Effects of silver nanoparticles on wastewater biofilms

The goal of this research is to understand the potential antibacterial effect of silver nanoparticles (Ag-NPs) on biological wastewater treatment processes. It was found that original wastewater biofilms are highly tolerant to the Ag-NP treatment. With an application of 200 mg Ag/L Ag-NPs, the reduction of biofilm bacteria measured by heterotrophic plate counts was insignificant after 24 h. After the removal of loosely bound extracellular polymeric substances (EPS), the viability of wastewater biofilms was reduced when treated under the same conditions. By contrast, when treated as planktonic pure culture, bacteria isolated from the wastewater biofilms were highly vulnerable to Ag-NPs. With a similar initial cell density, most bacteria died within 1 h with the application of 1 mg Ag/L Ag-NPs. The results obtained here indicate that EPS and microbial community interactions in the biofilms play important roles in controlling the antimicrobial effects of Ag-NPs. In addition, slow growth rates may enhance the tolerance of certain bacteria to Ag-NPs. The effects of Ag-NPs on the entire microbial community in wastewater biofilms were analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis, PCR-DGGE. The studies showed that the microbial susceptibility to Ag-NPs is different for each microorganism. For instance, Thiotrichales is more sensitive to Ag-NPs than other biofilm bacteria.     

Efficiency of phenol biodegradation by planktonic Pseudomonas pseudoalcaligenes (a constructed wetland isolate) vs. root and gravel biofilm

In the last two decades, constructed wetland systems gained increasing interest in wastewater treatment and as such have been intensively studied around the world. While most of the studies showed excellent removal of various pollutants, the exact contribution, in kinetic terms, of its particular components (such as: root, gravel and water) combined with bacteria is almost nonexistent.In the present study, a phenol degrader bacterium identified as Pseudomonas pseudoalcaligenes was isolated from a constructed wetland, and used in an experimental set-up containing: plants and gravel. Phenol removal rate by planktonic and biofilm bacteria (on sterile Zea mays roots and gravel surfaces) was studied. Specific phenol removal rates revealed significant advantage of planktonic cells (1.04 × 10⁻⁹ mg phenol/CFU/h) compared to root and gravel biofilms: 4.59 × 10⁻¹¹-2.04 × 10⁻¹⁰ and 8.04 × 10⁻¹¹-4.39 × 10⁻¹⁰ (mg phenol/CFU/h), respectively.In batch cultures, phenol biodegradation kinetic parameters were determined by biomass growth rates and phenol removal as a function of time. Based on Haldane equation, kinetic constants such as μ_max = 1.15/h, K_s = 35.4 mg/L and K_i = 198.6 mg/L fit well phenol removal by P. pseudoalcaligenes.Although P. pseudoalcaligenes planktonic cells showed the highest phenol removal rate, in constructed wetland systems and especially in those with sub-surface flow, it is expected that surface associated microorganisms (biofilms) will provide a much higher contribution in phenol and other organics removal, due to greater bacterial biomass.Factors affecting the performance of planktonic vs. biofilm bacteria in sub-surface flow constructed wetlands are further discussed.     

Bacterial response to a shock load of nanosilver in an activated sludge treatment system

The growing release of nanosilver into sewage systems has increased the concerns on the potential adverse impacts of silver nanoparticles (AgNPs) in wastewater treatment plants. The inhibitory effects of nanosilver on wastewater treatment and the response of activated sludge bacteria to the shock loading of AgNPs were evaluated in a Modified Ludzack-Ettinger (MLE) activated sludge treatment system. Before shock-loading experiments, batch extant respirometric assays determined that at 1 mg/L of total Ag, nitrification inhibitions by AgNPs (average size = 1-29 nm) and Ag⁺ ions were 41.4% and 13.5%, respectively, indicating that nanosilver was more toxic to nitrifying bacteria in activated sludge than silver ions. After a 12-h period of nanosilver shock loading to reach a final peak silver concentration of 0.75 mg/L in the MLE system, the total silver concentration in the mixed liquor decreased exponentially. A continuous flow-through model predicted that the silver in the activated sludge system would be washed out 25 days after the shock loading. Meanwhile, a prolonged period of nitrification inhibition (>1 month, the highest degree of inhibition = 46.5%) and increase of ammonia/nitrite concentration in wastewater effluent were observed. However, nanosilver exposure did not affect the growth of heterotrophs responsible for organic matter removal. Microbial community structure analysis indicated that the ammonium-oxidizing bacteria and nitrite-oxidizing bacteria, Nitrospira, had experienced population decrease while Nitrobacter was washed out after the shock loading.     

