Elisabetta de Alteriis

Toxicity Effects of Functionalized Quantum Dots, Gold and Polystyrene Nanoparticles on Target Aquatic Biological Models: A Review

Nano-based products are widespread in several sectors, including textiles, medical-products, cosmetics, paints and plastics. Nanosafety and safe-by-design are driving nanoparticle (NP) production and applications through NP functionalization (@NPs). Indeed, @NPs frequently present biological effects that differ from the parent material. This paper reviews the impact of quantum dots (QDs), gold nanoparticles (AuNPs), and polystyrene-cored NPs (PSNPs), evidencing the role of NP functionalization in toxicity definition. Key biological models were taken into consideration for NP evaluation: Saccharomyces cerevisiae, fresh- (F) and saltwater (S) microalgae (Raphidocelis subcapitata (F), Scenedesmus obliquus (F) and Chlorella spp. (F), and Phaeodactylum tricornutum (S)), Daphnia magna, and Xenopus laevis. QDs are quite widespread in technological devices, and they are known to induce genotoxicity and oxidative stress that can drastically change according to the coating employed. For example, AuNPs are frequently functionalized with antimicrobial peptides, which is shown to both increase their activity and decrease the relative environmental toxicity. P-NPs are frequently coated with NH2− for cationic and COOH− for anionic surfaces, but when positively charged toxicity effects can be observed. Careful assessment of functionalized and non-functionalized NPs is compulsory to also understand their potential direct and indirect effects when the coating is removed or degraded.

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

BackgroundSaccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain.ResultsOur results show that the concentrations of ACA in the feeding solution, corresponding to those routinely used in the literature, are limiting for the growth of S. cerevisiae BY4741 [PIR4-IL1β] in fed-batch reactor. Even in the presence of a proper ACA supplementation, S. cerevisiae BY4741 [PIR4-IL1β] did not achieve a high cell density. The Δyca1 deletion did not have a beneficial effect on the overall performance of the strain, but it had a clear effect on its viability, which was not impaired during fed-batch operations, as shown by the kdvalue (0.0045 h-1), negligible if compared to that of the parental strain (0.028 h-1). However, independently of their robustness, both the parental and the Δyca1 mutant ceased to grow early during fed-batch runs, both strains using most of the available carbon source for maintenance, rather than for further proliferation. The mathematical model used evidenced that the energy demand for maintenance was even higher in the case of the Δyca1 mutant, accounting for the growth arrest observed despite the fact that cell viability remained comparatively high.ConclusionsThe paper points out the relevance of a proper ACA formulation for the outcome of a fed-batch reactor growth carried out with S. cerevisiae BY4741 [PIR4-IL1β] strain and shows the sensitivity of this commonly used auxotrophic strain to aerated fed-batch operations. A Δyca1 disruption was able to reduce the loss of viability, but not to improve the overall performance of the process. A mathematical model has been developed that is able to describe the behaviour of both the parental and mutant producer strain during fed-batch runs, and evidence the role played by the energy demand for maintenance in the outcome of the process.

Invertase and acid phosphatase in free and gel-immobilized cells of Saccharomyces cerevisiae grown under different cultural conditions

Abstract A systematic investigation of the growth mode and appearance of two cell wall enzymes (invertase E.C. 3.1.1.26, and acid phosphatase E.C.3.1.3.2) in free and immobilized cells of Saccharomyces cerevisiae was carried out. Gel-entrapped yeast cells were stimulated into growth by incubation in media differing from one another in composition, under different experimental conditions. In immobilized cells, lower growth rates were observed which were mainly due to the resistance of the matrix to nutrient diffusion. Invertase activity reached levels higher than those of corresponding free cells. This result was independent of the cultural system adopted for growth and of the nature of the gel matrix chosen for immobilization, and could not be explained merely in terms of diffusional resistance. A possible explanation could be some physiological change related to the state of immobilization of the yeast cells. What was observed with invertase seems to be peculiar to this enzyme, as the extent of repression, in the case of acid phosphatase, was almost the same for both free and gel-immobilized cells.

Mechanical stability and diffusional resistance of a polymeric gel used for biocatalyst immobilization

The mechanical strength of gelatin gels insolubilized by crosslinking with formaldehyde was measured at various gelatin percentages and formaldehyde-to-gelatin ratios. This property was shown to be related to the characteristic sponge-like structure of the insolubilized gelatin gel, a structure that unexpectedly is also responsible for the resistance to substrate and product diffusion. A comparison between immobilizates of invertase and invertase-active yeast cells prepared with different gelatin concentrations showed that the enzyme, in contrast to cells, is deeply involved in the gel insolubilization process. The catalytic behavior of agar, kappa-carrageenan, alginate, and gelatin immobilizates was compared under the same conditions of cell loading.

Ethanolic fermentation by yeast cells immobilized in polyaldehyde-hardened gelatin beads

Abstract Viable cells of Saccharomyces cerevisiae were entrapped within gelatin beads by means of a novel technique using oxystarch as a harmless insolubilizing agent instead of formaldehyde or glutaraldehyde. The beads, after incubation in a nutrient medium, were examined in a packed-bed reactor for the continuous production of ethanol under different experimental conditions. Compared with Ca-alginate and κ-carrageenan, oxystarch-hardened gelatin showed the highest cell loading and mechanical stability.

