Cloud Point System Research Articles

Effect of Triton X-100 on Shiraia laccase production and its application in triclosan biodegradation

Shiraia bambusicola, a bambusicolous fungus exhibits an excellent capability to produce the novel photosensitizer hypocrellin for photodynamic therapy. Recently, Shiraia species attract increasingly more attention as fungal laccase producer due to its extensive application in bioremediation. In this study, the laccase production was enhanced by the addition of surfactants in mycelium culture of Shiraia sp. S9. We sought to explore the mechanism for the surfactant induction and apply the culture to biodegrade a micropollutant triclosan. Triton X-100 was found to induce the burst of reactive oxygen species (ROS), increase membrane permeability and up-regulate laccase-related gene expression, leading to the enhanced laccase production to 9.2 × 104 U/L, about 108-folds over the control. After 2 days of Shiraia mycelium culture under Triton X-100 treatment (0.8%, w/v), the degradation and adsorption rate of triclosan reached 80.71% and 10.21%, respectively. Furthermore, Triton X-100-induced cloud point system was established after the biodegradation, which could further enrich triclosan in the coacervate phase. With the help of a combination of triclosan biodegradation and the cloud point extraction, triclosan was almost completely removed (99.88%) from water phase in the culture. To our knowledge, this is the first report on the biodegradation of triclosan by Shiraia mycelium culture. Our study provided insights into elicitation of surfactants on fungal laccase production and a new strategy for triclosan biodegradation.

A polycyclic aromatic hydrocarbon degrading strain and its potential of degrading phenanthrene in various enhanced systems

Polycyclic aromatic hydrocarbons (PAHs) are a class of common environmental pollutants that pose threats to human health. In this study, a mesophilic bacterial strain CFP312 (grown at 15-37 °C, optimal at 30 °C) was isolated from PAHs-contaminated soil samples. It was identified as Moraxella sp. by morphological observation, physiological and biochemical test, and 16S rRNA gene phylogeny analysis. This is the first reported PAHs degrading strains in Moraxella. Degradation analysis showed that 84% and 90% of the loaded phenanthrene (400 mg/L) were degraded within 48 h and 60 h, and the degradation rates reached 1.21 and 1.29 mg/(L·h), respectively. During the degradation of phenanthrene, phenanthrene-3,4-dihydrodiol was detected as an intermediate. Based on this, it was proposed that double oxygenation at the positions 3 and 4 of phenanthrene was the first step of biodegradation. Adaptability of strain CFP312 to different enhanced phenanthrene-degradation systems was tested in aqueous-organic system, micellar aqueous system, and cloud point system. Strain CFP312 showed good adaptability to different systems. In addition, the bacterium can rapidly degrade the phenanthrene in contaminated soil in slurry-aqueous system, indicating great potential in environmental remediation.

Continuous degradation of phenanthrene in cloud point system by reuse of Sphingomonas polyaromaticivorans cells

Extractive biodegradation of phenanthrene by Sphingomonas polyaromaticivorans was previously carried out in cloud point system (CPS). In this study, we explored the possibility of further increasing the efficiency of the culture by repeatedly reusing cells and the system for biodegradation. Three different recycling strategies were adopted. In reuse of cells plus CPS, cells were reused for 3 times while maintaining high degradation rates (> 90%). Thereafter, the accumulation of metabolites in the dilute phase resulted in a decrease in cell viability. This inhibition was avoided in recycling the cells plus coacervate phase by replacing the dilute phase with fresh Medium. However, due to the slow adaptation of the cells to the new degradation environment and the reduction in the volume of the coacervate phase, the cells were only reused twice and their activity decreased. However, the same long degradation cycle (5 days) as the reuse of cells plus coacervate phase reduced the overall degradation efficiency of phenanthrene. Finally, a combined strategy of 3 times of cells plus CPS reuse and individual cells reuse once was employed and run for two cycles. 3 rounds of reuse of cells plus CPS improved cells utilization and phenanthrene degradation efficiency. Then, the subsequent round of reuse of cells alone relieved the effect of increasing metabolites on cell viability. This study provides a potential application for reusing cells to continuously degrade phenanthrene in soil and water in CPS.

