Biocatalysis in non-conventional media : kinetic and thermodynamic aspects

During the past decade biocatalysis in non-conventional media has gained a lot of interest. Especially in the field of bio-organic synthesis, where poorly water-soluble substrates and products are involved, these media are very attractive.Non-conventional media generally consist of an apolar solvent phase and an aqueous phase. In this thesis, mixtures of water with water-miscible organic solvents, or water- immiscible organic solvents or (near-)supercritical solvents are described. The conventional aqueous phase contains the cellular or enzymic biocatalyst. The aqueous phase can vary from a dilute aqueous solution, with a thermodynamic water activity a w close to 1, to a dried enzyme particle with only a monolayer of adsorbed water molecules (a w < 1).In non-conventional media biocatalytic processes are governed by the presence of a phase boundary when two phases are involved. This phase boundary not only influences the rate of the bioconversion (kinetics), but also the yield of the reaction (thermodynamic equilibrium). In this thesis, several factors are described which affect the (kinetics), and thermodynamics of biocatalytic porcessen in non-conventional media.Chapter 2 gives an overview of the recent developments in the field of medium engineering for biocatalysis in non-conventional media. In this chapter a few basic design rules for the rational design are formulated. These rules may serve as useful tools for optimization of biocatalytic processes in non-conventional media.A typical example of a non-conventional reaction medium is the mixture of water and water-immiscible organic solvent. Especially for this type of reaction media the liquid-impelled loop reactor has been developed. This reactor has been used for the bioconversion of tetralin, a very toxic apolar compound. In Chapter 3 the general strategy for the selection of a suitable solvent for the bioconversion of such toxic apolar compounds in the liquid-impelled loop reactor is given, where the tetralin conversion is used as a typical example. The water-immiscible solvents should be non-toxic and nonbiodegradable. Additionally, they should reduce the toxicity of the apolar substrate and they must be practical for use in the liquid-impelled loop reactor. All the steps in the selection procedure proved to be essential. Among the 57 solvents tested, only FC-40 proofs to be suitable for bioconversion of tetralin in the liquid-impelled loop reactor. In addition, the cellular biocatalyst needs to be immobilized, to reduce emulsion formation inside the bioreactor.For the bioconversion of tetralin in the liquid-impelled loop reactor oxygen is needed. Chapter 4 describes the mass transfer of tetralin and oxygen in the liquidimpelled loop reactor from the apolar solvent phase to the aqueous phase, where the bioconversion occurs. It is found that in case of mass-transfer limitation, tetralin is the rate-limiting substrate and not oxygen.One of the selection criteria of a suitable solvent for bioconversion of apolar substates is its non-toxicity for the biocatalyst. The log Poctanol , which describes the hydrophobicity of the solvent, is a good measure for the toxicity of the solvent in a twoliquid phase system. The toxicity of a water-immiscible solvent for cellular biocatalyst is caused by two factors, i.e. the presence of a phase boundery (phase toxicity) and by the solvent molecules that are dissolved in the aqueous phase (molecular toxicity). Chapter 5 describes these effects separately. When the solvent concentration in the membrane of the cellular biocatalyst reaches a critical concentration, the solvent becomes toxic. The toxic concentration in the membrane is constant and independent of the solvent used. It is directly related via the partition coefficient over the membrane and water, to the solvent concentration in the aqueous phase. This is in turn directly related to the log Poctanol of the solvent. If the critical membrane concentration of a certain microorganism is known, the toxicity of any solvent can be predicted with thelog Poctanol .Apart from the log Poctanol , also the Hildebrand solubility parameter δcan be used as a measure of the hydrophobicity of the solvent. In Chapter 6 this parameter has been used successfully as an indicator of the solubility of apolar compounds in near-supercritical carbon dioxide (SCCO 2 ). In addition, the effect of this parameter on the transesterification rate of Lypozyme in this non-aqueous reaction medium has been studied. The change in δof near-supercritical carbon dioxide hardly influences the reaction rate. The water content of the medium influences the kinetics much more.Water not only affects the kinetics of a synthetic reaction, but it also affects the equilibrium yield of these reactions. When the thermodynamic water activity a w is decreased, water-dependent side-reactions such as in transesterification reactions are suppressed (Chapter 6). In esterification reactions, a shift in equilibrium towards synthesis is expected upon decreasing the a w .Chapter 7 describes a new method to control the a w during esterification reactions. With this a w -control method the a w can be maintained at an optimal value, at which the biocatalyst still shows sufficient activity while a high thermodynamic product yield can be obtained.This thesis actually covers two central themes in biocatalysis in non-conventional media: kinetics and thermodynamics. In Chapter 8 a general discussion highlights how thermodynamics can be used as a basic tool to reveal the processes that govern biocatalysis in non-conventional media

On Energy Balance and Production Costs in Tubular and Flat Panel Photobioreactors

