Mining Marine Metagenomes Revealed a Quorum-Quenching Lactonase with Improved Biochemical Properties That Inhibits the Food Spoilage Bacterium Pseudomonas fluorescens.

The marine environment presents great potential as a source of microorganisms that possess novel enzymes with unique activities and biochemical properties. Examples of such are the quorum-quenching (QQ) enzymes that hydrolyze bacterial quorum-sensing (QS) signaling molecules, such as N-acyl-homoserine lactones (AHLs). QS is a form of cell-to-cell communication that enables bacteria to synchronize gene expression in correlation with population density. Searching marine metagenomes for sequences homologous to an AHL lactonase from the phosphotriesterase-like lactonase (PLL) family, we identified new putative AHL lactonases (sharing 30 to 40% amino acid identity to a thermostable PLL member). Phylogenetic analysis indicated that these putative AHL lactonases comprise a new clade of marine enzymes in the PLL family. Following recombinant expression and purification, we verified the AHL lactonase activity for one of these proteins, named moLRP (marine-originated lactonase-related protein). This enzyme presented greater activity and stability at a broad range of temperatures and pH, tolerance to high salinity levels (up to 5 M NaCl), and higher durability in bacterial culture, compared to another PLL member, parathion hydrolase (PPH). The addition of purified moLRP to cultures of Pseudomonas fluorescens inhibited its extracellular protease activity, expression of the protease encoding gene, biofilm formation, and the sedimentation process in milk-based medium. These findings suggest that moLRP is adapted to the marine environment and can potentially serve as an effective QQ enzyme, inhibiting the QS process in Gram-negative bacteria involved in food spoilage. IMPORTANCE Our results emphasize the potential of sequence and structure-based identification of new QQ enzymes from environmental metagenomes, such as from the ocean, with improved stability or activity. The findings also suggest that purified QQ enzymes can present new strategies against food spoilage, in addition to their recognized involvement in inhibiting bacterial pathogen virulence factors. Future studies on the delivery and safety of enzymatic QQ strategy against bacterial food spoilage should be performed.

The use of clay-polymer nanocomposites in wastewater pretreatment.

Some agricultural effluents are unsuitable for discharge into standard sewage-treatment plants: their pretreatment is necessary to avoid clogging of the filtering devices by colloidal matter. The colloidal stability of the effluents is mainly due to mutual repulsive forces that keep charged particles in suspension. Pretreatment processes are based on two separate stages: (a) neutralization of the charges ("coagulation") and (b) bridging between several small particles to form larger aggregates that sink, leaving clarified effluent ("flocculation"). The consequent destabilization of the colloidal suspension lowers total suspended solids (TSSs), turbidity, and other environmental quality parameters, making the treatments that follow more efficient. Clay-based materials have been widely used for effluent pretreatment and pollutant removal. This study presents the use of nanocomposites, comprised of an anchoring particle and a polymer, as "coagoflocculants" for the efficient and rapid reduction of TSS and turbidity in wastewater with a high organic load. The use of such particles combines the advantages of coagulant and flocculant by neutralizing the charge of the suspended particles while bridging between them and anchoring them to a denser particle (the clay mineral), enhancing their precipitation. Very rapid and efficient pretreatment is achieved in one single treatment step.

Sepiolite Clay Attenuates the Development of Hypercholesterolemia and Obesity in Mice Fed a High-Fat High-Cholesterol Diet.

