Muscodor albus and its biological promise

We have found a novel fungal genus that produces extremely bioactive volatile organic compounds (VOCs). This fungal isolate was initially discovered as an endophyte in Cinnamomum zeylanicum in a botanical garden in Honduras. This endophytic fungus, Muscodor albus, produces a mixture of VOCs that are lethal to a wide variety of plant and human pathogenic fungi and bacteria. It is also effective against nematodes and certain insects. The mixture of VOCs has been analyzed using GC/MS and consists primarily of various alcohols, acids, esters, ketones, and lipids. Final verification of the identity of the VOCs was carried out by using artificial mixtures of the putatively identified compounds and showing that the artificial mixture possessed the identical retention times and mass spectral qualities as those of the fungal derived substances. Artificial mixtures of the VOCs nicely mimicked the biological effects of the fungal VOCs when tested against a wide range of fungal and bacterial pathogens. Potential applications for “mycofumigation” by M. albus are currently being investigated and include uses for treating various plant parts, and human wastes. Another promising option includes its use to replace methyl bromide fumigation as a means to control soil-borne plant diseases.     

Recovery of biologically active enzymes after HPLC separation

The mass and activity recovery of eight different enzymes (two monomeric, six oligomeric) with molecular masses between 25,000 and 240,000 daltons were tested after HPLC separation on three different HPLC instruments (two with stainless steel and one with titanium flow paths). Most of the tested proteins are known to be sensitive to heavy metal ions. Eight wide pore, ion-exchange columns, two size-exclusion columns and two hydrophobic-interaction columns were used. Both stainless steel and glass column hardware were used in all three separation modes. The elution times were between 8 and 12 minutes. In almost all cases, the activity recovery was between 90% and 100% compared with a control sample incubated in the chromatographic elution buffer for the same time at the same temperature. A severe activity loss (about 30%) was observed with only one ion-exchange column and one enzyme. Neither the column hardware nor the material of the HPLC equipment had any negative effect on the activity recovery of the enzymes tested.     

A carbon dioxide trap for prolonged sampling ofIxodes ricinus L. populations

A carbon dioxide trap designed to captureIxodes ricinus over periods of up to 7 days is described. The trap compared favourably with blanket dragging and flagging in areas of high tick density, particularly on rough ground and for adult ticks; it was also surprisingly efficient for larvae. Ticks appeared to be captured throughout the 7-day trapping period. The maximum attraction distance recorded for adult female ticks was 3.5 m and for nymphs 1.0 m. Trapping rates were influenced by air temperature.     

Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real‐time PCR

Aim: We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. Methods and Results: We evaluated the sample recovery efficiencies of two collection methods – swabs and wipes – for both nonvirulent and virulent strains of Bacillus anthracis and Yersinia pestis from four types of nonporous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using real‐time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface‐dependent for virulent strains than nonvirulent strains. For the two nonvirulent strains, collection efficiency was similar between all four surfaces, albeit B. anthracis Sterne exhibited higher levels of recovery compared to Y. pestis A1122. In contrast, recovery of B. anthracis Ames spores and Y. pestis CO92 from the hydrophilic glass or stainless steel surfaces was generally more efficient compared to collection from the hydrophobic vinyl and plastic surfaces. Conclusions: Our results suggest that surface hydrophobicity may play a role in the strength of pathogen adhesion. The surface‐dependent collection efficiencies observed with the virulent strains may arise from strain‐specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. Significance and Impact of the Study: These findings contribute to the validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.     

The influence of trap density and sampling duration on the detection of small mammal species richness

Assessing species richness of small mammal communities is an important research objective for many live-trapping studies designed to assess or monitor biological diversity. We tested the effectiveness and efficiency of various trap densities for determining estimates and counts of small mammal species richness. Trapping was conducted in grassland habitats in northeastern Kansas during spring and fall of 2002 and 2003. Estimates and counts of species richness were higher at increased trap densities. This effect appeared to be primarily due to the higher number of individuals sampled at higher trap densities. At least 3 nights duration was needed to produce a stable estimate of species richness for the range of trap densities tested (9–144 trap stations/ha). Higher trap densities generally reached stable richness estimates in fewer nights than low density trapping arrangements. Given that counts and estimates of species richness were influenced by trap density and sampling duration, it is critical that these parameters are selected to most effectively meet research objectives.     

