Comparison of European and US biological indicators for ethylene oxide sterilization

Summary Biological indicators (BIs) are used to monitor ethylene oxide (EO) gas sterilization processes for medical devices. Several European and United States BIs for EO sterilization were evaluated for resistance according to both United States Pharmacopeia (USP) XXI and United Kingdom's (UK) tests for D-values. US BIs areB. subtilis var. niger spores on paper strips or disc carriers while European BIs use aluminum strips, quartz sand, or cotton yarn. Numerous BIs per run and runs per lot, as well as 2–3 different lots of BIs from each manufacturer, were examined. Both British and US BIs met their respective label claims for rates of inactivation when tested against British and USP EO test parameters, respectively. However, Danish BIs, on cotton yarn or quartz sand, were not inactivated following USP specifications during the exposure dwell times tested (600 mg L^–1 EO, 54°C, 60% RH, 0–110 min). The Danish BIs will require further testing in order for us to determine if theirB. subtilis spores are unusually resistant to EO or if the spore carrier substrates protect the spores from the sterilizing gas. In conclusion, the British and American BIs for EO sterilization are equivalent in resistance despite differences in carrier substrate, recovery conditions, calculation methods for D-values, and the labeled sterilization conditions for use.     

Survival of Spacecraft-Associated Microorganisms under Simulated Martian UV Irradiation

Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA+B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three Bacillus spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m⁻² of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, Bacillus subtilis 168. In addition, the exposure time required for UVA+B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the Bacillus species tested, spores of a Bacillus pumilus strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific; B. pumilus SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like B. pumilus SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the sanitation capabilities of UV irradiation are determined.     

Role of Dipicolinic Acid in Survival of Bacillus subtilis Spores Exposed to Artificial and Solar UV Radiation

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of Bacillus subtilis spore dry weight and has been shown to play a significant role in the survival of B. subtilis spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon dpaAB were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the gerA, gerB, and gerK nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.     

Development of an aerosol surface inoculation method for bacillus spores

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10(7) CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.     

Chlorine Dioxide Gas Sterilization under Square-Wave Conditions

Experiments were designed to study chlorine dioxide (CD) gas sterilization under square-wave conditions. By using controlled humidity, gas concentration, and temperature at atmospheric pressure, standard biological indicators (BIs) and spore disks of environmental isolates were exposed to CD gas. The sporicidal activity of CD gas was found to be concentration dependent. Prehumidification enhanced the CD activity. The D values (time required for 90% inactivation) of Bacillus subtilis subsp. niger ATCC 9372 BIs were estimated to be 1.5, 2.5, and 4.2 min when exposed to CD concentrations of 30, 15, and 7 mg/liter, respectively, at 23°C and ambient (20 to 40%) relative humidity (RH). Survivor tailings were observed. Prehumidification of BIs to 70 to 75% RH in an environmental chamber for 30 min resulted in a D value of 1.6 min after exposure to a concentration of 6 to 7 mg of CD per liter at 23°C and eliminated survivor tailing. Prolonging prehumidification at 70 to 75% RH for up to 16 h did not further improve the inactivation rate. Prehumidification by ultrasonic nebulization was found to be more effective than prehumidification in the environmental chamber, improving the D value to 0.55 min at a CD concentration of 6 to 7 mg/liter. Based on the current observations, CD gas is estimated, on a molar concentration basis, to be 1,075 times more potent than ethylene oxide as a sterilant at 30°C. A comparative study showed B. subtilis var. niger BIs were more resistant than other types of BIs and most of the tested bacterial spores of environmental isolates.     

Photodynamic Inactivation of Bacillus Spores, Mediated by Phenothiazinium Dyes

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.     

