Editorial: Insights in bioprocess engineering 2021/22: novel developments, current challenges, and future perspectives

Editorial on the Research Topic Insights in bioprocess engineering 2021/22: novel developments, current challenges, and future perspectives.SNF -Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung(FNS IZJFZ2_185638)info:eu-repo/semantics/publishedVersio

Impédancemètre et fluidique: validation d'un nouveau système pour l'évaluation in vitro de barrières biologiques: travail de bachelor : diplôme 2016

Le but de ce projet est de mettre en avant l’importance de la fluidique dans les modèles in vitro de barrières biologiques en validant biologiquement un impédancemètre intégrant de la fluidique et pouvant mesurer jusqu’à 24 puits

Pressure to kill or pressure to boost: a review on the various effects and applications of hydrostatic pressure in bacterial biotechnology

Much knowledge has been gained for the last 30years about the effects of pressure on bacteria, and various pressure-based technologies have been designed. The development of modern molecular biology techniques (e.g., DNA microarrays) as well as the technological advances realized in the manufacturing of robust sampling and high-pressure devices has allowed these advances. Not only the direct effects on cell components (membranes, proteins, and nucleic acids) have been unraveled, but also the cellular response to pressure has been investigated by means of transcriptome and proteome analyses. Initially, research was performed by marine biologists who studied the microorganisms living in the deep sea at pressures of 1,000bar. In parallel, food technologists developed pressure-based methods for inactivating microorganisms without altering the food properties as much as with temperature treatment. The preservation of specific product properties is also the rationale for pressure-based methods for the disinfection of biomaterials and for vaccine production. Therefore, attention was first focused on the "killing” potential of high pressure. On the other hand, there has been a growing interest in using elevated pressures (up to ~10bar) for enhancing the productivity of bioprocesses. In this case, no killing effect was sought, but pressure was applied to "boost” the process by enhancing the oxygen transfer to the cell culture. This paper gives an overview on the effects of pressures in the range of 1bar to 10kbar on bacteria and presents the major and most recent achievements realized in the development of pressure-based biotechnological application

A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate

<p>Abstract</p> <p>Background</p> <p>The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is <it>Pseudomonas putida </it>KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, <it>P. putida </it>KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription.</p> <p>Results</p> <p>To assess PHA production in <it>P. putida </it>KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate), PHA-unrelated (gluconate) and poor PHA substrate (citrate). The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively) but not on octanoate. In addition, <it>P</it>. <it>putida </it>KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded.</p> <p>Conclusions</p> <p>The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes <it>P. putida </it>KT2440 and KT2442 two different strains in terms of mcl-PHA production. The differences include the onset of mcl-PHA production (nitrogen limitation) and the resulting strain performance (growth rate). It remains difficult to predict a priori<it/>where such major changes might occur, as illustrated by the comparable behavior on octanoate. Consequently, experimental data on mcl-PHA production acquired for <it>P. putida </it>KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.</p

Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives

The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA productio

Encapsulated Zosteric Acid Embedded in Poly[3-hydroxyalkanoate] Coatings—Protection against Biofouling

Summary : The natural, non-toxic antifouling compound zosteric acid (ZA, p-coumaric acid sulfate) was encapsulated in polystyrene (PS) microcapsules (30 mg ZA /1 g PS) with an efficiency of 30 % via an in-liquid drying process. Electron micrographs showed microcapsules with smooth surfaces and a mean diameter of 200 μm. The FIB method was used to cross-section a microcapsule in order to visualize the inner capsule structure and to localize ZA via element analysis. Coatings of a biocompatible polyester, poly[3-hydroxyalkanoate-co-3-hydroxyalkenoate] (PHAE), were prepared on microscopic slides. These coatings contained dispersed ZA (PHAE/ZA) or ZA-loaded PS microcapsules (PHAE/PS(ZA)). The release of ZA was monitored via conductivy measurements in water and was 4 μgcm-2d-1 for PHAE/ZA and 0.9 μgcm-2d-1 for PHAE/PS(ZA) coatings. To follow the initial steps of biofilm formation, coated slides were exposed to activated sludge and analyzed for cell adhesion with ESEM. ZA was effective during the burst release time of the PHAE/ZA coating, but no significant differences in biofouling were observed after 48 h. This was attributed to the minimal effective release rate of ZA, which is approximately 10 μgcm-2d-

Application of Activated Charcoal in the Downstream Processing of Bacterial Olefinic Poly(3-hydroxyalkanoates)