Extractive biodegradation of diphenyl ethers in a cloud point system: Pollutant bioavailability enhancement and surfactant recycling

The biodegradation of diphenyl ethers (DEs) in the environment is limited by their high hydrophobicity. The enhancement of DE bioavailability by a cloud point system (CPS) was investigated in this study. Three CPSs (i.e., Triton X-114, Triton X-114 + Triton X-45, and Brij30 + TMN-3) were tested to promote DE biodegradation. Biocompatibility tests showed that the biodegradation of DE and 4-bromodiphenyl ether (4-BDE) was inhibited by TX-114, unaffected by TX-114 + TX-45, and promoted by Brij30 + TMN-3 over 48 h of cultivation with Cupriavidus basilensis and 4% (w/v) nonionic surfactants. Further optimization with 2% (w/v) Brij30 + TMN-3 yielded residual DE and 4-BDE quantities of 143 and 154 mg/L, respectively, lower than quantities in the control. During degradation, DE content did not decrease in the dilute phase, but sharply decreased in the coacervate phase, indicating that the DEs gradually diffused and transferred from the coacervate phase to the dilute phase for degradation by microbial cells. This behavior also enhanced the bioavailability of DEs in the CPS. By removing the cell-rich dilute phase and adding fresh degradation medium and DE to the coacervate phase, surfactants were successfully recovered and reused twice without affecting DE biodegradation. Results demonstrated that a CPS with 2% (w/v) Brij30 + TMN-3 not only enhanced the bioavailability of DEs, but also decreased the treatment cost through surfactant recycling, which is beneficial for large-scale applications.

Biotoxicity and bioavailability of hydrophobic organic compounds solubilized in nonionic surfactant micelle phase and cloud point system.

A recent work has shown that hydrophobic organic compounds solubilized in the micelle phase of some nonionic surfactants present substrate toxicity to microorganisms with increasing bioavailability. However, in cloud point systems, biotoxicity is prevented, because the compounds are solubilized into a coacervate phase, thereby leaving a fraction of compounds with cells in a dilute phase. This study extends the understanding of the relationship between substrate toxicity and bioavailability of hydrophobic organic compounds solubilized in nonionic surfactant micelle phase and cloud point system. Biotoxicity experiments were conducted with naphthalene and phenanthrene in the presence of mixed nonionic surfactants Brij30 and TMN-3, which formed a micelle phase or cloud point system at different concentrations. Saccharomyces cerevisiae, unable to degrade these compounds, was used for the biotoxicity experiments. Glucose in the cloud point system was consumed faster than in the nonionic surfactant micelle phase, indicating that the solubilized compounds had increased toxicity to cells in the nonionic surfactant micelle phase. The results were verified by subsequent biodegradation experiments. The compounds were degraded faster by PAH-degrading bacterium in the cloud point system than in the micelle phase. All these results showed that biotoxicity of the hydrophobic organic compounds increases with bioavailability in the surfactant micelle phase but remains at a low level in the cloud point system. These results provide a guideline for the application of cloud point systems as novel media for microbial transformation or biodegradation.

Nonionic surfactants induced cloud point extraction of Polyhydroxyalkanoate from Cupriavidus necator

ABSTRACTPolyhydroxyalkanoate synthesized by Cupriavidus necator DSM 428 was purified from the crude fermentation broth as such by performing nonionic surfactants (Triton X100, Triton X114 & Tergitol 6) induced cloud point extraction. Polyhydroxyalkanoate was extracted into the micelle-rich bottom phase (coacervate phase), while most of the cellular impurities partitioned into the aqueous phase. Cloud point temperatures and the extraction efficiency of different cloud point systems were studied at different pH value and in the presence of additives. Maximum extraction of biopolymer was achieved (recovery of 84.4%) with a purity of 92.49% at 3 pH with the addition of 0.1 M ammonium chloride in the mixed surfactant system at a reduced cloud point temperature of 33°C.