Investigation of the electrostatic charge of basidiospores of the Phellinus igniarius group

The polarity and magnitude of primary electric charges carried by basidiospores in the airborne state were investigated in living fungal fruiting bodies under natural forest conditions using a portable experimental device designed by the author. The operating principle was the falling of spores in the homogeneous horizontal electric field. The vertical and horizontal components of the trajectories of the spores were determined according to their deposition sites on electrodes (vertical metal plates). Altogether 33 samples of spores were examined for polarity, 10 of these samples (with 10⁴–10⁶ spores per sample) also were used to calculate the mean spore charge-to-mass quotient and the mean spore charge. The detection limits of spore charge-to-mass quotient varied in the range from (4.9±2.3)×10^?5 to (1.36±0.33)×10^?4 C kg^?1. Basidiospores (subglobose, smooth, diameter of 4–6 μm) of the closely related (sibling) species Phellinus alni, P. nigricans, P. populicola and P. tremulae (Hymenochaetales, Basidiomycota) carried positive electrical charges that have mean values from 48 to 305 elementary charges. The intraspecies variation of the spore charge could depend on the natural variation in spore size.     

Description of Chytridiopsis Trichopterae N. Sp. (Microspora, Chytridiopsidae), A Microsporidian Parasite of the Caddis Fly Polycentropus Flavomaculatus (Trichoptera, Polycentropodidae), With Comments On Relationships Between the Families Chytridiopsidae and Metchnikovellidae

ABSTRACT. The microsporidium Chytridiopsis trichopterae n. sp., a parasite of the midgut epithelium of larvae of the caddis fly Polycentropus flavomaculatus found in southern Sweden, is described based on light microscopic and ultrastructural characteristics. All life cycle stages have isolated nuclei. Merogonial reproduction was not observed. the sporogony comprises two sequences: one with free spores in parasitophorous vacuoles, the other in spherical, 5.6-6.8 μm wide, sporophorous vesicles which lie in the cytoplasm. the free sporogony yields more than 20 spores per sporont. the vesicle-bound sporogony produces 8, 12 or 16 spores. the envelope of the sporophorous vesicle is about 82 nm thick and layered. the internal layer is the plasma membrane of the sporont; the surface layer is electron dense with regularly arranged translucent components. Both spore types are spherical. They have an ～ 35-nm thick spore wall, with a plasma membrane, an electron-lucent endospore, and an ～ 14-nm thick electron-dense exospore. the polar sac is cup-like and lacks a layered anchoring disc. the polar filament is arranged in two to three isofilar coils in the half of the spore opposite the nucleus. the coupling between the polar sac and the polar filament is characteristic. the surface of the polar filament is covered with regularly arranged membraneous chambers resembling a honeycomb. There is no polaroplast of traditional type. the cytoplasm lacks polyribosomes. the nucleus has a prominent, wide nucleolus. the two spore types have identical construction, but differ in dimensions and electron density. Free living spores are about 3.2 μm wide, the diameter of the polar filament proper is 102-187 nm, the chambers of the honeycomb are 70-85 nm high, and the polar sac is up to 425 nm wide. Living spores in the vesicle-bound sporogony are about 2.1 μm wide, the polar filament measures 69-102 nm, the chambers of the honeycomb are about 45 nm high, and these spores are more electron dense. Comparisons of cytology (especially the construction of the spore wall and the polar filament and associated structures) and life cycles reveal prominent differences among the Chytridiopsis-like microsporidia, and close relationships between the families Chytridiopsidae and Metchnikovellidae.     

Levels of Germination Proteins in Bacillus subtilis Dormant,Superdormant, and Germinating Spores

Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.     

Deposition of Bacillus subtilis spores using an airbrush-spray or spots to study surface decontamination by pulsed light

Microbial contamination on surfaces of food processing equipment is a major concern in industries. A new method to inoculate a single-cell layer (monolayer) of microorganisms onto polystyrene was developed, using a deposition with an airbrush. A homogeneous dispersion of Bacillus subtilis DSM 402 spores sprayed on the surface was observed using both plate count and scanning electron microscopy. No clusters were found, even with high spore concentrations (10⁷ spores/inoculated surface). A monolayer of microorganisms was also obtained after deposition of 10 μL droplets containing 3 × 10⁴ spores/spot on polystyrene disks, but not with a higher spore concentration. Pulsed light (PL) applied to monolayers of B. subtilis spores allowed log reductions higher than 6. As a consequence of clusters formation in spots of 10 μL containing more than 3 × 10⁵ spores, log reductions obtained by PL were significantly lower. The comparative advantages of spot and spray depositions were discussed.     