Pulsed ultraviolet light inactivation of Pseudomonas aeruginosa and Staphylococcus aureus biofilms

Microbial biofilms are complex communities that form when planktonic bacterial species attach to surfaces in many settings where they can provide a source of pathogenicity. The relative ineffectiveness of conventional disinfectants such as free chlorine and monochloramine for the inactivation of some species found in water has led to evaluation of alternative disinfectants for drinking and wastewater treatment. In recent years, novel pulsed power electrotechnologies have been introduced and are being considered as possible alternatives to current methods for inactivating problematic species in water. This study focuses on the ultraviolet (UV) inactivation of bacterial biofilms using a pulsed UV light approach as a potential disinfection method for water treatment operating systems. Biofilms were stimulated to form attached to polyvinyl chloride coupons using a recommended Centre for Disease Control biofilm reactor followed by exposure to a range of UV doses. Findings show that this method is highly effective at inactivating both planktonic and biofilm cells with significant inactivation rates obtained for both test species. Specifically, a 7.2 and 5.9 log₁₀ inactivation was achieved with up to 21.6 μJ/cm² UV for Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Findings from this study highlight the effectiveness of pulsed UV for the inactivation of Pseudomonas biofilms among other test species. Research conducted by this group suggests that this pulsed UV system may offer a useful method for the disinfection of drinking and wastewater supplies.     

Control of biofilm formation in water using molecularly capped silver nanoparticles

Control of biofouling and its negative effects on process performance of water systems is a serious operational challenge in all of the water sectors. Molecularly capped silver nanoparticles (Ag-MCNPs) were used as a pretreatment strategy for controlling biofilm development in aqueous suspensions using the model organism Pseudomonas aeruginosa. Biofilm control was tested in a two-step procedure: planktonic P. aeruginosa was exposed to the Ag-MCNPs and then the adherent biofilm formed by the surviving cells was monitored by applying a model biofilm-formation assay. Under specific conditions, Ag-MCNPs retarded biofilm formation, even when high percentage of planktonic P. aeruginosa cells survived the treatment. For example, Ag-MCNPs (10 μg mL⁻¹) retarded biofilm formation (>60%), when 50 percent of the planktonic P. aeruginosa cells survived the treatment. Moreover, stable low value of relative biomass has been formed in the presence of fixed Ag-MCNPs concentrations at various biofilm incubation times. Our results showed that Ag-MCNPs pretreated cells were able to produce EPS although they succeeded to form relatively low adherent biofilm. These pretreated cells appear well preserved and undamaged under TEM HPH/freeze micrographs, yet the intra cellular material seems to be pushed towards the peripheral parts of the cell, possibly indicating a survival strategy to the presence of Ag-MCNPs. The lower value of relative biomass formed in the presence of Ag-MCNPs could be associated with molecular mechanisms related to biofilm formation or continuous release of silver ions in the sample. However, further research is required to examine these factors.     

Role of sulfide and ligand strength in controlling nanosilver toxicity

Nanosilver has been used broadly in nanotechnology enhanced consumer products because of its strong antimicrobial properties. Silver nanoparticles (AgNPs) released from these products will likely enter wastewater collection and treatment systems. This research evaluated the role of sulfide and ligand strength in controlling nanosilver toxicity to nitrifying bacteria that are important in wastewater treatment. The nanosilver toxicity in the absence and presence of ligands (SO₄²⁻, S²⁻, Cl⁻, PO₄³⁻, and EDTA⁻) commonly present in wastewater was determined from the oxygen uptake rate measurements. Sulfide appeared to be the only ligand to effectively reduce nanosilver toxicity. By adding a small aliquot of sulfide that was stoichiometrically complexed with AgNPs, the nanosilver toxicity to nitrifying organisms was reduced by up to 80%. Scanning electron microscopy coupled with energy dispersive X-ray analysis indicated that AgNPs were highly reactive with sulfide to form new Ag_xS_y complexes or precipitates. These complexes were not oxidized after a prolonged period of aeration (18 h). This information is useful for wastewater treatment design and operation to reduce nanosilver toxicity via sulfide complexation. While the biotic ligand model was successful in predicting the toxicity of Ag⁺ ions, it could not accurately predict the toxicity of AgNPs. Nevertheless, it could be one of the many tools useful in predicting and controlling nanosilver toxicity to wastewater microorganisms.     