An integrated process for thein siturecovery of prodigiosin using micellar ATPS from a culture ofSerratia marcescens

BACKGROUND In situ product recovery (ISPR), or extractive fermentation, is a strategy for the primary recovery of metabolites. A micellar aqueous two-phase system (ATPS) composed of a surfactant-rich phase was coupled to batch and continuous fermentations for the production and in situ recovery of the red alkaloid prodigiosin from Serratia marcescens. A cloud point ATPS was formed at 24 °C using triton X-114 and used as a production/extraction system for this metabolite, which shows therapeutic properties as antibiotic, anti-proliferative and anti-malarial activities. RESULTS The integration of the micellar ATPS in batch fermentation resulted in the production of 342 mg prodigiosin L−1 with a recovery of 81% in the extractive phase. When a continuous fermentation was performed, based on a cloud point system, 1.2-fold prodigiosin content was obtained compared with batch mode and a recovery of 88% of the metabolite. This continuous operation resulted in 0.43 g of purified prodigiosin from a 1.5 L bioreactor with a concentration of 872 mg prodigiosin L−1 of extractive phase and a purity of 82%, demonstrating particular selectivity of the system for prodigiosin as a primary recovery method. This is the first report on the continuous production of this generally recognized secondary metabolite. CONCLUSION The experimental approach proposed proved that an ISPR system could lead to the establishment of a more efficient process for continuous large-scale prodigiosin production. The proposal of a primary recovery strategy with only one bioprocess step enhances this metabolite production by lowering the inhibiting properties of prodigiosin for the microorganism. © 2016 Society of Chemical Industry

Extractive biodegradation and bioavailability assessment of phenanthrene in the cloud point system by Sphingomonas polyaromaticivorans.

The biological treatment of polycyclic aromatic hydrocarbons is an important issue. Most microbes have limited practical applications because of the poor bioavailability of polycyclic aromatic hydrocarbons. In this study, the extractive biodegradation of phenanthrene by Sphingomonas polyaromaticivorans was conducted by introducing the cloud point system. The cloud point system is composed of a mixture of (40g/L) Brij 30 and Tergitol TMN-3, which are nonionic surfactants, in equal proportions. After phenanthrene degradation, a higher wet cell weight and lower phenanthrene residue were obtained in the cloud point system than that in the control system. According to the results of high-performance liquid chromatography, the residual phenanthrene preferred to partition from the dilute phase into the coacervate phase. The concentration of residual phenanthrene in the dilute phase (below 0.001mg/L) is lower than its solubility in water (1.18mg/L) after extractive biodegradation. Therefore, dilute phase detoxification was achieved, thus indicating that the dilute phase could be discharged without causing phenanthrene pollution. Bioavailability was assessed by introducing the apparent logP in the cloud point system. Apparent logP decreased significantly, thus indicating that the bioavailability of phenanthrene increased remarkably in the system. This study provides a potential application of biological treatment in water and soil contaminated by phenanthrene.

Transferring of red Monascus pigments from nonionic surfactant to hydrophobic ionic liquid by novel microemulsion extraction

Nonionic surfactant micelle aqueous solution as well as cloud point system is a potential separation/reaction medium. Downstream processing for recovery of product and regeneration of surfactant is indispensable. Adjustment of pH in the dilute phase of cloud point extraction is a common strategy for separation of ionizable organic compound from nonionic surfactant. However, this strategy is no longer suitable for recovery of ionizable organic compound with strong hydrophobicity due to the high solubilization of the ionic state of hydrophobic organic compound in micelles. Setting extractive fermentation of intracellular Monascus pigments in nonionic surfactant micelle aqueous solution to produce high color hue of red Monascus pigments as an example, transferring of hydrophobic Monascus pigments from nonionic surfactant to ionic liquid by novel hydrophobic ionic liquid–nonionic surfactant–water Winsor I microemulsion extraction and then back-extraction of the ionizable Monascus pigments from the ionic liquid by ionic liquid–water two-phase extraction were carried out. Nearly 80% yield was achieved in the complete process for recovery of Monascus pigments. It provides a novel strategy for stripping of hydrophobic ionizable organic compound from nonionic surfactant micelle aqueous solution.