Medium chain length poly(hydroxyalkanoates) (mcl-PHAs) are bacterial thermoplastic elastomers with a large potential in medical applications. The present study provides a novel process to isolate and purify poly([ R]3-hydroxy-w-undecenoate-co-3-hydroxy-w-nonenoate-co-3-hydroxy-w-heptenoate) (PHUE) and poly([R]-3hydroxy-w-undecenoate-co-3-hydroxy-w-nonenoate-co-3-hydroxyoctanoate- co-3-hydroxy-w-heptenoate-co 3-hydroxyhexanoate) (PHOUE) from Pseudomonas putida species. Three different types of activated charcoal were compared with regard to their capability to selectively remove impurities. The product 'Charcoal activated, powder, pure' from Merck was found to be most suitable. Using ethyl acetate as solvent, the polyesters were extracted from freeze-dried biomass at room temperature and simultaneously purified by addition of activated charcoal at the beginning of the extraction. The period of extraction was one hour and the ratio solvent to biomass was 15:1 (vol/wt). After extraction, the solids were separated by pressure filtration through a metallic lace tissue. The filtrate was again passed through the previously accumulated filter cake, followed by a second filtration through a 0.45 mm membrane to remove finest coal particles. The resulting filtrate was concentrated, thus yielding polyesters whose quality and yield depended on the quantity of activated charcoal applied. For highly pure PHUE and PHOUE with low endotoxin levels, the optimum ratio of activated charcoal to solvent for extraction (V/V) was found to be 0.5 for PHUE and 0.25 for PHOUE. The yields with regard to the raw polymers amounted to 55 wt% for PHUE and 75 wt% for PHOUE, which are acceptable for polymers that can be used for medical applications

Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applie

Overexpression and characterization of medium-chain-length polyhydroxyalkanoate granule bound polymerases from Pseudomonas putida GPo1

<p>Abstract</p> <p>Background</p> <p>Polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA biosynthesis are PHA polymerases, which catalyze the covalent linkage of 3-hydroxyacyl coenzymeA thioesters by transesterification with concomitant release of CoA. <it>Pseudomonas putida </it>GPo1 and many other <it>Pseudomonas </it>species contain two different class II polymerases, encoded by <it>phaC1 </it>and <it>phaC2</it>. Although numerous studies have been carried out on PHA polymerases and they are well characterized at the molecular level, the biochemical properties of the class II polymerases have not been studied in detail. Previously we and other groups purified the polymerases, however, the activities of the purified enzymes were several magnitude lower than the granule-bound enzymes. It is problematic to study the intrinsic properties of these enzymes with such low activities, although they are pure.</p> <p>Results</p> <p>PHA polymerase 1 (PhaC1) and PHA polymerase 2 (PhaC2) from <it>P. putida </it>GPo1 were overexpressed in the PHA-negative host <it>P. putida </it>GPp104 and purified from isolated PHA granules. Only minor activity (two to three orders of magnitude lower than that of the granule bound proteins) could be recovered when the enzymes were purified to homogeneity. Therefore, kinetic properties and substrate ranges were determined for the granule bound polymerases. The polymerases differed significantly with respect to their association with PHA granules, enzyme kinetics and substrate specificity. PhaC2 appeared to bind PHA granules more tightly than PhaC1. When <it>R</it>-3-hydroxyoctanoic acid was used as substrate, the granule-bound PhaC1 exhibited a <it>Km </it>of 125 (± 35) μM and a <it>V</it>max of 40.8 (± 6.2) U/mg PhaC1, while a <it>Km </it>of 37 (± 10) μM and a <it>V</it>max of 2.7 (± 0.7) U/mg PhaC2 could be derived for the granule-bound PhaC2. Granule-bound PhaC1 showed a strong preference for medium chain length (mcl-) 3-hydroxyacly-CoAs, with highest affinity towards 3-hydroxydecanoyl-CoA (40 U/mg PhaC1). Granule-bound PhaC2 demonstrated a far broader specificity ranging from short chain length up to long chain length substrates. Activity increased with increasing chain length with a maximum activity for 3-hydroxyacyl-CoAs containing 12 or more C-atoms.</p> <p>Conclusion</p> <p>The kinetic properties and substrate ranges were determined for both granule bound polymerases. Evidence was provided for the first time that two PHA polymerases exhibited significant differences in granule release and in vitro activity profiles, suggesting that there are substantial functional differences between granule bound PhaC1 and PhaC2.</p

Influence of growth stage on activities of polyhydroxyalkanoate (PHA) polymerase and PHA depolymerase in Pseudomonas putida U

<p>Abstract</p> <p>Background</p> <p>Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for <it>in vitro </it>activity measurements of PhaC and PhaZ in crude cell extracts are necessary.</p> <p>Results</p> <p>Two <it>in vitro </it>assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of <it>Pseudomonas putida </it>U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.</p> <p>Conclusion</p> <p>Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in <it>P. putida </it>PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.</p