The strong biocidal effect of free nitrous acid on anaerobic sewer biofilms

Several recent studies showed that nitrite dosage to wastewater results in long-lasting reduction of the sulfate-reducing and methanogenic activities of anaerobic sewer biofilms. In this study, we revealed that the quick reduction in these activities is due to the biocidal effect of free nitrous acid (FNA), the protonated form of nitrite, on biofilm microorganisms. The microbial viability was assessed after sewer biofilms being exposed to wastewater containing nitrite at concentrations of 0-120 mg-N/L under pH levels of 5-7 for 6-24 h. The viable fraction of microorganisms was found to decrease substantially from approximately 80% prior to the treatment to 5-15% after 6-24 h treatment at FNA levels above 0.2 mg-N/L. The level of the biocidal effect has a much stronger correlation with the FNA concentration, which is well described by an exponential function, than with the nitrite concentration or with the pH level, suggesting that FNA is the actual biocidal agent. An increase of the treatment from 6 to 12 and 24 h resulted in only slight decreases in microbial viability. Physical disrupted biofilm was more susceptible to FNA in comparison with intact biofilms, indicating that the biocidal effect of FNA on biofilms was somewhat reduced by mass transfer limitations. The inability to achieve 2-log killing even in the case of disrupted biofilms suggests that some microorganisms may be more resistant to FNA than others. The recovery of biofilm activities in anaerobic reactors after being exposed to FNA at 0.18 and 0.36 mg-N/L, respectively, resembled the regrowth of residual sulfate-reducing bacteria and methanogens, further confirming the biocidal effects of FNA on microorganisms in biofilms.     

Rotating disk electrodes to assess river biofilm thickness and elasticity

The present study examined the relevance of an electrochemical method based on a rotating disk electrode (RDE) to assess river biofilm thickness and elasticity. An in situ colonisation experiment in the River Garonne (France) in August 2009 sought to obtain natural river biofilms exhibiting differentiated architecture. A constricted pipe providing two contrasted flow conditions (about 0.1 and 0.45 m s⁻¹ in inflow and constricted sections respectively) and containing 24 RDE was immersed in the river for 21 days. Biofilm thickness and elasticity were quantified using an electrochemical assay on 7 and 21 days old RDE-grown biofilms (t₇ and t₂₁, respectively). Biofilm thickness was affected by colonisation length and flow conditions and ranged from 36 ± 15 μm (mean ± standard deviation, n = 6) in the fast flow section at t₇ to 340 ± 140 μm (n = 3) in the slow flow section at t₂₁. Comparing the electrochemical signal to stereomicroscopic estimates of biofilms thickness indicated that the method consistently allowed (i) to detect early biofilm colonisation in the river and (ii) to measure biofilm thickness of up to a few hundred μm. Biofilm elasticity, i.e. biofilm squeeze by hydrodynamic constraint, was significantly higher in the slow (1300 ± 480 μm rpm^1/2, n = 8) than in the fast flow sections (790 ± 350 μm rpm^1/2, n = 11). Diatom and bacterial density, and biofilm-covered RDE surface analyses (i) confirmed that microbial accrual resulted in biofilm formation on the RDE surface, and (ii) indicated that thickness and elasticity represent useful integrative parameters of biofilm architecture that could be measured on natural river assemblages using the proposed electrochemical method.     