A novel process-based model of microbial growth: self-inhibition in Saccharomyces cerevisiae aerobic fed-batch cultures

AbstractBackgroundMicrobial population dynamics in bioreactors depend on both nutrients availability and changes in the growth environment. Research is still ongoing on the optimization of bioreactor yields focusing on the increase of the maximum achievable cell density.ResultsA new process-based model is proposed to describe the aerobic growth of Saccharomyces cerevisiae cultured on glucose as carbon and energy source. The model considers the main metabolic routes of glucose assimilation (fermentation to ethanol and respiration) and the occurrence of inhibition due to the accumulation of both ethanol and other self-produced toxic compounds in the medium. Model simulations reproduced data from classic and new experiments of yeast growth in batch and fed-batch cultures. Model and experimental results showed that the growth decline observed in prolonged fed-batch cultures had to be ascribed to self-produced inhibitory compounds other than ethanol.ConclusionsThe presented results clarify the dynamics of microbial growth under different feeding conditions and highlight the relevance of the negative feedback by self-produced inhibitory compounds on the maximum cell densities achieved in a bioreactor.

Effect of gelatin-immobilization on the catalytic activity of enzymes and microbial cells

The immobilization of enzymes and microbial cells within insolubilized gelatin involves both physical entrapment and covalent crosslinking, each one playing its role. The effect of this dual type of bonding on the kinetic parameters and activity yield of three enzymes (acid phosphatase, invertase and α-glucosidase) and of whole microbial cells belonging to three yeast species (Saccharomyces cerevisiae,Candida utilis andKluyveromyces marxianus) have been investigated.

Effect of auxotrophies on yeast performance in aerated fed-batch reactor

A systematic investigation on the effects of auxotrophies on the performance of yeast in aerated fed-batch reactor was carried out. Six isogenic strains from the CEN.PK family of Saccharomyces cerevisiae, one prototroph and five auxotrophs, were grown in aerated fed-batch reactor using the same operative conditions and a proper nutritional supplementation. The performance of the strains, in terms of final biomass decreased with increasing the number of auxotrophies. Auxotrophy for leucine exerted a profound negative effect on the performance of the strains. Accumulation of reactive oxygen species (ROS) in the cells of the strain carrying four auxotrophies and its significant viability loss, were indicative of an oxidative stress response induced by exposure of cells to the environmental conditions. The mathematical model was fundamental to highlight how the carbon flux, depending on the number and type of auxotrophies, was diverted towards the production of increasingly large quantities of energy for maintenance.

Expression of human interleukin-1β in Saccharomyces cerevisiae using PIR4 as fusion partner and production in aerated fed-batch reactor

To circumvent cell wall retention commonly associated to Saccharomyces cerevisiae when used as a host for heterologous protein production, we have created a translational fusion of human interleukin-1β (IL-1β) to the Pir4 cell wall protein, so as to drive the secretion of the recombinant product to the growth medium. The auxotrophic S. cerevisiae BY4741 was used as host to express the Pir4-IL1β fusion protein. Once it was ascertained that the fusion protein was secreted to the culture medium and behaved as a growth-linked product, S. cerevisiae BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor to achieve high cell density and, consequently, high product concentration in the medium. Two cultivation media were employed, a rich complex and a defined mineral medium, the latter suitably supplemented with bacto-casamino acids as ACA (auxotrophy-complementing amino acid) source. The rich complex medium allowed a good performance of the producer strain only during batch growth, but was revealed to be inadequate for long-term fed-batch operations. The defined mineral medium ensured a better performance, even though not yet satisfactory in spite of a proper ACA supplementation. The behaviour of BY4741 was attributed to an intrinsic sensitivity of the producer strain to long-term aerated fed-batch operations.

D-Amino acid oxidase from Trigonopsis variabilis : Immobilization of whole cells in natural polymeric gels for glutaryl-7-aminocephalosporanic acid production

Abstract The enzymatic oxidation of cephalosporin C to glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) was carried out utilizing permeabilized whole cells of the yeast Trigonopsis variabilis entrapped in Ca-alginate beads. The biomass, cultured in a rich medium containing dl -methionine and harvested after 72 h of growth, exhibited high levels of d -amino acid oxidase activity. Prior to use, the whole cells were permeabilized with four freeze-thawing cycles and immobilized in polysaccharide matrices, such as Ca-alginate and κ-carrageenan, and in an insolubilized gelatin gel. The best results in terms of activity yield and storage stability were obtained with cells entrapped in Ca-alginate beads. These cells were utilized for glutaryl-7-ACA production in a continuous stirred batch reactor (CSTR) and in a packed bed reactor working as a plug flow reactor (PFR), using 50 mM cephalosporin C as substrate. The performances of the two systems were compared. The overall productivities (calculated on a void volume basis) were 1.64 g and 255 mg of glutaryl-7-ACA l −1 h −1 in the PFR and in the CSTR, respectively.