Reducing mixing in both flat panel and tubular photobioreactors can result in a positive net energy balance with state-of-the-art technology and Dutch weather conditions. In the tubular photobioreactor, the net energy balance becomes positive at velocities < 0.3 ms-1, at which point the biomass production cost is 3.2 €/kg dry weight. In flat panel reactors, this point is at an air supply rate < 0.25 vol vol-1 min-1, at which the biomass production cost is 2.39 €/kg dry weight. To achieve these values in flat panel reactors, cheap low pressure blowers must be used, which limits the panel height to a maximum of 0.5 m, and in tubular reactors the tubes must be hydraulically smooth. For tubular reactors, it is important to prevent the formation of wall growth in order to keep the tubes hydraulically smooth. This paper shows how current production costs and energy requirement could be decreased.Reducing mixing in both flat panel and tubular photobioreactors can result in a positive net energy balance with state-of-the-art technology and Dutch weather conditions. In the tubular photobioreactor, the net energy balance becomes positive at velocities < 0.3 ms-1, at which point the biomass production cost is 3.2 €/kg dry weight. In flat panel reactors, this point is at an air supply rate < 0.25 vol vol-1 min-1, at which the biomass production cost is 2.39 €/kg dry weight. To achieve these values in flat panel reactors, cheap low pressure blowers must be used, which limits the panel height to a maximum of 0.5 m, and in tubular reactors the tubes must be hydraulically smooth. For tubular reactors, it is important to prevent the formation of wall growth in order to keep the tubes hydraulically smooth. This paper shows how current production costs and energy requirement could be decreased

Cultivation of shear stress sensitive and tolerant microalgalspecies in a tubular photobioreactor equipped with a centrifugal pump

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Effect of oxygen concentration on the growth of Nannochloropsis sp. at low light intensity

In large-scale microalgal production in tubular photobioreactors, the build-up of O2 along the tubes is one of the major bottlenecks to obtain high productivities. Oxygen inhibits the growth, since it competes with carbon dioxide for the Rubisco enzyme involved in the CO2 fixation to generate biomass. The effect of oxygen on growth of Nannochloropsis sp. was experimentally determined in a fully controlled flat-panel photobioreactor operated in turbidostat mode using an incident photon flux density of 100 µmol photons m-2 s-1 and with only the oxygen concentration as variable parameter. The dissolved oxygen concentration was varied from 20 to 250% air saturation. Results showed that there was no clear effect of oxygen concentration on specific growth rate (mean of 0.48¿±¿0.40 day-1) upon increasing the oxygen concentration from 20% to 75% air saturation. Upon further increasing the oxygen concentration, however, a linear decrease in specific growth rate was observed, ranging from 0.48¿±¿0.40 day-1 at a dissolved oxygen concentration of 75% air saturation to 0.18¿±¿0.01 day-1 at 250% air saturation. In vitro data on isolated Rubisco were used to predict the quantum yield at different oxygen concentrations in the medium. The predicted decrease in quantum yield matches well with the observed decrease that was measured in vivo. These results indicate that the effect of oxygen on growth of Nannochloropsis sp. at low light intensity is only due to competitive inhibition of the Rubisco enzyme. At these sub-saturating light conditions, the presence of high concentrations of oxygen in the medium induced slightly higher carotenoid content, but the increased levels of this protective antioxidant did not diminish the growth-inhibiting effects of oxygen on the Rubisco

Cultivation of shear stress sensitive and tolerant microalgal species in a tubular photobioreactor equipped with a centrifugal pump

The tolerance to shear stress of Tetraselmis suecica, Isochrysis galbana, Skeletonema costatum, and Chaetoceros muelleri was determined in shear cylinders. The shear tolerance of the microalgae species strongly depends on the strain. I. galbana, S. costatum, and C. muelleri exposed to shear stress between 1.2 and 5.4 Pa resulted in severe cell damage. T. suecica is not sensitive to stresses up to 80 Pa. The possibility to grow these algae in a tubular photobioreactor (PBR) using a centrifugal pump for recirculation of the algae suspension was studied. The shear stresses imposed on the algae in the circulation tubes and at the pressure side of the pump were 0.57 and 1.82 Pa, respectively. The shear stress tolerant T. suecica was successfully cultivated in the PBR. Growth of I. galbana, S. costatum, and C. muelleri in the tubular PBR was not observed, not even at the lowest pumping speed. For the latter shear sensitive strains, the encountered shear stress levels were in the order of magnitude of the determined maximum shear tolerance of the algae. An equation was used to simulate the effect of possible damage of microalgae caused by passages through local high shear zones in centrifugal pumps on the total algae culture in the PBR. This simulation shows that a culture of shear stress sensitive species is bound to collapse after only limited number of passages, confirming the importance of considering shear stress as a process parameter in future design of closed PBRs for microalgal cultivation

Pulsed Electric Field for protein release of the microalgae Chlorella vulgaris and Neochloris oleoabundans

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Energetics of smooth muscle taenia caecum of guinea-pig: a 31P-NMR study