Thermal Death of Bacillus subtilis var. niger Spores on Selected Lander Capsule Surfaces

Dry-heat sterilization of planetary lander capsules requires a knowledge of the thermal resistivity of microorganisms in the environment to which they will be subjected during sterilization of the space hardware. The dry-heat resistance of Bacillus subtilis var. niger spores on various lander capsule materials was determined at 125 C. Eight surface materials were evaluated, including a reference material, stainless steel. Survivor curves were computed, and decimal reduction times (D values) were obtained by a linear regression analysis. In four tests on stainless steel, the average value of D at 125 C was 17.07 min. The D values for the other seven materials tested ranged from 18.64 min on magnesium surfaces to 20.83 min on conversion-coated magnesium. Of the materials evaluated, the results indicate that there is only a significant difference in the thermal resistance of B. subtilis var. niger spores on conversion-coated magnesium and conversion-coated aluminum from that on the reference material, stainless steel. The differences in D values for all the test surfaces may be the result of variations in test procedures rather than the effect of the surfaces on the thermal resistivity of the spores.     

Determination of the release rate of aldehyde pheromones from insect lures by cold trapping and direct bioluminescence analysis

A rapid and sensitive procedure for measuring the release rate of aldehyde pheromones from insect lures has been developed. Using cold traps to condense aldehydes released into an airstream, the amount of pheromone in the aqueous condensate could be directly analyzed using a luminescence assay based on the response of bacterial luciferase to long chain aldehydes. A high recovery (approximately 70%) of aldehyde pheromone in the cold trap was obtained even when amounts as low as 10 ng were released into the airstream. Trapping times as low as 5 min can be used and analysis requires only a few seconds after temperature equilibration of the sample. This approach was applied to measure the release rate of 11-tetradecenal from different spruce budworm lures as well as to demonstrate that the release of aldehyde from a lure containing [14C]-cis-9-hexadecenal correlated closely to the release rate of radioactive material.     

Comparative Study on Caustic and Enzymatic Cleanings of Stainless Steel Surface Fouled with β-Lactoglobulin

Desorption behavior of β-lactoglobulin (β-Lg), one of the main constituents of fouling deposits in milk processing, from stainless steel surfaces during caustic and enzymatic cleanings was studied by using a glass column packed with stainless steel particles fouled with β-Lg. Both in caustic and enzymatic cleanings, the amount of β-Lg remaining on the stainless steel particles decreased according to first-order kinetics at the initial stage, and gradually reached a constant value. The desorption rate constant at the initial stage and the residual amount of β-Lg after 2 h of cleaning were evaluated as the measures of cleaning efficiency under various conditions. In caustic cleaning, these two values were strongly affected by the concentration of NaOH. The initial desorption rate increased with increasing flow rate of the caustic solution, suggesting a mass transfer effect. In enzymatic cleaning, the maximum desorption rate constant was obtained at around the optimum pH for the enzyme reaction. The temperature dependence of the initial desorption rate constant was stronger in caustic cleaning than in enzymatic cleaning.     

Trap Induction and Trapping in Eight Nematode-trapping Fungi (Orbiliaceae) as Affected by Juvenile Stage of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

This study measured trap induction and trapping on agar disks as affected by juvenile stages (J1, J2, J3, and J4) of the nematode Caenorhabditis elegans and by species of nematode-trapping fungi. Eight species of nematode-trapping fungi belonging to the family Orbiliaceae and producing four kinds of traps were studied: adhesive network-forming Arthrobotrys oligospora, A. vermicola, and A. eudermata, constricting ring-forming Drechslerella brochopaga, and Dr. stenobrocha, adhesive column-forming Dactylellina cionopaga, and adhesive knob-forming Da. ellipsospora, and Da. drechsleri. The number of traps induced generally increased with increasing juvenile stages of C. elegans. The ability to capture the juveniles tended to be similar among isolates that produced the same kind of trap but differed among species that produced different kinds of traps. Trapping by Dr. stenobrocha and Da. cionopaga was correlated with trap number and with juvenile stage. A. oligospora and A. vermicola respectively captured more than 92 and 88% of the J1, J3, and J4 but captured a lower percentage of J2. The knob-producing isolates captured more younger than elder juveniles. Partial correlation analyses demonstrated that the trap induction of the most fungal species positively correlated with the juvenile size and motility, which was juvenile stage dependent. Overall, trap induction and trapping correlated with C. elegans juvenile stage (size and motility) in six species of trapping fungi.     

