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College of Environmental Sciences and Engineering

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1 College of Environmental Sciences and Engineering
A review on bioaerosol science, technology and engineering: current and beyond Maosheng Yao College of Environmental Sciences and Engineering Peking University

2 Outline Bioaerosol Emissions Air Sampling Techniques
Biological Assessment, Detection and Control Current State of Bioaerosol Research

3 Biological Agents in the Environments
Particles of biological origins --bacteria, fungi, viruses , pollen --their derivatives such as endotoxins, glucans, and mycotoxins --fungal allergens, indoor house dust mites, dog, cat allergens 0.5-2μm for bacteria & 2-5μm for fungi commonly found, viruses are usually below 0.3 μm

4 Bioaerosol Emissions Natural environments
Human and animal are sources of bioaerosol Waste recycling, bio-solid land application, composting, agriculture, pharmaceutical and bio-tech activities Hospital settings (surgery, organ transplant, and dental treatment) Bioterrorism events

5 Why Are We Concerned about These Biological Agents?
Exposure to the biological agents presents a serious health challenge both for public and private sectors Respiratory diseases such as asthma, pneumonia, and allergies. Infectious diseases such as SARS, and Bird Flu. SARS outbreak in 2003 Influenza (bird flu) Outbreak

6 Global Asthma Impacts USA Brazil Canada Australia China According to WHO estimates, 300 million people suffer from asthma and 255, 000 people died of asthma in 2005

7 Pneumonia Death in Children < 5 Years Old
WHO estimates that up to 1 million children under 5 die each year from pneumonia.

8 MMWR 2003; 52 (RR-8); Thompson et al. JAMA 2003; 289:179
Annual Impacts of Epidemic Influenza Estimates for the US Cases: millions Days of illness: millions Work & school loss: tens of millions Hospitalizations: 85, ,000 Deaths: 34,000-50,000 Economic loss: billions of dollars MMWR 2003; 52 (RR-8); Thompson et al. JAMA 2003; 289:179 Thompson et al., JAMA 2004; 292:1333; Adams PF et al. Vital Health Stat 1999; 10(200) In addition, infectious diseases took a tremendous toll both on human and economy

9 Global Outbreak of SARS in 2003

10 Social Impacts of SARS

11 Increasing Threat of Bio-terrorism That May Release Lethal Airborne Biological Agents

12 The first critical step
Bio-Sampling The first critical step for monitoring, assessment, or control strategy for the biological inhalation exposure

13 Air Sampling Techniques
Impactors Liquid impingers Filters Electrostatic precipitator

14 Impactors It was used in anthrax investigation
Andersen six-stage impactor was developed in the 50s and widely used as a standard for bioaerosol sampling BioStage Impactor (SKC, Inc., Eighty Four, PA) --collect microorganisms onto agar surface Liter/min with an impaction velocity of 24m/s BioStage Velocity is about 24m/s It was used in anthrax investigation

15 Principle of Collection by Impactor

16 Portable Microbial Impactors
Becoming more popular for sampling airborne biological agents -- Battery-powered, portable, easy to handle -- High volume sampling, more than 100 L/min

17 Particle Collection of Portable Samplers

18 Portable Microbial Impactors
Bio-Culture 120 L/min Microflow 120 L/min SMA MicroPortable 28.3/141.5 L/min

19 Portable Microbial Impactors
RCS High Flow 100 L/min SAS Super 180 180L/min Millipore Air Tester 140/180 L/min They have been used in military sites

20 Portable Microbial Impactors
These samplers are increasingly being used for bio-sampling Their sampling performances are not fully described or investigated MAS-100 100 L/min Investigation of physical and biological collection efficiencies

21 Sampler Testing System
Yao, M. and Mainelis, G. Aerosol Sci. Technology, 2006, 40:1-13.

22 Physical Collection Efficiencies & Cutoff Sizes of Seven Portable Microbial Samplers When Sampling PSL Particles Virus anthrax Yao, M. and Mainelis, G. Aerosol Sci. Technology, 2006, 40:1-13.

