Two-liquid Phase Research Articles

Styrene removal from polluted air in one and two-liquid phase biotrickling filter: Steady and transient-state performance and pressure drop control

A Sporothrix variecibatus-inoculated biotrickling filter (BTF) was examined for styrene removal, without and with the addition of silicone oil, at different empty bed residence times. The highest elimination capacities (ECs) were 172.8 (without silicone oil) and 670 g m −3 h −1 (with silicone oil), respectively, corresponding to a 4-fold improvement in presence of oil. The addition of silicone oil formed a well-coalesced emulsion of fungi and silicone oil, resulting in filter-bed clogging. Clogging prevention strategies adopted were; (i) lowering the volume ratio of silicone oil from 10% to 2% (v/v), and (ii) periodic increase in trickling rate of the medium from 50 to 190 mL min −1. During shock-load experiments, the BTF with silicone oil (2% v/v) could withstand high styrene loads, of up to 1900 g m −3 h −1, when compared to the BTF without silicone oil (400 g m −3 h −1).

Performance Evaluation and Neural Modeling of Gas-Phase Styrene Removal in One- and Two-Liquid Phase Suspended-Growth Bioreactors

The removal of gas-phase styrene was studied in both one- and two-liquid phase continuous stirred tank bioreactors (CSTBs) inoculated with Sporothrix variecibatus. Experiments were carried out at different gas residence times and inlet styrene concentrations to reach inlet loading rates varying between 10 and 838 g m−3 h−1 in the bioreactors. The addition of 10% (v/v) silicone oil acted as a buffer for high styrene loads and improved the two-liquid phase CSTB performance 3.1-fold in comparison to the one-liquid phase CSTB, with maximum elimination capacities of 426 and 137 g m−3 h−1, respectively. The CSTBs performance was modeled using artificial neural networks (ANNs) with inlet concentration (g m−3) and unit flow (h−1) as the input variables. The best network topology, selected by a trial and error approach and by estimating the determination coefficient (R2) values, was found to be 2−5−1 and 2−6−1, respectively, for the one- and two-liquid phase CSTB.

Effects of Autoclaving and Mercuric Chloride Sterilization on PAHs Dissipation in a Two-Liquid-Phase Soil Slurry

A two-liquid-phase (TLP) soil slurry system was employed to quantify the efficiencies of autoclaving and mercuric chloride sterilization in the dissipation of polycyclic aromatic hydrocarbons (PAHs). The fates of 11 PAHs (naphthalene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo( a)anthracene, benzo( a)pyrene, benzo( b)fluoranthene, benzo( k)fluoranthene, dibenzo( a, h)anth-racene) were recorded over 113 days of incubation. No microorganisms were detected in the HgCl 2-sterilized soil slurries during the whole incubation period, indicating very effective sterilization. However, about 2%–36% losses of PAHs were observed in the HgCl 2-sterilized slurry. In contrast to the HgCl 2-sterilized soil slurry, some microorganisms survived in the autoclaved soil slurries. Moreover, significant biodegradation of 6 PAHs (naphthalene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene) was observed in the autoclaved soil slurries. This indicated that biodegradation results of PAHs in the soil slurries, calculated on basis of the autoclaved control, would be underestimated. It could be concluded that the sterilization efficiency and effectiveness of HgCl 2 on soil slurry was much higher than those of autoclaving at 121 °C for 45 min.

Regioselective aromatic hydroxylation of quinaldine by water using quinaldine 4-oxidase in recombinant Pseudomonas putida

Biocatalytic hydrocarbon oxyfunctionalizations are typically accomplished using oxygenases in living bacteria as biocatalysts. These processes are often limited by either oxygen mass transfer, cofactor regeneration, and/or enzyme instabilities due to the formation of reactive oxygen species. Here, we discuss an alternative approach based on molybdenum (Mo)-containing dehydrogenases, which produce, rather than consume, reducing equivalents in the course of substrate hydroxylation and use water as the oxygen donor. Mo-containing dehydrogenases have a high potential for overcoming limitations encountered with oxygenases. In order to evaluate the suitability and efficiency of a Mo-containing dehydrogenase-based biocatalyst, we investigated quinaldine 4-oxidase (Qox)-containing Pseudomonas strains and the conversion of quinaldine to 4-hydroxyquinaldine. Host strain and carbon source selection proved to be crucial factors influencing biocatalyst efficiency. Resting P. putida KT2440 (pKP1) cells, grown on and induced with benzoate, showed the highest Qox activity and were used for process development. To circumvent substrate and product toxicity/inhibition, a two-liquid phase approach was chosen. Without active aeration and with 1-dodecanol as organic carrier solvent a productivity of 0.4 g l (tot) (-1) h(-1) was achieved, leading to the accumulation of 2.1 g l (tot) (-1) 4-hydroxyquinaldine in 6 h. The process efficiency compares well with values reported for academic and industrially applied biocatalytic oxyfunctionalization processes emphasizing the potential and feasibility of the Qox-containing recombinant cells for heteroaromatic carbon oxyfunctionalizations without the necessity for active aeration.