ANALYSIS OF STARCH ACCUMULATION AND GERMINATION IN ONOCLEA SPORES

Previous studies have shown that low fluences of light accelerate starch accumulation and enhance germination of Onoclea spores. Fluence response curves for induction of starch accumulation were compared with fluence response curves for enhancement of germination in order to determine if the two photoresponses in Onoclea spores have a common photoreceptor. Fluence response curves indicate that both responses were proportional to the log of the fluence and that the relative fluence efficiencies of the four wavelength regions tested were similar for induction of both germination and starch accumulation. Red (600–720 nm) irradiation was the most efficient, while green (500–600 nm), blue (400–520 nm), and far-red (720–900 nm) irradiations showed a decreasing order of efficiency for induction of the responses. A correlation coefficient between the amount of starch accumulated as a result of red irradiation and the final percent germination was calculated to be 0.964. These results support the hypothesis that a common photoreceptor mediates both photoinduced germination and starch accumulation. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibits photosynthesis by blocking the flow of electrons from Photosystem II to Photosystem I. At 0.1 mM DCMU failed to inhibit both photoinduced starch acumulation and germination. This result and the greater efficiency of red than blue light, the low fluence functional for induction, and the fluence dependency argue against the participation of photosynthesis in photoinduced starch accumulation. A similar conclusion has been previously drawn for photoenhancement of Onoclea spore germination. Additionally, the effects 0.01–1.0 mm cycloheximide and 100 μl/l ethylene on photoinduced starch accumulation were investigated. Neither agent inhibited starch accumulation, whereas both substances inhibited germination 70–90% when applied at a time coincidental with the period of rapid starch accumulation. These results indicate that the photoinduction of starch accumulation does not have an ethylene sensitive stage nor does it require protein synthesis as does photoenhancement of germination of Onoclea spores.     

Effect of Light on Uredospore Germination and Germ Tube Growth of Soybean Rust (Phakopsora pachyrhizi Syd.)

The effect of light on uredospore germination and germ tube growth of Phakopsora pachyrhizi was studied. Frequency of uredospore germination was only partially reduced by high light intensity (> 1,9 * 10⁴ mW * m^?2). In uredospores unilaterally irradiated with polychromatic light germ tubes always emerged from the shadowed side. Already developed germ tubes showed a negative phototropic response. Both effects were inducible by low light intensities. Negative phototropism of germ tubes was a blue light effect. Light of 441 nm was more effective than that of 422 nm or 372 nm. Red light (> 600 nm) was ineffective, green light (513 nm) induced medium responses. In half-side illumination studies longitudinal halves of germ tube tips and spores were irradiated under a microscope. The tips of the germ tubes bent into the illuminating beam. In half-side illumination studies germ tubes always emerged from the illuminated spore halves. Under unilateral illumination liquid paraffin reversed this light “polarization” of spores and the negative phototropism of germ tubes. These results suggest that during unilateral illumination spores and germ tube tips act as a lens focussing the light on the wall farthest away from the light source., There, in uredospores emergence of germ tubes is stimulated and in germ tubes growth is inhibited. As a consequence, under unilateral illumination germ tubes emerge at the shadowed side of the spores and grow away from the light.     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

Light germination ofAnthoceros miyabeanus spores

The effects of light on the spore germination of a hornwort species,Anthoceros miyabeanus Steph., were investigated. Spores of this species were photoblastic, but their sensitivities to light quality were different. Under either continuous white, red or diffused daylight, more than 80% of the spores germinated, but under blue light none or a few of them germinated. Under continuous far-red light or in total darkness, the spores did not germinate at all.Anthoceros spores required red light irradiation for a very long duration, i.e., over 12–24 hr of red light for saturated germination. However, the spore germination showed clear photo-reversibility by repeated irradiation of red and far-red light. The germination pattern clearly varied with the light quality. There were two fundamental patterns; (1) cell mass type in white or blue light: spores divide before germination, and the sporelings divide frequently and form 1–2 rhizoids soon after germination, and (2) germ tube type in red light: spores germinate without cell division, and the single-cell sporelings elongate without cell division and rhizoid formation.     

