Effect of oxidizing and non-oxidizing biocides on biofilm at different substrate levels in the model recirculating cooling water system

With the reducing of water resources, using advanced treated refinery wastewater as recirculating cooling water is an effective method to save water and to reduce the pollution of petroleum and petrochemical industry. However, the control of biofilm is a bottleneck in the application of this technology. To resolve the problem of biofilm formation and development, antimicrobial characteristics of chlorine dioxide and benzyldimethyldodecyl-ammonium chloride on biofilm at different substrate levels were investigated. Biofilm detachment ratio and TTC-dehydrogenase activity (DHA) were two indexes to discuss the antimicrobial effects. The results showed that at the high substrate level, the biofilms characteristics (biomass, the content of protein, polysaccharide and EPS) were the higher than those at the medium and low substrate levels, however biofilm’s DHA at the medium substrate level (12.97 μgTF/(g h)) was higher than those at the medium substrate level (7.64 μgTF/(g h)) and low substrate level (1.94 μgTF/(g h). The difference of substrate level in the media resulted in different biofilm structure. By contrast with the control experiment, biofilm detachment ratios were all increased in three media with ClO₂ and BDMDAC addition. After ClO₂ addition, MITs were 30, 120 and 240 min and MIC was 1, 4 and 6 mg/L, respectively, at the low, medium and high substrate level. After BDMDAC addition, MITs in three media were all longer than those after ClO₂ addition, MIC was 200, 300 and 400 mg/L, respectively, at the low, medium and high substrate level.     

Potentiometry of the Reaction of Residual Lignin of Lignocellulose Powder Material with Chlorine Dioxide

The consumption rate of chlorine dioxide in the reaction with the residual lignin was studied using measurements of the potential of a redox couple ClO₂/ClO ₂ ^? in an aqueous suspension of lignocellulose powder material. The methodology was developed for the study of reaction kinetics of chlorine dioxide with the residual lignin using the first order reaction model with the initial lignin concentration in the reaction medium varying in the range of (1–17) × 10^–4 М at the initial chlorine dioxide concentration of 4.48 × 10^–4 М. The monochronic rate constant of the second order reaction of chlorine dioxide with the residual lignin was calculated by the dependence k_eff(I) = k_eff(II)[L]₀ under the lignin excess conditions, it was 1462 ± 108 М^–1 s^–1. Considering the initial concentrations of reagents, it was found that, with increasing the degree of lignin conversion Θ from ≈0.27 to 0.54, its reactivity in the interaction with the chlorine dioxide is reduced, and the value of k_eff(II) decreased from ≈1280 to 900 М^–1 s^–1, which according to the theory of polychronic kinetics was a manifestation of the kinetic nonequivalence of the various ensembles of macromolecules of the residual lignin. Thus, it was experimentally shown for the first time that residual lignin of lignocellulose powder material had a kinetic nonequivalence in the interaction with chlorine dioxide.     

Micropropagation of gerbera using chlorine dioxide (ClO2) to sterilize the culture medium

One of the alternatives to autoclaving culture media is chemical sterilization, which may cause fewer changes to the chemical composition of the media. In this study, the effect of chemical sterilization by inclusion of chlorine dioxide (ClO₂) in the culture medium on the in vitro development of gerbera (Gerbera jamesonii) cv. AL101, cultured at different stages of micropropagation, was evaluated. The following five concentrations of ClO₂ were tested: 0%, 0.0025%, 0.0050%, 0.0075%, and 0.010%. Autoclaved medium was used as the control. ClO₂ in the culture medium reduced contamination at rates comparable to autoclaving when tested at three stages of the culture process: in vitro establishment, multiplication, and rooting. Plantlets grown in culture media sterilized with ClO₂ showed similar or better development than those grown in autoclaved culture medium. Use of 0.0025% ClO₂ to sterilize the culture medium resulted in better plantlet development than autoclaved medium, regardless of the stage of micropropagation.     

Chlorine dioxide disinfection of single and dual species biofilms,detached biofilm and planktonic cells

Disinfection efficacy testing is usually done with planktonic cells or more recently, biofilms. While disinfectants are much less effective against biofilms compared to planktonic cells, questions regarding the disinfection tolerance of detached biofilm clusters remain largely unanswered. Burkholderia cepacia and Pseudomonas aeruginosa were grown in chemostats and biofilm tubing reactors, with the tubing reactor serving as a source of detached biofilm clusters. Chlorine dioxide susceptibility was assessed for B. cepacia and P. aeruginosa in these three sample types as monocultures and binary cultures. Similar doses of chlorine dioxide inactivated samples of chemostat and tubing reactor effluent and no statistically significant difference between the log₁₀ reductions was found. This contrasts with chlorine, shown previously to be generally less effective against detached biofilm particles. Biofilms were more tolerant and required chlorine dioxide doses ten times higher than chemostat and tubing reactor effluent samples. A second species was advantageous in all sample types and resulted in lower log₁₀ reductions when compared to the single species cultures, suggesting a beneficial interaction of the species.     