Decontamination of laboratory microbiological waste by steam sterilization.

A steam sterilizer (autoclave) was tested to determine the operating parameters that affected sterilization of microbiological waste. Tests involved standardized loads (5, 10 ad 15 lb [ca. 2.27, 4.54, and 6.80 kg, respectively]) contaminated petri plates in autoclave bags placed in polypropylene or stainless steel containers. Thermal and biological data were obtained by using a digital potentiometer and a biological indicator containing spores of Bacillus stearothermophilus, respectively. The transfer of heat was more efficient when smaller loads of microbiological waste were tested and stainless steel rather than polypropylene containers were used. A single bag with the sides rolled down to expose the top layer of petri plates allowed heat to pass better than did a single bag with the top constricted by a twist-tie. The presence of water in the autoclave bag did not significantly improve heat-up time in stainless steel or polypropylene containers. The results of biological tests substantiated the temperature data. When 10 or 15 lb of microbiological waste was exposed to various test conditions, the only condition that ensured the destruction of B. stearothermophilus involved the use of a stainless steel container (with or without water) for 90 min. Autoclaving for 45 min resulted in the destruction of bacteria included in 10 lb (136 +/- 3 plates) or 15 lb (205 +/- 6 plates) of microbiological waste when stainless steel containers with or without water or polypropylene containers with water used, whereas 60 min was required to kill all bacteria if polypropylene containers without water were used.     

Effects of double bands on Magellanic Penguins

ABSTRACT Banding penguins is controversial because bands can alter the survival, reproduction, and behavior of marked individuals. The effects of bands are not consistent among band types and, although stainless steel is thought to be better than other materials, tests of the long‐term impact of bands on tag‐loss rates and the reproduction and survival of individuals are needed. We tested three types of external tags on Magellanic Penguins (Spheniscus magellanicus) to measure band effects and tag‐loss rates. In 1993, we double‐tagged 300 penguins with aluminum flipper bands, stainless‐steel flipper bands, or small (2 mm × 10 mm) metal tags attached to foot webbing. We searched for double‐tagged birds for 13 of 15 yrs (1994–2008). Aluminum bands deformed, caused feather wear, injured and killed some penguins, and were lost more often than stainless‐steel bands or web tags. During the first 2 yrs of our study, at least nine penguins lost one aluminum band (N= 71 penguins resighted), but no penguins lost a stainless‐steel band (N= 84) or a web tag (N= 88). During the next 13 yrs, five penguins lost one of their two web tags (N= 89), but none lost a stainless‐steel band (N= 84). Females laid eggs of similar size before they carried a band and in the year following tagging (P= 0.09). The type of tags a female carried did not significantly change egg size (P > 0.22). During the first breeding season after tagging, penguins with aluminum bands had lower reproductive success than penguins with stainless‐steel bands or web‐tags (P= 0.04). The annual survival of females with two stainless‐steel bands was lower (0.79) than that of males with two stainless‐steel bands or males and females with two web‐tags (0.87). Aluminum bands injured Magellanic Penguins, were lost at high rates, and should not be used. Double stainless‐steel bands had no apparent effects on adult male Magellanic Penguins, but reduced survival rates of adult females. A single stainless‐steel band would likely have less impact than two bands, and our results suggest that the impact of a single band would be difficult to measure.     

Harnessing endophytes for industrial microbiology

Endophytic microorganisms exist within the living tissues of most plant species. They are most abundant in rainforest plants. Novel endophytes usually have associated with them novel secondary natural products and/or processes. Muscodor is a novel endophytic fungal genus that produces bioactive volatile organic compounds (VOCs). This fungus, as well as its VOCs, has enormous potential for uses in agriculture, industry and medicine. Muscodor albus produces a mixture of VOCs that act synergistically to kill a wide variety of plant and human pathogenic fungi and bacteria. This mixture of gases consists primarily of various alcohols, acids, esters, ketones and lipids. Artificial mixtures of the VOCs mimic the biological effects of the fungal VOCs when tested against a wide range of fungal and bacterial pathogens. Many practical applications for 'mycofumigation' by M. albus have been investigated and the fungus is now in the market place.     