Patterns of primary spore discharge of Entomophthora spp from the blue green aphid,Acyrthosiphon kondoi

Experiments were conducted to study the effects of time, temperature, and light regime on primary spore formation at 100% RH for the three major pathogens of Acyrothosiphon kondoi. Only small differences were detected between the continuous light and continuous dark regimes. Entomophthora obscura produced between 6 and 10 × 10³ primary spores mostly during the first 48 hr. Total primary spore production was similar at the five temperatures tested from 5° to 25°C. Entomophthora planchoniana produced large numbers of primary spores (about 5 × 10⁴ per aphid) only at temperatures between 10° and 20°C. The majority of primary spores were formed during the first 24 hr. Primary spore production with Entomophthora nr. exitialis ranged from about 10⁵ per aphid at 5° and 10°C to 3 or 4 × 10⁵ at 15° to 25°C, with most spores being formed during the first 48 hr. It is suggested that rainfall is more likely to be important for transmission of E. obscura and E. nr. exitialis than for transmission of E. planchoniana, and that E. obscura is likely to be the most important pathogen during cool or cold weather.     

Photosynthetic action spectra of trees: I. Comparative photosynthetic action spectra of one deciduous and four coniferous tree species as related to photorespiration and pigment complements

Comparative isoenergetic action spectra of net photosynthesis for intact, current year foliage of five tree species were determined from 400 to 710 nm by CO₂ exchange analysis. The blue (400 to 500 nm) peak of net photosynthetic activity for the green broadleaves of red alder (Alnus rubra Bong.) was reduced to a plateau for the green needle-leaves of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) and Sitka spruce (Picea sitchensis [Bong.] Carr.), a shoulder for the blue-green needles of Colorado spruce (Picea pungens Engelm.), and a reduced shoulder for the blue-white needles of Blue spruce (Picea pungens var. hoospii). These differences were attributable neither to a differential blue light stimulation of photorespiration nor to a differential presence of a nonplastid screening pigment. The conifers all had similar carotenoid-chlorophyll ratios, with approximately 50% more carotenoid relative to chlorophyll as compared to red alder. Blue light absorption and low efficiency of energy transfer by the carotenoids probably accounts for the low net photosynthetic activity of the green conifers in blue light as compared to red alder. Leaf form per se (broad versus needle) had no distinguishable influence on these results.     

Control of Mitosis by Phytochrome and a Blue-Light Receptor in Fern Spores

The first mitosis in spores of the fern A. capillus-veneris was observed under a microscope equipped with Nomarski optics with irradiation from a safelight at 900 nm, and under a fluorescent microscope after staining with 4[prime],6-diamidino-2-phenylindole. During imbibition the nucleus remained near one corner of each tetrahedron-shaped dormant spore, and asymmetric cell division occurred upon brief irradiation with red light. This red light-induced mitosis was photoreversibly prevented by subsequent brief exposure to far-red light and was photo-irreversibly prevented by brief irradiation with blue light. However, neither far-red nor blue light affected the germination rate when spores were irradiated after the first mitosis. Therefore, the first mitosis in the spores appears to be the crucial step for photoinduction of spore germination. Furthermore, experiments using a microbeam of red or blue light demonstrated that blue light was effective only when exposed to the nucleus, and no specific intracellular photoreceptive site for red light was found in the spores. Therefore, phytochrome in the far-red absorbing form induces the first mitosis in germinating spores but prevents the subsequent mitosis in protonemata, whereas a blue-light receptor prevents the former but induces the latter.     