Microemulsion Extraction of Monascus Pigments from Nonionic Surfactant using High Polarity of Diethyl Ether as Excess Oil Phase

Stripping of organic compound from nonionic surfactant micelle aqueous solution is indispensable for many industrial processes. In this paper, a relatively high polarity diethyl ether was screened for forming Winsor I microemulsion, which was used for stripping of organic compound from nonionic surfactant. Setting up extractive fermentation of Monascus pigments in Triton X-100 aqueous solution as a model, cloud point extraction of Monascus pigments from fermentation broth, and back-extraction of Monascus pigments from the coacervate phase of cloud point system by Winsor I microemulsion were conducted. Monascus pigments were successfully separated from nonionic surfactant into the excess diethyl ether phase.

Cloud Point Extraction of Four Triphenylmethane Dyes by Triton X-114 as Nonionic Surfactant

A method for removing four triphenylmethane dyes from wastewater by cloud point extraction with the nonionic surfactant Triton X-114 (TX-114) was developed. The triphenylmethane dyes were crystal violet, ethyl violet, malachite green and brilliant green. The cloud point of TX-114 generally increased in the presence of any of the four dyes. In the cloud point system, these dyes were solubilized into a coacervate phase that left a color-free dilute phase. The extraction efficiency of the dyes increased with the temperature, TX-114 concentration, and salt (NaCl and CaCl2) concentration. More than 97% TX-114 in the dilute phase was recovered by adjusting the volume ratio of dichloromethane to the dilute phase. The Langmuir-type adsorption isotherm was used to describe the dye solubilization. The Langmuir constants m and n were calculated as functions of temperature. The results showed that the solubilization of the triphenylmethane dyes in the cloud point system was related to the partition coefficient and their molecular structures.

Partition coefficients of ionizable solutes in aqueous micellar two-phase systems

The potential of micellar cloud point systems for extraction processes has been demonstrated for a broad range of different applications. The extraction is based on the phenomenon that aqueous non-ionic surfactant solutions split into two phases if the temperature is increased beyond a certain temperature, the so called cloud point temperature (CPT). Recently, aqueous micellar two-phase systems have been implemented in a counter current extraction. However, almost equally important for large scale applications is the recovery of the solute from micellar solutions, which remains a serious concern considering micellar cloud point systems as alternative to conventional solvents. In conventional solvent extraction processes the influence of the pH on the partitioning of ionizable solutes is an important parameter to control the extraction and back extraction efficiency. However, the partitioning of ionizable solutes such as organic acids and bases in micellar two-phase systems has not been studied so far. In this work the partitioning of various ionizable solutes including phenolic as well as pharmaceutical active compounds has been investigated. Lipophilicity profiles of all solutes were analyzed for the system TritonX-114/water in a pH range between 2 and 12. Further, the influence of temperature, solute concentration, as well as different salt concentrations on the partitioning of charged solutes was investigated. Experimental results are verified by means of predicted values using the thermodynamic model COSMO-RS. The results reveal that COSMO-RS can be applied to predict the partitioning of ionizable solutes in micellar biphasic systems, when the phase behavior of the pure surfactant is known.Based on these data optimal strategies for large scale micellar extraction processes are proposed and validated by means of a continuous pilot plant micellar extraction column.