Direct measurement of chlorine penetration into biofilms during disinfection.

Transient chlorine concentration profiles were measured in biofilms during disinfection by use of a microelectrode developed for this investigation. The electrode had a tip diameter of ca. 10 microm and was sensitive to chlorine in the micromolar range. The biofilms contained Pseudomonas aeruginosa and Klebsiella pneumoniae. Chlorine concentrations measured in biofilms were typically only 20% or less of the concentration in the bulk liquid. Complete equilibration with the bulk liquid did not occur during the incubation time of 1 to 2 h. The penetration depth of chlorine into the biofilm and rate of penetration varied depending on the measurement location, reflecting heterogeneity in the distribution of biomass and in local hydrodynamics. The shape of the chlorine profiles, the long equilibration times, and the dependence on the bulk chlorine concentration showed that the penetration was a function of simultaneous reaction and diffusion of chlorine in the biofilm matrix. Frozen cross sections of biofilms, stained with a redox dye and a DNA stain, showed that the area of chlorine penetration overlapped with nonrespiring zones near the biofilm-bulk fluid interface. These data indicate that the limited penetration of chlorine into the biofilm matrix is likely to be an important factor influencing the reduced efficacy of this biocide against biofilms as compared with its action against planktonic cells.     

Ab initio double-ζ (D95) valence bond calculations for the ground states of NO2, O3, and ClO2

The results of some double-ζ D95 valence-bond (VB) calculations are reported for the ground states of nitrogen dioxide, NO₂ (17 valence electrons), ozone, O₃ (18 valence electrons), and chlorine dioxide, ClO₂ (19 valence electrons). The Mulliken, Löwdin and Hiberty structural weights are reported for nine (NO₂), six (O₃), and three (ClO₂) Lewis structures that differ in the locations of the π electrons. The most important Lewis structures for NO₂ are the uncharged spin-paired diradical structure VII and the two equivalent structures that carry no formal charges (II and V). For O₃ and ClO₂, the primary Lewis structures are, respectively, the uncharged singlet diradical structure III, and I with the odd electron located in the chlorine 3pπ atomic orbital.For ClO₂, the results of some STO-6G calculations, with 16 canonical Lewis type structures included, give a much smaller value for the chlorine odd-electron charge than does the D95 vb2000 calculation with a Hartree-Fock core. However, the structural weights obtained from the STO-6G calculations do reflect the expectation that small atomic formal charge separations, together with some Cl-O covalent σ-bonding, are associated with large structural weights.     

Inactivation and mechanisms of chlorine dioxide on Nosema bombycis

Biological tests demonstrated that the inactivation of Nosema bombycis (N. bombycis) spores by chlorine dioxide (ClO₂) occurs very fast and is highly sensitive. The lowest effective inactivation dosage and time was 15 mg/mL for 30 min. The inactivation of spores was additionally verified by using double color fluorescence stain and spore germination testing. A series of biological changes, including a large number of substrates that were leaked out from the spores included proteins, DNA, polysaccharide, K⁺, and Ca²⁺, occurred a short time after N. bombycis spores were treated with ClO₂. In addition, the lipid of spores was disrupted and ATPase activity was inhibited, which resulted in the destruction of the inner structure of the spores.     

Detoxification of cyanide using titanium dioxide and hydrocyclone sparger with chlorine dioxide

Abstract

The extraction of gold and silver from minerals and concentrates with cyanide is an important hydrometallurgy process that has been studied for more than 120 years. This technology, which consists of the dissolutions of the precious metals in cyanide solutions, followed by the recovery of the values by cementation, activated carbon or ion exchange resin. Most of the wastes in the industrial effluents’ milling are known to contain high contents of free cyanide as well as metallic cyanide complexes, which give them a high degree of toxicity. Appropriate methods for the treatment of cyanide solutions include cyanide destruction by oxidation using a photoelectrocatalytic detoxification technique with titanium dioxide microelectrodes. This is one of the most innovative ways for the treatment of wastewater containing cyanide. Another is the use of chlorine dioxide (ClO₂) with a gas-sparged hydrocyclone as the reactor. The results show that photodegradation of cyanide was 93% in 30 minutes using a 450 W halogen lamp, and in the case of ClO₂ the destruction of cyanides was 99% in 1 minute. In both cases, excellent performances can be achieved with the high capacity of these technologies.     

