White-rot fungi are versatile in degrading xenobiotics; however, the key problem encountered with their application in actual scenarios is competition with indigenous microorganisms, mainly bacteria. To address this barrier, two different strategies were implemented in the present study. One strategy was to set up a trickle bed with Trametes versicolor immobilized on pine wood, and another strategy was to employ a T. versicolor-pelleted, fluidized-bed reactor to remove diuron and bentazon from actual wastewater under non-sterile conditions. The residence time in the trickle bed was estimated using three methodologies. With 10 batches of a 3-day cycle operation, although the trickle-bed reactor possessed a shorter contact time (8.5 h per cycle) and lower laccase activity compared with those of the fluidized-bed reactor, it demonstrated a higher removal yield and lower bacterial counts. In addition, the utilization of pine wood as a carrier obviously reduced the cost since no additional nutrients were required. Hence, after evaluating all advantages and limitations of both bioreactors, for the purpose of treating over the long term and scaling up, a trickle-bed reactor is the preferred choice

Ús de fongs per a la degradació de filtres solars

Els compostos que trobem en cremes solars o d'altres productes i que s'utilitzen per reduir l'efecte de les radiacions ultraviolades en el nostre cos s'alliberen cada vegada més al medi ambient, sense que els procediments habituals per eliminar compostos no desitjats, com les depuradores, estiguin encara preparats per eliminar-los. Aquesta investigació, duta a terme al Departament d'Enginyeria Química de la UAB en un projecte en col·laboració amb l'IDAEA-CSIC, ha utilitzat fongs ligninolítics per eliminar aquests productes. La investigació conclou que aquests fongs són prometedors per a l'eliminació d'aquests nous contaminats però que encara som lluny de la seva implementació definitiva.Los compuestos que podemos encontrar en cremas solares o de otros productos y que se utilizan para reducir el efecto de las radiaciones ultravioletas en nuestro cuerpo se liberan cada vez más al medio ambiente, sin que los procedimientos habituales para eliminar compuestos no deseados, como las depuradoras, estén aún preparados para eliminarlos. Esta investigación, llevada a cabo en el Departamento de Ingeniería Química de la UAB en un proyecto en colaboración con el IDAEA-CSIC, ha utilizado hongos ligninolíticos para eliminar estos productos. La investigación concluye que estos hongos son prometedores para la eliminación de estos nuevos contaminantes pero que todavía estamos lejos de su implementación definitiva.The compounds found in sunscreens and other products used to reduce the effect of ultraviolet radiations in our body are released into the environment, without the usual procedures to remove unwanted compounds such as treatment facilities, are prepared to remove them. This research, carried out at the Department of Chemical Engineering at UAB in collaboration with the IDAEA-CSIC, has used ligninolytic fungi to remove these products. This research concludes that these fungi are promising for the elimination of these new pollutants but we are still far from its implementation

Can white-rot fungi be a real wastewater treatment alternative for organic micropollutants removal? A review

Micropollutants are a diverse group of compounds that are detected at trace concentrations and may have a negative effect on the environment and/or human health. Most of them are unregulated contaminants, although they have raised a concern in the scientific and global community and future regulation might be written in the near future. Several approaches have been tested to remove micropollutants from wastewater streams. In this manuscript, a focus is placed in reactor biological treatments that use white-rot fungi. A critical review of white-rot fungal-based technologies for micropollutant removal from wastewater has been conducted, several capabilities and limitations of such approaches have been identified and a range of solutions to overcome most of the limitations have been reviewed and/or proposed. Overall, this review argues that white-rot fungal reactors could be an efficient technology to remove micropollutants from specific wastewater streams

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli

Ketoisophorone synthesis with an immobilized alcohol dehydrogenase

Altres ajuts: Authors also thank COST Action CM 1303-Systems Biocatalysis for financial support.The monoterpenoid α-isophorone is sourced from the available and renewable plant dry matter, as well as a waste recovery operation from acetone. This compound, can be hydroxylated to 4-hydroxy-isophorone which is the main precursor for the synthesis of ketoisophorone. On its turn, ketoisophorone is a key intermediate for the production of carotenoids and Vitamin E. Here, the enzymatic oxidation of 4-hydroxy-isophorone to ketoisophorone is demonstrated employing an alcohol dehydrogenase (ADHaa) from Artemisia annua and a NADPH oxidase (NOX), as a cofactor regeneration enzyme. After 24 h of reaction and an initial substrate concentration of 50 mM, 95.7 % yield and a space time yield of 6.52 g L⁻¹ day⁻¹ could be obtained. Furthermore, the immobilization of the alcohol dehydrogenase was studied on 17 different supports. An epoxy-functionalized agarose resulted in the highest metrics, 100±0% immobilization yield and 58.2±3.5 % retained activity. Finally, the immobilized ADHaa was successfully implemented in 4 reaction cycles (96 h operation) presenting a biocatalyst yield of 23.4 g product g⁻¹ of enzyme. It represents a 2.5-fold increase compared with the reaction with soluble enzymes

Enzymatic Synthesis of Trimethyl-ϵ-caprolactone : Process Intensification and Demonstration on a 100 L Scale

