Endotoxin inactivation in water by using medium-pressure UV lamps

Deionized water was spiked with various concentrations of endotoxin and exposed to UV irradiation from medium-pressure UV lamps to assess endotoxin inactivation. It was found that endotoxin inactivation was proportional to the UV dose under the conditions examined. The inactivation rate was determined to be approximately 0.55 endotoxin unit/ml per mJ/cm(2) of irradiation delivered.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Inactivation of Cryptosporidium parvum Oocysts in Fresh Apple Cider by UV Irradiation

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm². Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.     

Enhanced UV Inactivation of Adenoviruses under Polychromatic UV Lamps

Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The U.S. EPA has stipulated that a UV fluence (dose) of 186 mJ cm⁻² is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date published in the peer-reviewed literature have been based on UV disinfection experiments using UV irradiation at 253.7 nm produced from a conventional low-pressure UV source. The work reported here presents inactivation data for adenovirus based on polychromatic UV sources and details the significant enhancement in inactivation achieved using these polychromatic sources. When full-spectrum, medium-pressure UV lamps were used, 4-log inactivation of adenovirus type 40 is achieved at a UV fluence of less than 60 mJ cm⁻² and a surface discharge pulsed UV source required a UV fluence of less than 40 mJ cm⁻². The action spectrum for adenovirus type 2 was also developed and partially explains the improved inactivation based on enhancements at wavelengths below 230 nm. Implications for water treatment, public health, and the future of UV regulations for virus disinfection are discussed.     

Ultraviolet Irradiation Produces Loss of Saxitoxin Binding to Sodium Channels in Rat Synaptosomes

Ultraviolet irradiation (UV) has been shown to cause an electrophysiologically measured inactivation of the rapid, transient sodium conductance system in nerve. Tritiated saxitoxin ([³H]STX) was used as a structural probe to assess the possibility of a corresponding perturbation in the conformation of the STX binding site. UV irradiation caused an irreversible decrease in the total number of high-affinity [³H]STX binding sites in rat synaptosomes, while the dissociation constant of the remaining sites did not change. The receptor loss followed first-order kinetics, and the rate of loss was independent of temperature. The action spectrum for binding loss indicated a peak in spectral sensitivity near 280 nm. A²²Na flux assay in irradiated synaptosomes directly demonstrated that [³H]STX binding sites and veratridinestimulated, STX-blocked ²²Na efflux had similar sensitivities to UV radiation. We conclude that the UV inactivation of functional channels includes a modification of the STX binding-site structure.     

Efficacy of UV Irradiation in Inactivating Cryptosporidiumparvum Oocysts

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log₁₀ reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm² at 20°C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm² for a 2-log₁₀ reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm². Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10°C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log₁₀ reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.     

Lethal and mutational effects of solar and UV radiation on Staphylococcus aureus

Strains of Staphylococcus aureus, an opportunistic pathogen commonly found on human skin, were exposed to sunlight and UV C radiation, and the lethal and mutational effects measured. Sunlight killed cells with an inactivation constant of 3×10^-5 per joule per square metre; UV C was much more lethal, giving an inactivation constant of approximately 0.1 per joule per square metre. Some strains tested showed a sensitivity to sunlight that was dependent on the growth phase of the cells, exponentially growing cells showing a greater sensitivity. Mutational effects of irradiation were measured by the appearance of mutants sensitive to methicillin following irradiation of a multiresistant strain. Mutants appeared at a frequency of 10^-3; this high frequency of mutation in the region of the mec gene has also been observed when multiresistant strains are subjected to nutritional or thermal stress. Mutants showed the same chromosomal alteration (seen in pulse-field gel electrophoresis of Smal-digested DNA) whether induced by solar or UV C irradiation.     