Development of a continuous steroid biotransformation process and product extraction within microchannel system

Results of an integrated approach for the improvement of progesterone 11α-hydroxylation by Rhizopus nigricans, which is one of the key steps in the production of corticosteroid drugs and hormones, are presented. Several issues have been addressed in order to increase process productivity, including biocatalyst immobilization, improvement in the solubility of the lipophilic substrate, setting-up of continuous reaction process and easing downstream processing. After preliminary studies on the effects on Rhizopus nigricans morphology, fungal pellets of a desired size and structure could be obtained. The mycelia grown in spherical agglomerates further enabled its continuous use in the process of biotransformation. The problem of low steroid solubility was addressed by the addition of organic solvents or β-cyclodextrin. In order to develop an integrated biotransformation process, a continuous two-liquid phase extraction within microchannel device has been set up, where very efficient steroid extraction was achieved within a few seconds.

Lattice Boltzmann modelling of liquid-liquid phase separation of monotectic alloys

本文建立了一个模拟在弥散相液滴的扩散长大、碰撞凝并和Ostwald熟化等因素的作用下偏晶合金液-液相分离过程的二维格子玻尔兹曼方法 (lattice Boltzmann method, LBM) 模型.该模型结合了Shan-Chen的两相流模型和Qin的介观粒子相互作用势模型的优点，并在LB演化方程中引入了反映相变的源项.应用该模型模拟研究了偏晶合金液-液相分离过程中单液滴的生长、两液滴的合并和多液滴的生长规律.结果表明在两液相区中第二相单个液滴的生长是一个通过扩散从非平衡态到平衡态过渡的过程.两液滴合并

Intensification and economic and ecological assessment of a biocatalytic oxyfunctionalization process

Bio-based processes are commonly accepted to be environmentally preferable to chemical alternatives. Reasons include high selectivities, the avoidance of heavy metals, and mild reaction conditions. However, ecological benefits and economic viability have to be verified in each case. Oxygenases are a very attractive enzyme class, allowing selective oxyfunctionalization by introduction of molecular oxygen into hydrocarbons at optical purities unparalleled by traditional chemical methods. Here, styrene monooxygenase from Pseudomonas sp. strain VLB120 was used in recombinant Escherichia coli for the production of enantiopure (S)-styrene oxide from styrene. Substrate and product toxicity was attenuated using a two-liquid phase system with bis(2-ethylhexyl)phthalate as organic carrier solvent. By maintaining previously reported productivities for a longer time period, product concentrations were doubled to 36.3 g Ltot−1 making it one of the most productive biocatalytic oxyfunctionalization processes. This biotransformation was incorporated along with an appropriate upstream and downstream processing into a complete process scheme and compared with chemical process alternatives. Ecological assessment showed the bioprocess to be superior to two chemical process alternatives and slightly inferior to the third one, with solvent use being the most critical factor. However, the bioprocess performed best in terms of production costs ($10.2 kg−1). This study underlines the importance of a detailed ecological and economic assessment of bioprocesses to verify their sustainability and to identify weak points with respect to environmental and/or economical sustainability.

Application of Young’s equation to two immiscible liquids resting on a solid substrate

The geometry of two immiscible liquid drops (water and mercury) resting on a solid substrate is reported. The problem under investigation is a very simple situation of a non-miscible two-phase liquid system which is properly documented. The simple experimental observation of water micro drop on top of a mercury drop on polypropylene substrate is discussed. The static situation of the drops is explored, and a new equation is derived for such situation by applying Young’s equation to the contact angles. The obtained equation simply represents the additive summation of Young equations for two independent drops, and may be applied for the case when the interfaces of two drops are close to each other and hence interact. The forces at the contact lines of each drop are treated separately as well as in correlation. This paper shows a schematic representation of two-liquid phase contact angle system. The value of this work may be appreciated from a pedagogical point of view.

Waste gas treatment in bioreactors: environmental engineering aspectsThis article is one of a selection of papers published in this Special Issue on Biological Air Treatment.