Probing young drinking water biofilms with hard and soft particles

The aim of our study was to investigate, through the use of soft (Escherichia coli) and hard (polystyrene microspheres) particles, the distribution and persistence of allochthonous particles inoculated in drinking water flow chambers. Biofilms were allowed to grow for 7-10 months in tap water from Nancy's drinking water network and were composed of bacterial aggregates and filamentous fungi. Both model particles adhered almost exclusively on the biofilms (i.e. on the bacterial aggregates and on the filamentous structures) and not directly on the uncolonized walls (glass or Plexiglas). Biofilm age (i.e. bacterial density and biofilm properties) and convective-diffusion were found to govern particle accumulation: older biofilms and higher wall shear rates both increased the velocity and the amount of particle deposition on the biofilm. Persistence of the polystyrene particles was measured over a two-month period after inoculation. Accumulation amounts were found to be very different between hard and soft particles as only 0.03‰ of the soft particles inoculated accumulated in the biofilm against 0.3-0.8% for hard particles.     

Differential effect of chlorine on the oxidative stress generation in dormant and active cells within colony biofilm

In an effort to better control bacterial biofilm, we examined the effects of various oxidative antimicrobial chemicals including silver, paraquat, hydrogen peroxide, and chlorine depending on the physiological status of cells in biofilm. The metabolically heterogeneous cells within colony biofilm were physically fractionated and the oxidative stress generated in each fraction was monitored by soxS and oxyS promoter reporter systems. Chlorine induced soxS to a greater degree in the dormant cells than active cells of biofilm. In addition, chlorine-dependent induction of soxS was more prominent in aerobically grown cells compared with anaerobically grown cells. On the contrary, the soxS induction by other chemicals such as paraquat and silver, and the oxyS induction by hydrogen peroxide were higher in active biofilm cells and aerobically grown cells. Our results suggest that chlorine might generate strong oxidative stress by direct modification of the 2Fe-2S cluster in an O₂-independent manner, which provides the molecular basis of our previous report showing that chlorine has a more efficient killing effect on dormant cells in biofilm and cells grown under unaerobic conditions. This study shows that chlorine may be particularly promising for the control of anaerobic bacteria and biofilm where dormant cells are hard to control.     

Effective disinfection of airborne microbial contamination in hospital wards using a zero‐valent nano‐silver/TiO2‐chitosan composite

A novel antimicrobial composite of zero‐valent silver nanoparticles (AgNPs), titania (TiO₂), and chitosan (CS) was prepared via photochemical deposition of AgNPs on a CS‐TiO₂ matrix (AgNPs@CS‐TiO₂). Electron microscopy showed that the AgNPs were well dispersed on the CS‐TiO₂, with diameters of 6.69‐8.84 nm. X‐ray photoelectron spectra indicated that most of the AgNPs were reduced to metallic Ag. Fourier‐transform infrared spectroscopy indicated that some AgNPs formed a chelate with CS through coordination of Ag⁺ with the CS amide II groups. The zones of inhibition of AgNPs@CS‐TiO₂ for bacteria (Escherichia coli and Staphylococcus epidermidis) and fungi (Aspergillus niger and Penicillium spinulosum) were 6.72‐11.08 and 5.45‐5.77 mm, respectively, and the minimum (critical) concentrations of AgNPs required to inhibit the growth of bacteria and fungi were 7.57 and 16.51 µg‐Ag/mm², respectively. The removal efficiency of a AgNPs@TiO₂‐CS bed filter for bioaerosols (η) increased with the packing depth, and the optimal filter quality (qF) occurred for packing depths of 2‐4 cm (qF = 0.0285‐0.103 Pa^?1; η = 57.6%‐98.2%). When AgNPs@TiO₂‐CS bed filters were installed in the ventilation systems of hospital wards, up to 88% of bacteria and 97% of fungi were removed within 30 minutes. Consequently, AgNPs@TiO₂‐CS has promising potentials in bioaerosol purification.     