Biodegradation of VOCs from printing press air by an on-site pilot plant bioscrubber and laboratory scale continuous yeast cultures

The volatile organic compound composition (VOCs) of printing press air wasfound to contain mostly ethanol, but also ethyl acetate, 1-propanol, 2-propanol,1-methoxy-2-propanol and 3-ethoxy-1-propanol. A pilot plant bioscrubber inoculatedwith a mixed microbial population was constructed on-site. The bioscrubber wasable to treat the polluted gas efficiently. It, however, suffered from strong wall growthand blockages in the column. The efficiencies of the pilot plant and a bioreactor iscompared. The yeasts Candida guilliermondii and Saccharomyces cerevisiae known to tolerate ethanol were selected instead of mixed population to avoid the wall growth and blockages in the bioreactor. The removal of the VOCs both individually and as a complex mixture was tested in a microcultivation system and in continuous chemostat cultures with and without cell recycling. The Candida yeast could use all the compounds as a carbon source while growth of S. cerevisiae was markedly slower on the methoxylated and ethoxylated propanols. Best total removal of the VOCs was 99% and achieved by C. guilliermondii. The only compound that was not totally removable in the chemostat experiment with C. guilliermondii was 1-methoxy-2-propanol. In laboratory scale the total and volumetric removal of VOCs by C. guilliermondii was more efficient compared to the pilot plant encouraging to scale up and applying the yeast bioreactor to real field conditions.     

The Volatome of Aspergillus fumigatus

Early detection of invasive aspergillosis is absolutely required for efficient therapy of this fungal infection. The identification of fungal volatiles in patient breath can be an alternative for the detection of Aspergillus fumigatus that still remains problematic. In this work, we investigated the production of volatile organic compounds (VOCs) by A. fumigatus in vitro, and we show that volatile production depends on the nutritional environment. A. fumigatus produces a multiplicity of VOCs, predominantly terpenes and related compounds. The production of sesquiterpenoid compounds was found to be strongly induced by increased iron concentrations and certain drugs, i.e., pravastatin. Terpenes that were always detectable in large amounts were α-pinene, camphene, and limonene, as well as sesquiterpenes, identified as α-bergamotene and β-trans-bergamotene. Other substance classes that were found to be present in the volatome, such as 1-octen-3-ol, 3-octanone, and pyrazines, were found only under specific growth conditions. Drugs that interfere with the terpene biosynthesis pathway influenced the composition of the fungal volatome, and most notably, a block of sesquiterpene biosynthesis by the bisphosphonate alendronate fundamentally changed the VOC composition. Using deletion mutants, we also show that a terpene cyclase and a putative kaurene synthase are essential for the synthesis of volatile terpenes by A. fumigatus. The present analysis of in vitro volatile production by A. fumigatus suggests that VOCs may be used in the diagnosis of infections caused by this fungus.     

AFM study of the colonisation of stainless steel by Aquabacterium commune

Aquabacterium commune, a member of the beta proteobacteria family that is a recently isolated, predominant member of various European drinking water distribution system biofilms, was selected as a test organism in this study. Attachment of A. commune cells onto two increasingly popular pipe materials, stainless steel EN1.4307 and medium density polyethylene (MDPE) was studied at 15 °C, 150 rpm, and a hydraulic retention time of 10.5 h in a rotating annular biofilm (RAB) reactor. Planktonic and sessile growth was monitored by spread plate technique. Atomic force microscopy (AFM) was used to obtain information about surface topography and biofilm formation pattern. Our study has shown that: (i) Steady-state conditions were reached after ca. 100 h for both materials; (ii) biofilm cell density on MDPE slides is four times greater than on stainless; (iii) the primary colonization of MDPE and stainless steel occurred at the edge of the slides; and (iv) no preferential attachment to stainless steel grain boundaries was observed. Stainless-steel manufacturers and suppliers, researchers, and companies working in the drinking-water sector will benefit from this paper. It is suggested that electropolishing of stainless-steel pipes for drinking water installations is not necessary to remove specific biofilm formation sites (i.e. grain boundaries). Furthermore, this paper provides, for the first time, some fundamental information for the continuous cultivation of the recently isolated drinking water microorganism, A. commune.     