Reactivation of Giardia lamblia cysts after exposure to polychromatic UV light

Aims: In this study, we determined the ability of a promising alternative UV technology – a polychromatic emission from a medium‐pressure UV (MP UV) technology – to inhibit the reactivation of UV‐irradiated Giardia lamblia cysts. Methods and Results: A UV‐collimated beam apparatus was used to expose shallow suspensions of purified G. lamblia cysts in PBS (pH 7·2) or filtered drinking water to a low dose (1 mJ cm^?2) of MP UV irradiation. After UV irradiation, samples were exposed to two repair conditions (light or dark) and two temperature conditions (25°C or 37°C for 2–4 h). The inactivation of G. lamblia cysts by MP UV was very extensive, and c. 3 log₁₀ inactivation was achieved with a dose of 1 mJ cm^?2. Meanwhile, there was no apparent reactivation (neither in vivo nor in vitro) of UV‐irradiated G. lamblia under the conditions tested. Conclusion: The results of this study indicated that, unlike the traditional low‐pressure (LP) UV technology, an alternative UV technology (MP UV) could inhibit the reactivation of UV‐irradiated G. lamblia cysts even when the cysts were exposed to low UV doses. Significance and Impact of the Study: It appears that alternative UV technology has some advantages over the traditional LP UV technology in drinking water disinfection because of their high level of inactivation against G. lamblia cysts and also effective inhibition of reactivation in UV‐irradiated G. lamblia cysts.     

Calicivirus Inactivation by Nonionizing (253.7-Nanometer-Wavelength [UV]) and Ionizing (Gamma) Radiation

Noroviruses (previously Norwalk-like viruses) are the most common viral agents associated with food- and waterborne outbreaks of gastroenteritis. In the absence of culture methods for noroviruses, animal caliciviruses were used as model viruses to study inactivation by nonionizing (253.7-nm-wavelength [UV]) and ionizing (gamma) radiation. Here, we studied the respiratory feline calicivirus (FeCV) and the presumed enteric canine calicivirus (CaCV) and compared them with the well-studied bacteriophage MS2. When UV irradiation was used, a 3-log₁₀ reduction was observed at a fluence of 120 J/m² in the FeCV suspension and at a fluence of 200 J/m² for CaCV; for the more resistant phage MS2 there was a 3-log₁₀ reduction at a fluence of 650 J/m². Few or no differences were observed between levels of UV inactivation in high- and low-protein-content virus stocks. In contrast, ionizing radiation could readily inactivate MS2 in water, and there was a 3-log₁₀ reduction at a dose of 100 Gy, although this did not occur when the phage was diluted in high-protein-content stocks of CaCV or FeCV. The low-protein-content stocks showed 3-log₁₀ reductions at a dose of 500 Gy for FeCV and at a dose of 300 for CaCV. The inactivation rates for both caliciviruses with ionizing and nonionizing radiation were comparable but different from the inactivation rates for MS2. Although most FeCV and CaCV characteristics, such as overall particle and genome size and structure, are similar, the capsid sequences differ significantly, making it difficult to predict human norovirus inactivation. Adequate management of UV and gamma radiation processes for virus inactivation should limit public health risks.     

Increased inactivation of Saccharomyces cerevisiae by protraction of UV irradiation.

The principle of equi-effectivity of the product of intensity and exposure time (principle of Bunsen-Roscoe) of UV irradiation has been assumed to be valid for the inactivation of microorganisms in general. Earlier studies claimed higher survival of Escherichia coli B/r with fractionated irradiation compared with single-exposure survival. However, data on the inactivation effect of protraction of UV irradiation are not available. By means of a specially designed UV irradiation apparatus which secured absolute UV dose measurements throughout the experiments, the effects of variation of UV irradiation intensities (253.7 nm) and exposure times were tested on the inactivation of a bacterial virus (Staphylococcus aureus phage A994), a vegetative bacterial strain (E. coli ATCC 25922), and bacterial spores (Bacillus subtilis ATCC 6633) as well as three haploid laboratory strains (RC43a, YNN281, and YNN282) and two diploid strains (commercial bakery yeast strain and laboratory strain YNN281 x YNN282) or yeast (Saccharomyces cerevisiae) and spores of the latter diploid yeast strain. Each test organism was exposed to three UV intensities (0.02, 0.2, and 2 W/m2), with corresponding exposure times resulting in three dose levels for each intensity. Differences in inactivation rates were tested by analyses of variance and Newman-Keuls tests. Virus and bacteria showed no differences in inactivation rates by variation of intensities and exposure times within selected UV doses; hence, the principle of Bunsen-Roscoe could not be rejected for these strains. However, in the eukaryotic test strains of S. cerevisiae longer exposure times with lower intensities led to enhanced inactivation in both haploid and diploid strains, with a more pronounced effect in the diploid yeast strains, whereas in yeast spores in this dose rate effect could not be observed.     

