pH-dependent electrostatic interactions between enzymes and nanoparticles in Pickering emulsions - Influence on activity and droplet size.

Pickering emulsions (PE) are liquid-liquid systems that are stabilized by solid (nano)particles at the fluid interface. They offer higher stability, easier separation and lower toxicity compared to classical emulsions stabilized by surfactants. Common applications range from food science to drug delivery. In the last decade they have become of more interest in the field of multiphasic biocatalysis. First, this study aims to present the influence of pH, salt strength and particle charge on enzyme activity. The different behavior of two lipases (CaLA and CRL) is shown. While the activity optimum of CaLA changed from pH 6.5 to pH 5 by applying particles with negative instead of positive surface charge, the CRL activity optimum stayed at pH 5-5.5. This enables particle charge as an additional parameter to optimize biocatalytic reactions in PEs. Second, the resulting drop sizes were measured to elucidate further interactions between the enzymes and particles in PEs. Drop sizes in PEs prepared with CaLA were not influenced by pH, but increased for positively and decreased for negatively charged particles upon the addition of CaLA. Electrostatic attraction between particles and CRL increased the droplet diameter from 10 µm up to 30 µm and therefore destabilized the PE for both particle types.

Mobilization of Unexploded Ordnance on the Seabed.

Unexploded ordnance devices (UXO) pose a potential threat to human life and material during offshore construction activities. Extensive survey activities are conducted to locate, identify, and clear these objects as necessary. For the period thereafter, it is necessary to investigate whether areas that have already been cleared, or even objects that remain in place, may be affected by mobilization under tidal currents or waves, and could thus have an impact on operation and maintenance during the lifetime of the offshore installation. In this study, model simulations based on fluid mechanics are described to derive the loads on the objects caused by currents and waves and combined with knowledge of the known burial condition of the objects. Within the model, the hydrodynamic and hydrostatic loads on the object caused by waves and currents are balanced with inertia and rolling resistance. Thus, the critical current velocity and critical wave conditions for the mobilization of different objects are calculated and compared with the environmental conditions prevailing in the North Sea. As a result, a recurrence interval for the potential mobilization of objects on the seafloor is given, which can now be used to optimize route surveys and thus help accelerate offshore construction work. It is shown that currents are not able to mobilize the objects investigated in the study in almost all regions of the North Sea. Waves can mobilize certain objects in very shallow and extreme conditions.

Organic Solvent Nanofiltration of Water-in-Oil Pickering Emulsions-What Influences Permeability?

Pickering emulsions (PEs) have received increasing interest for their application in catalytic multiphase reactions. Organic solvent nanofiltration of PEs was shown to be a promising procedure for efficient and effective catalyst recycling. In this work, a systematic parameter study to identify the main influencing parameters on PE filtration was conducted for a large variety of PE compositions for the first time. In addition to temperature, only the type of organic solvent significantly influenced the filtration performance, which could be mathematically modeled via a combination of the solution-diffusion and the resistance in the series model. Particle type and concentration, dispersed phase fraction and the presence of reaction (by-)products did not show any significant impact on the permeability. The stirrer speed only became important when emulsions stabilized by particles without the tendency to form 3D network structures were filtered in long-term filtration experiments. These results pave the way towards the application of PE membrane filtration for catalyst recovery in continuous liquid/liquid multiphase reactions and enable broad operation windows. As the mechanical separation of PEs was shown to be a very robust process, the emulsion composition can now be tuned to meet the needs of the reaction without any (significant) loss in filtration performance.

The quantitative impact of fluid vs. solid interfaces on the catalytic performance of pickering emulsions.

