Spore Deactivation Research Articles

Hexane abatement and spore emission control in a fungal biofilter-photoreactor hybrid unit

The performance of a fungal perlite-based biofilter coupled to a post-treatment photoreactor was evaluated over 234 days in terms of n-hexane removal, emission and deactivation of fungal spores. The biofilter and photoreactor were operated at gas residence times of 1.20 and 0.14min, respectively, and a hexane loading rate of 115±5gm−3h−1. Steady n-hexane elimination capacities of 30–40gm−3h−1 were achieved, concomitantly with pollutant mineralization efficiencies of 60–90%. No significant influence of biofilter irrigation frequency or irrigation nitrogen concentration on hexane abatement was recorded. Photolysis did not support an efficient hexane post-treatment likely due to the short EBRT applied in the photoreactor, while overall hexane removal and mineralization enhancements of 25% were recorded when the irradiated photoreactor was packed with ZnO-impregnated perlite. However, a rapid catalyst deactivation was observed, which required a periodic reactivation every 48h. Biofilter irrigation every 3 days supported fungal spore emissions at concentrations ranging from 2.4×103 to 9.0×104CFUm−3. Finally, spore deactivation efficiencies of ≈98% were recorded for the photolytic and photocatalytic post-treatment processes. This study confirmed the potential of photo-assisted post-treatment processes to mitigate the emission of hazardous fungal spores and boost the abatement performance of biotechnologies.

Inactivation of Bacillus subtilis var. niger of both spore and vegetative forms by means of corona discharges applied in water

Spores are dormant units of bacteria resistant to numerous disinfection methods. Additionally, the effects on bacteria of repetitive electrical discharges in water by used of the so-called “corona discharges” or streamer are poorly described. In this study vegetative and spore forms of Bacillus subtilis var. niger were subjected to these discharges. To generate corona discharges in water, a Marx generator capable of delivering 60–90 kV was used with a coaxial chamber of treatment. Vegetative and spore form reductions were defined using colony-forming unit counting. Proteins extracts were separated by two-dimensional electrophoresis and spots of interest were characterized by mass spectrometry. Shock waves were assessed by the diminution of liposome size and OD400 nm. The results show a decrease in bacteria viability of 2 log10 after 1000 discharges on the vegetative form and 4 log10 after 10,000 discharges on the spores. Two-dimensional electrophoresis showed that the streamers impact the regulation of several proteins in the vegetative forms with UniProt ID: P80861, Q06797, P80244, C0ZI91, respectively. The reduction appears to be due, in part, to hydrogen peroxide (H2O2) generated by the corona discharges while spore deactivation remained insensitive to these chemicals. The spore eradication was associated to shock waves induced by the discharges but not H2O2. Corona discharges appear as a prospective method for eradication of spores in water. The corona discharges can be an efficient method for decontamination processes of waste water.

Thermo-structural studies of spores subjected to high temperature gas environments

One of the major mechanisms of deactivating spores is to expose them to elevated temperatures. Experiments carried out in an exposure tube to study the effects on spores in a high temperature gas environment provide evidence of spore deactivation, although the deactivation mechanism is not clear. Numerical studies have been performed to complement the experimental work and to study the effects of various parameters, such as gas temperature, exposure time, and internal pressure on spore survivability. The laminar and turbulent flow fields of a sudden expansion inside the thermal exposure system were simulated by computational fluid dynamics. A thermo-structural model with wall porosity was developed to take into account the dynamic response of the spore wall structure to the internal pressure exerted by the heated vapor on the spore wall. Finally, the results of the transient heat transfer and the thermo-structural models are discussed in the context of the uncertainties in biological system parameters and spore survivability.

Deactivation of Highly Resistant Microorganisms in Water Using Solar Driven Photocatalytic Processes

Inactivation of Bacillus subtilis spores was carried out using solar-driven Advanced Oxidation Processes (AOPs) alone and coupled with free chlorine in sequential processes. Experimental results showed that Fenton-like processes are more efficient under the tested conditions than heterogeneous photocatalytic processes using suspended titanium dioxide. Experimental data were fitted using modified Chick-Watson kinetics and the accumulated energy as the radiation dose needed for spore deactivation. It was demonstrated that the deactivation of B. subtilis spores using chlorine can be enhanced by primary treatment with photocatalysis. This study also suggests that AOPs could be an effective alternative to control the occurrence of highly resistant waterborne pathogens.