Reactivity of lignin-related phenols with haemoglobin and NaClO3

Summary Haemoglobin (Hb), in the presence of sodium chlorate (NaClO₃), promoted the decraboxylation of vanillic acid, the oxidation of guaiacol and the demethoxylation of vanillic and ferulic acids with maximal activity at pH 3.5–4.0. These reactions were also mediated by Hb + chlorine dioxide (ClO₂) with a similar pH profile. However, differences in the activity of sodium chlorite (NaClOP₂) in the presence or absence of Hb were observed, especially at low pH values. It is speculated that ClO₂ is mainly responsible for acitivities observedc. The Hb-dependent activity was more stable in the presence of NaClO₃ than with hydrogen peroxide (H₂O₂). *** DIRECT SUPPORT *** AG903028 00008     

Inactivation Kinetics and Mechanism of a Human Norovirus Surrogate on Stainless Steel Coupons via Chlorine Dioxide Gas

Acute gastroenteritis caused by human norovirus is a significant public health issue. Fresh produce and seafood are examples of high-risk foods associated with norovirus outbreaks. Food contact surfaces also have the potential to harbor noroviruses if exposed to fecal contamination, aerosolized vomitus, or infected food handlers. Currently, there is no effective measure to decontaminate norovirus on food contact surfaces. Chlorine dioxide (ClO₂) gas is a strong oxidizer and is used as a decontaminating agent in food processing plants. The objective of this study was to determine the kinetics and mechanism of ClO₂ gas inactivation of a norovirus surrogate, murine norovirus 1 (MNV-1), on stainless steel (SS) coupons. MNV-1 was inoculated on SS coupons at the concentration of 10⁷ PFU/coupon. The samples were treated with ClO₂ gas at 1, 1.5, 2, 2.5, and 4 mg/liter for up to 5 min at 25°C and a relative humidity of 85%, and virus survival was determined by plaque assay. Treatment of the SS coupons with ClO₂ gas at 2 mg/liter for 5 min and 2.5 mg/liter for 2 min resulted in at least a 3-log reduction in MNV-1, while no infectious virus was recovered at a concentration of 4 mg/liter even within 1 min of treatment. Furthermore, it was found that the mechanism of ClO₂ gas inactivation included degradation of viral protein, disruption of viral structure, and degradation of viral genomic RNA. In conclusion, treatment with ClO₂ gas can serve as an effective method to inactivate a human norovirus surrogate on SS contact surfaces.     

Effect of low-concentration chlorine dioxide gas against bacteria and viruses on a glass surface in wet environments

Aims: To evaluate the efficacy of low‐concentration chlorine dioxide (ClO₂) gas against model microbes in the wet state on a glass surface. Methods and Results: We set up a test room (39 m³) and the ClO₂ gas was produced by a ClO₂ gas generator that continuously releases a constant low‐concentration ClO₂ gas. Influenza A virus (Flu‐A), feline calicivirus (FCV), Staphylococcus aureus and Escherichia coli were chosen as the model microbes. The low‐concentration ClO₂ gas (mean 0·05 ppmv, 0·14 mg m^?3) inactivated Flu‐A and E. coli (>5 log₁₀ reductions) and FCV and S. aureus (>2 log₁₀ reductions) in the wet state on glass dishes within 5 h. Conclusions: The treatment of wet environments in the presence of human activity such as kitchens and bathrooms with the low‐concentration ClO₂ gas would be useful for reducing the risk of infection by bacteria and viruses residing on the environmental hard surfaces without adverse effects. Significance and Impact of the Study: This study demonstrates that the low‐concentration ClO₂ gas (mean 0·05 ppmv) inactivates various kinds of microbes such as Gram‐positive and Gram‐negative bacteria, enveloped and nonenveloped viruses in the wet state.     