This paper gives an overview of the most important bioreactor configurations used in waste gas treatment, i.e., conventional and trickling biofilters, the bioscrubber, suspended-growth bioreactors, as well as two-liquid phase systems (stirred tank bioreactor and biotrickling filter). The historical evolution, main characteristics, and recent developments are described in each case.

Modelling gas–liquid VOCs transport in two-liquid phase partitioning bioreactors

A mechanistic model capable to accurately describe the mass transport of hexane in a two-liquid phase partitioning bioreactor (TLPB) constructed with silicone oil was developed. This work constitutes the first step in the development of simple and reliable models for the mathematical description of the off-gas treatment of volatile organic contaminants in TLPBs. The model (based on general mass balances and transport equations over off-gas rising bubbles) predicted a negative linear relationship between the fraction of organic phase and the logarithm of the pollutant fraction that remains in the gas phase. The average relative errors of the model predictions were lower than 7%. Under the tested range of operational conditions (organic phase fractions and stirring rates ranging from 5% to 30% and from 100 to 300 rpm, respectively) the maximum hexane fraction transferred from the gas to the liquid (aqueous + organic) phase increased at increasing stirring rates and silicone oil fractions up to 200 rpm and 20%, respectively. In addition, the proposed modelling approach quantified the proximity of the dispersion to thermodynamic equilibrium conditions, predicting thus the degree of mixing in the systems.

Development of a recombinant Escherichia coli-based biocatalyst to enable high styrene epoxidation activity with high product yield on energy source

A highly active recombinant whole-cell biocatalyst, Escherichia coli pETAB2/pG-KJE1, was developed for the efficient production of (S)-styrene oxide from styrene. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL–GroES and DnaK–DnaJ–GrpE). The styrene monooxygenases were produced to ca. 40% of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180 U/g CDW. The high StyAB activity in turn appeared to direct cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40%. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400 mM in the organic phase within 6 h, resulting in an average specific and volumetric productivity of 6.4 mmol/g dry cells/h (106 U/g dry cells) and 67 mM/h (1110 U/L aq), respectively, under mild reaction conditions. These results indicated that the high productivity and the high product yield on energy source were driven by the high enzyme activity. Therefore, it was concluded that oxygenase activity of whole-cell biocatalysts is one of the critical factors to determine their catalytic performance.

Enhanced degradation of bioremediation residues in petroleum-contaminated soil using a two-liquid-phase bioslurry reactor

A study was performed to determine the potential of two-liquid-phase (TLP) bioslurry reactors using silicon oil as solvent for degradation of residual contaminants in petroleum-contaminated soil. The residues were characterized by gas chromatography–mass spectrometry and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. This allowed for the identification of a mixture of residual biomarkers, metabolic byproducts, oxygenated and hetero-polynuclear aromatic hydrocarbons in the contaminated soil. The removal amount of total extractable organics (TEO) was 15 900 mg kg −1 soil in the TLP reactor within 12 weeks. However, TEO remained intact in the bioslurry reactor without the addition of silicon oil for the duration of the experiment, due to high toxicity of metabolites to the microorganisms. The availability of TEO was calculated using a mild extraction with Triton X-100, and the amount of TEO extracted was in accord with the amount of biodegraded TEO. Significantly reduced toxicity in soil was observed at week 12 through TLP remediation. Dehydrogenase activity in the bioslurry reactor was strongly suppressed. Fluorescein diacetate was significantly hydrolyzed by the composition of bioremediation residues in the contaminated soil. Microbial adhesion to the solvent was revealed by the determination of microbial activity in the water-immiscible-liquid.

Buffer interactions: Densities and solubilities of some selected biological buffers in water and in aqueous 1,4-dioxane solutions

The densities of aqueous 1,4-dioxane solutions of tris(hydroxymethyl)aminomethane (TRIS), N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), and N-tris[hydroxymethyl]-4-amino-butanesulfonic acid (TABS) were measured at T = 298.15, 303.15, 310.15, and 318.15 K, using a high precision vibrating tube digital densitometer. On the basis of these data, the isobaric thermal expansibilities, α( m, T), have been evaluated. The solubilities of these buffers in water and at several concentrations of 1,4-dioxane at 298.15 K have also been determined from the experimental results of density measurements. Two-liquid phase behavior was found from the binary system containing the TABS buffer over a given composition range. The solubility data were further used to calculate the free energy of transfer ( Δ G ′ tr ) of these buffers from water to aqueous 1,4-dioxane solutions at 298.15 K. We observed positive values of ( Δ G ′ tr ) for the buffers from water to 1,4-dioxane, indicating that the interactions between these biological buffers and 1,4-dioxane are unfavorable. The contribution of transfer free energies ( Δ g ′ tr ) of various functional groups from water to the aqueous 1,4-dioxane solutions, have been estimated.

