Adrian Ponce

Bacterial diversity in hyperarid Atacama Desert soils

[1] Surface and subsurface soil samples analyzed for this investigation were collected from the hyperarid Yungay region in the Atacama Desert, Chile. This report details the bacterial diversity derived from DNA and PLFA extracted directly from these extremely desiccated soils. Actinobacteria, Proteobacteria, Firmicutes and TM7 division bacteria were detected. Ninety-four percent of the 16S rRNA genes cloned from these soils belong to the Actinobacteria phylum, and the majority of these were most closely related to the genus Frankia. A 24-hour water activity (a w ) time course showed a diurnal cycle that peaked at 0.52 in the early predawn hours, and ranged from 0.01-0.08 during the day. All measured water activity values were below the levels required for microbial growth or enzyme activity. Total organic carbon (TOC) concentrations were above the limit of detection and below the limit of quantification (i.e., 200 μg/g < TOC < 1000 μg/g), and phospholipid fatty acid (PLFA) concentrations ranged from 2 x 105 to 7 x 10 cell equivalents per gram of soil. Soil extracts analyzed for culturable biomass yielded mostly no growth on R2A media; the highest single extract yielded 47 colony forming units (CFU) per gram of soil.

Microbial life at −13 °C in the brine of an ice-sealed Antarctic lake

The permanent ice cover of Lake Vida (Antarctica) encapsulates an extreme cryogenic brine ecosystem (−13 °C; salinity, 200). This aphotic ecosystem is anoxic and consists of a slightly acidic (pH 6.2) sodium chloride-dominated brine. Expeditions in 2005 and 2010 were conducted to investigate the biogeochemistry of Lake Vida’s brine system. A phylogenetically diverse and metabolically active Bacteria dominated microbial assemblage was observed in the brine. These bacteria live under very high levels of reduced metals, ammonia, molecular hydrogen (H2), and dissolved organic carbon, as well as high concentrations of oxidized species of nitrogen (i.e., supersaturated nitrous oxide and ∼1 mmol⋅L−1 nitrate) and sulfur (as sulfate). The existence of this system, with active biota, and a suite of reduced as well as oxidized compounds, is unusual given the millennial scale of its isolation from external sources of energy. The geochemistry of the brine suggests that abiotic brine-rock reactions may occur in this system and that the rich sources of dissolved electron acceptors prevent sulfate reduction and methanogenesis from being energetically favorable. The discovery of this ecosystem and the in situ biotic and abiotic processes occurring at low temperature provides a tractable system to study habitability of isolated terrestrial cryoenvironments (e.g., permafrost cryopegs and subglacial ecosystems), and is a potential analog for habitats on other icy worlds where water-rock reactions may cooccur with saline deposits and subsurface oceans.

Detection of Bacterial Spores with Lanthanide-Macrocycle Binary Complexes

The detection of bacterial spores via dipicolinate-triggered lanthanide luminescence has been improved in terms of detection limit, stability, and susceptibility to interferents by use of lanthanide-macrocycle binary complexes. Specifically, we compared the effectiveness of Sm, Eu, Tb, and Dy complexes with the macrocycle 1,4,7,10-tetraazacyclododecane-1,7-diacetate (DO2A) to the corresponding lanthanide aquo ions. The Ln(DO2A)(+) binary complexes bind dipicolinic acid (DPA), a major constituent of bacterial spores, with greater affinity and demonstrate significant improvement in bacterial spore detection. Of the four luminescent lanthanides studied, the terbium complex exhibits the greatest dipicolinate binding affinity (100-fold greater than Tb(3+) alone, and 10-fold greater than other Ln(DO2A)(+) complexes) and highest quantum yield. Moreover, the inclusion of DO2A extends the pH range over which Tb-DPA coordination is stable, reduces the interference of calcium ions nearly 5-fold, and mitigates phosphate interference 1000-fold compared to free terbium alone. In addition, detection of Bacillus atrophaeus bacterial spores was improved by the use of Tb(DO2A)(+), yielding a 3-fold increase in the signal-to-noise ratio over Tb(3+). Out of the eight cases investigated, the Tb(DO2A)(+) binary complex is best for the detection of bacterial spores.

Substrate induced phosphorescence from cyclodextrin·lumophore host-guest complexes

Hydrogen bonding substrates (S) trigger bright phosphorescence upon their addition to aqueous solutions containing 1-bromonaphthalene (1-BrNp) and a glucosyl modified cyclodextrin (Gβ-CD). Steady-state and time-resolved luminescence measurements establish that the phosphorescence arises from 1-BrNp as part of a 1-BrNp·Gβ-CD·S complex. In all cases, the substrate comprises a bulky t-butyl or cyclohexyl group spaced from the functional group of an alcohol, amine, carboxylic acid, ester, or aldehyde. Association constants, molecular models, and substrate binding studies are consistent with the substrate hydrogen bonding to the rim of the CD cup, with its aliphatic end flipped over the hydrophobic interior of the CD. The phosphorescence enhancement induced by substrate is related to its effectiveness in shielding photoexcited 1-BrNp from quenching by oxygen. The enhancements can be extremely large, approaching a 105 increase in intensity, depending on the fit of the substrate to the top of the CD. We have explored the topological features of the substrate that lead to the best shielding and hence greatest triggered luminescence response.