Pickering emulsions (PEs), i.e. particle stabilized emulsions, are used as reaction environments in biphasic catalysis for the hydroformylation of 1-dodecene into tridecanal using the catalyst rhodium (Rh)-sulfoxantphos (SX). The present study connects the knowledge about particle-catalyst interactions and PE structure with the reaction results. It quantifies the efficiency of the catalytic performance of the catalyst localized in the voids between the particles (liquid-liquid interface) and the catalyst adsorbed on the particle surface (liquid-solid interface) using a new numerical approach. First, it is ensured that the overall packing density and geometry at the droplet interface and the size of the water droplets of the resulting w/o PEs are predictable. Second, it is shown that approximately all particles assemble at the droplet surface after emulsion preparation and neither the packing parameter nor the droplet size change with the particle surface charge or size when the total particle cross section is kept constant. Third, studies on the influence of the catalyst on the emulsion structure reveal that irrespective of the particle charge the surface active and negatively charged catalyst Rh-SX reduces the PE droplet size significantly and decreases the particle packing parameter from s = 0.91 (hexagonal packing in 2D) to s = 0.69 (shattered structure). In this latter case, large voids of the free w/o interface form and become covered with the catalyst. With a deep knowledge about the PE structure the reaction efficiencies of the liquid-liquid vs. the solid-liquid interface are quantified. By excluding any other influence factors, it is shown that the activity of the catalyst is the same at the fluid and solid interface and the performance of the reaction is explained by the geometry of the system. After the reaction, the catalyst retention via membrane filtration is shown to be successfully achieved without damaging the emulsions. This enables the continuous recovery of the catalyst, i.e. the most expensive compound in PE-based catalytic reactions, being a crucial criterion for industrial applications.

W/O Pickering emulsion preparation using a batch rotor-stator mixer - Influence on rheology, drop size distribution and filtration behavior.

HYPOTHESIS: Pickering emulsions (PE) are becoming of increasing interest for catalytic multiphase processes. Ultrafiltration of PE is a promising procedure for catalyst recovery to enable continuous processes. Dispersing conditions during production of PE are expected to significantly influence PE characteristics, and control of these properties is essential for robust process design. However, while the impact of PE composition has been studied before, knowledge on dispersing conditions is surprisingly scarce. EXPERIMENTS: The influence of dispersing time, speed and emulsion volume during the preparation of PE with an UltraTurrax (2 dispersing tools) on the drop size distribution, rheology, stability and filtration was investigated. FINDINGS: In this first systematic study of PE preparation conditions, obtained Sauter mean diameters were correlated with energy density (R2 = 0.80), energy dissipation rate (R2 = 0.85) and tip speed (R2 = 0.86). All emulsions were stable for at least 10 weeks. With increasing tip speed (4-13 m/s), the dynamic viscosity first decreased, passed through a plateau value and then increased again. Filtration of concentrated PE was successful but strong membrane-particle-solvent interactions were revealed. This work contributes to a better understanding of PE properties that are essential for a sound application of PE in continuous multiphase catalysis.

The impact of lipases on the rheological behavior of colloidal silica nanoparticle stabilized Pickering emulsions for biocatalytical applications.

The use of Pickering emulsions for biocatalytical applications has recently received increased attention in cases where hydrophobic reactants are involved. For process applications, knowledge of the emulsion's rheology is crucial for the fluid dynamical design of equipment and selection of operating conditions. Colloidal silica nanoparticle stabilized Pickering emulsions usually exhibit shear-thinning behavior caused by a complex particle-particle network. While this has been observed by many authors, no publication has yet dealt with the rheology of silica nanoparticle stabilized Pickering emulsions containing enzymes. Thus, the aim of this study was to investigate the impact of the commonly used biocatalyst lipase (type and concentration), the dispersed phase volume fraction and the silica particle concentration on the rheological behavior of water-in-oil Pickering emulsions. For this purpose, the impact of the named parameters on the viscosity curves were measured. Lipases reduced the viscosities and transferred the rheological behavior from shear-thinning to Newtonian, which might be due to interactions of the lipase molecules via the formation of intermolecular disulfide bonds, which disturb the hydrogen-bond based silica particle-particle network. However, by increasing the dispersed phase volume fraction or the silica particle concentration the rheological behavior of emulsions became again shear-thinning. This work will help to produce bioactive Pickering emulsions with tailor-made characteristics.