Obesity reduces the quality of life and life expectancy, whereas nonoperative interventions have shown poor results so far. Statins effectively combat hypercholesterolemia but are not well tolerated at high doses, raising the need for coprescription with cholesterol sorbents and/or absorption inhibitors. Montmorillonite (MMT) clay was found to attenuate hypercholesterolemia and obesity by reducing cholesterol and fat absorption. However, acicular clay-like sepiolite may offer better results due to its more substantial adsorption of nonpolar molecules. We herein aimed at (1) assessing in vitro the capacity of sepiolite to adsorb edible oil and cholesterol compared with that of MMT and (2) assessing in vivo the effect of continuous feeding on a high-fat high-cholesterol diet (HFD) (53.6% w/w fat and 0.2% cholesterol) supplemented with 5% (w/w) edible sepiolite, on diet-induced obesity rate, hypercholesterolemia, and hyperlipidemia. Fourier transform infrared spectroscopy showed in vitro that sepiolite adsorption of olive oil and cholesterol was five to eight times greater than that of MMT clay. Sepiolite supplementation to HFD fed to mature mice for 12.5 weeks resulted in lower total blood cholesterol and triacylglycerol levels and attenuated body weight gain, by reducing fat gain. Sepiolite supplementation did not affect energy intake but increased fecal extraction of sterols and lipids, without notable side effects. These results demonstrate that supplementing a HFD with sepiolite attenuates gastrointestinal absorption of dietary lipids and sterols, thus mitigating obesity, hyperlipidemia, and hypercholesterolemia. Further exploration of the efficacy, mechanism of action, and safety of sepiolite as a food supplement for combating the metabolic syndrome is needed.

Hybrid Clay-Polymer Nanocomposites for the Clarification of Water and Effluents.

BACKGROUND: Freshwater resources will not be able to meet all requirements and water should not be considered a self-renewable, low cost resource. Thus, the needs for increased amount of water imply adopting several approaches that include intercepting and transferring water, desalination and water re-use. In all three approaches removal of turbidity and total suspended solids (TSS) is a crucial step, as one of the essential parameters limiting water quality. This paper reviews a series of patents focusing on the use of clay-polymer nanocomposites to obtain very fast and efficient turbidity and suspended solids removal in water and effluents. METHOD: The rational beyond the use of nanoparticles based on an anchoring denser core (for example a clay mineral) to which chains of a polyelectrolyte with charge that opposes the colloidal charges, is that it induces fast formation of neutralized flocs, that are denser than organic colloids. In such a way all three colloidal stability factors (size, charge and density) are addressed achieving very fast clarification. RESULTS: This paper summarizes several applications and examples of the procedure: removal of algae or microbes in surface water, clarification of saline effluents or sea water before desalination, and almost complete removal of turbidity and suspended solids in several industrial very turbid effluents. In all cases, clarification is obtained in seconds by the formation of 20-500 µm flocs, and their further separation by sedimentation or filtration, within a very wide pH range (3-11). CONCLUSION: Clay polymer nanocomposites can offer very effective clarification yielding reduction of more than 90% of the turbidity and the suspended solids in water or effluents, including in some cases deactivation of microorganisms that could offer an environmentally oriented alternative to chlorination. The good performance observed is presumably due to the broad versatility that stems from the use of different polymers and clays, and even different clay-polymer ratios yielding a wide range of charges.

Production of biochar from olive mill solid waste for heavy metal removal.

Commercial activated carbon (CAC) and biochar are useful adsorbents for removing heavy metals (HM) from water, but their production is costly. Biochar production from olive solid waste from two olive cultivars (Picual and Souri) and two oil production process (two- or three-phase) and two temperatures (350 and 450°C) was tested. The biochar yield was 24-35% of the biomass, with a surface area of 1.65-8.12m2g-1, as compared to 1100m2g-1 for CAC. Picual residue from the two-phase milling technique, pyrolysed at 350°C, had the best cumulative removal capacity for Cu+2, Pb+2, Cd+2, Ni+2 and Zn+2 with more than 85% compared to other biochar types and CAC. These results suggest that surface area cannot be used as a sole predictor of HM removal capacity. FTIR analysis revealed the presence of different functional groups in the different biochar types, which may be related to the differences in absorbing capacities.

Using the dual-mode model to describe adsorption of organic pollutants onto an organoclay.