Cloud point extraction of microalgae cultures

The micellar in situ extraction of hydrophobic compounds from microalgae cultures (Chlamydomonas reinhardtii, Chlorella vulgaris, Scenedesmus obliquus) using nonionic surfactants (Triton X-114, Tergitol TMN 6, Tergitol 15-s-7) was studied in lab- and pilot scale. Therefore, phase separation behavior of the cloud point systems as well as the biocompatibility of the surfactants was investigated. It was shown that algae cells are concentrated in the aqueous phase, whereas the hydrophobic compounds (fatty acids) are enriched in the micellar phase. The thermodynamic model COSMO-RS was used to calculate partitioning coefficients of fatty acids between the phases. The biocompatibility of the surfactants applied was studied by monitoring the photosynthetic activity of algae upon contact with surfactants using puls amplitude modulation. Significant differences in growth and the photosynthetic activity of different algae strains after exposure to surfactants were determined. Whereas the photosynthetic activity of C. reinhardtii decreased rapidly upon contact with surfactants, the photosynthetic activity of S. obliquus, was 90% in comparison to a control after exposure for 240min to all tested surfactants. C. vulgaris showed an intermediate decrease. Thus, besides extraction, surfactants might be applied to control the growth of certain algae enabling the monoalgal culturing in outdoor cultivation of microalgae since the growth of the less surfactant-tolerant microalgae could be suppressed. Overall, the results indicate that a micellar extraction with nonionic surfactants offers the opportunity for an in situ extraction of hydrophobic substances directly from the culture medium and might be a promising alternative to a classic solvent extraction of dried algal biomass.

Extractive biodecolorization of triphenylmethane dyes in cloud point system by Aeromonas hydrophila DN322p

The biological treatment of triphenylmethane dyes is an important issue. Most microbes have limited practical application because they cannot completely detoxicate these dyes. In this study, the extractive biodecolorization of triphenylmethane dyes by Aeromonas hydrophila DN322p was carried out by introducing the cloud point system. The cloud point system is composed of a mixture of nonionic surfactants (20 g/L) Brij 30 and Tergitol TMN-3 in equal proportions. After the decolorization of crystal violet, a higher wet cell weight was obtained in the cloud point system than that of the control system. Based on the results of thin-layer chromatography, the residual crystal violet and its decolorized product, leuco crystal violet, preferred to partition into the coacervate phase. Therefore, the detoxification of the dilute phase was achieved, which indicated that the dilute phase could be discharged without causing dye pollution. The extractive biodecolorization of three other triphenylmethane dyes was also examined in this system. The decolorization of malachite green and brilliant green was similar to that of crystal violet. Only ethyl violet achieved a poor decolorization rate because DN322p decolorized it via adsorption but did not convert it into its leuco form. This study provides potential application of biological treatment in triphenylmethane dye wastewater.

Assessing bioavailability of the solubilization of organic compound in nonionic surfactant micelles by dose–response analysis

It is uncertain in some extent that organic compounds solubilized in micelles of a nonionic surfactant aqueous solution are bioavailable directly by the microbes in an extractive microbial transformation or biodegradation process. In this work, a dose-response method, where a bioequivalence concept is introduced to evaluate the synergic toxicity of the nonionic surfactants and the organic compounds, was applied to analyze the inhibition effect of organic compounds (naphthalene, phenyl ether, 2-phenylethanol, and 1-butanol) in nonionic surfactant Triton X-100 micelle aqueous solutions and Triton X-114 in aqueous solutions forming cloud point systems. Based on the result, a mole solubilization ratio of organic compounds in micelle was also determined, which consisted very well with those of classic semi-equilibrium dialysis experiments. The results exhibit that bioavailability of organic compounds solubilized in micelles to microbial cells is negligible, which provides a guideline for application of nonionic surfactant micelle aqueous solutions or cloud point systems as novel media for microbial transformations or biodegradations.

Stripping of nonionic surfactants from the coacervate phase of cloud point system for lipase separation by Winsor II microemulsion extraction with the direct addition of alcohols

Extractive microbial fermentation of lipase by Serratia marcescens ECU1010 in cloud point system was previously carried out in the cloud point system. The direct addition of different alcohols, including iso-butanol, 2-phenylethanol and 1-octanol, into the coacervate phase of the clear supernatant of the fermentation broth formed microemulsion, where the nonionic surfactants and lipase were unevenly partitioned between the different phases in the microemulsion system. The polarity of alcohols strongly affected the microemulsion type at room temperature condition. The results indicated that the Winsor II microemulsion, formed by the addition of iso-butanol or 2-phenylethanol as the organic solvent, favored the stripping of the nonionic surfactant into the O m phase, whereas the lipase was left in the excess aqueous phase. However, the Winsor I microemulsion, formed by the addition of 1-octanol as the organic solvent, failed to separate the lipase from the nonionic surfactant in the coacervate phase of cloud point system, because the nonionic surfactant and lipase were partitioned into the W m phase at the same time. Moreover, in the Winsor II microemulsion extraction with 2-phenylethanol as the organic solvent, in which case the protein–surfactant complexes were absent at the interface between the O m phase and the excess aqueous phase, the high lipase recovery (above 80%) and good nonionic surfactant removal were achieved. The effect of nonionic surfactants on lipase activity was also presented.