Stopping Autoinducer-2 Chatter by Means of an Indigenous Bacterium ( Acinetobacter sp. DKY-1): A New Antibiofouling Strategy in a Membrane Bioreactor for Wastewater Treatment.

Bacterial quorum quenching (QQ) by means of degrading signaling molecules has been applied to antibiofouling strategies in a membrane bioreactor (MBR) for wastewater treatment. However, the target signaling molecules have been limited to N-acyl homoserine lactones participating in intraspecies quorum sensing. Here, an approach to disrupting autoinducer-2 (AI-2) signaling molecules participating in interspecies quorum sensing was pursued as a next-generation antibiofouling strategy in an MBR for wastewater treatment. We isolated an indigenous QQ bacterium ( Acinetobacter sp. DKY-1) that can attenuate the expression of the quorum-sensing (QS) response through the inactivation of an autoinducer-2 signaling molecule, 4,5-dihydroxy-2,3-pentanedione (DPD), among four kinds of autoinducer-2 QS bacteria. DKY-1 released AI-2 QQ compounds, which were verified to be hydrophilic with a molecular weight of <400 Da. The addition of DKY-1 entrapping beads into an MBR significantly decreased DPD concentration and remarkably reduced membrane biofouling. This new approach, combining molecular biology with wastewater engineering, could enlarge the range of QQ-MBR for antibiofouling and energy savings in the field of wastewater treatment.

A continuous single organic phase process for the lipase catalyzed synthesis of peroxy acids increases productivity.

The design of an optimal process is particularly crucial when the reactants deactivate the biocatalyst. The reaction cascades of the chemo-enzymatic epoxidation where the intermediate peroxy acid is produced by an enzyme are still limited by enzyme inhibition and deactivation by hydrogen peroxide. To avoid additional effects caused by interfaces (aq/org) and to reduce the process limiting deactivation by the substrate hydrogen peroxide, a single-phase concept was applied in a fed-batch and a continuous process (stirred tank), without the commonly applied addition of a carrier solvent. The synthesis of peroxyoctanoic acid catalyzed by Candida antarctica lipase B was chosen as the model reaction. Here, the feasibility of this biocatalytic reaction in a single-phase system was shown for the first time. The work shows the economic superiority of the continuous process compared to the fed-batch process. Employing the fed-batch process reaction rates up to 36 mmol h-1 per gramcat, and a maximum yield of 96 % was achieved, but activity dropped quickly. In contrast, continuous operation can maintain long-term enzyme activity. For the first time, the continuous enzymatic reaction could be performed for 55 h without any loss of activity and with a space-time yield of 154 mmol L-1 h-1, which is three times higher than in the fed-batch process. The higher catalytic productivity compared to the fed-batch process (34 vs. 18 gProd g-1 cat) shows the increased enzyme stability in the continuous process.

Recent advances on prebiotic lactulose production.

Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic. The production of lactulose is important in the dairy industry, as it is regarded as a high value-added derivative of whey or lactose. The industrial production of lactulose is still mainly done by chemical isomerization. Due to concerns on the environmental and tedious separation processes, the enzymatic-based lactulose synthesis has been regarded as an interesting alternative. This work aims at comparing chemical and enzyme-catalyzed lactulose synthesis. With an emphasis on the latter one, this review discusses the influences of the critical operating conditions and the suited operation mode on the transgalactosylation of lactulose using microbial enzymes. As an update and supplement to other previous reviews, this work also summarizes the recent reports that highlighted the enzymatic isomerization of lactose using cellobiose 2-epimerase to produce lactulose at elevated yields.

Influences of operating conditions on continuous lactulose synthesis in an enzymatic membrane reactor system: A basis prior to long-term operation.