Endotoxin inactivation via steam‐heat treatment in dilute simethicone emulsions used in biopharmaceutical processes

Simethicone emulsion is used to regulate foaming in cell culture operations in biopharmaceutical processes. It is also a potential source of endotoxin contamination. The inactivation of endotoxins in dilute simethicone emulsions was assessed as a function of time at different steam temperatures using a Limulus amebocyte lysate kinetic chromogenic technique. Endotoxin inactivation from steam‐heat treatment was fit to a four‐parameter double exponential decay model, which indicated that endotoxin inactivation was biphasic, consisting of fast and slow regimes. In the fast regime, temperature‐related effects were dominant. Transitioning into the slow regime, the observed temperature dependence diminished, and concentration‐related effects became increasingly significant. The change in the Gibbs free energy moving through the transition state indicated that a large energy barrier must be overcome for endotoxin inactivation to occur. The corresponding Arrhenius pre‐exponential factor was >>10¹² s^?1 suggesting that endotoxins in aqueous solution exist as aggregates. The disorder associated with the endotoxin inactivation reaction pathway was assessed via the change in entropy moving through the transition state. This quantity was positive indicating that endotoxin inactivation may result from hydrolysis of individual endotoxin molecules, which perturbs the conformation of endotoxin aggregates, thereby modulating the biological activity observed. Steam‐heat treatment decreased endotoxin levels by 1–2 logarithm (log) reduction (LRV), which may be practically relevant depending on incoming raw material endotoxin levels. Antifoam efficiency and cell culture performance were negligibly impacted following steam‐heat treatment. The results from this study show that steam‐heat treatment is a viable endotoxin control strategy that can be implemented to support large‐scale biopharmaceutical manufacturing. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1145–1160, 2014     

Assessing the effectiveness of low‐pressure ultraviolet light for inactivating Mycobacterium avium complex (MAC) micro‐organisms

Aims: To assess low‐pressure ultraviolet light (LP‐UV) inactivation kinetics of Mycobacterium avium complex (MAC) strains in a water matrix using collimated beam apparatus. Methods and Results: Strains of M. avium (n = 3) and Mycobacterium intracellulare (n = 2) were exposed to LP‐UV, and log₁₀ inactivation and inactivation kinetics were evaluated. All strains exhibited greater than 4 log₁₀ inactivation at fluences of less than 20 mJ cm^?2. Repair potential was evaluated using one M. avium strain. Light repair was evaluated by simultaneous exposure using visible and LP‐UV irradiation. Dark repair was evaluated by incubating UV‐exposed organisms in the dark for 4 h. The isolate did not exhibit light or dark repair activity. Conclusions: Results indicate that MAC organisms are readily inactivated at UV fluences typically used in drinking water treatment. Differences in activation kinetics were small but statistically significant between some tested isolates. Significance and Impact of the Study: Results provide LP‐UV inactivation kinetics for isolates from the relatively resistant MAC. Although UV inactivation of Mycobaterium species have been reported previously, data collected in this effort are comparable with recent UV inactivation research efforts performed in a similar manner. Data were assessed using a rigorous statistical approach and were useful towards modelling efforts.     

Biofouling control in water by various UVC wavelengths and doses

UV light irradiation is being increasingly applied as a primary process for water disinfection, effectively used for inactivation of suspended (planktonic) cells. In this study, the use of UV irradiation was evaluated as a pretreatment strategy to control biofouling. The objective of this research was to elucidate the relative effectiveness of various targeted UV wavelengths and a polychromatic spectrum on bacterial inactivation and biofilm control. In a model system using Pseudomonas aeruginosa, the inactivation spectra corresponded to the DNA absorption spectra for all wavelengths between 220 and 280 nm, while wavelengths between 254 nm and 270 nm were the most effective for bacterial inactivation. Similar wavelengths of 254-260-270 nm were also more effective for biofilm control in most cases than targeted 239 and 280 nm. In addition, the prevention of biofilm formation by P. aeruginosa with a full polychromatic lamp was UV dose-dependent. It appears that biofilm control is improved when larger UV doses are given, while higher levels of inactivation are obtained when using a full polychromatic MP lamp. However, no significant differences were found between biofilms produced by bacteria that survived UV irradiation and biofilms produced by control bacteria at the same microbial counts. Moreover, the experiments showed that biofilm prevention depends on the post-treatment incubation time and nutrient availability, in addition to targeted wavelengths, UV spectrum and UV dose.     