23 Comparison of Sampler Performance with Particle Inhalation and Deposition in Human Lung
Yao and Mainelis, J. of Exposure Analysis and Env Epi, (2007), 17, 31–38

24 Biological Collection Efficiency
the ability of the sampler to not only collect, but also keep the viability of the bio-particles

25 Influences of Jet Velocity and Jet-to-plate Distance on Biological Collection Efficiency

26 Air sampling Techniques
filtration gelatin filter Anthrax surrogate

27 Air Sampling Techniques
BioSampler 1) Liquid Impinger, use of centrifuge and impaction to collect aerosol particles with a sampling flow rate of 12.5 L/min 2) Longer sampling time up to 8 hours 3) Transferring aerosols into hydrosols BioSampler Powerful aerosol-2-hydrosol sampling techniques are needed

28 Bio- sampling Challenges
Impaction-based sampling techniques were shown to cause damages to the viability of microorganisms Virus is too small to be collected by these techniques & their sampling method is significantly lacking There is a need to develop a more advanced sampling strategy

29 Electrostatic Collection
Electrostatic collection is a mechanism of collecting the airborne charged particles using the electrical force Collection velocity is about 2 to 4 orders of magnitude lower than that of BioStage impactor (24 m/s) Lower mechanical stress and less desiccation upon the microorganisms being sampled

30 Yao and Mainelis, Journal of Aerosol Science, 2006, 37:513-527
Electrosampler was designed to investigate if natural charges of microorganisms can be used for their effective electrostatic collection Yao and Mainelis, Journal of Aerosol Science, 2006, 37:

31 Physical Collection Efficiency of Electrosampler
Collection efficiency decreases with increase of flow rates 70% collection efficiency can be achieved at 1.2L/min Larger particles have lower concentrations Electrostatic field may have the ability to collect viruses Electrostatic field, 5kV/cm, was used

32 Comparisons of outdoor bacteria sampling using Electrosampler and BioStage impactor
Electrostatic method provides a better biological quantification

33 Use of Electrostatic Field in Collecting Airborne Toxins

34 Use of Electrostatic Field in Collecting Airborne Allergens

35 Bioaerosol Detection and Assessment (Combining Physical , Biochemical and Molecular Techniques )
The electrostatic method demonstrates ability in collecting viruses from the air. Virus concentration could be very low in the air, even collected, might not be enough to be detected. Combination of electrostatic method with advanced molecular techniques such as qPCR and ELISA may offer a solution, e.g., for detecting influenza A virus.

36 Globally confirmed human cases of H5N1 avian influenza since 2003

37 (magnified approximately 70,000 times) in May 1997
Influenza A Virus Commonly known as flu, is an infectious disease of birds and mammals caused by an RNA virus Typically, influenza is transmitted from infected mammals through the air by coughs or sneezes, creating bioaerosols containing the virus Currently, the strand is only limited to animals, but it is very likely to mutate further becoming a human-to-human case. Hong Kong Flu (magnified approximately 70,000 times) in May 1997

38 Spanish Flu in 1918 “In 1918, Spanish flu killed 675,000 people in the U.S. and an estimated 20–50 million people worldwide”

39 Detection of influenza A Virus
Metal Plate Virus particles Air Out Air In E qPCR ELISA Metal Plate Biosensor + 96-well-plate Endotoxin/Glucan Yao et al. (2007) Integration of Technologies for Constant Monitoring of Exposure [E-Letter], Science.

40 Environmental Allergens
Common Allergens? House dust allergens (Der p 1 and Der f 1), cat allergens Fel d 1 (cat), dog allergen Can f 1 (dog), Bla g 1 (cockroach), Bla g 2 (mouse) fungal allergens, e.g., Alternaria alternata allergen Alt1 Enzyme-Linked ImmunoSorbent Assay (ELISA) is often used to analyze allergens

41 ELISA Sample Processing
Dissolve into 1.5 mL PBS 0.05% Tween 20 shaking 2 h Dust Sieving (>30 mg) centrifuge 20 min supernatant 96-well plate Antibody coated plate

42 Procedure of ELISA tests
ELISA can be used together with air sampling technique for measuring airborne viruses and allergens

43 House Dust Mite (Der p & Der f 1)

44 Principle of LAL/Glucatell Assay (airborne endotoxin and glucan)
Activates Endotoxin (LPS) (1,3)-β-D-glucan Factor C Factor G LAL LAL Factor B LAL Preclotting Enzyme LAL Substrate Ac-Ile-Glu-Ala-Arg-pNA. pNA (yellow) Horseshoe crab LAL (Limulus Amoebocyte Lysate)

45 LAL/Glucatell 分析方法的流程
Add sample extracts, standards into 96 well plate Filter Extraction Dilution (10-3) 0.05% Tween 20 for Endotoxin 0.5 N NaOH for glucan, neutralized by Tris-HCL Incubation (15 min at 37 oC) add LAL or Glucatell Agents Placed inside spectraphotometer Log(Y)=A+Log(X) 60-80 min

46 Results for Road Dust In collaboration with Lovelace Respiratory Research Laboratory

47 Results for Road Dust In collaboration with Lovelace Respiratory Research Laboratory

48 qPCR for quantification of microbial species
Primary tasks include: Selection or design of primer sets for specific microbial species (alternaria spp) Design of probes for specific allergens & develop standard curves quantitative-PCR tests for DNA extracts from environmental samples DNA extraction Primers & Probes Standard Curve qPCR tests

49 qPCR for quantification of microbial species
reaction mixture: Template DNA Forward/Reverse Primers, Probes dNTPs DNA Polymerase Buffer Tris, KCl, Mg2+ , BAS , etc.