Bioproduction of the aroma compound 2‐Phenylethanol in a solid–liquid two‐phase partitioning bioreactor system by Kluyveromyces marxianus

The rose-like aroma compound 2-phenylethanol (2-PE) is an important fragrance and flavor ingredient. Several yeast strains are able to convert l-phenylalanine (l-phe) to 2-PE among which Kluyveromyces marxianus has shown promising results. The limitation of this process is the low product concentration and productivity primarily due to end product inhibition. This study explored the possibility and benefits of using a solid-liquid Two-Phase Partition Bioreactor (TPPB) system as an in situ product removal technique. The system applies polymer beads as the sequestering immiscible phase to partition 2-PE and reduce the aqueous 2-PE concentration to non-inhibitory levels. Among six polymers screened for extracting 2-PE, Hytrel 8206 performed best with a partition coefficient of 79. The desired product stored in the polymer was ultimately extracted using methanol. A 3 L working volume solid-liquid batch mode TPPB using 500 g Hytrel as the sequestering phase generated a final overall 2-PE concentration of 13.7 g/L, the highest reported in the current literature. This was based on a polymer phase concentration of 88.74 g/L and aqueous phase concentration of 1.2 g/L. Even better results were achieved via contact with more polymers (approximately 900 g) with the aqueous phase applying a semi-continuous reactor configuration. In this system, a final 2-PE concentration (overall) of 20.4 g/L was achieved with 1.4 g/L in the aqueous and 97 g/L in the polymer phase. The overall productivities of these two reactor systems were 0.38 and 0.43 g/L h, respectively. This is the first report in the literature of the use of a polymer sequestering phase to enhance the bioproduction of 2-PE, and exceeds the performance of two-liquid phase systems in terms of productivity as well as ease of operation (no emulsions) and ultimate product recovery.

Phase-separated microstructures and shear-banding behavior in a designed Zr-based glass-forming alloy

We have employed a thermodynamic-computation approach to identify the composition of the Zr–Cu–Ni–Al alloy system exhibiting a two-liquid miscibility phase equilibrium in the liquid-temperature region, which tends to favor the occurrence of the liquid-phase separation. Guided by these calculated diagrams, a Zr-based alloy with a 10 at.% Al is designed, and its bulk-metallic glass (BMG) is prepared successfully by the copper-mould suction casting. A heterogeneous microstructure characterized by the existence of phase-separated regions with several to decades micrometers in size forms in the BMG. Under uniaxial compressive loading, the designed Zr-based BMG demonstrates the continuous “work hardening” and remarkable macroscopic plastic strain at room temperature. The improvement of mechanical properties is attributed to the unique glassy structure correlated with both the heterogeneous microstructure and the micro-scaled phase separation, leading to the extensive shear-band formation, interaction, and multiplication.

Efficient epoxidation of alkenes with hydrogen peroxide, lactone, and lipase

A green and efficient oxidation system containing hydrogen peroxide, lactone, and lipase was developed for the epoxidation of alkenes. A variety of alkenes was oxidized with this system, giving 87–95% analytical yield of the corresponding epoxides. The epoxidation occurred vialipase-catalyzed formation of hydroxy peroxy acid from lactone, without release of any harmful short-chain acid and alcohol, and in situ chemical oxidation of alkenes. Both hydrophilic ε-caprolactone and hydrophobic δ-decanolactone were shown to be good substrates to produce hydroxy peracids and good reaction solvents, and the method is suitable for the oxidation in either single phase or two-liquid phase. In comparison with other lipase-mediated oxidation systems, the new oxidation system gave higher yield, higher efficiency, and higher enzyme stability.