Adsorption of 2,4,5-trichlorophenol (TCP) and picric acid (PA) by montmorillonite preloaded with crystal violet (CV) up to 80, 100, and 125% of the cation exchange capacity was studied. The adsorption isotherms indicated a strong interaction of TCP and PA with the organoclay. While the adsorption isotherms of both pollutants can be described by the Langmuir model with relatively good fit, the adsorption of TCP is more adequately described by the dual-mode model (DMM), which combines the Langmuir equation with a partition mechanism. Competitive adsorption experiments and adsorption experiments in water:ethanol mixed medium as a solvent confirmed the assumption that the removal of TCP from water by the organoclay follows two different mechanisms: site-specific adsorption occurs in the low concentration range, whereas a partition mechanism dominates the high concentration range. This double mechanism in organoclays exhibits both complete removal at low pollutant concentrations and high sorption capacity in the high concentration range, and therefore may be effective in water treatment for a wide range of pollution levels.

Sulfometuron incorporation in cationic micelles adsorbed on montmorillonite.

The aim of this study was to understand the interactions between alkylammonium cations present as monomers and micelles and a clay mineral, montmorillonite, to develop slow release formulations of anionic herbicides, such as sulfometuron (SFM) whose leaching in soils is an environmental and economic problem. In the proposed formulation the herbicide is incorporated in positively charged micelles of quaternary amine cations, which in turn adsorb on the negatively charged clay. The adsorption of hexadecyltrimethylammonium (HDTMA) and octadecyltrimethylammonium (ODTMA) on montmorillonite was studied above and below their critical micelle concentrations (CMC). At concentrations above the CMC, the loading exceeded the clay's cation exchange capacity (CEC) and indicated higher affinity of the cation with the longer alkyl chain. An adsorption model could adequately simulate adsorption at concentrations below the CMC, and yield fair predictions for the effect of ionic strength. The model indicated that above the CMC adsorbed micelles contributed significantly to the amount of ODTMA adsorbed. Evidence for adsorption of ODTMA micelles on montmorillonite was provided by X-ray diffraction, freeze-fracture electron microscopy, and dialysis bag measurements. SFM was not adsorbed directly on the clay mineral, and adsorbed at low levels, when the organic cation was adsorbed as monomers. In contrast, a large fraction of SFM adsorbed on the clay mineral when incorporated in micelles that adsorbed on the clay.

Addition of a monovalent cationic pesticide to improve efficacy of bipyridyl herbicides in Hulah Valley soils.

Bipyridyl herbicides are widely used in agriculture and gardening for non-selective weed control. Since they are toxic and relatively expensive, it is ecologically and economically desirable to reduce the amounts applied. A decrease in efficacy of these herbicides is caused by dust accumulated on leaves of weeds. This inactivation arises from the adsorption of the herbicides on dust particles, mainly made of clay minerals, lime and soil organic matter. In order to improve the efficacy and so lower the amounts applied, formulations were developed which include cationic pesticides approved for agricultural use, such as mepiquat or difenzoquat. Such addition restored the efficacy of the bipyridyl herbicides by reducing their binding to dust particles. The proposed formulations, which were tested on a number of different dust-covered plants, allowed the amounts of herbicide applied to be reduced to 50% of the minimum recommended rate. Neither mepiquat or difenzoquat had any herbicidal activity when sprayed alone at the added rates. The results suggest a procedure that may lower the required rates of contact herbicides, reducing costs and toxicity. This procedure, which can be applied immediately, may have broad implications in farming and gardening.

Metachromasy as an indicator of photostabilization of methylene blue adsorbed to clays and minerals.

The influence of methylene blue adsorption to different clays on its photodegradation was studied. Methylene blue in solution was decomposed by sunlight in a zero-order process. Adsorption to some clay minerals (sepiolite and vermiculite) and a zeolite (clinoptilolite) accelerated the degradation process, and converted it to a first-order reaction. On the other hand, adsorption to other clay minerals (palygorskite and montmorillonite) stabilized the dye and prevented its degradation. Interestingly, in the clay-dye complexes that exhibited stability, clear metachromasy of the adsorbed methylene blue occurred, whereas the effect was not observed in the clay-dye complexes that underwent photodegradation.