Lactulose synthesis was performed in a continuous stirred enzymatic membrane reactor. Each investigated operating condition (agitation, pH, feed molar ratio of lactose to fructose (mL/mF ratio), hydraulic residence time (HRT)) had an influence on reaction performances, in terms of lactulose concentration, productivity and selectivity. Lactulose concentration was maximum at an mL/mF ratio of 1/2. Higher than this ratio, synthesis of galactooligosaccharides was promoted rather than lactulose. At mL/mF ratios lower than 1/2, enzyme inhibition was pronounced to the detriment of lactulose production. At 7 or 9h HRT, higher lactulose concentrations were obtained than at shorter HRTs. Applying an mL/mF ratio of 1/2 and an HRT of 9h in a long-term operation, nearly constant lactulose concentration was reached after 23h and lasted up to 32h with a mean concentration of 14.51±0.07g/L and a reaction selectivity of 0.075-0.080mollactulose/molcons.lactose. After 7d, lactulose concentration reduced by 31%. A continuous synthesis of lactulose at lab-scale was shown to be amenable using a membrane reactor process. Moreover, for process evaluation, this study can bridge the gap between batch laboratory scale and continuous full-scale operation regarding lactulose synthesis.

Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose secondary hydrolysis.

Newly developed parallel small-scale enzymatic membrane reactors (EMRs) were used to enhance the synthesis of lactulose using ß-galactosidase. Under batch operation, the productivity of lactulose decreased abruptly from 2.72 down to 0.04 mg lactulose/(Uenzymeh) over 35 h of reaction. This was presumably caused by the action of ß-galactosidase which performed secondary hydrolysis upon the produced lactulose. The continuous operations of an EMR system led to continuous removal of lactulose in the reactors restricting lactulose degradation caused by secondary hydrolysis. Therefore, continuous lactulose syntheses in the EMRs yielded significantly higher specific productivities under "steady state" conditions. Approximately 0.70 and 0.50 mg lactulose/(U enzyme h) for hydraulic residence times of 5 and 7h were reached, respectively. Continuous lactulose synthesis performed in an EMR system conclusively can circumvent the drawbacks (e.g., secondary hydrolysis) of lactulose synthesis encountered in batch operation. It is, therefore, beneficial in terms of enhanced lactulose productivity and reduced enzyme consumption.

The importance of fluid dynamics for MBR fouling mitigation.

The importance of the multiphase fluid dynamics for fouling mitigation in MBR systems has been widely acknowledged with air sparging having been applied commercially for about 20 years. However, the effects of air scouring are still not fully understood since the transient orthogonal and parallel flows as well as turbulent eddies created by bubbling generate complex hydrodynamic flow fields in the vicinity of a membrane. There is no generally valid model that describes the relationship between fouling rate and fluid dynamics. So, a reliable and universally applicable model to optimize membrane module and tank geometries, air scouring and filtration cycles is still pending. In addition to providing a discussion on the importance of multiphase fluid dynamics for fouling control, this review aims at developing guidelines to choose appropriate experimental and numerical methods for fluid dynamics investigations in MBR systems.

Continuous membrane-based screening system for biocatalysis.

The use of membrane reactors for enzymatic and co-factor regenerating reactions offers versatile advantages such as higher conversion rates and space-time-yields and is therefore often applied in industry. However, currently available screening and kinetics characterization systems are based on batch and fed-batch operated reactors and were developed for whole cell biotransformations rather than for enzymatic catalysis. Therefore, the data obtained from such systems has only limited transferability for continuous membrane reactors. The aim of this study is to evaluate and to improve a novel screening and characterization system based on the membrane reactor concept using the enzymatic hydrolysis of cellulose as a model reaction. Important aspects for the applicability of the developed system such as long-term stability and reproducibility of continuous experiments were very high. The concept used for flow control and fouling suppression allowed control of the residence time with a high degree of precision (±1% accuracy) in a long-term study (>100 h).

Continuous screening system for inhibited enzyme catalysis: a membrane reactor approach.