50 qPCR Application Curves
Sample 19 Vesper et al, 2005, American Laboratory, pp

51 Quantification of Environmental Sample
Eff=10(-1/slope) -1 Xn=X0(1+E)n qPCR can be used for detecting airborne low concentration biological agents

52 Other Bioaerosol Detection Techniques
1) Bioaerosol mass spectrometry (BAMS) (Herbert et al., 2005) 2) Surface-enhanced Raman spectroscopy (Sengupta et al., 2007) 3) Flow cytometry with fluorochrome (Chen and Li, 2005 4) Bio-functional oligonucleotides based techniques such as aptamer (Brody et al., 1999) 5) Nanowire-based detection techniques All these techniques can be also used for detecting airborne low concentration biological agents

53 Microorganism Inactivation
> Bacteria Preparation In this research, we have tested both sensitive microorganisms and hardy microorganisms (P. fluorescens and Bacillus subtilis ) > Deposition of Bacteria on filters >Exposure of filter-borne bacteria MCE filter (Mixed Cellulose Ester) Yao and Mainelis, Environmental Science Technology, 2005, 39:

54 Survival rates of P. fluorescens bacteria when deposited on MCE filter and exposed to the electrostatic field Survival rate of Pseudomonas fluorescens bacteria deposited on MCE filter and exposed to positive electrostatic field Yao and Mainelis, Environmental Science Technology, 2005, 39:

55 Use of nano-scale Zero Valent iron particles in Inactivating Microbial Species
Characterization of nanoscale iron particles FE-SEM Images NZVI particles Iron Oxides 所有被表征的与微生物混合后的铁溶液都与细菌或者真菌悬浊液振荡混合5分钟。 Shaking-oxidization

56 Inactivation of B. subtilis by NZVI particles
关键!![1] BST原来只有20%-30%灭活。 [2]

57 Characterization of the Inactivation of B. subtilis
Pure BST NZVI+BST anaerobic (c) (d) FeOOH+BST Aerobic NZVI+BST Aerobic

58 Inactivation of P. fluorescence by NZVI

59 Inactivation Mechanisms by NZVI
Gram-positive Matthew T. Cabeen & Christine Jacobs-Wagner,Nature Reviews Microbiology 3, (August 2005) Gram-negative

60 Inactivation of A. versicolor by NZVI

61 Characterization of the Inactivation of A. versicolor
Submitted to Applied & Environmental Microbiology

62 Summary of NZVI Inactivation
Inactivation was fast and efficient, within 5 minutes, all B. subtilis were inactivated Due to its small size, the inactivation efficiency was very high, and 10mg/ml can achieve good results The inactivation depended on the membrane type of the microbial species, e.g., no effects on fungi species tested

63 Other Biological Control Technologies
Biofiltration (Sanchez-Monedero et al., 2003) X-Ray enhanced electrostatic field Photocatalytic materials such as TiO2 have been investigated (Pal et al., 2005). Cold plasma (Birmingham and Hammerstrom, 2000) and UV light (Tseng and Li, 2005) Control of air stream, e.g., negative pressure rooms

64 Current Research Areas of BioAerosol Science
Integration of bioaerosol science with molecular science such as qPCR, PCR, and ELISA High volume of sampling: Portable Microbial Sampler, aerosol-2-hydrosol techniques Investigation of the link between bioaerosol exposure and health effects Development of high throughput environmental bio-sensor

65 Current Research Areas of BioAerosol Science
Combining bioaerosol with physics, chemistry, bio-medical engineering and molecular techniques Drug delivery to the lung using aerosol technology Human early disease detection such as lung cancer Inactivation of BioAerosols

66 Biological Exposure Assessment and Control
Air Sampling Bioaerosol Emission Exposure Assessment Detection Biological Control Prevent Minimize Human Biological Exposure

67 Biological Exposure Assessment and Control
Detect Control Collect CDC

68 Bioaerosol field is multidisciplinary ,
and requires many areas of expertise

69 Thank you !!! Maosheng Yao,PhD PKU “100 Scholar Program” Professor

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