Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus aureus

In this study, the microbial toxicities of metal oxide nanoparticles were evaluated for Escherichia coli, Bacillus subtilis, and Streptococcus aureus in laboratory experiments. The nanoparticles tested were CuO, NiO, ZnO, and Sb₂O₃. The metal oxide nanoparticles were dispersed thoroughly in a culture medium, and the microorganisms were cultivated on Luria-Bertani agar plates containing different concentrations of metal oxide nanoparticles. The bacteria were counted in terms of colony forming units (CFU). The CFU was reduced in a culture medium containing metal oxide NP, and the dose-response relationship was characterized. CuO nanoparticles were found to be the most toxic among the tested nanoparticles, followed by ZnO (except S. aureus), NiO, and Sb₂O₃ nanoparticles. We determined that the intrinsic toxic properties of heavy metals are also associated with the toxicity of metal oxide nanoparticles. Ion toxicity was also evaluated to determine the effects of metal ions dissolved from metal oxide NPs, and the toxicity induced from the dissolved ions was determined to be negligible herein. To the best of our knowledge, this is the first study of the toxicity of NiO and Sb₂O₃ NPs on microorganisms. We also discuss the implications of our findings regarding the effects of the intrinsic toxic properties of heavy metals, and concluded that the apparent toxicities of metal oxide NPs can largely be understood as a matter of particle toxicity.     

The inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth

Emerging nanomaterials are of great concern to wastewater treatment utilities and the environment. The inhibitory effects of silver nanoparticles (Ag NPs) and other important Ag species on microbial growth were evaluated using extant respirometry and an automatic microtiter fluorescence assay. Using autotrophic nitrifying organisms from a well-controlled continuously operated bioreactor, Ag NPs (average size=14+/-6 nm), Ag(+) ions (AgNO(3)), and AgCl colloids (average size=0.25 microm), all at 1mg/L Ag, inhibited respiration by 86+/-3%, 42+/-7%, and 46+/-4%, respectively. Based on a prolonged microtiter assay, at about 0.5mg/L Ag, the inhibitions on the growth of Escherichia coli PHL628-gfp by Ag NPs, Ag(+) ions, and AgCl colloids were 55+/-8%, 100%, and 66+/-6%, respectively. Cell membrane integrity was not compromised under the treatment of test Ag species by using a LIVE/DEAD Baclight bacterial viability assay. However, electron micrographs demonstrated that Ag NPs attached to the microbial cells, probably causing cell wall pitting. The results suggest that nitrifying bacteria are especially susceptible to inhibition by Ag NPs, and the accumulation of Ag NPs could have detrimental effects on the microorganisms in wastewater treatment.     

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.     

Size effect in reactivity of copper nanoparticles to carbon tetrachloride degradation

Surface area-normalized rate constants (k(SA)) of reaction between metallic nanoparticles and reducible contaminants, such as chlorinated hydrocarbons, heavy metals, and nitrate, have been reported to be dramatically increased as compared to that of commercial metallic powder. However, k(SA) for individual pollutants in previously published data vary by as much as 1-2 orders of magnitude and much of this variability is due to the effect of various sizes. The size dependence of the reactivity of nanoparticles is not yet fully understood; however, yielding nanoparticles with uniform size and without agglomeration during the period of reaction would demonstrate the effect of varying particle size. In this study, resin-supported zerovalent copper with average particle size of 7, 10, 18, 26, and 29, respectively, were successfully synthesized and evidenced no agglomeration during the reaction period of 10h. The k(SA) of copper nanoparticles (k(n,SA)) was 110-120 times higher than that of powdered copper particles (k(p,SA)) when the copper particle size was about 10nm. However, for diameters of 18-29 nm, the ratio of k(n,SA)/k(p,SA) was around 10-20, indicating that the reactivity of small copper nanoparticles (approximately 10nm) varies discontinuously. Thus, most variability in previous k(SA) is attributed to the presence of small nanoparticles.     

