Bioaerosol Research Research Articles

Characterization of steady-state fluorescence properties of polystyrene latex spheres using off- and online spectroscopic methods

Abstract. Fluorescent dyed polystyrene latex spheres (PSLs) are commonly used for characterization and calibration of instruments detecting fluorescence signals from particles suspended in the air and other fluids. Instruments like the Ultraviolet Aerodynamic Particle Sizer (UV-APS) and the Waveband Integrated Bioaerosol Sensor (WIBS) are widely used for bioaerosol research, but these instruments present significant technical and physical challenges requiring careful characterization with standard particles. Many other research communities use flow cytometry and other instruments that interrogate fluorescence from individual particles, and these also frequently rely on fluorescent PSLs as standards. Nevertheless, information about physical properties of commercially available PSLs provided by each manufacturer is generally proprietary and rarely available, making their use in fluorescence validation and calibration very difficult. This technical note presents an overview of steady-state fluorescence properties of fluorescent and non-fluorescent PSLs, as well as of polystyrene-divinylbenzene (PS-DVB) particles, by using on- and offline spectroscopic techniques. We show that the “fluorescence landscape” of PSLs is more complex than the information typically provided by manufacturers may imply, especially revealing multimodal emission patterns. Furthermore, non-fluorescent PSLs also exhibit defined patterns of fluorescent emission originating from a mixture of polystyrene and detergents, which becomes a crucial point for fluorescence threshold calibrations and qualitative comparison between instruments. By comparing PSLs of different sizes, but doped with the same dye, changes in emission spectra from bulk solutions are not immediately obvious. On a single-particle scale, however, fluorescence intensity values increase with increasing particle size. No significant effect in the fluorescence signatures was detectable by comparing PSLs in dry vs. wet states, indicating that solvent water may only play a minor role as a fluorescence quencher. Because information provided by manufacturers of commercially available PSLs is generally very limited, we provide the steady-state excitation–emission matrices (EEMs) of PSLs as open-access data within the Supplement. Detergent and solvent effects are also discussed in order to provide information not available elsewhere to researchers in the bioaerosol and other research communities. These data are not meant to serve as a fundamental library of PSL properties because of the variability of fluorescent properties between batches and as a function of particle aging and agglomeration. The data presented, however, provide a summary of spectral features which are consistent across these widely used fluorescent standards. Using these concepts, further checks will likely be required by individual researchers using specific lots of standards.

Bioaerosol research: Yesterday, today and tomorrow

Bioaerosols have important impacts on many scientific fields, including atmospheric environment, ecology, climate change, food safety, anti-bioterrorism, infectious diseases, public health and environmental health. This review was carried out based on the scientific outcomes from the 600th Xiangshan Science Conference held in Beijing, for whcih 47 scientists including those overseas were invited to discuss the bioaerosol research progress. Over the past 10 years, great strides have been made in the bioaerosol field, such as the expansion of research directions, research broadness and also scientists. For example, atmospheric chemistry researchers have also started to carry out studies about bioaerosol impacts on the atmospheric environment, including those on the cloud droplets, the climate and bioaerosol dynamics during haze events. In addition, new methods and instruments for bioaerosol studies are increasingly being developed in bioaerosol monitoring and capture systems, various biosensors, microfluidics, high-throughput sequencing, and they have helped the scientific community obtain a better understanding of microbes in the air. In terms of health effects, human and indoor bioaerosol emissions have become one of the hot research areas. In addition, bioaerosols were also shown to directly affect the oxidation potential of atmospheric particles and thus on the human health, however, the relevant mechanisms remain to be very limited and could be far more complex than what we have known so far. On the other hand, respiratory infections/outbreaks, caused largely by the spread of bioaerosols, continue to be a major killer for humans, especially for younger children. Currently, although a significant progress has been made in high-throughput screening of respiratory pathogens, the airborne disease transmission mechanisms are still not well understood, including the influences on the pathogen viability and effective transmission distance. The risk of military conflicts in certain regions is increasing, which could potentially lead to possible use of biological weapons via bioaerosol for mass human destruction. In addition, the airborne transmission of antibiotic resistant genes has also received wide attention as an emerging biological pollutant. By integrating sampling, microfluidics, biosensors and electronics, the scientists have taken an important step towards real-time online bioaerosol screening and biological hazard early warning. As a special type of bioaerosol, biomarkers in exhaled breath are also increasingly being studied for early disease diagnosis and screening. At the same time, surgical and nosocomial infections caused by the spread of bioaerosols in the hospital settings are also receiving wide attention. The scientific researchers have also made significant advances in the controlling airborne biological agents, e.g., microwave irradiation, cold plasma and nanomaterial filtration. This review intends to broadly review current state-of-the-art advances in bioaerosol studies, to summarize and discuss the existing problems, correspondingly providing references for the future studies. Despite of great progress made in many aspects of bioaerosols, our understanding of its significance and relevant impacts on our surroundings and health is still quite limited. In the years to come, bioaerosol research is expected to turn a new chapter if multidisciplinary collaboration can be further strengthened.