Inactivation of Single-Celled Ascaris suum Eggs by Low-Pressure UV Radiation

Intact and decorticated single-celled Ascaris suum eggs were exposed to UV radiation from low-pressure, germicidal lamps at fluences (doses) ranging from 0 to 8,000 J/m² for intact eggs and from 0 to 500 J/m² for decorticated eggs. With a UV fluence of 500 J/m², 0.44- ± 0.20-log inactivation (mean ± 95% confidence interval) (63.7%) of intact eggs was observed, while a fluence of 4,000 J/m² resulted in 2.23- ± 0.49-log inactivation (99.4%). (The maximum quantifiable inactivation was 2.5 log units.) Thus, according to the methods used here, Ascaris eggs are the most UV-resistant water-related pathogen identified to date. For the range of fluences recommended for disinfecting drinking water and wastewater (200 to 2,000 J/m²), from 0- to 1.5-log inactivation can be expected, although at typical fluences (less than 1,000 J/m²), the inactivation may be less than 1 log. When the eggs were decorticated (the outer egg shell layers were removed with sodium hypochlorite, leaving only the lipoprotein ascaroside layer) before exposure to UV, 1.80- ± 0.32-log reduction (98.4%) was achieved with a fluence of 500 J/m², suggesting that the outer eggshell layers protected A. suum eggs from inactivation by UV radiation. This protection may have been due to UV absorption by proteins in the outer layers of the 3- to 4-μm-thick eggshell. Stirring alone (without UV exposure) also inactivated some of the Ascaris eggs (~20% after 75 min), which complicated determination of the inactivation caused by UV radiation alone.     

Inactivation ofSaccharomyces cells by 8-methoxypsoralen plus pulsed laser irradiation in the wavelength range 308 nm-380 nm

Summary Two different strains ofSaccharomyces cerevisiae, one diploid wild type and one haploid mutant deficient in excision repair were irradiated with laser pulses in the range 308 nm to 380 nm after 8-MOP treatment. Both the shoulder (Dq) and the final slope (Do) of the inactivation curves were dependent on wavelength which showed a broad minimum around 355 nm. No differences in inactivation were recorded after pulsed irradiations between the repetition rates of 5 Hz and 35 Hz. Irradiations with pulses of the energy density from 0.1 mJ/cm² up to 26 mJ/cm² resulted in a final slope increasing with pulse energy density. This was in contrast to the effects of irradiation alone.Abbreviations 8-MOP 8-methoxypsoralen - UV ultraviolet - PUVA therapy withPsoralen plusUV-A     

Induced androgenesis in acipenserids may be obtained by ultraviolet radiation

The results are presented on haploid androgenesis in Siberian sturgeon and sterlet induced by UV irradiation of ovicells. During irradiation, the cells in Ringer solution were rotated around a UV lamp. The efficiency of genetic inactivation of ovicells was estimated by the following parameters: manifestation of Hertwig effect, the fraction of embryos demonstrating haploid syndrome at final developmental stages, by arrest of embryonic development in hybrids Siberian sturgeon × great sturgeon, and by absence of maternal alleles of microsatellite loci in embryos. The dose-effect curve suggests that, during UV irradiation of ovicells of Siberian sturgeon, the complete genetic inactivation is attained at exposition of 120 s, while that in sterlet is 90 or 105 s. The results show a principal possibility of inactivation of ovicells by UV irradiation and use of such cells for producing androgenetic progeny of acipenserids.     

Inactivation of transfecting DNA by physical and chemical agents: influence of genotypes of phage lambda and host cells

Inactivation of λ₁₁c and its purified DNA by UV irradiation, γ-rays of ¹³⁷Cs (in conditions of indirect action), nitrous acid, hydroxylamine and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was studied. The biological activity of isolated phage DNA was measured by the calcium transfection procedure. 14 different recipient strains of Escherichia coli K12 were used, including mutants deficient in excision and recombination repair (uvrA6, uvrB5, uvrC34, polA1, recA13, recC38, recD34, recA13B21C22, recA56uvrA6, exrA and recB21C22sbcB15).Whole phage was more resistant to the action of γ-rays than was isolated DNA. On the other hand, the chemical agents HNO₂ and MNNG inactivated phage much faster than isolated DNA. Of all mutations of the host cell only polA1 considerably increased the sensitivity of phage DNA to UV irradiation, γ-rays and MNNG. The mutations uvr^? affected the inactivation kinetics under UV action. In all other cases the genotype of the host cell was indifferent for the inactivation kinetics of phage DNA, even if it belonged to recombination deficient mutant λ red3 int6 (in which only UV and γ inactivation was studied). Possible reasons for the low efficiency of the host-cell repair toward the damage caused to λ DNA by different agents are discussed.