Inactivation of Bacillus subtilis spores and formation of bromate during ozonation

Inactivation of B. subtilis spores with ozone was investigated to assess the effect of pH and temperature, to compare the kinetics to those for the inactivation of C. parvum oocysts, to investigate bromate formation under 2-log inactivation conditions, and to assess the need for bromate control strategies. The rate of B. subtilis inactivation with ozone was independent of pH, decreased with temperature (activation energy of 42,100 Jmol(-1)), and was consistent with the CT concept. B. subtilis was found to be a good indicator for C. parvum at 20-30 degrees C, but at lower temperatures B. subtilis was inactivated more readily than C. parvum. Bromate formation increased as both pH and temperature increased. For water with an initial bromide concentration of 33 microgl(-1), achieving 2-logs of inactivation, without exceeding the 100 microg l(-1) bromate standard, was most difficult at 30 degrees C for B. subtilis and at midrange temperatures (10-20 degrees C) for C. partum. pH depression and ammonia addition were found to reduce bromate formation without affecting B. subtilis inactivation, and may be necessary for waters containing more than 50 microgl(-1) bromide.     

Genotoxicity of silver nanoparticles in Allium cepa

Potential health and environmental effects of nanoparticles need to be thoroughly assessed before their widespread commercialization. Though there are few studies on cytotoxicity of nanoparticles on mammalian and human cell lines, there are hardly any reports on genotoxic and cytotoxic behavior of nanoparticles in plant cells. This study aims to investigate cytotoxic and genotoxic impacts of silver nanoparticles using root tip cells of Allium cepa as an indicator organism. A.cepa root tip cells were treated with four different concentrations (25, 20, 75, and 100 ppm) of engineered silver nanoparticles (below 100 nm size) dispersion, to study endpoints like mitotic index, distribution of cells in mitotic phases, different types of chromosomal aberrations, disturbed metaphase, sticky chromosome, cell wall disintegration, and breaks. For each concentration five sets of microscopic observations were carried out. No chromosomal aberration was observed in the control (untreated onion root tips) and the mitotic index (MI) value was 60.3%. With increasing concentration of the nanoparticles decrease in the mitotic index was noticed (60.30% to 27.62%). The different cytological effects including the chromosomal aberrations were studied in detail for the treated cells as well as control. We infer from this study that silver nanoparticles could penetrate plant system and may impair stages of cell division causing chromatin bridge, stickiness, disturbed metaphase, multiple chromosomal breaks and cell disintegration. The findings also suggest that plants as an important component of the ecosystems need to be included when evaluating the overall toxicological impact of the nanoparticles in the environment.     

Impact of exopolysaccharides on the stability of silver nanoparticles in water

The stability of commercial silver nanoparticles (SNPs) in aquatic environment plays a significant role in its toxicity to the environment and to human health. Here, we have studied the impact of bacterial exopolysaccharides (EPS) to the stability of engineered SNPs. When nanoparticles are present in neutral water, the nanoparticles exhibited low zeta potential and are least stable. However, in the presence of EPS (10-250 mg/L), the negative surface charge of nanoparticles increased and therefore the propensity of nanoparticles to aggregate is reduced. In UV-visible spectroscopic analysis a decrease in absorbance at plasmon peak of SNPs (425 nm) was observed till the addition of 50 mg/L of EPS, beyond that a blue shift towards 417 nm was observed. The adsorption of EPS was confirmed by Fourier-transform infrared spectroscopy. The EPS adsorbed SNPs were more stable and exhibited the zeta potential of higher than −30 mV.     

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.     

The effect of humic acids on the cytotoxicity of silver nanoparticles to a natural aquatic bacterial assemblage

The effect of a terrestrial humic acid (HA) and a river HA on the cytotoxicity of silver nanoparticles (AgNPs) to natural aquatic bacterial assemblages (0 μM, 2.5 μM and 5 μM) was measured with spread plate counting. The effect of HA (20 and 40 ppm) on the cytotoxicity of AgNPs ranging in size between 15 and 25 nm was tested in the presence and in the absence of natural sunlight. The experiment was a full factorial, completely randomized design and the results were analyzed using the General Linear Model in SAS. LSMEANS was used to separate the means or combinations of means. Significant main effects of all independent variables, plus interaction effects in all cases except HA/LI and HA/AgNPs/LI were observed. The toxicity of AgNPs to natural aquatic bacterial assemblages appears to be concentration dependent for concentrations between 0 μM and 5 μM. The data indicate that the light exposure inhibited viability more than the darkness exposure. The HA treatment groups in the presence of light showed greater reduced viability count compared to darkness exposure groups. The inhibition of bacterial viability counts by AgNPs exposure was less in the light treatment groups containing a terrestrial HA compared to that with a river HA. Difference in the extent of reactive oxygen species formation and adsorption/binding of AgNPs was speculated to account for the observed phenomenon.     