The screening of catalysts, substrates or conditions in the early stages of bioprocess development requires an enormous number of experiments and is a tedious, expensive and time-consuming task. Currently available screening systems can only be operated in batch or fed-batch mode, which can lead to severe misinterpretations of screening results. For example, catalysts that are inhibited by substrates or accumulating products will be excluded from further investigations in the early stages of process development despite the fact that they might be superior to other candidates in a different operational mode. Important and advantageous properties such as turnover stability can also be overshadowed by product inhibition. The aim of this study was to develop a novel screening system that enables continuous feeding of substrates and continuous removal of products. A prototype based on the membrane reactor concept was designed and operated for a model reaction, the hydrolysis of cellulose.

Filterability assessment in membrane bioreactors using an in-situ filtration test cell.

A new method for the assessment of the filterability in membrane bioreactors was tested for five months in four MBR units in Berlin. The new method BFM (Berlin Filtration Method) for filterability assessment uses a small membrane filtration test cell which can be submerged directly in the biological tanks to determine the filterability of the activated sludge in-situ. The test cell contains an aerated flat-sheet membrane which operates at similar conditions as in the plant. Filterability is expressed in terms of critical flux obtained by performing flux-stepping experiments. The ultimate goal of monitoring the filterability with the device is to detect in real time fouling occurrences due to changes in sludge composition and to adapt accordingly the operating conditions. The usefulness of the device for this purpose was evaluated for five months after monitoring four MBR plants in Berlin with different activated sludge characteristics (MLSS from 5 to 21 g/L, SRT 12-35 d and COD in the supernatant 30-400 mg/L). The first results show a good agreement between the filterability of the sludge with the portable filtration test cell and the filtration performance of the plant. Critical flux values varied between 3 and 30 L/m(2) h during the studied period. Useful information concerning the irreversibility of the fouling was provided by looking at the hysteresis curve of the flux-stepping experiments.

Occurrence, source, and fate of dissolved organic matter (DOM) in a pilot-scale membrane bioreactor.

In this study, the fate of carbohydrates, proteins, and humic substances in feedwater, sludge supernatant, and permeate of a pilot-scale membrane bioreactor (MBR) was investigated. Over 10 months, carbohydrates were observed to have a lower bioelimination degree (45%) and higher rejection degree (79%) than those of proteins (81% and 44%, respectively), which led to a high carbohydrate/protein ratio of dissolved organic matter (DOM) in sludge supernatant. The batch tests showed that DOM derived from feedwater and bound extracellular polymeric substances (EPS) was eliminated by activated sludge via biosorption and biodegradation. The proteins in bound EPS and feedwater were also found to have much higher biosorption potential (27% and 31%, respectively) than humic substances (11% and 17%, respectively) and carbohydrates (16% and 14%, respectively), indicating that proteins had a high affinity with sludge flocs. The results also showed that carbohydrates and humic substances in bound EPS were more difficult to be eliminated by activated sludge. In addition, the batch tests confirmed that feedwater was mainly composed of readily biodegradable matter, and bound EPS was mainly composed of slowly biodegradable matter. Size exclusion chromatography with continuous organic carbon and UV(254) detection (LC-OCD) showed that large-size substances (i.e., carbohydrates and macromolecular proteins) in sludge supernatant might originate from bound EPS, which can be rejected by membranes. The LC-OCD analysis, together with the results obtained from batch tests, suggested bound EPS might be the most important source of DOM in the sludge suspension.

Recent advances in membrane bioreactors (MBRs): membrane fouling and membrane material.

Membrane bioreactors (MBRs) have been actively employed for municipal and industrial wastewater treatments. So far, membrane fouling and the high cost of membranes are main obstacles for wider application of MBRs. Over the past few years, considerable investigations have been performed to understand MBR fouling in detail and to develop high-flux or low-cost membranes. This review attempted to address the recent and current developments in MBRs on the basis of reported literature in order to provide more detailed information about MBRs. In this paper, the fouling behaviour, fouling factors and fouling control strategies were discussed. Recent developments in membrane materials including low-cost filters, membrane modification and dynamic membranes were also reviewed. Lastly, the future trends in membrane fouling research and membrane material development in the coming years were addressed.