Recent research progress and perspective of characteristics of ambient bioaerosols during hazy pollution in China

Since the haze event occurred in many areas of central and eastern China in 2013, haze pollution has become one of the most important environmental issues and has attracted more and more attention from government, industry circle, scholars and the general public in China. Haze pollution is characterized by atmospheric particular matter (PM) being one of the top pollutants. A large number of studies have been conducted to investigate the chemical components of atmospheric PM during haze pollution, while little is known about the biological components and characteristics of PM in the atmosphere, especially during haze pollution. Biological particles in the atmosphere, generally defined as bioaerosols, are airborne particles or large molecules carrying living organisms or released from living organisms (e.g., bacteria, fungi, viruses, pollen, bacterial endotoxins and mycotoxins, etc.). Bioaerosols not only pose potential risks on air quality and human health, but also have direct effects on the alteration of global climate and atmospheric physical and chemical processes. Up to now, however, the concentration and size distribution, diversity, and abundance of different types of bioaerosols as well as their temporal and spatial variability are not well characterized on hazy days. Moreover, due to the changeable community structure, flexible biological activity and comprehensive influential factors, the knowledge involving how haze pollution affects bioaerosols, to what extent it affects, and the influence mechanism is still unclear so far. Therefore, this review first has presented an overview of the state of bioaerosol research during haze pollution, including concentration level, size distribution and community structure of microorganisms in bioaerosols. Subsequently, the current understanding and open questions have been emphatically discussed concerning the characteristics of bioaerosols at different air pollution levels and effects of air pollution on airborne microbes. Finally, we suggest that the following research activities should be pursued in future studies of bioaerosols during haze pollution: (1) It is extremely urgent to develop and establish uniform standardized sampling and identification criterion to better judge and measure the numerous monitoring data from different researchers. It is also essential to develop advanced, sensitive, real-time and online detection techniques to quantify the biological properties of bioaerosols. (2) The nationwide field monitoring of bioaerosols covering more climate types and pollution levels at various time scales should be carried out to obtain more comprehensive data for fully understanding the temporal and spatial variability of bioaerosols and its influential factors. Some other meteorological influential factors (e.g., boundary-layer height, atmospheric extreme temperature, extreme wind speed, and atmospheric pressure, etc.), and pollution factors such as the concentration of heavy metal and polycyclic aromatic hydrocarbon (PAHs) in PM should be taken into account to deeply understand the effect of haze pollution on bioaerosols. (3) Current studies have shown that many gaseous and particulate pollutants are related to the concentration of bioaerosols. However, little is known about the influence mechanisms of pollutants. Therefore, in addition to field monitoring, it is also necessary to design and conduct the laboratory experiment of smog chamber to determine the influence mechanism of each single environmental factor or pollution factor on bioaerosols. (4) In addition to different degrees of haze pollution, there are different kinds of haze pollution including the formation of haze by photochemical reaction in summer and liquid phase reaction at high RH in winter. Therefore, it is also essential to examine the distribution and variation of bioaerosols during different haze formation processes in order to improve our understanding of haze pollution effects on bioaerosols. (5) It is necessary to develop a biological source-tracking technology based on the bioaerosol species and community composition to reveal the possible sources of bioaerosols during haze episodes.

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions.

The ability to perform controlled experiments with bioaerosols is a fundamental enabler of many bioaerosol research disciplines. A practical alternative to using hazardous biothreat agents, e.g., for detection equipment development and testing, involves using appropriate model organisms (simulants). Several species of Gram-negative bacteria have been used or proposed as biothreat simulants. However, the appropriateness of different bacterial genera, species, and strains as simulants is still debated. Here, we report aerobiological stability characteristics of four species of Gram-negative bacteria (Pantoea agglomerans, Serratia marcescens, Escherichia coli, and Xanthomonas arboricola) in single-cell particles and cell clusters produced using four spray liquids (H2O, phosphate-buffered saline[PBS], spent culture medium[SCM], and a SCM-PBS mixture). E. coli showed higher stability in cell clusters from all spray liquids than the other species, but it showed similar or lower stability in single-cell particles. The overall stability was higher in cell clusters than in single-cell particles. The highest overall stability was observed for bioaerosols produced using SCM-containing spray liquids. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research.IMPORTANCE The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns.