Temperature control in solid substrate fermentation through discontinuous rotation

A laboratory-scale system for controlled dynamic solid substrate fermentation was developed and tested. The fermentation takes place in a stainless steel discontinuously rotating drum reactor, under controlled conditions of temperature, gas composition, relative humidity and direction and rate of rotation. The system was tested on a model fermentation of soya beans with Rhizopus oligosporus. In contrast to the traditional tempe fermentation, a granular product is obtained and build-up of heat and mass gradients is restricted. Despite the discontinuous rotation, the fungal growth continues, as evidenced by the production of heat. The rate of cooling depends on the temperature of the gas flushed through the reactor, the gas flow rate and the lenght of the rotation period. As a consequence of the homogeneous temperature control, the fungal heat development continued up to 70 h of fermentation. This is in clear contrast with the traditional tempe fermentation, which is already limited after 36 h by its own heat accumulation. Correspondence to: M. J. R. Nout     

Matter-tag: A universal immobilization platform for enzymes on polymers,metals, and silicon-based materials

Enzyme immobilization is extensively studied to improve enzyme properties in catalysis and analytical applications. Here, we introduce a simple and versatile enzyme immobilization platform based on adhesion-promoting peptides, namely Matter-tags. Matter-tags immobilize enzymes in an oriented way as a dense monolayer. The immobilization platform was established with three adhesion-promoting peptides; Cecropin A (CecA), liquid chromatography peak I (LCI), and Tachystatin A2 (TA2), that were genetically fused to enhanced green fluorescent protein and to two industrially important enzymes: a phytase (from Yersinia mollaretii) and a cellulase (CelA2 from a metagenomic library). Here, we report a universal and simple Matter-tag–based immobilization platform for enzymes on various materials including polymers (polystyrene, polypropylene, and polyethylene terephthalate), metals (stainless steel and gold), and silicon-based materials (silicon wafer). The Matter-tag–based enzyme immobilization is performed at ambient temperature within minutes (<10 min) in an aqueous solution harboring the phytase or cellulase by immersing the targeted material. The peptide LCI was identified as universal adhesion promoter; LCI immobilized both enzymes on all investigated materials. The attachment of phytase-LCI onto gold was characterized with surface plasmon resonance spectroscopy obtaining a dissociation constant value (K_D) of 2.9·10⁻⁸ M and a maximal surface coverage of 504 ng/cm².     

The effect of molybdenum on biofilm development

Little is known about the formation and effects of biofilms on stainless steel pipes in freshwater environments, particularly as they are considered as a direct replacement for copper pipes for ‘problem’ water. There is some cause for concern especially as stainless steel cannot claim the inherent biocidal potential of copper. As molybdenum is known to be leached out of stainless steel grade 316, in very small amounts, a study was set up to see if molybdenum could retard the development of biofilms. When a comparison of biofilm viable and total cell counts was made between pure molybdenum metal and stainless steel grade 304, it was found that cell counts were significantly higher (P < 0.05) on grade 304 stainless steel after 5 weeks exposure to flowing water (0.64 m s⁻¹). Molybdenum (above a concentration of 1 g L⁻¹) affected the growth rate of Acinetobacter sp, a pioneering bacterium of biofilms in potable water. Received 18 February 1998/ Accepted in revised form 17 May 1999     

Extracellular polysaccharides from Desulfovibrio desulfuricans and Pseudomonas fluorescens in the presence of mild and stainless steel

Summary This communication reports the presence of polysaccharides in biofilms formed by pure and mixed cultures of Desulfovibrio desulfuricans and Pseudomonas fluorescens on mild and stainless steel surfaces. The results of colorimetric assays, indicating significant differences between the amounts of neutral sugars present in these biofilms, were supported by gas chromatographic (GC)-mass spectrophotometric and GC-flame ionisation detection analyses. Neutral sugars in biofilms grown on mild steel surfaces were identified and quantified, revealing glucose as a major carbohydrate followed by mannose and galactose in all types of biofilm. Extracellular polymeric substances (EPS) precipitated from bacterial cultures grown with and without steel surfaces were also analysed for their carbohydrate content. The influence of the surfaces present in the cultures on the amount and type of sugars released into the bulk phase was established. There was significantly more carbohydrate in EPS harvested from pure and mixed cultures of D. desulfuricans incubated mild and stainless steel coupons than in EPS obtained from coupon-free cultures. No significant difference in sugar quantities was observed in EPS precipitated from cultures of P. fluorescens grown under different conditions (absence or presence of steel surfaces). The main carbohydrates identified in all types of EPS samples were mannose, glucose and galactose in order of prevalence. Offprint requests to: I. B. Beech