Flow injection system for the on-line preconcentration of Pb by cloud point extraction coupled to USN–ICP OES

A novel method for the determination of Pb by on-line cloud point extraction coupled to inductively coupled plasma optical emission spectrometry with ultrasonic nebulization (USN–ICP OES) is presented. The cloud point system was formed in the presence of non-ionic micelles of polyethyleneglycolmono- p-nonylphenylether (PONPE 7.5) and was retained in a minicolumn filled with particles of PTFE. Since the micelles of PONPE were able to extract Pb from the solutions, the use of a complexing reagent was not necessary. Afterwards, the surfactant-rich phase containing the analyte was removed from the minicolumn with nitric acid and introduced into the ultrasonic nebulizer. A total enhancement factor of 150 was obtained for a preconcentration time of 3.3 min, with respect to the direct determination of Pb by conventional ICP OES. The values of the detection (3 σ) and quantification (10 σ) limits for the preconcentration of 10 mL of sample solution were 0.09 µg L − 1 and 0.2 µg L − 1 respectively. The precision, expressed as the relative standard deviation (RSD), for 10 replicate determinations at 5.0 µg L − 1 Pb level was 6.0%. Verification of the accuracy was carried out by analysis of two certified reference materials (NIST SRM 1640e and VKI QC Metal LL1). The method was successfully applied to the determination of Pb in drinking water samples.

Extractive fermentation in cloud point system for lipase production by Serratia marcescens ECU1010

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.

A Closed Concept of Extractive Whole Cell Microbial Transformation of Benzaldehyde into l-Phenylacetylcarbinol by Saccharomyces cerevisiae in Novel Polyethylene-Glycol-Induced Cloud-Point System

Extractive microbial transformation of benzaldehyde into L-phenylacetylcarbinol (L-PAC) by Saccharomyces cerevisiae (Baker's yeast) has been carried out in a novel polyethylene-glycol-induced cloud-point system (PEG-CPS). The extractive microbial transformation in the PEG-CPS and a downstream process for stripping of the product from the microbial transformation broth with microemulsion extraction are demonstrated. The results indicate that the PEG-CPS maintains the advantage of CPS for in situ extraction of polar product in the microbial transformation. At the same time, the utilization of hydrophilic nonionic surfactant in the PEG-CPS is favorable for stripping of product from the nonionic surfactant in the microbial transformation broth by Winsor I microemulsion extraction. Thus, a closed concept of in situ extraction of polar product in microbial transformation and its downstream process of product recovery are fulfilled at the same time.

Novel polyethylene glycol induced cloud point system for extraction and back-extraction of organic compounds

A novel polyethylene glycol (PEG) induced cloud point system (PEG-CPS) with high hydrophile–lipophile balance (HLB) value nonionic surfactant and high molecular weight PEG had been developed for extraction of organic compounds. The main advantage of this novel system is that the back extraction process of stripping of nonvolatile organic compounds from the nonionic surfactant aqueous solution can be carried out with Winsor I microemulsion extraction. Thus a separation of organic compounds from the nonionic surfactants in aqueous solution become possible, which is potential for cloud point extraction (CPE) or extractive microbial transformation in cloud point system from economical and environmental consideration. With Triton X-100 as a model hydrophilic nonionic surfactant, the phase diagram of PEG-CPS was determined. High boiling point organic compounds, such as phenol, p-nitrophenol and 1-naphthol phenol, were extracted with PEG-CPS. Then the organic compounds in the surfactant rich phase of PEG-CPS were back-extracted with microemulsion.