Aerobiology: Experimental Considerations, Observations, and Future Tools.

ABSTRACTUnderstanding airborne survival and decay of microorganisms is important for a range of public health and biodefense applications, including epidemiological and risk analysis modeling. Techniques for experimental aerosol generation, retention in the aerosol phase, and sampling require careful consideration and understanding so that they are representative of the conditions the bioaerosol would experience in the environment. This review explores the current understanding of atmospheric transport in relation to advances and limitations of aerosol generation, maintenance in the aerosol phase, and sampling techniques. Potential tools for the future are examined at the interface between atmospheric chemistry, aerosol physics, and molecular microbiology where the heterogeneity and variability of aerosols can be explored at the single-droplet and single-microorganism levels within a bioaerosol. The review highlights the importance of method comparison and validation in bioaerosol research and the benefits that the application of novel techniques could bring to increasing the understanding of aerobiological phenomena in diverse research fields, particularly during the progression of atmospheric transport, where complex interdependent physicochemical and biological processes occur within bioaerosol particles.

Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis.

IntroductionAs bioaerosol research attracts increasing attention, there is a need for additional efforts that focus on method development to deal with different environmental samples. Bioaerosol environmental samples typically have very low biomass concentrations in the air, which often leaves researchers with limited options in choosing the downstream analysis steps, especially when culture-independent methods are intended.ObjectivesThis study investigates the impacts of three important factors that can influence the performance of culture-independent DNA-based analysis in dealing with bioaerosol environmental samples engaged in this study. The factors are: 1) enhanced high temperature sonication during DNA extraction; 2) effect of sampling duration on DNA recoverability; and 3) an alternative method for concentrating composite samples. In this study, DNA extracted from samples was analysed using the Qubit fluorometer (for direct total DNA measurement) and quantitative polymerase chain reaction (qPCR).Results and FindingsThe findings suggest that additional lysis from high temperature sonication is crucial: DNA yields from both high and low biomass samples increased up to 600% when the protocol included 30-min sonication at 65°C. Long air sampling duration on a filter media was shown to have a negative impact on DNA recoverability with up to 98% of DNA lost over a 20-h sampling period. Pooling DNA from separate samples during extraction was proven to be feasible with margins of error below 30%.

Application of ATP-based bioluminescence for bioaerosol quantification: Effect of sampling method

An adenosine triphosphate (ATP)-based bioluminescence has potential to offer a quick and affordable method for quantifying bioaerosol samples. Here we report on our investigation into how different bioaerosol aerosolization parameters and sampling methods affect bioluminescence output per bacterium, and implications of that effect for bioaerosol research. Bacillus atrophaeus and Pseudomonas fluorescens bacteria were aerosolized by using a Collison nebulizer (BGI Inc., Waltham, MA) with a glass or polycarbonate jar and then collected for 15 and 60min with: (1) Button Aerosol Sampler (SKC Inc., Eighty Four, PA) with polycarbonate, PTFE, and cellulose nitrate filters, (2) BioSampler (SKC Inc.) with 5 and 20mL of collection liquid, and (3) our newly developed Electrostatic Precipitator with Superhydrophobic Surface (EPSS). For all aerosolization and sampling parameters we compared the ATP bioluminescence output per bacterium relative to that before aerosolization and sampling. In addition, we also determined the ATP reagent storage and preparation conditions that do not affect the bioluminescence signal intensity.Our results show that aerosolization by a Collison nebulizer with a polycarbonate jar yields higher bioluminescence output per bacterium compared to the glass jar. Interestingly enough, the bioluminescence output by P. fluorescens increased substantially after its aerosolization compared to the fresh liquid suspension. For both test microorganisms, the bioluminescence intensity per bacterium after sampling was significantly lower than that before sampling suggesting negative effect of sampling stress on bioluminescence output. The decrease in bioluminescence intensity was more pronounces for longer sampling times and significantly and substantially depended on the sampling method. Among the investigated method, the EPSS was the least injurious for both microorganisms and sampling times.While the ATP-based bioluminescence offers a quick bioaerosol sample analysis method, this works demonstrates that the method output depends on bioaerosol generation and sampling methods, as well as reagent storage.