Chlorine Dioxide for Reduction of Postharvest Pathogen Inoculum during Handling of Tree Fruits

Alternatives to hypochlorous acid and fungicides are needed for treatment of fruit and fruit-handling facilities. Chlorine dioxide was evaluated and found effective against common postharvest decay fungi and against filamentous fungi occurring on fruit packinghouse surfaces. In vitro tests with conidial or sporangiospore suspensions of Botrytis cinerea, Penicillium expansum, Mucor piriformis, and Cryptosporiopsis perennans demonstrated >99% spore mortality within 1 min when the fungi were exposed to aqueous chlorine dioxide at 3 or 5 μg · ml^-1. Longer exposure times were necessary to achieve similar spore mortalities with 1 μg · ml^-1. Of the fungi tested, B. cinerea and P. expansum were the least sensitive to ClO₂. In comparison with the number recovered from untreated control areas, the number of filamentous fungi recovered was significantly lower in swipe tests from hard surfaces such as belts and pads in a commercial apple and pear packinghouse after treatment of surfaces with a 14.0- to 18.0-μg · ml^-1 ClO₂ foam formulation. Chlorine dioxide has desirable properties as a sanitizing agent for postharvest decay management when residues of postharvest fungicides are not desired or allowed.     

Life after treatment: detecting living microorganisms following exposure to UV light and chlorine dioxide

Rapid analytical methods are needed to quantify living microorganisms to determine if ships’ discharged ballast water is in compliance with national and international standards. Traditionally, regrowth assays and microscope counts of stained organisms—which are time-consuming, require expensive equipment, and require extensive staff training—are used to assess microorganisms. The goal of this study was to evaluate other approaches. Both ambient microorganisms from an oligotrophic marine environment and laboratory cultures of marine algae were evaluated following exposure to two types of ballast water treatment: ultraviolet (UV) light and chlorine dioxide (ClO₂). Microorganisms in two size classes (<10 and ≥10 to <50 μm) were quantified using regrowth assays and vital staining, and samples were evaluated using two rapid approaches: (1) chlorophyll a fluorescence and photochemical yield were measured using a pulse amplitude modulated fluorometer and (2) the concentration of adenosine triphosphate (ATP) was measured with a handheld luminometer. The response of microorganisms to UV and ClO₂ was evident in measurements of photochemical yield, as photochemical yield decreased at high doses. However, initial values of photochemical yield were variable and species-specific. Oddly, in some trials, initial fluorescence increased at intermediate UV doses; this phenomenon could lead to overestimation of total biomass. In samples treated with UV light, ATP was not significantly different among any of the doses used; however, concentrations of ATP were significantly lower at the highest dose of ClO₂ than control samples. These results demonstrate that approaches used for ballast water testing can be treatment-specific, and compliance approaches should be validated to determine their utility with the appropriate treatments.     

Modelling inactivation by aqueous chlorine dioxide of Dothiorella gregaria Sacc. and Fusarium tricinctum (Corda) Sacc. spores inoculated on fresh chestnut kernel

Aims: To model survival curves of Dothiorella gregaria Sacc. and Fusarium tricinctum (Corda) Sacc. spores inoculated on fresh chestnut kernel exposed to aqueous chlorine dioxide (ClO₂). Methods and Results: Spores of two dominant spoilage fungi, D. gregaria and F. tricinctum, were inoculated onto chestnut kernel and treated with ClO₂. The inactivation efficacy of ClO₂ treatment increased with ClO₂ concentration and treatment time. The Weibull model was the best model to describe the ClO₂ survival curves of D. gregaria, while the modified Gompertz model was most appropriate for fitting the survival curves of F. tricinctum. Within the range of ClO₂ concentration from 3 to 7 mg l^?1, the n values in the Weibull model were similar. The b value in the Weibull model and decimal logarithms of the M, B and C values in the modified Gompertz model had linear relationships with ClO₂ concentration. After simplification, these two models still provided acceptable predictions. Conclusion: Applying models for describing survival curves of fungal spores on chestnut kernel by aqueous ClO₂ was feasible. Significance and Impact of the Study: This work would promote the application of ClO₂ sanitizing technique and mathematical models when preventing the occurrence of chestnut kernel decay.     