Simulation of sterilization of canned liquid food using sucrose degradation as an indicator

Numerical simulation of natural convection as well as changes of invert sugar concentration during sterilization of canned liquid food using CFX are presented in this study. The invert sugar, which resulted from sucrose inversion, was used as an indicator of thermal deactivation of microorganisms. The effect of surrounding temperature and F 0 calculation methods were also investigated. The simulation was validated with experiment by using a 300 × 407 can fully filled with 20% (w/w) sucrose syrup and subjected to steam at 100 °C. The temperature history, at 3.6 and 5.4 cm from the bottom of the can along the longitudinal axis, showed a good fit against the experimental data with relative deviations of 0.957% and 0.717%, respectively. The relative deviation of the average invert sugar concentration was 0.997%. At a low heating temperature (100 °C), the slowest heating zone did not affect to accumulative F 0 (at 2.0 min). The accumulative F 0 value obtained from the deactivation of spores increased slower than that obtained from the General Method at 100 and 110 °C but a reversed effect was found at 120 °C.

Mechanisms of Bacterial Spore Deactivation Using Ambient Pressure Nonthermal Discharges

Atmospheric pressure nonthermal plasmas have demonstrated rapid deactivation of bacterial spores on surfaces. For example, the lethal Ames strain of Bacillus anthracis, known as anthrax, has been deactivated after 1 min of ionized gas exposure. The hypothesized plasma-produced spore deactivation processes, such as ultraviolet light, hot gases, and intense electric fields, were isolated. Exposure to each of these assumed deactivation mechanisms has demonstrated little to no effect on the sterilization of bacterial spores at plasma discharge exposure levels. The focus of this study has therefore, been on the determination of the mechanism of spore lysis and resultant deactivation from the interaction of the ionized gas with the biological substances within the spore. The rapid lysis of bacterial spores and other cells by nonthermal plasma has led to the integration of this approach with pathogen detection instruments, such as mass spectrometers. The identification by mass spectrometry of the extracted proteins from plasmalyzed spores has facilitated the discovery and subsequent sequencing of small acid soluble proteins (SASP) within the spore responsible for protecting bacterial spores. The identified SASP surrounds the nucleic acids providing some measure of protection from insult in the environment. The plasma discharge rapidly interacts with this SASP causing deactivation of the spore.

High cAMP in spores of Dictyostelium discoideum: association with spore dormancy and inhibition of germination.

Signalling mechanisms involving cAMP have a well-documented role in the coordination of multicellular development and differentiation leading to spore formation in the social amoeba, Dictyostelium discoideum. The involvement of cAMP in the poorly understood developmental stages of spore dormancy and germination have been investigated in this study. Dormant spores contained up to 11-fold more cAMP than nascent amoebae. The spore cAMP levels were not constant, but typically underwent a surge at 14-18 d when spores acquired the ability to germinate spontaneously. The high cAMP levels decreased only during successful spore germination, i.e. emergence of nascent amoebae. The temporal pattern of cAMP decrease was complex and unique to the method of spore activation, supporting our hypothesis that exogenously (e.g. heat) activated and autoactivated spores germinate by different mechanisms. During heat-induced activation, transcription of acg (a gene encoding adenylyl cyclase associated with germination) correlated well with spore cAMP content. Young wild-type spores, incapable of spontaneous germination, maintained a uniformly high cAMP level, and spore cAMP levels also remained high if germination was inhibited. When activated spores were deactivated by applying increased osmotic pressure, cAMP concentrations rose and ultimately levelled off at the high levels typical of dormant spores. The correlation between high cAMP and failure to germinate was also evident when autoactivation was inhibited by the cAMP analogue, 8-bromo-cAMP. Also, spores from a strain (HTY217) with unrestrained protein kinase A activity were incapable of spontaneous germination. Overall, our experiments provide evidence for continued cAMP signalling in spores up to 18 d after sporulation and for linkages between elevated cAMP, spore deactivation and inhibition of spontaneous germination.