Adsorption of Bacillus subtilis on single-walled carbon nanotube aggregates, activated carbon and NanoCeram

Adsorption equilibrium and kinetics of Bacillus subtilis spores on single-walled carbon nanotube aggregates were investigated to explore the possibility of using single-walled carbon nanotubes for concentration, detection and removal of pathogens from contaminated water sources. Batch adsorption experiments were conducted to determine adsorption kinetics and adsorption equilibrium of B. subtilis spores on single-walled carbon nanotube aggregates, activated carbon and NanoCeram™. The adsorption kinetics data were analyzed with both the Lagergren pseudo first order and a pseudo second order models. The adsorption equilibrium data on three porous media were quantified by the Henry's law constant. It was observed that both the Lagergren first order rate model and the pseudo second order model correlate the adsorption kinetic data well although the calculated adsorption rate constants vary with adsorbate concentrations. The Henry's law adsorption equilibrium constant of B. subtilis spores on single-walled carbon nanotube aggregates is about 27-37 times higher than those on activated carbon and NanoCeram™. The high adsorption affinity of carbon nanotubes towards the B. subtilis spores is due to the mesoporous structure and unique surface properties of carbon nanotubes. These results suggest that single-walled carbon nanotube aggregates are good candidates as biosensors and adsorbent media for concentrating, detecting and removal of pathogens from contaminated water resources.     

Disinfection of Bacillus subtilis spores with chlorine dioxide: a bench-scale and pilot-scale study

Chlorine dioxide (ClO2) inactivation of Bacillus subtilis ATCC 19659 spores was examined at pilot-scale during periods representative of winter and summer temperature extremes at the Britannia Water Treatment Plant in Ottawa, Canada. In addition, bench-scale experiments using the same source water (Ottawa River, Ontario, Canada), as well as buffered and unbuffered laboratory waters were conducted using B. subtilis spores. Bench-scale inactivation of B. subtilis spores by ClO2 was similar to reported values for Cryptosporidium parvum (both organisms being more resistant to ClO2 than Giardia lamblia), suggesting the possibility that these spores may be used as potential indicators for protozoan parasites. Additionally, spore inactivation was observed to be influenced by pH in laboratory (distilled deionised water) water but not in Ottawa River water. At pilot-scale, spore inactivation was influenced by water temperature: a ClO2 dose of 2.5 mg/L resulted in a spore inactivation of approximately 2.0 log10 and 0.5 log10 at water temperatures of 23.2d egrees C and 5.2 degrees C, respectively. Chlorite concentrations remained below both the US EPA maximum contaminant level of 1.0 mg/L and the maximum contaminant level goal of 0.8 mg/L for up to 2.0log10 B. subtilis inactivation.     

Pulsed ultra-violet inactivation spectrum of Escherichia coli

Inactivation of Escherichia coli is examined using ultra-violet (UV) radiation from a pulsed xenon flashlamp. The light from the discharge has a broadband emission spectrum extending from the UV to the infrared region with a rich UV content. The flashlamp provides high-energy UV output using a small number of short-duration pulses (30 micros). The flashlamp is used with a monochromator to investigate the wavelength sensitivity of E. coli to inactivation by the pulsed UV light. Using 8 nm wide pulses of UV radiation, the most efficient inactivation is found to occur at around 270 nm and no inactivation is observed above 300 nm. A pyroelectric detector allows the energy dose to be determined at each wavelength, and a peak value for E. coli population reduction of 0.43 log per mJ/cm(2) is measured at 270 nm. The results are compared with the published data available for continuous UV light sources.     