Optimization and scaling up of the Baeyer-Villiger oxidation of 3,3,5-trimethyl-cyclohexanone to trimethyl-ε-caprolactones (CHLs) were studied to demonstrate this technology on a 100 L pilot plant scale. The reaction was catalyzed by a cyclohexanone monooxygenase from Thermocrispum municipale that utilizes the costly redox cofactor nicotinamide adenine dinucleotide phosphate (reduced form), which was regenerated by a glucose dehydrogenase (GDH). As a first stage, different cyclohexanone monooxygenase formulations were tested: cell-free extract, whole cells, fermentation broth, and sonicated fermentation broth. Using broth resulted in the highest yield (63%) and required the least biocatalyst preparation effort. Two commercial glucose dehydrogenases (GDH-105 and GDH-01) were evaluated, resulting in similar performances. Substrate dosing rates and biocatalyst loadings were optimized. On a 30 mL scale, the best conditions were found when 30 mM h-1 dosing rate, 10% (v/v) cyclohexanone monooxygenase broth, and 0.05% (v/v) of glucose dehydrogenase (GDH-01) liquid enzyme formulation were applied. These same conditions (with oxygen instead of air) were applied on a 1 L scale with 92% conversion, achieving a specific activity of 13.3 U gcell wet weight (cww)-1, a space time yield of 3.4 gCHL L-1 h-1, and a biocatalyst yield of 0.83 gCHL gcww-1. A final 100 L demonstration was performed in a pilot plant facility. After 9 h, the reaction reached 85% conversion, 12.8 U gcww-1, a space time yield of 2.7 g L-1 h-1, and a biocatalyst yield of 0.60 gCHL gcww-1. The extraction of product resulted in 2.58 kg of isolated final product. The overall isolated CHL yield was 76% (distal lactone 47% and proximal lactone 53%)

Bioremediation of PAHs-contaminated soil through composting : influence of bioaugmentation and biostimulation on the contaminants biodegradation

The degradation of several polycyclic aromatic hydrocarbons (PAHs) in soil through composting was investigated. The selected PAHs included: fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, and chrysene, with concentrations simulating a real creosote sample. The degradation of PAHs (initial concentration 1 g of total PAHs kg⁻¹ dry soil) was assessed applying bioaugmentation with the white-rot fungi Trametes versicolor and biostimulation using compost of the source-selected organic fraction of municipal solid waste (OFMSW) and rabbit food as organic co-substrates. The process performance during 30 days of incubation was evaluated through different analyses including: dynamic respiration index (DRI), cumulative oxygen consumption during 5 days (AT₅), enzymatic activity, and fungal biomass. These analyses demonstrated that the introduced T. versicolor did not significantly enhance the degradation of PAHs. However, biostimulation was able to improve the PAHs degradation: 89% of the total PAHs were degraded by the end of the composting period (30 days) compared to the only 29.5% that was achieved by the soil indigenous microorganisms without any co-substrate (control, not amended). Indeed, the results showed that stable compost from the OFMSW has a greater potential to enhance the degradation of PAHs compared to non-stable co-substrates such as rabbit food

A comparison between biostimulation and bioaugmentation in a solid treatment of anaerobic sludge : drug contentent and microbial evaluation

Emerging pollutants can reach the environment through the sludge of Wastewater Treatment Plants. In this work, the use of Trametes versicolor in biopiles at lab-scale was studied, evaluating its capacity to remove the most hydrophobic Pharmaceuticals and assessing the evolution of the biopiles microbial communities. The total removal of drugs at real concentrations from sewage sludge was assessed for non-inoculated and fungal inoculated biopiles, testing if the re-inoculation of the biopiles after 22 days of treatment would improve the removal yields. It was found that 2 out of the 15 initially detected pharmaceuticals were totally degraded after 22 days, and re-inoculated fungal biopiles achieved higher removal rates than non-re-inoculated fungal biopiles for single compounds and for all the drugs simultaneously: 66.45% and 49.18% re-inoculated and non-re-inoculated biopiles, respectively. Finally, the study of the bacterial and fungal communities revealed that fungal inoculated and non-inoculated biopiles evolved to similar communities adapted to the presence of those drugs

Continuous treatment of non-sterile hospital wastewater by Trametes versicolor : how to increase fungal viability by means of operational strategies and pretreatments

Hospital wastewaters have a high load of pharmaceutical active compounds (PhACs). Fungal treatments could be appropriate for source treatment of such effluents but the transition to non-sterile conditions proved to be difficult due to competition with indigenous microorganisms, resulting in very short-duration operations. In this article, coagulation-flocculation and UV-radiation processes were studied as pretreatments to a fungal reactor treating non-sterile hospital wastewater in sequential batch operation and continuous operation modes. The influent was spiked with ibuprofen and ketoprofen, and both compounds were successfully degraded by over 80%. UV pretreatment did not extent the fungal activity after coagulation-flocculation measured as laccase production and pellet integrity. Sequential batch operation did not reduce bacteria competition during fungal treatment. The best strategy was the addition of a coagulation-flocculation pretreatment to a continuous reactor, which led to an operation of 28days without biomass renovation