Moshe Herzberg

Antibacterial effects of carbon nanotubes: size does matter!

We provide the first evidence that the size (diameter) of carbon nanotubes (CNTs) is a key factor governing their antibacterial effects and that the likely main CNT-cytotoxicity mechanism is cell membrane damage by direct contact with CNTs. Experiments with well-characterized single-walled carbon nanotubes (SWNTs) and multiwalled carbon nanotubes (MWNTs) demonstrate that SWNTs are much more toxic to bacteria than MWNTs. Gene expression data show that in the presence of both MWNTs and SWNTs, Escherichia coli expresses high levels of stress-related gene products, with the quantity and magnitude of expression being much higher in the presence of SWNTs.

Physiology and genetic traits of reverse osmosis membrane biofilms: a case study with Pseudomonas aeruginosa

Biofilm formation of Pseudomonas aeruginosa on the surface of a reverse osmosis (RO) membrane was studied using a synthetic wastewater medium to simulate conditions relevant to reclamation of secondary wastewater effluent. P. aeruginosa biofilm physiology and spatial activity were analyzed following growth on the membrane using a short-life green fluorescent protein derivative expressed in a growth-dependent manner. As a consequence of the limiting carbon source prevailing in the suspended culture of the RO unit, a higher distribution of active cells was observed in the biofilm close to the membrane surface, likely due to the higher nutrient levels induced by concentration polarization effects. The faster growth of the RO-sessile cells compared to the planktonic cells in the RO unit was reflected by the transcriptome of the two cultures analyzed with DNA microarrays. In contrast to the findings recently reported in gene expression studies of P. aeruginosa biofilms, in the RO system, genes related to stress, adaptation, chemotaxis and resistance to antibacterial agents were induced in the planktonic cells. In agreement with the findings of previous P. aeruginosa biofilm studies, motility- and attachment-related genes were repressed in the RO P. aeruginosa biofilm. Supported by the microarray data, an increase in both motility and chemotaxis phenotypes was observed in the suspended cells. The increase in nutrient concentration in close proximity to the membrane is suggested to enhance biofouling by chemotaxis response of the suspended cells and their swimming toward the membrane surface.

Impact of microfiltration treatment of secondary wastewater effluent on biofouling of reverse osmosis membranes

The effects of microfiltration (MF) as pretreatment for reverse osmosis (RO) on biofouling of RO membranes were analyzed with secondary wastewater effluents. MF pretreatment reduced permeate flux decline two- to three-fold, while increasing salt rejection. Additionally, the oxygen uptake rate (OUR) in the biofouling layer of the RO membrane was higher for an RO system that received pretreated secondary wastewater effluent compared to a control RO system that received untreated secondary effluent, likely due to the removal of inert particulate/colloidal matter during MF. A higher cell viability in the RO biofilm was observed close to the membrane surface irrespective of pretreatment, which is consistent with the biofilm-enhanced concentration polarization effect. Bacterial 16S rRNA gene clone library analysis revealed dominant biofilm communities of Proteobacteria and Bacteroidetes under all conditions. The Cramer-von Mises test statistic showed that MF pretreatment did not significantly change the bacterial community structure of RO membrane biofilms, though it affected bacterial community structure of non-membrane-associated biofilms (collected from the feed tank wall). The finding that the biofilm community developed on the RO membrane was not influenced by MF pretreatment may imply that RO membranes select for a conserved biofilm community.

The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment

Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layers thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.

Pseudomonas aeruginosa attachment on QCM-D sensors: the role of cell and surface hydrophobicities.