Sequential UV- and chlorine-based disinfection to mitigate Escherichia coli in drinking water biofilms

This study was designed to examine the potential downstream benefits of sequential disinfection to control the persistence of Escherichia coli under conditions relevant to drinking water distribution systems. Eight annular reactors (four polycarbonate and four cast iron) were setup in parallel to address various factors that could influence biofilm growth in distribution systems. Eight reactors were treated with chlorine, chlorine dioxide and monochloramine alone or in combination with UV to examine the effects on Escherichia coli growth and persistence in both the effluent and biofilm. In general, UV-treated systems in combination with chlorine or chlorine dioxide and monochloramine achieved greater log reductions in both effluent and biofilm than systems treated with chlorine-based disinfectants alone. However, during UV-low chlorine disinfection, E. coli was found to persist at low levels, suggesting that the UV treatment had instigated an adaptive mutation. During UV-chlorine-dioxide treatment, the E. coli that was initially below the detection limit reappeared during a low level of disinfection (0.2 mg/L) in the cast iron systems. Chloramine was shown to be effective in disinfecting suspended E. coli in the effluent but was unable to reduce biofilm counts to below the detection limit. Issues such as repair mechanism of E. coli and nitrification could help explain some of these aberrations. Improved understanding of the ability of chlorine-based disinfectant in combination with UV to provide sufficient disinfection will ultimately effect in improved management and safety of drinking water.     

Investigating synergism during sequential inactivation of Bacillus subtilis spores with several disinfectants

The sequential application of ozone, chlorine dioxide, or UV followed by free chlorine was performed to investigate the synergistic inactivation of Bacillus subtilis spores. The greatest synergism was observed when chlorine dioxide was used as a primary disinfectant followed by secondary disinfection with free chlorine. A lesser synergistic effect was observed when ozone was used as the primary disinfectant, but no synergism was observed when UV was used as the primary disinfectant. When free chlorine was used as the primary disinfectant (i.e., sequential application in the reverse order), the synergistic effect was shown only when chlorine dioxide was applied as the secondary disinfectant. The synergistic effect observed could be related to damage to the spore coat during primary disinfection, suggested by the loss of proteins from spores during disinfectant treatment. The greatest synergism observed by the chlorine dioxide/free chlorine pair suggested that common reaction sites might exist for these disinfectants. The concept of percent synergistic effect was introduced to quantitatively compare the extent of synergistic effects in the sequential disinfection processes.     

Phenotypic and genotypic characterization of human and nonhuman Escherichia coli

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is one of several fecal coliform bacteria that inhabit the intestines of many warm-blooded animals that sometimes contaminate water. Its presence does not specifically implicate human fecal input, therefore it is necessary to differentiate contamination sources to accurately assess health risks. E. coli were isolated from human sources (HS) and nonhuman sources (NHS) in the Apalachicola National Estuarine Research Reserve and analyzed for fatty acid methyl ester (FAME), O-serogroup, and pulsed-field gel electrophoresis (PFGE) profiles. For FAME and PFGE analyses, there was no relationship between profile and isolate source. Human source PFGE profiles were less diverse than NHS isolates, and conversely for FAME. In contrast, O-serogrouping showed less diversity for HS vs. NHS isolates, and the predominant HS O-serogroups differed significantly (P < 0.01) from those of NHS isolates.     

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.     

Transport and deposition of sediment-associated Escherichia coli in natural streams

The association of microorganisms with sediment particles is one of the primary complicating factors in assessing microbial fate in aquatic systems. The literature indicates that the majority of enteric bacteria in aquatic systems are associated with sediments and that these associations influence their survival and transport characteristics. Yet, the nature of these associations has not been fully characterized. In this study, a combination of field experiments and mathematical modeling were used to better understand the processes which control the fate and transport of enteric bacteria in alluvial streams. An experimental procedure, involving the use of a tracer-bacteria, was developed to simulate the transport and deposition of bacteria-laden bed sediments in a small alluvial stream during steady flow conditions. The experimental data and mathematical model were used to determine dispersion coefficients, deposition rates, and partitioning coefficients for sediment-associated bacteria in two natural streams. The results provided evidence that bacterial adsorption can be modeled as an irreversible process in freshwater environments. Net settling velocities of fine sediments and associated bacteria were typically two orders of magnitude lower than those predicted from Stokes equation, due to re-entrainment of settled particles. The information presented in this study will further the development of representative microbial water quality models.     