A systematic comparison of four bioaerosol generators: Affect on culturability and cell membrane integrity when aerosolizing Escherichia coli bacteria

Bioaerosol research requires stable and reliable aerosol generators that can produce high particle concentrations with minimal damage to microorganisms. This study compared Collison nebulizer, Liquid Sparging Aerosolizer (LSA), C-Flow nebulizer, and a newly designed Single-Pass Aerosolizer with respect to their physical performance, and ability to preserve the culturability and structural integrity of bacteria. Escherichia coli bacteria were aerosolized at different air pressures, collected by a BioSampler and their Cell Membrane Damage Index (ID), expressed as the fraction of 16S rRNA gene copies in the supernatant liquid versus the amount of 16S rRNA gene copies in the total sample (cell pellet plus supernatant), was determined. The ID of E. coli aerosolized by the Collison and C-Flow nebulizers at 40psi compared to aerosolization at 5 and 15psi was significantly higher (p<0.05). However, the ID of E. coli aerosolized with the LSA and Single-Pass Aerosolizer did not seem to significantly depend on aerosolization pressure. The ID of E. coli collected with a BioSampler was found to positively and significantly correlate (p=0.043) with the presence of airborne bacterial fragments (aerodynamic size range 0.37–0.523µm) as measured by the Aerodynamic Particle Sizer. Increased loss of culturability was observed for bacteria aerosolized by the Collison nebulizer and Single-Pass Aerosolizer with increasing aerosolization pressure (p<0.05), while no significant change in culturability was found for the other two generators as a function of aerosolization pressure. At particle output concentration of ~100particles/cm3, the Single-Pass Aerosolizer preserved the culturability of bacteria significantly better than the other three generators (p<0.05). It also exhibited a significantly lower ID (p<0.001) and less culturability reduction (p=0.03) compared to the Collison nebulizer at particle output concentrations of ~1000particles/cm3. It is hoped that this study will help bioaerosol researchers select a bioaerosol generator and method best suiting their studies.

NanoPCR detection of bacterial aerosols

Bioaerosol detection is of great importance in many fields, yet their low quantity in complex samples makes such an effort difficult. Here, we have investigated the use of nanoparticles (AgNPs, TiO2NPs and their combination) assisted PCR (nanoPCR) in enhancing the efficiency. Pure bacterial species Escherichia coli and Bacillus subtilis with serial dilutions were tested for obtaining optimal parameters such as nanoparticle concentration and the volume of the nanoparticle mixture. These optimal parameters were then applied to detecting indoor and outdoor bacterial aerosols collected using a liquid impinger. The PCR products were subsequently subjected to agarose gel electrophoresis and DNA stain.The results showed that the enhancements varied with nanoparticle type, concentration level, and DNA template concentration. Data with E. coli and B. subtilis revealed that the optimal parameters were: 0.6nM for TiO2NPs, 0.9nM for AgNPs, 2µL for the mixture, and diluted DNA templates. Our data also suggest that the mixture of AgNPs and TiO2NPs performed better compared to their individual ones especially for lower DNA levels when coupled with qPCR. BacLight DNA stain results showed that use of nanoparticles could form nanoparticle–DNA clusters, which led to concentrating local DNA and thus enhancing PCR detection. Our environmental bioaerosol monitoring data suggest that NanoPCR technique can be immediately applied to detecting low quantity of airborne biological materials with up to 500 times enhancement with a detection limit down to 40pg/μL. The developments in this work can further broaden qPCR applications in bioaerosol research fields.

Bioaerosol Science, Technology, and Engineering: Past, Present, and Future

Poor air hygiene as a result of bioaerosol contamination has caused diverse forms of adverse health effects and diseases. In addition, global biosecurity is threatened by purposeful use of biowarfare agents and the vulnerability of people to the infectious agents. Accordingly, developments in high-volume biosampling, including aerosol-to-hydrosol techniques with low cut-off size, real-time bioaerosol detection, adequate biological quantification, and exposure control, as well as the investigation of the link between disease outcome and bioaerosol exposure, are current areas of bioaerosol research. Although milestone progress has been achieved both in bioaerosol sampling and analysis techniques since late 1800s, compared to atmospheric chemistry the bioaerosol field is still understudied. This is partially because of the lack of both bioaerosol scientists and multidisciplinary collaboration. It is becoming necessary to develop a pool of scientists with different expertise, e.g., bioaerosol scientists, environmental engineers, biomedical engineers, epidemiologists, microbiologists, chemists, physicists, as well as researchers in other engineering fields, in mitigating bioaerosol-related adverse health effects, eliminating diseases, and preventing and controlling epidemic outbreaks. This work is conducted to broadly review current state-of-the-art sciences and technologies in the bioaerosol field. In tackling the challenges ahead, the review also provides perspectives for bioaerosol research needs, and further reminds bioaerosol scientists of those existing technologies in other fields that can be leveraged. In view of the past, forward-looking hypotheses and revolutional perspectives are needed to be formed in order to allow the bioaerosol research have major impacts in the academic community in this new millennium.