Failure to decontaminate Glomus clarum NT4 spores is due to spore wall-associated bacteria

 Exposure of spores of Glomus clarum NT4 to solutions of chloramine-T (2.5–10% w/v) for 10–120 min failed to fully decontaminate all spores. Scanning electron microscopy did not show the presence of contaminants on treated spores, but transmission electron microscopy revealed bacterial cells embedded within the outer spore wall layer. Bacteria that remained protected within the spore walls were detected only when the spores were placed on appropriate media. Nutrient agar and tryptic soy agar supported relatively high levels of contaminant growth and were regarded as good media for assessing contamination, whereas the detection of contaminant growth on water agar required prolonged incubation. Contamination and germination of G. clarum NT4 spores following decontamination treatments were dependent on spore age. Generally, lower concentrations of chloramine-T and shorter incubation periods were required to reduce contamination of freshly harvested spores than of mature spores. Exposure to 10% chloramine-T for 120 min was required to reduce the levels of contamination of mature spores to ≤10%. Unfortunately, spore germination was compromised by rigorous decontamination treatments, thus the success of any decontamination procedure should be evaluated prior to its routine use. Moreover, if the interpretation of experimental results rests on the assumption of true surface sterility of VAMF spores, we suggest that the axenic condition of spores be confirmed prior to experimentation on a medium that encourages contaminant growth. Accepted: 12 July 1995     

Effect of Shadowing on Survival of Bacteria under Conditions Simulating the Martian Atmosphere and UV Radiation

Spacecraft-associated spores and four non-spore-forming bacterial isolates were prepared in Atacama Desert soil suspensions and tested both in solution and in a desiccated state to elucidate the shadowing effect of soil particulates on bacterial survival under simulated Martian atmospheric and UV irradiation conditions. All non-spore-forming cells that were prepared in nutrient-depleted, 0.2-μm-filtered desert soil (DSE) microcosms and desiccated for 75 days on aluminum died, whereas cells prepared similarly in 60-μm-filtered desert soil (DS) microcosms survived such conditions. Among the bacterial cells tested, Microbacterium schleiferi and Arthrobacter sp. exhibited elevated resistance to 254-nm UV irradiation (low-pressure Hg lamp), and their survival indices were comparable to those of DS- and DSE-associated Bacillus pumilus spores. Desiccated DSE-associated spores survived exposure to full Martian UV irradiation (200 to 400 nm) for 5 min and were only slightly affected by Martian atmospheric conditions in the absence of UV irradiation. Although prolonged UV irradiation (5 min to 12 h) killed substantial portions of the spores in DSE microcosms (~5- to 6-log reduction with Martian UV irradiation), dramatic survival of spores was apparent in DS-spore microcosms. The survival of soil-associated wild-type spores under Martian conditions could have repercussions for forward contamination of extraterrestrial environments, especially Mars.     

Surface Charge Properties of and Cu(II) Adsorption by Spores of the Marine Bacillus sp. Strain SG-1

Spores of marine Bacillus sp. strain SG-1 are capable of oxidizing Mn(II) and Co(II), which results in the precipitation of Mn(III, IV) and Co(III) oxides and hydroxides on the spore surface. The spores also bind other heavy metals; however, little is known about the mechanism and capacity of this metal binding. In this study the characteristics of the spore surface and Cu(II) adsorption to this surface were investigated. The specific surface area of wet SG-1 spores was 74.7 m² per g of dry weight as measured by the methylene blue adsorption method. This surface area is 11-fold greater than the surface area of dried spores, as determined with an N₂ adsorption surface area analyzer or as calculated from the spore dimensions, suggesting that the spore surface is porous. The surface exchange capacity as measured by the proton exchange method was found to be 30.6 μmol m⁻², which is equal to a surface site density of 18.3 sites nm⁻². The SG-1 spore surface charge characteristics were obtained from acid-base titration data. The surface charge density varied with pH, and the zero point of charge was pH 4.5. The titration curves suggest that the spore surface is dominated by negatively charged sites that are largely carboxylate groups but also phosphate groups. Copper adsorption by SG-1 spores was rapid and complete within minutes. The spores exhibited a high affinity for Cu(II). The amounts of copper adsorbed increased from negligible at pH 3 to maximum levels at pH >6. Their great surface area, site density, and affinity give SG-1 spores a high capability for binding metals on their surfaces, as demonstrated by our experiments with Cu(II).