Luminescence from supramolecules triggered by the molecular recognition of substrates

Abstract Supramolecules containing a photoluminescent center (PLC) exhibit prompt and intense luminescence upon the molecular recognition of substrates. Luminescence from supramolecular assemblies is triggered by substrates that are light-harvesting and able to coordinate metal ion PLCs, light-harvesting and non-coordinating to PLCs, and non-absorbing and non-coordinating. Our efforts to elaborate the photophysical schemes for these three classes of substrates are described herein.

An anthrax "smoke" detector

We report a method for automated monitoring of airborne endospores, which combines an aerosol capture technique with endospore detection based on terbium luminescence turn-on. We have demonstrated quantification of aerosolized bacterial spores with a response time of /spl sim/15 min, a sensitivity of 10/sup 4/ spores/ml, and a dynamic range of 4 orders of magnitude using a bioaerosol sampler, a microwave, and a lifetime-gated fluorimeter. Ultimately, the most attractive feature we have demonstrated is the unattended monitoring of aerosolized bacterial spores for the duration of a workday (/spl sim/8 hours).

Applications of a Rapid Endospore Viability Assay for Monitoring UV Inactivation and Characterizing Arctic Ice Cores

ABSTRACT We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by l-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% ± 3.8% and 48.9% ± 4.5%, respectively), while only 27.8% ± 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 ± 19 germinable spores/ml and 369 ± 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% ± 9.3%), and the second core contained 131 ± 4 germinable spores/ml and 162 ± 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% ± 8.8%), whereas only 2 CFU/ml were detected by culturing.

Fast Sterility Assessment by Germinable-Endospore Biodosimetry

ABSTRACT The increased demand for sterile products has created the need for rapid technologies capable of validating the hygiene of industrial production processes. Bacillus endospores are in standard use as biological indicators for evaluating the effectiveness of sterilization processes. Currently, culture-based methods, requiring more than 2 days before results become available, are employed to verify endospore inactivation. We describe a rapid, microscopy-based endospore viability assay (μEVA) capable of enumerating germinable endospores in less than 15 min. μEVA employs time-gated luminescence microscopy to enumerate single germinable endospores via terbium-dipicolinate (Tb-DPA) luminescence, which is triggered under UV excitation as 108 DPA molecules are released during germination on agarose containing Tb3+ and a germinant (e.g., l-alanine). Inactivation of endospore populations to sterility was monitored with μEVA as a function of thermal and UV dosage. A comparison of culturing results yielded nearly identical decimal reduction values, thus validating μEVA as a rapid biodosimetry method for monitoring sterilization processes. The simple Tb-DPA chemical test for germinability is envisioned to enable fully automated instrumentation for in-line monitoring of hygiene in industrial production processes.

Rapid endospore viability assay of Clostridium sporogenes spores.

A rapid Endospore Viability Assay (EVA), previously developed for Bacillus spores, was modified for enumeration of germinable Clostridium sporogenes spores. The EVA is based on the detection of dipicolinic acid (DPA), which is released during stage I germination and quantified by terbium (III) ion Tb-DPA luminescence. Germination of C. sporogenes spores in aqueous suspension was induced by L-alanine and NaHCO(3) addition, and germinable endospore numbers were determined by reference to a standard curve. Determination of the fractions of germinable C. sporogenes spores by EVA and phase-contrast microscopy yielded comparable results of 54.0%+/-2.9% and 59.3%+/-2.6%, respectively, while only 32.3%+/-5.3% of spores produced colonies on reinforced clostridial medium (RCM). Rates of germination were measured as a function of temperature (30 degrees C-60 degrees C) using EVA, yielding a linear relationship between the square root of the rate constant and inverse temperature.

Luminescent lanthanide sensors

Abstract Luminescent lanthanide optical sensors have been developed that utilize ancillary ligands to enhance detection of a target analyte. In these systems, the lanthanide (ligand) binary complex serves as the receptor, which upon analyte binding forms a ternary complex resulting in detectable change in lanthanide luminescence ( Fig. 1 ). The ancillary ligand improves many properties of analyte detection by protecting the lanthanide and strengthening analyte binding affinity. Encapsulation shields the lanthanide ion from solvent-quenching effects and interfering ions, improving assay sensitivity and selectivity. The ligand-induced enhancement in binding affinity appears to be the result of an increase in positive charge at the analyte binding site due to the electronegative ancillary ligand bound on the opposite hemisphere of the lanthanide. We have elucidated the effects of ancillary ligands for various lanthanide/analyte systems and shown how such effects can greatly improve sensor performance for medical, planetary science, and biodefense applications.