While biofilms are ubiquitous in nature, the mechanism by which they form is still poorly understood. This study investigated the process by which bacteria deposit and, shortly after, attach irreversibly to surfaces by reorienting to create a stronger interaction, which leads to biofilm formation. A model for attachment of Pseudomonas aeruginosa was developed using a quartz crystal microbalance with dissipation monitoring (QCM-D) technology, along with a fluorescent microscope and camera to monitor kinetics of adherence of the cells over time. In this model, the interaction differs depending on the force that dominates between the viscous, inertial, and elastic loads. P. aeruginosa, grown to the midexponential growth phase (hydrophilic) and stationary phase (hydrophobic) and two different surfaces, silica (SiO(2)) and polyvinylidene fluoride (PVDF), which are hydrophilic and hydrophobic, respectively, were used to test the model. The bacteria deposited on both of the sensor surfaces, though on the silica surface the cells reached a steady state where there was no net increase in deposition of bacteria, while the quantity of cells depositing on the PVDF surface continued to increase until the end of the experiments. The change in frequency and dissipation per cell were both positive for each overtone (n), except when the cells and surface are both hydrophilic. In the model three factors, specifically, viscous, inertial, and elastic loads, contribute to the change in frequency and dissipation at each overtone when a cell deposits on a sensor. On the basis of the model, hydrophobic cells were shown to form an elastic connection to either surface, with an increase of elasticity at higher overtones. At lower overtones, hydrophilic cells depositing on the hydrophobic surface were shown to also be elastic, but as the overtone increases the connection between the cells and sensor becomes more viscoelastic. In the case of hydrophilic cells interacting with the hydrophilic surface, the connection is viscous at each overtone measured. It could be inferred that the transformation of the viscoelasticity of the cell-surface connection is due to changes in the orientation of the cells to the surface, which allow the bacteria to attach irreversibly and begin biofilm formation.

Surface properties and reduced biofouling of graft-copolymers that possess oppositely charged groups.

Microbial biofilms and their components present a major obstacle for ensuring the long-term effectiveness of membrane processes. Graft polymerization on membrane surfaces, in general, and grafting with oppositely charged monomers, have been shown to reduce biofouling significantly. In this study, surface forces and macromolecular properties of graft copolymers that possess oppositely charged groups were related to their potent antibiofouling behavior. Graft polymerization was performed using the negatively charged 3-sulphopropyl methacrylate (SPM) and positively charged [2-(methacryloyloxy)ethyl]-trimethylammonium (MOETMA) monomers to yield a copolymer layer on polyvinylidene fluoride (PVDF) surface. Quartz crystal microbalance with dissipation monitoring (QCM-D) technology was used to monitor the reduced adsorption of extracellular polymeric substances (EPS) extracted from a membrane bioreactor (MBR) wastewater treatment facility. Complemented measurements of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy provided evaluation of the antifouling properties of the surface. Increase in water content in grafted layer exposed to 100 mM aqueous NaCl solution was observed by QCM-D. Therefore, the grafted copolymer layer is swelled in the presence of 100 mM NaCl because of reversing of polymer self-association by counterions. Force measurements by atomic force microscopy (AFM) showed an increased repulsion between a carboxylate-modified latex (CML) particle probe and a modified PVDF surface, especially in the presence of 100 mM NaCl. The hydration and swelling of the grafted polymer layer are shown to repel EPS and reduce their adsorption. Delineating the surface properties of antifouling grafted layers may lead to the design of novel antifouling surfaces.

Bacterial Attachment and Viscoelasticity: Physicochemical and Motility Effects Analyzed Using Quartz Crystal Microbalance with Dissipation (QCM-D)

This investigation is focused on the combined effect of bacterial physicochemical characteristics and motility on cell adhesion and deposition using a flow-through quartz crystal microbalance with dissipation (QCM-D). Three model flagellated strains with different degrees of motility were selected, including a highly motile Escherichia coli K12 MG1655, an environmental strain Sphingomonas wittichii RW1, and a nonmotile (with paralyzed flagella) Escherichia coli K12 MG1655 ΔmotA that is incapable of encoding the motor torque generator for flagellar movement. Of the three strains, S. wittichii RW1 is highly hydrophobic, while E. coli strains are equally hydrophilic. Consideration of the hydrophobicity provides an alternative explanation for the bacterial adhesion behavior. QCM-D results show that motility is a critical factor in determining bacterial adhesion, as long as the aquatic chemical conditions are conducive for motility and the substratum and bacterial surface are similarly hydrophobic or hydrophilic. Once their properties are not similar, the contribution of hydrophobic interactions becomes more pronounced. QCM-D results suggest that during adhesion of the hydrophobic bacterium, S. wittichii RW1, the initial step of adhesion and maturation of bacteria-substratum interaction on hydrophilic surface includes a dynamic change of the viscoelastic properties of the bond bacterium-surface becoming more viscously oriented.

The influence of antiscalants on biofouling of RO membranes in seawater desalination.