AbstractSmooth muscle cell energetics of taenia caeci during relaxation, activity and maximal contraction were investigated using 31P-NMR. In relaxed muscle obtained in calcium-free medium, [ATP], [phosphocreatine] and [sugar phosphate] were 4.4 mM, 7.7 mM and 2.8 mM, respectively. There was only a small difference in the energetics of spontaneously active and maximally contracted muscles, but under both conditions substantial changes occurred as compared with relaxed muscles. The internal pH in relaxed muscle was found to be 7.05, which acidified to 6.5 during contraction. The level of sugar phosphates was found to be not a limiting factor in energetics

Harvesting of microalgae by bio-flocculation

The high-energy input for harvesting biomass makes current commercial microalgal biodiesel production economically unfeasible. A novel harvesting method is presented as a cost and energy efficient alternative: the bio-flocculation by using one flocculating microalga to concentrate the non-flocculating microalga of interest. Three flocculating microalgae, tested for harvesting of microalgae from different habitats, improved the sedimentation rate of the accompanying microalga and increased the recovery of biomass. The advantages of this method are that no addition of chemical flocculants is required and that similar cultivation conditions can be used for the flocculating microalgae as for the microalgae of interest that accumulate lipids. This method is as easy and effective as chemical flocculation which is applied at industrial scale, however in contrast it is sustainable and cost-effective as no costs are involved for pre-treatment of the biomass for oil extraction and for pre-treatment of the medium before it can be re-used

Escherichia coli mar and acrAB Mutants Display No Tolerance to Simple Alcohols

The inducible Mar phenotype of Escherichia coli is associated with increased tolerance to multiple hydrophobic antibiotics as well as some highly hydrophobic organic solvents such as cyclohexane, mediated mainly through the AcrAB/TolC efflux system. The influence of water miscible alcohols ethanol and 1-propanol on a Mar constitutive mutant and a mar deletion mutant of E. coli K-12, as well as the corresponding strains carrying the additional acrAB deletion, was investigated. In contrast to hydrophobic solvents, all strains were killed in exponential phase by 1-propanol and ethanol at rates comparable to the parent strain. Thus, the Mar phenotype does not protect E. coli from killing by these more polar solvents. Surprisingly, AcrAB does not contribute to an increased alcohol tolerance. In addition, sodium salicylate, at concentrations known to induce the mar operon, was unable to increase 1-propanol or ethanol tolerance. Rather, the toxicity of both solvents was increased in the presence of sodium salicylate. Collectively, the results imply that the resilience of E. coli to water miscible alcohols, in contrast to more hydrophobic solvents, does not depend upon the AcrAB/TolC efflux system, and suggests a lower limit for substrate molecular size and functionality. Implications for the application of microbiological systems in environments containing high contents of water miscible organic solvents, e.g., phage display screening, are discussed

Gemeenplaats en illusie : De structuur van de receptie van mimetische revoluties

This study examines the reception of various image techniques with the help of memorable expressions (topoi,commonplaces) that offer qualitative descriptions of these techniques’ realism. The image techniques in question are perspective painting, the novel, photography, television, and film. The commonplaces under discussion consist, respectively, of comparisons of works of art to mirrors and windows, associations with Zeuxis and his selection method, the ‘condemnation’ of images as lies, and the appropriation of pictured reality. These commonplaces derive from Renaissance theorists no less than antique writings and are alike in the manner in which they arise, fall out of use, and are then reintroduced. The present thesis tries to trace and explain this development of the commonplace. Every newly invented image technique is approached with the same enthusiasm and approving metaphors. Images are compared to windows and mirrors and, in the case of the photograph, to shadows, to depict their exactitude. These comparisons implicitly contain the promise of total representation, the bringing forth of the perfect illusion of reality in color and relief, and later also in movement and sound. Although initial reviews are not always positive (too much realism is regarded as a potential danger for mental health), the procedure is widely embraced and adaptations are made. One of these adaptations is the method of selection made famous by the legend of Zeuxis: since a single model did not contain all the desired features, the artist combined separate fragments of different models into one whole. The notion of exact representation recedes into the background, and the images begin to be compared to lies. Less and less can the artist point to the window through which he/she looked in making the image or fragments for it. More and more the image becomes an unreconstructable collage of many models or artefacts of nature. But drawing a parallel between an artwork and a lie is not detrimental to the reputation of the arts. To the contrary, by selecting and combining typical things the artist penetrates to the essence of reality. The public recognizes this. In the end the public appropriates the images, unconsciously, into their mental set and their behaviour. This is what is meant by ‘life imitates art’. Apart from being a re-examination of old and established art-theoretical convictions, this dissertation is also meant to contribute to the discussion of media-archaeology. It will not be a ‘polishing and perfecting of the great archaic ideas’ (Zielinski) nor will it fall into ‘the temptation to clean and polish [the story] until it will sparkle in the unambiguous light of certainty’ (Huhtamo). The ideas and stories in this study are not clear and distinct, still less immutable. The reception of each new image technique seems to resemble that of an older one, irrespective of cultural period, art-historical theories and historical movements. Furthermore, the reception of digital social media seems to fit within the structure demonstrated here. Therefore this study should be a constructive contribution to the discussion of old and new media