Clarification of olive mill and winery wastewater by means of clay-polymer nanocomposites.

Highly polluted effluents from olive mills and wineries, among others, are unsuitable for discharge into standard sewage-treatment plants due to the large amounts of organic and suspended matter. Efficiency of all management practices for such effluents depends on an effective pretreatment that lowers the amount of suspended solids. Such pretreatments are usually based on three separate stages, taking a total of 2 to 6h: coagulation-neutralizing the colloids, flocculation-aggregating the colloids into larger particles, and separation via filtration or decanting. Previous studies have presented the concept of coagoflocculation based on the use of clay-polymer nanocomposites. This process adds a higher density clay particle to the flocs, accelerating the process to between 15 and 60 min. This study examined suitable nanocomposites based on different clays and polymers. The charge of the compounds increased proportionally to the polymer-to-clay ratio. X-ray diffraction (XRD) measurements indicated that in sepiolite-based nanocomposites there is no change in the structure of the mineral, whereas in smectite-based nanocomposites, the polymer intercalates between the clay layers and increases the spacing depending on the polymer-to-clay ratio. Efficiency of the coagoflocculation process was studied with a dispersion analyzer. Sequential addition of olive mill or winery effluents with a boosting dose of nanocomposites may yield a very efficient and rapid clarification pretreatment.

Improved colorimetric determination of chitosan concentrations by dye binding.

The increasing use of chitosan encourages the search for fast and sensitive methods to quantify its concentrations in water solutions. Recent colorimetric studies have suggested quantification by binding chitosan to Cibacron Brilliant Red 3B-A (BR) dye and measuring the absorbance at 575 nm related to the complex that is formed. This study presents an improved colorimetric technique based on the complexation reaction between BR and chitosan. However, instead of measuring the spectra of the dispersed chitosan-dye complex, the solution is centrifuged for the sedimentation of the colloids, and the concentration of the uncomplexed dye in the supernatant is measured. By performing this simple procedure, sensitivity can be improved to >2 ppm. Equilibration time did not influence the measurements. Charge measurements and bathochromic shift of the measured spectra might yield information about the mechanism of the interaction between the dye and the polymer.

Using a Matlab implemented algorithm for UV-vis spectral resolution for pKa determination and multicomponent analysis.

A Matlab implemented computer code for spectral resolution is presented. The code enables the user to resolve the UV-visible absorption spectrum of a mixture of up to 3 previously known components, to the individual components, thus, evaluating their quantities. The resolving procedure is based on searching the combination of the components which yields the spectrum which is the most similar (minimal RMSE) to the measured spectrum of the mixture. Examples of using the software for pK(a) value estimation and multicomponent analysis are presented and other implementations are suggested.

Modeling adsorption-desorption processes of Cu on edge and planar sites of montmorillonite.

The effect of the ionic strength on adsorption of Cu on calcium montmorillonite was studied at concentrations ranging from 31 to 516 microM. An adsorption model was employed in the analysis of the data. When the background electrolyte was NaClO4, the ionic exchange was suppressed at 0.5 M concentration, and Cu adsorption took place on edge sites, reaching a plateau at about 24 mmol/kg. A further increase in ionic strength did not have any effect on Cu adsorption, suggesting that the heavy metal was adsorbed by inner-sphere complexes on the edge sites of the clay. A binding coefficient for Cu2+ on the edge sites K = 2 x 10(4) M(-1) was determined, indicating very high affinity of Cu2+ for these sites. When the electrolyte used was NaCl, the amounts of Cu adsorbed were reduced. The model predicted well the adsorption data by considering the adsorption of CuCl+ species. Adsorption-desorption processes of Cu on calcium montmorillonite in media of 0.01 and 0.1 M NaCl showed hysteresis. Model calculations also predict the desorption fairly well. According to the model, the hysteresis is mainly attributed to the heterogeneity of sites for the adsorption of Cu. The hysteresis arising from the planar sites is largely due to reduced competition of ion species for adsorption and enhancement in the magnitude of the surface potential.