Impacts of membrane flux enhancers on activated sludge respiration and nutrient removal in MBRs.

This paper presents the findings of experimental investigations regarding the influence of 13 different flux enhancing chemicals (FeCl3, polyaluminium chloride, 2 chitosans, 5 synthetic polymers, 2 starches and 2 activated carbons) on respirometric characteristics and nitrification/denitrification performance of membrane bioreactor (MBR) mixed liquor. Flux enhancing chemicals are a promising method to reduce the detrimental effects of fouling phenomena via the modification of mixed liquor characteristics. However, potentially inhibiting effects of these chemicals on mixed liquor biological activity triggered the biokinetic studies (in jar tests) conducted in this work. The tested polyaluminium chloride (PACl) strongly impacted on nitrification (-16%) and denitrification rate (-43%). The biodegradable nature of chitosan was striking in endogenous and exogenous tests. Considering the relatively high costs of this chemical, an application for wastewater treatment does thus not seem to be advisable. Also, addition of one of the tested activated carbons strongly impacted on the oxygen uptake rate (-28%), nitrification (-90%) and denitrification rate (-43%), due to a decrease of pH. Results show that the changes in kLa values were mostly not significant, however, a decrease of 13% in oxygen transfer was found for sludge treated with PACl.

Correlating TMP increases with microbial characteristics in the bio-cake on the membrane surface in a membrane bioreactor.

Subcritical flux operation is widely practiced in membrane bioreactors (MBRs) to avoid severe membrane fouling and, thus, to maintain sustainable permeability. Filtration at a constant subcritical flux, however, usually leads to a two-stage increase in the transmembrane pressure (TMP): initially slowly, then abruptly. We have investigated the mechanism of this two-stage TMP increase through analyses of the structure and microbial characteristics of the bio-cake formed on the membrane. The MBR was operated under various subcritical and supercritical flux conditions. Under subcritical conditions, we observed the typical two-stage TMP increase. When a constant flux augmented and reached the supercritical conditions, however, the dual TMP change gradually transformed into a steeper, one-stage TMP increase. The second stage TMP increase under the subcritical flux was closely related to the sudden increase in the concentration of extra-cellular polymeric substances (EPSs) at the bottom layer of the bio-cake; we attribute the one-stage TMP increase under the supercritical conditions to the accumulation of microbial flocs and the reduced porosity of the bio-cake under compression. We explain the variation of the EPS concentration in the bio-cake in terms of the spatial and temporal changes of the live-to-dead ratio along the depth of the bio-cake.

Effect of anionic and nonionic surfactants on the kinetics of the aerobic heterotrophic biodegradation of organic matter in industrial wastewater.

While using the contemporary mathematical models for activated sludge systems, it is necessary to describe quantitatively the kinetics of microbiological processes and to characterise substrate (wastewater components in the influent) as well as biomass (activated sludge). In this paper, the kinetic parameters of the aerobic biodegradation of organic matter in wastewater containing synthetic surfactants in an activated sludge system were determined and discussed. Also, the composition of the tested wastewater was estimated and expressed as COD fractions. Five synthetic surfactants, three anionic and two nonionic, of different chemical structure were investigated. Each of them was tested separately and dissolved in wastewater to obtain a concentration of 50 mgl(-1), which can be found in some industrial wastewater. The presence of the elevated amount of synthetic surfactants in wastewater decreased the affinity of biomass to substrate. Nevertheless, maximum specific growth rates (micromax) of heterotrophic biomass exposed to wastewater containing surfactants were high but usually lower than micromax estimated for wastewater without surfactant. Surfactants, which contain a benzene ring, were the most likely to deteriorate wastewater treatment processes in the activated sludge systems.