Development of an online biosensor for in situ monitoring of chlorine dioxide gas disinfection efficacy

A prototype bioluminescence-based biosensor was designed and constructed to evaluate the antimicrobial efficacy of chlorine dioxide (ClO₂) gas under various treatment conditions. The biosensor consisted of a bioluminescent bioreporter (Pseudomonas fluorescens 5RL), an optical transducer (photomultiplier tube), and a light-tight chamber housing, the bioreporter and the transducer. The bioluminescent recombinant P. fluorescens 5RL in the biosensor allowed for online monitoring of bioluminescence during ClO₂ gas disinfection. Experiments were performed to evaluate the effects of the two key physical parameters associated with ClO₂ disinfection: relative humidity (40, 60, 80%) and ClO₂ gas concentration (0.5, 1.0, 1.6, 2.1 mg/l) on the bioreporter. Results showed that increasing concentrations of ClO₂ gas corresponded to a faster decrease in luminescence. The rates of luminescence decrease from P. fluorescens 5RL, and the log reduction time (LRT, time required to obtain 1-log reduction in luminescence) were calculated for each treatment tested. The LRT values of luminescence were 103, 78, 53, and 35 s for 0.5, 1.0, 1.6, and 2.1 mg/l of ClO₂ gas treatment, respectively, at 78% relative humidity. The gas concentration which caused a tenfold change in LRT (z value) for luminescence of P. fluorescens 5RL was 3.4 mg/l of ClO₂. The prototype biosensor showed potential for many applications, such as monitoring real-time microbial inactivation and understanding parameters that influence the efficacy of gaseous decontamination procedures. This paper is journal article # 2008-18279 of the Purdue University Agricultural Research Program.     

Chlorine dioxide inactivation of bacterial threat agents

Aims: To evaluate the efficacy of chlorine dioxide (ClO₂) against seven species of bacterial threat (BT) agents in water. Methods and Results: Two strains of Bacillus anthracis spores, Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Burkholderia mallei and Brucella species were each inoculated into a ClO₂ solution with an initial concentration of 2·0 (spores only) and 0·25 mg l^?1 (all other bacteria) at pH 7 or 8, 5 or 25°C. At 0·25 mg l^?1 in potable water, six species were inactivated by at least three orders of magnitude within 10 min. Bacillus anthracis spores required up to 7 h at 5°C for the same inactivation with 2·0 mg l^?1 ClO₂. Conclusions: Typical ClO₂ doses used in water treatment facilities would be effective against all bacteria tested except B. anthracis spores that would require up to 7 h with the largest allowable dose of 2 mg l^?1 ClO₂. Other water treatment processes may be required in addition to ClO₂ disinfection for effective spore removal or inactivation. Significance and Impact of Study: The data obtained from this study provide valuable information for water treatment facilities and public health officials in the event that a potable water supply is contaminated with these BT agents.     

Fungicidal mechanism of chlorine dioxide on Saccharomyces cerevisiae

The fungicidal mechanism of chlorine dioxide on Saccharomyces cerevisiae was investigated. During S. cerevisiae inactivation by ClO₂, protein, DNA, and ion leakage, enzyme activity, genomic DNA structure, and cell ultrastructure were examined. Protein and DNA leakages were not observed, while ion leakages of K⁺, Ca²⁺, and Mg²⁺ were detected and were related to the inactivation rate. The glucose-6-phosphate dehydrogenase, citrate synthase, and phosphofructokinase activities were inhibited and were also correlated with the inactivation rate. Genomic DNA structure was not damaged except for an extremely high ClO₂ concentration (100 mg L^-1). Electron micrographs showed that cell surface damage was pronounced and disruption in inner cell components was also apparent. The ion leakage, the inhibition of key enzyme activities of metabolic pathway, and the alteration of cell structure were critical events in S. cerevisiae inactivation by ClO₂.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.     

Listeria monocytogenes Behaviour in Presence of Non-UV-Irradiated Titanium Dioxide Nanoparticles

Listeria monocytogenes is the agent of listeriosis, a food-borne disease. It represents a serious problem for the food industry because of its environmental persistence mainly due to its ability to form biofilm on a variety of surfaces. Microrganisms attached on the surfaces are a potential source of contamination for environment and animals and humans. Titanium dioxide nanoparticles (TiO₂ NPs) are used in food industry in a variety of products and it was reported that daily exposure to these nanomaterials is very high. Anti-listerial activity of TiO₂ NPs was investigated only with UV-irradiated nanomaterials, based on generation of reactive oxigen species (ROS) with antibacterial effect after UV exposure. Since both Listeria monocytogenes and TiO₂ NPs are veicolated with foods, this study explores the interaction between Listeria monocytogenes and non UV-irradiated TiO₂ NPs, with special focus on biofilm formation and intestinal cell interaction. Scanning electron microscopy and quantitative measurements of biofilm mass indicate that NPs influence both production and structural architecture of listerial biofilm. Moreover, TiO₂ NPs show to interfere with bacterial interaction to intestinal cells. Increased biofilm production due to TiO₂ NPs exposure may favour bacterial survival in environment and its transmission to animal and human hosts.