Reversible and irreversible activation of Phycomyces blakesleeanus spores

Phycomyces spores do not germinate in a suitable culture medium unless they are activated by one of a range of treatments. Heating the spores for 3 min at 44°C resulted in nearly complete germination in culture medium; however, when such spores were incubated in water, rapid deactivation occurred. Deactivation of spores treated for 3 min at 50°C was much slower and only partial. After reversible activation (44°C) RNA and protein synthesis increased rapidly in culture medium but not in water. Immediately after irreversible activation (50°C) uridine and leucine incorporation was severely reduced but increased rapidly upon further incubation in both water and culture medium. Activation of spores at 44 or 50°C in culture medium had a similar effect on nucleoside triphosphate content: the ATP level was high and did not change markedly after activation; the UTP and GTP content, however, showed a clear-cut increase shortly after activation. Spores incubated in water had a much lower nucleoside triphosphate content but upon irreversible activation (50°C) the pattern of the nucleoside triphosphates was similar to that in culture medium. Reversible activation (44°C) of spores incubated in water yielded only a temporary increase in ATP level. The pattern of respiration was the same for reversibly and irreversibly activated spores in culture medium. During incubation in water, however, irreversibly activated spores had a higher respiration than reversibly activated spores. This suggests that the irreversibility of activation at 50°C was caused by the occurrence of the initial phases of germination, even in water, whereas after reversible activation external carbon sources were required to start germination and to maintain the activated state. Respiration was insensitive to azide in dormant spores but became progressively more sensitive during germination.

The physiological effects of restrictive environmental conditions on Dictyostelium discoideum spore germination

Spores may be reversibly activated by the application of heat, dimethyl sulfoxide, urea, or ethylene glucol. Severe changes in four environmental variables (high osmotic pressure, low oxygen tension, low or high pH, and low or high temperature) interfere with the germination process. Spores at the end of the postactivation lag phase of germination were usually deactivated if exposed to severe environmental conditions and thus did not swell; spores in the swelling and oxygen uptake which began during spore activation was primarily attributable to a cyanide-sensitive pathway and secondarily to a salicylhydroxamic acid (SHAM) sensitive pathway. Inhibition of the SHAM-sensitive pathway did not cause spore deactivation while the addition of cyanide resulted in rapid spore deactivation. Treatment of activated spores with azide or environmental shifts also resulted in inhibition of oxygen uptake and spore deactivation. Deactivating spores did not demonstrate the amino acid incorporation, uridine incorporation, and expression of trehalase activity which is found in the later stages of germinating control spores. Protein synthesis inhibitors did not cause spore deactivation or a decrease in oxygen uptake but they inhibited amino acid incorporation and the expression trehalase activity in swollen spores. It is concluded that control of respiratory activity is involved in regulation of reversible activation.

The effects of osmotic pressure changes on the germination of Dictyostelium discoideum spores.

Polyalcohols such as ethylene glycol and glycerol at 3 M penetrate and activate spores of Dictyostelium discoideum incubated at room temperature. Higher concentrations of ethylene glycol result in lysis upon suspension of spores in dilute phosphate buffer. Erythritol and arabitol at 3 M do not penetrate or activate D. discoideum spores.Air-dried spores or those incubated in 2 M sucrose solutions are not activated with the usual heat treatment of 45 °C for 30 min. The plasmolyzed spores are activated at temperatures above 45 °C when heated in the presence of 2 M sucrose for 30 min. The temperature for maximum activation and the temperature for thermal inactivation of spores are raised 7–10 °C in high sucrose concentrations. Long-term incubation of heat-activated spores in 2 M sucrose solutions does not result in a return to dormancy.Moderate sucrose concentrations near 0.2 M do not block the heat-induced activation process but must be removed from the spore population to prevent a return to dormancy within 6 h. Other polyhydric compounds at 0.25 M concentration also cause spore deactivation within 6 h of room temperature incubation. Oxygen uptake of spores undergoing deactivation in 0.18 M sucrose is inhibited as compared to control levels. Moderate concentrations of sucrose do not block the early events of postactivation lag and the spores accumulate at the end of the lag phase. The longer the spores remain unswollen at the end of the postactivation lag phase, the greater the percentage of spores which return to dormancy. The effects of moderate sucrose concentration (lowered water activity) are not duplicated by the same quantity of Ficoll, indicating that the colligative properties of the sucrose solutions are responsible for deactivation.