Antiscalants are surface active polyelectrolyte compounds commonly used in reverse osmosis (RO) desalination processes to avoid membrane scaling. In spite of the significant roles of antiscalants in preventing membrane scaling, they are prone to enhance biofilm growth on RO membranes by either altering membrane surface properties or by serving as nutritional source for microorganisms. In this study, the contribution of antiscalants to membrane biofouling in seawater desalination was investigated. The effects of two commonly used antiscalants, polyphosphonate- and polyacrylate-based, were tested. The effects of RO membrane (DOW-Filmtec SW30 HRLE-400) exposure to antiscalants on its physico-chemical properties were studied, including the consequent effects on initial deposition and growth of the sessile microorganisms on the RO membrane surface. The effects of antiscalants on membrane physico-chemical properties were investigated by filtration of seawater supplemented with the antiscalants through flat-sheet RO membrane and changes in surface zeta potential and hydrophobicity were delineated. Adsorption of antiscalants to polyamide surfaces simulating RO membranes polyamide layer and their effects on the consequent bacterial adhesion was tested using a quartz crystal microbalance with dissipation monitoring technology (QCM-D) and direct fluorescent microscopy. A significant increase in biofilm formation rate on RO membranes surface was observed in the presence of both types of antiscalants. Polyacrylate-based antiscalant was shown to enhance initial cell attachment as observed with the QCM-D and a parallel plate flow cell, due to rendering the polyamide surface more hydrophobic. Polyphosphonate-based antiscalants also increased biofilm formation rate, most likely by serving as an additional source of phosphorous to the seawater microbial population. A thicker biofilm layer was formed on the RO membrane when the polyacrylate-based antiscalant was used. Following these results, a wise selection of antiscalants for scaling control should take into account their contribution to membrane biofouling propensity.

Reduced bacterial deposition and attachment by quorum-sensing inhibitor 4-nitro-pyridine-N-oxide: the role of physicochemical effects.

Surface-attached chemical groups that resist protein adhesion are commonly characterized as being hydrophilic, H-bond acceptors, non-H-bond donors, and electrically neutral. Quorum-sensing (QS) inhibitor 4-nitropyridine-N-oxide (4-NPO) that previously was found to decrease Pseudomonas aeruginosa biofilm formation possesses all of these characteristics, making this molecule an ideal antiadhesive compound. It was hypothesized that once 4-NPO adsorbs to either the solid surface or bacteria, resultant changes in the physical-chemical surface properties of the solid surface and bacteria will reduce the extent of bacterial adhesion. These physical-chemical effects take place prior to the commencement of already well-established QS biofilm-inhibition mechanisms. Bacterial adhesion experiments to silica conducted in quartz crystal microbalance with dissipation (QCM-D) and parallel plate flow cells demonstrated that 4-NPO reduces bacterial adhesion to silica-coated surfaces by the adsorption of 4-NPO to the silica surface as well to the outer membrane of both gram-negative P. aeruginosa PAO1 and gram-positive Staphylococcus aureus. 4-NPO effectively neutralizes both the bacterial and silica surface charge, and it is proposed that this neutralization of local surface charge heterogeneities by 4-NPO adsorption is the mechanism responsible for decelerating rates of bacterial deposition.

‘Should I stay or should I go?’ Bacterial attachment vs biofilm formation on surface-modified membranes

A number of techniques are used for testing the anti-biofouling activity of surfaces, yet the correlation between different results is often questionable. In this report, the correlation between initial bacterial deposition (fast tests, reported previously) and biofilm growth (much slower tests) was analyzed on a pristine and a surface-modified reverse osmosis membrane ESPA-1. The membrane was modified with grafted hydrophilic polymers bearing negatively charged, positively charged and zwitter-ionic moieties. Using three different bacterial strains it was found that there was no general correlation between the initial bacterial deposition rates and biofilm growth on surfaces, the reasons being different for each modified surface. For the negatively charged surface the slowest deposition due to the charge repulsion was eventually succeeded by the largest biofilm growth, probably due to secretion of extracellular polymeric substances (EPS) that mediated a strong attachment. For the positively charged surface, short-term charge attraction by quaternary amine groups led to the fastest deposition, but could be eventually overridden by their antimicrobial activity, resulting in non-consistent results where in some cases a lower biofilm formation rate was observed. The results indicate that initial deposition rates have to be used and interpreted with great care, when used for assessing the anti-biofouling activity of surfaces. However, for a weakly interacting ‘low-fouling’ zwitter-ionic surface, the positive correlation between initial cell deposition and biofilm growth, especially under flow, suggests that for this type of coating initial deposition tests may be fairly indicative of anti-biofouling potential.