Polysulfone ultrafiltration membranes impregnated with silver nanoparticles show improved biofouling resistance and virus removal

Biofouling and virus penetration are two significant obstacles in water treatment membrane filtration. Biofouling reduces membrane permeability, increases energy costs, and decreases the lifetime of membranes. In order to effectively remove viruses, nanofiltration or reverse osmosis (both high energy filtration schemes) must be used. Thus, there is an urgent demand for low pressure membranes with anti-biofouling and antiviral properties. The antibacterial properties of silver are well known, and silver nanoparticles (nAg) are now incorporated into a wide variety of consumer products for microbial control. In this study, nAg incorporated into polysulfone ultrafiltration membranes (nAg-PSf) exhibited antimicrobial properties towards a variety of bacteria, including Escherichia coli K12 and Pseudomonas mendocina KR1, and the MS2 bacteriophage. Nanosilver incorporation also increased membrane hydrophilicity, reducing the potential for other types of membrane fouling. XPS analysis indicated a significant loss of silver from the membrane surface after a relatively short filtration period (0.4 L/cm²) even though ICP analysis of digested membrane material showed that 90% of the added silver remained in the membrane. This silver loss resulted in a significant loss of antibacterial and antiviral activity. Thus, successful fabrication of nAg-impregnated membranes needs to allow for the release of sufficient silver ions for microbial control while preventing a rapid depletion of silver.     

The sulfide electrode in bacterial studies

The applicability of a glass/Ag, Ag₂S (silver sulfide coated silver) electrode, which is capable of selectively detecting the H₂S fraction of total reduced inorganic sulfur (S_tot) concentration in biological systems is demonstrated. This electrode was used to monitor photosynthetic sulfide oxidation by Chlorobium phaeobacteroides. The electrode was resistant to Pfennig's medium and bacterial attack and after five months of use showed a potential drift within ±2 mV corresponding to ±13% maximum. The results obtained in a batch type experiment of growth of Chlorobium at different pH values suggest that Chlorobium cells preferentially take up the HS⁻ ion rather than H₂S.     

Interactions of 1 μm latex particles with pseudomonas aeruginosa biofilms

Fluorescently labelled latex microbeads were used to study the interaction of particles with Pseudomonas aeruginosa biofilms in a continuous flow annular reactor. Beads were readily distinguished and enumerated in both intact and disaggregated biofilm samples. The fraction of beads that attached to biofilm during a 24 h period ranged from 0.001 to 0.01 and was proportional to biofilm cell carbon and to the standard deviation of biofilm thickness. Microbeads added to biofilm of steady state thickness (30 μm) were observed to be located throughout the entire biofilm depth in 24 h. Many of the microbeads that attached to biofilm shortly after bacterial inoculation (thickness of 2 μm) remained near the substratum as cells grew past and covered them. Microbeads were observed near the biofilm-substratum interface for up to 5 days after bead addition. Beads formed aggregates on biofilms, but not in bulk water. Beads captured by biofilm remained in the reactor system longer than beads that never attached to biofilm.     

Factors influencing 4-fluorobenzoate degradation in biofilm cultures of Pseudomonas knackmussii B13

Membrane aerated biofilm reactors (MABRs) have potential in wastewater treatment as they permit simultaneous COD minimisation, nitrification and denitrification. Here we report on the application of the MABR to the removal of fluorinated xenobiotics from wastewater, employing a Pseudomonas knackmussii monoculture to degrade the model compound 4-fluorobenzoate. Growth of biofilm in the MABR using the fluorinated compound as the sole carbon source occurred in two distinct phases, with early rapid growth (up to 0.007 h⁻¹) followed by ten-fold slower growth after 200 h operation. Furthermore, the specific 4-fluorobenzoate degradation rate decreased from 1.2 g g⁻¹ h⁻¹ to 0.2 g g⁻¹ h⁻¹, indicating a diminishing effectiveness of the biofilm as thickness increased. In planktonic cultures stoichiometric conversion of substrate to the fluoride ion was observed, however in the MABR, approximately 85% of the fluorine added was recovered as fluoride, suggesting accumulation of ‘fluorine’ in the biofilm might account for the decreasing efficiency. This was investigated by culturing the bacterium in a tubular biofilm reactor (TBR), revealing that there was significant fluoride accumulation within the biofilm (0.25 M), which might be responsible for inhibition of 4-fluorobenzoate degradation. This contention was supported by the observation of the inhibition of biofilm accumulation on glass cover slips in the presence of 40 mM fluoride. These experiments highlight the importance of fluoride ion accumulation on biofilm performance when applied to organofluorine remediation.     