紫外线强度及剂量对大肠杆菌光复活的影响

以水中大肠杆菌为研究对象,考察了不同紫外线强度及剂量对大肠杆菌的灭活效果以及对其光复活的影响.结果表明:大肠杆菌的灭活率随着紫外线剂量的增加而增加,而相同剂量下高强度的紫外线对大肠杆菌的灭活效果要好于低强度的,且高紫外线强度有利于控制大肠杆菌的光复活程度.当紫外线强度为0.153 mW/cm2、紫外线剂量为60 mJ/cm2时,其对大肠杆菌的灭活率高达5个对数级.高剂量紫外线照射下大肠杆菌发生光复活的能力明显弱于低剂量下的,紫外线剂量为120 mJ/cm2时的大肠杆菌光复活率较5 mJ/cm2时的低了3个对数级.     

Phenotypic characterization of Escherichia coli through whole-cell fatty acid profiling to investigate host specificity

The objective of the study was to investigate whole-cell fatty acid methyl ester (FAME) profiles of 605 Escherichia coli isolates to determine their host specificity. The isolates were cultured from six possible sources of fecal pollution; 180 isolates from sewage, 85 from dairy cow, 98 from chicken, 76 from swine, 94 from deer, and 72 from waterfowl, mostly geese and ducks. The FAME profiles were presented as the relative masses of 12 FAMEs identified in the isolates and it was found that none of the six hosts carried a "signature" FAME, a FAME that is uniquely associated with a particular host category. However, two-sample t-test analyses indicated that the mean relative masses of seven FAMEs out of the 12 identified showed statistically significant differences (95% confidence interval) between isolates of human and non-human origins. In addition, a linear discriminant function based on mean relative mass variations in individual FAMEs classified the known-source isolates into their respective host categories with a 47.6% average rate of correct classification (ARCC) in a six-way discriminant analysis. The ARCC increased to 61.3% when the individual hosts were pooled into larger categories of human, livestock, and wildlife. The accuracy was 75.5% when isolates of human origin were discriminated against those of non-human origins. Random cluster formation analysis indicated that the library size was sufficient to prevent random grouping among the isolates.     

Effect of particles and bioflocculation on ultraviolet disinfection of Escherichia coli

Presence of particles is known to decrease the effectiveness of ultraviolet (UV) disinfection by shielding the targeted microorganisms from UV light. This study aims to provide an in-depth understanding on the effect of particles and flocs on UV disinfection by using a stable, well-defined and well-controlled synthetic system that can simulate the bioflocculation of particles and microorganisms in water and wastewater samples. The synthetic system was created by using Escherichia coli, latex particles (1, 3.2, 11, 25, and 45 μm), alginate, and divalent cations; and the bioflocculation of particles was achieved naturally, as it would occur in the environment, without using chemical coagulants. E. coli was quantified before and after UV disinfection using membrane filtration. Even in the absence of particles, some of the self-aggregated E. coli could survive a UV dose of 90 mJ/cm². E. coli inactivation levels measured in the presence of particles were lower than the inactivation levels measured in the absence of particles. At low UV doses (<9 mJ/cm²), neither particle size nor degree of flocculation had a significant effect on the inactivation of E. coli. Particle size had a significant effect on the inactivation of E. coli only at high UV doses (80 mJ/cm²), and larger particles (e.g., 25 μm) protected bacteria more compared to smaller particles (e.g., 3.2 and 11 μm). What size of particles flocs were made of (3.2, 11, and 25 μm) did not make a significant difference on the inactivation levels of E. coli. For 3.2 μm particles, there was no significant difference in E. coli inactivation between non-flocculated and flocculated samples at any UV dose. For 11 and 25 μm particles, there was a significant difference in E. coli inactivation between non-flocculated and flocculated samples at 80 mJ/cm². Degree of flocculation became a significant factor in determining the number of surviving bacteria only at high UV doses and only for larger particles.