Assessment of Bioaerosol Sampling Techniques for ViableLegionella pneumophilaby Ethidium Monoazide Quantitative PCR

Legionella pneumophila causes severe pneumonia and Pontiac fever in humans. Rapid and sensitive bioaerosol monitoring techniques for viable L. pneumophila are unavailable. Coupled with a newly developed viable assay called ethidium monoazide with quantitative PCR (EMA-qPCR), this study applies EMA-qPCR to aerobiology for the first time to evaluate the effects of the method of sampling (all-glass impinger (AGI-30), BioSampler, and MAS-100 sampler) and sampling time (3, 30, 60 min) on the collection of viable L. pneumophila. The effects of the collection fluid (deionized water (DW) and Tween mixture) and the replenishment of DW every 15 min during 60-min sampling were also assessed. Escherichia coli, as a model microorganism in bioaerosol research, was also tested. Using the Tween mixture (DW containing 1% peptone, 0.01% Tween 80, and 0.005% antifoam), the AGI-30 and BioSampler performed significantly better than the MAS-100 sampler for collecting viable L. pneumophila and viable E. coli (P < 0.05). An incr...

Life Comes from the Air: A Short Review on Bioaerosol Control

ABSTRACTAir is filled with numerous tiny organisms, with sizes ranging from 50 nm to 10 μm. These organisms are called airborne biological particles or bioaerosols. In the human history of investigating the origin of life and fighting against contagious diseases, the recognition of bioaerosols and the development of control methods against them have played crucial roles. The pandemic outbreak of flu due to the influenza A H1N1 virus in 2009 and the bio-terror incidents in 2001 have alerted us to the importance of bioaerosol research. Here, control methods against bioaerosols are briefly reviewed, and suggestions are offered for future research on airborne biological particles.

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

The generation of airborne microorganisms from concentrated animal-feeding operations (CAFOs) is a concern from a human and animal health perspective. To better understand the airborne microorganisms found in these environments, a number of collection and analytical techniques have been utilized and will be discussed in this review. The most commonly used bioaerosol collection method is the liquid impingement format, which is suitable with a number of culture-based and non-culture molecular-based approaches, such as polymerase chain reaction. However, the vast majority of airborne microorganism studies conducted at CAFOs utilize culture-based analyses. Because of the limitations often associated with culture-based analyses, we focused our discussion on the application of molecular-based techniques to identify and/or quantify microorganisms, as they have promising application in bioaerosol research. The ability to rapidly characterize airborne microorganisms will help to ensure protection of public and environmental health.

The Effect of Phosphate Buffer on Aerosol Size Distribution of Nebulized Bacillus subtilis and Pseudomonas fluorescens Bacteria

During bioaerosol research microorganisms are frequently aerosolized from phosphate-buffered suspension via air-jet nebulization. Buffer will therefore be present in organism-free droplets (''empties) as solutes in hydrous surface coatings of airborne organisms or as dry surface coatings on organisms. It might be expected that the buffer would influence the resulting aerosol size distribution and that the influence might vary with relative humidity. In this work we examined the effect of various buffer concentrations on the aerosol size distribution of Bacillus subtilis (B. subtilis) and Pseudomonas fluorescens ( P. fluorescens ) at various relative humidities. Microorganism suspensions in distilled deionized water and phosphate-buffered water were prepared and nebulized from a 3-jet Collison nebulizer into a sampling chamber maintained at 10% relative humidity. The aerosol was then conducted to a second chamber to which moist air was added to achieve the target relative humidity. Particle aerodynamic si...

Artificial wind-gust liberation of microbial bioaerosols previously deposited on plants

A bioaerosol research chamber was constructed and used to evaluate wind-gust release of previously depositedPseudomonas syringae, spores ofBacillus subtilis var.niger, and fluorescent microspheres (FM) on oat plants, and the airborne survival of the releasedP. syringae. Observations of wind gusts on the releasedBacillus spores and FM showed they had similar particle size distributions and therefore FM could act as bacterial sized surrogates in particle dispersion. Microscopic examination of the released FM containing particles revealed that 84% were associated with either fungal spores and hyphae were epiphytic on the plant, or with plant and soil debris. The release rate ofBacillus spores decreased as the number of gusts experienced by the plants increased, with a greater proportion of larger particles removed. The larger the particle associated with the releasedP. syringae, the longer the bacteria survived.