Application of nanosilver surface modification to RO membrane and spacer for mitigating biofouling in seawater desalination

Biofouling is one the most critical problems in seawater desalination plants and science has not yet found effective ways to control it. Silver compounds and ions are historically recognized for their effective antimicrobial activity. Nanosilver particles have been applied as a biocide in many aspects of disinfection, including healthcare products and water treatment. This study proposes an innovative biofouling control approach by surface modification of the RO membrane and spacer with nanosilver coating. A chemical reduction method was used for directly coating nanosilver particles on the membrane sheet and spacer. The surface-modified membrane and spacer were tested for their antifouling performance in a cross-flow flat-sheet membrane cell, which is a part of a pilot plant in Wukan desalination plant. The silver-coating membranes and spacers, along with an unmodified membrane sheet, were tested in the membrane cell and compared on the basis of their antifouling performance. Permeate flux decline and salt rejection was continuously monitored through the testing period. Meanwhile regrowth of microbial populations on the membrane cell was quantified by a unique microbial counting every three to four days. The results showed that both silver-coated membrane (Ag-cM) with uncoated spacer and silver-coated spacer (Ag-cS) with uncoated membrane perforemed better than the unmodified membrane and spacer (Un-MS), in terms of much slower decrease in permeate flux and TDS rejection. However, the effect of silver-coated spacer on antimicrobial activity was more lasting. In the silver-coated spacer test, there was almost no multiplication of cells detected on the membrane during the whole testing period. Besides, the cells adhering to the membrane seemed to lose their activity quickly. According to the RO performance and microbial growth morphology, the nanosilver coating technology is valuable for use in biofouling control in seawater desalination.     

Potential nanosilver impact on anaerobic digestion at moderate silver concentrations

Silver nanoparticles (AgNPs, nanosilver) entering the sewers and wastewater treatment plants (WWTPs) are mostly accumulated in the sludge. In this study, we determined the impact of AgNPs on anaerobic glucose degradation, sludge digestion and methanogenic assemblages. At ambient (22 °C) and mesophilic temperatures (37 °C), there was no significant difference in biogas and methane production between the sludge treated with AgNPs at the concentrations up to 40 mg Ag/L (13.2 g silver/Kg biomass COD) and the control. In these anaerobic digestion samples, acetate and propionic acid were the only detectable volatile fatty acids (VFAs) and they were depleted in 3 days. On the other hand, more than 90% of AgNPs was removed from the liquid phase and associated with the sludge while almost no silver ions were released from AgNPs under anaerobic conditions. Quantitative PCR results indicated that Methanosaeta and Methanomicrobiales were the dominant methanogens, and the methanogenic diversity and population remained largely unchanged after nanosilver exposure and anaerobic digestion. The results suggest that AgNPs at moderate concentrations (e.g., ≤40 mg/L) have negligible impact on anaerobic digestion and methanogenic assemblages because of little to no silver ion release.     

Bacterial activity in plant (Schoenoplectus validus) biofilms of constructed wetlands

Biofilm-bacterial communities have been exploited in the treatment of wastewater in ‘fixed-film’ processes. Our understanding of biofilm dynamics requires a quantitative knowledge of bacterial growth-kinetics in these microenvironments. The aim of this paper was to apply the thymidine assay to quantify bacterial growth without disturbing the biofilm on the surfaces of emergent macrophytes (Schoenoplectus validus) of a constructed wetland. The isotope was rapidly and efficiently taken-up and incorporated into dividing biofilm-bacteria. Isotope diffusion into the biofilm did not limit the growth rate measurement. Isotope dilution was inhibited at >12 μM thymidine. Biofilm-bacterial biomass and growth rates were not correlated to the plant surface area (r² < 0.02). The measurements of in situ biofilm-bacterial growth rates both displayed, and accommodated, the inherent heterogeneity of the complex wetland ecosystem. Biofilm-bacterial respiratory activities, measured using the redox dye CTC, and growth rates were measured simultaneously. The dye did not interfere with bacterial growth. Biofilm-bacterial specific growth rates ranged from 1.4 ± 0.6 d⁻¹ to 3.3 ± 1.3 d⁻¹. In the constructed wetlands of this study biofilm-bacterial specific growth rates, compared to those of natural ecosystems, could be markedly improved through changes in wetland design that increased bacterial respiration while minimising biofilm growth.