Improved production of ethyl-(R)-2-hydroxy-4-phenylbutyrate with pretreated Saccharomyces cerevisiae in water/organic solvent two-liquid phase systems

Baker's yeast pretreated with α-phenacyl chloride was employed to improve the enantioselectivity of the asymmetric reduction of ethyl-2-oxo-4-phenylbutyrate (EOPB) to ethyl-(R)-2-hydroxy-4-phenylbutyrate ((R)-EHPB) and maintain a high activity of the yeast. A water/organic solvent two-liquid phase system was also introduced to overcome the strong substrate and product inhibition of the enzyme; the highest catalytic activity and enantioselectivity were obtained in a water/benzene two-liquid phase system. When the reduction was catalyzed with pretreated yeast (300 mg mL−1 buffer) in the water/benzene two-liquid phase system (Vaq/Vben=20:40), 41.9% molar conversion of EOPB and 87.5% e.e. of (R)-EHPB were obtained in 48 h, using pH 8.0 phosphate buffer with 1.5% (v/v) of ethanol added as a co-substrate at 30°C, even with an initial EOPB concentration of 400 mM and a final EHPB concentration as high as 167.7 mM.

Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase

A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logP(ow) solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using (13)C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only approximately 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth.

Enrichment of a dioxin‐dehalogenating Dehalococcoides species in two‐liquid phase cultures

Enrichment cultures capable of reductively dechlorinating 1,2,4-trichlorodibenzo-p-dioxin (1,2,4-TrCDD) were shown to dechlorinate 1,2,3-trichlorobenzene (1,2,3-TrCB) to 1,3-dichlorobenzene. To test if this activity can be used to enrich for dioxin-dechlorinating bacteria, a two-liquid phase cultivation with 200 mM 1,2,3-TrCB dissolved in hexadecane was established. During the dechlorination of 1,2,3-TrCB, the number of 1,2,4-TrCDD-dechlorinating bacteria increased by four orders of magnitude, eventually accounting for 11% of the total cell number. Characterization of the bacterial communities of the initial dioxin-dechlorinating culture and of the trichlorobenzene enrichments by restriction fragment length polymorphism (RFLP) analysis of cloned 16S rRNA genes revealed a proportional increase of nine different sequence types, one representing a Dehalococcoides strain. Inhibition of methanogens further enhanced the rate of chlorobenzene dehalogenation and also resulted in a rapid dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin that was applied via a hexadecane phase. The further enrichment was monitored by terminal RFLP, quantitative real-time PCR and microscopy, and aimed at the reduction of the accompanying non-dehalogenating populations by using different combinations of electron donors and the application of antibiotics. Hydrogen as the sole electron donor proved to be less efficient due to the co-enrichment of acetogens. The novel Dehalococcoides strain DCMB5 was enriched up to 50% by the cultivation with organic acids, hydrogen and vancomycin, and was finally purified by conventional isolation techniques.

Bacterial Diversity of a Consortium Degrading High-Molecular-Weight Polycyclic Aromatic Hydrocarbons in a Two-Liquid Phase Biosystem

High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments. Although several PAH-degrading bacterial species have been isolated, it is not expected that a single isolate would exhibit the ability to degrade completely all PAHs. A consortium composed of different microorganisms can better achieve this. Two-liquid phase (TLP) culture systems have been developed to increase the bioavailability of poorly soluble substrates for uptake and biodegradation by microorganisms. By combining a silicone oil-water TLP system with a microbial consortium capable of degrading HMW PAHs, we previously developed a highly efficient PAH-degrading system. In this report, we characterized the bacterial diversity of the consortium with a combination of culture-dependent and culture-independent methods. Polymerase chain reaction (PCR) of part of the 16S ribosomal RNA gene (rDNA) sequences combined with denaturing gradient gel electrophoresis was used to monitor the bacterial population changes during PAH degradation of the consortium when pyrene, chrysene, and benzo[a]pyrene were provided together or separately in the TLP cultures. No substantial changes in bacterial profiles occurred during biodegradation of pyrene and chrysene in these cultures. However, the addition of the low-molecular-weight PAHs phenanthrene or naphthalene in the system favored one bacterial species related to Sphingobium yanoikuyae. Eleven bacterial strains were isolated from the consortium but, interestingly, only one-IAFILS9 affiliated to Novosphingobium pentaromativorans-was capable of growing on pyrene and chrysene as sole source of carbon. A 16S rDNA library was derived from the consortium to identify noncultured bacteria. Among 86 clones screened, 20 were affiliated to different bacterial species-genera. Only three strains were represented in the screened clones. Eighty-five percent of clones and strains were affiliated to Alphaproteobacteria and Betaproteobacteria; among them, several were affiliated to bacterial species known for their PAH degradation activities such as those belonging to the Sphingomonadaceae. Finally, three genes involved in the degradation of aromatic molecules were detected in the consortium and two in IAFILS9. This study provides information on the bacterial composition of a HWM PAH-degrading consortium and its dynamics in a TLP biosystem during PAH degradation.