Bacteria-Polymer Composite Material for Glycerol Valorization.

Bacterial immobilization is regarded as an enabling technology to improve the stability and reusability of biocatalysts. Natural polymers are often used as immobilization matrices but present certain drawbacks, such as biocatalyst leakage and loss of physical integrity upon utilization in bioprocesses. Herein, we prepared a hybrid polymeric matrix that included silica nanoparticles for the unprecedented immobilization of the industrially relevant Gluconobacter frateurii (Gfr). This biocatalyst can valorize glycerol, an abundant by-product of the biodiesel industry, into glyceric acid (GA) and dihydroxyacetone (DHA). Different concentrations of siliceous nanosized materials, such as biomimetic Si nanoparticles (SiNps) and montmorillonite (MT), were added to alginate. These hybrid materials were significantly more resistant by texture analysis and presented a more compact structure as seen by scanning electron microscopy. The preparation including 4% alginate with 4% SiNps proved to be the most resistant material, with a homogeneous distribution of the biocatalyst in the beads as seen by confocal microscopy using a fluorescent mutant of Gfr. It produced the highest amounts of GA and DHA and could be reused for up to eight consecutive 24 h reactions with no loss of physical integrity and negligible bacterial leakage. Overall, our results indicate a new approach to generating biocatalysts using hybrid biopolymer supports.

New perspectives into Gluconobacter-catalysed biotransformations.

Different from other aerobic microorganisms that oxidise carbon sources to water and carbon dioxide, Gluconobacter catalyses the incomplete oxidation of various substrates with regio- and stereoselectivity. This ability, as well as its capacity to release the resulting products into the reaction media, place Gluconobacter as a privileged member of a non-model microorganism class that may boost industrial biotechnology. Knowledge of new technologies applied to Gluconobacter has been piling up in recent years. Advancements in its genetic modification, application of immobilisation tools and careful designs of the transformations, have improved productivities and stabilities of Gluconobacter strains or enabled new bioconversions for the production of valuable marketable chemicals. In this work, the latest advancements applied to Gluconobacter-catalysed biotransformations are summarised with a special focus on recent available tools to improve them. From genetic and metabolic engineering to bioreactor design, the most recent works on the topic are analysed in depth to provide a comprehensive resource not only for scientists and technologists working on/with Gluconobacter, but for the general biotechnologist.

Increased paclitaxel recovery from Taxus baccata vascular stem cells using novel in situ product recovery approaches.

In this study, several approaches were tested to optimise the production and recovery of the widely used anticancer drug Taxol® (paclitaxel) from culturable vascular stem cells (VSCs) of Taxus baccata, which is currently used as a successful cell line for paclitaxel production. An in situ product recovery (ISPR) technique was employed, which involved combining three commercial macro-porous resin beads (HP-20, XAD7HP and HP-2MG) with batch and semi-continuous cultivations of the T. baccata VSCs after adding methyl jasmonate (Me-JA) as an elicitor. The optimal resin combination resulted in 234 ± 23 mg of paclitaxel per kg of fresh-weight cells, indicating a 13-fold improved yield compared to the control (with no resins) in batch cultivation. This resin treatment was further studied to evaluate the resins' removal capacity of reactive oxygen species (ROS), which can cause poor cell growth or reduce product synthesis. It was observed that the ISPR cultivations had fourfold less intracellular ROS concentration than that of the control; thus, a reduced ROS concentration established by the resin contributed to increased paclitaxel yield, contrary to previous studies. These paclitaxel yields are the highest reported to date using VSCs, and this scalable production method could be applied for a diverse range of similar compounds utilising plant cell culture.

Dihydroxyacetone production via heterogeneous biotransformations of crude glycerol.

In this work, several immobilization strategies for Gluconobacter oxydans NBRC 14819 (Gox) were tested in the bioconversion of crude glycerol to dihydroxyacetone (DHA). Agar, agarose and polyacrylamide were evaluated as immobilization matrixes. Glutaraldehyde crosslinked versions of the agar and agarose preparations were also tested. Agar immobilized Gox proved to be the best heterogeneous biocatalyst in the bioconversion of crude glycerol reaching a quantitative production of 50 g/L glycerol into DHA solely in water. Immobilization allowed reutilization for at least eight cycles, reaching four times more DHA than the amount obtained by a single batch of free cells which cannot be reutilized. An increase in scale of 34 times had no impact on DHA productivity. The results obtained herein constitute a contribution to the microbiological production of DHA as they not only attain unprecedented productivities for the reaction with immobilized biocatalysts but also proved that it is feasible to do it in a clean background of solely water that alleviates the cost of downstream processing.

Stabilization of ω-transaminase from Pseudomonas fluorescens by immobilization techniques.

Transaminases are a class of enzymes with promising applications for the preparation and resolution of a vast diversity of valued amines. Their poor operational stability has fueled many investigations on its stabilization due to their biotechnological relevance. In this work, we screened the stabilization of the tetrameric ω-transaminase from Pseudomonas fluorescens (PfωTA) through both carrier-bound and carrier-free immobilization techniques. The best heterogeneous biocatalyst was the PfωTA immobilized as cross-linked enzyme aggregates (PfωTA-CLEA) which resulted after studying different parameters as the precipitant, additives and glutaraldehyde concentrations. The best conditions for maximum recovered activity (29 %) and maximum thermostability at 60 ºC and 70 ºC (100 % and 71 % residual activity after 1 h, respectively) were achieved by enzyme precipitation with 90% acetone or ethanol, in presence of BSA (100 mg/mL) and employing glutaraldehyde (100 mM) as cross-linker. Studies on different conditions for PfωTA-CLEA preparation yielded a biocatalyst that exhibited 31 and 4.6 times enhanced thermal stability at 60 °C and 70 °C, respectively, compared to its soluble counterpart. The PfωTA-CLEA was successfully used in the bioamination of 4-hydroxybenzaldehyde to 4-hydroxybenzylamine. To the best of our knowledge, this is the first report describing a transaminase cross-linked enzyme aggregates as immobilization strategy to generate a biocatalyst with outstanding thermostability.

Efficient glycerol transformation by resting Gluconobacter cells.

In the present work, glycerol biotransformation using Gluconobacter strains was studied with a process intensification perspective that facilitated the development of a cleaner and more efficient technology from those previously reported. Starting from the industrial by-product, crude glycerol, resting cells of Gluconobacter frateurii and Gluconobacter oxydans were able to convert glycerol under batch reactor conditions in water with no other additive but for the substrate. The study of strains, biomass:solution ratio, pH, growth stage, and simplification of media composition in crude glycerol bioconversions facilitated productivities of glyceric acid of 0.03 g/L.h and 2.07 g/L.h (71.5 g/g % pure by NMR) of dihydroxyacetone (DHA). Productivities surmounted recent reported fermentative bioconversions of crude glycerol and were unprecedented for the use of cell suspended solely in water. This work proposes a novel approach that allows higher productivities, cleaner production, and reduction in water and energy consumption, and demonstrates the applicability of the proposed approach.