ARTICLE
Received 25 Apr 2016
| Accepted 23 Jan 2017 | Published 7 Mar 2017
Bioaerosol generation by raindrops on soil
Young Soo Joung
1,2
, Zhifei Ge
1
& Cullen R. Buie
1
Aerosolized microorganisms may play an important role in climate change, disease
transmission, water and soil contaminants, and geographic migration of microbes. While it is
known that bioaerosols are generated when bubbles break on the surface of water containing
microbes, it is largely unclear how viable soil-based microbes are transferred to the
atmosphere. Here we report a previously unknown mechanism by which rain disperses
soil bacteria into the air. Bubbles, tens of micrometres in size, formed inside the raindrops
disperse micro-droplets containing soil bacteria during raindrop impingement. A single
raindrop can transfer 0.01% of bacteria on the soil surface and the bacteria can survive more
than one hour after the aerosol generation process. This work further reveals that bacteria
transfer by rain is highly dependent on the regional soil profile and climate conditions.
DOI: 10.1038/ncomms14668
OPEN
1
Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
2
Division of Mechanical Systems Engineering, Sookmyung Women’s University, 100, Cheongpa-ro 47-gil, Yongsan-gu, Seoul, Republic of Korea.
Correspondence and requests for materials should be addressed to Y.S.J. (email: ysjoung@sookmyung.ac.kr) or to Z.G. (email: zhifeige@mit.edu)
or to C.R.B. (email: crb@mit.edu).
NATURE COMMUNICATIONS | 8:14668 | DOI: 10.1038/ncomms14668 | www.nature.com/naturecommunications 1
B
ioaerosols play an important role in climate change, human
health and agricultural productivity
1–5
. In the atmosphere,
bioaerosols can influence on the global climate, promoting
cloud formation and ice nucleation even though their fraction is
relatively small compared to all the atmospheric aerosols
6–10
.On
the ground, bioaerosols can change micro-biogeography faster
than many other transport mechanisms
11,12
. Because bioaerosols
can lead to dispersion of biological contaminants over long
distances relative to terrestrial transport mechanisms
13,14
, they
significantly affect changes in biodiversity and ecology as well
as the propagation of biological pollutants
10,15
. Furthermore,
bioaerosols can be effective carriers of pathogenic organisms to
plants, animals and humans, resulting in the spread of disease
4,16
.
To date, aerosols generated at water/air interfaces are considered
one of the main mechanisms for transferring microbes to the
environment
17–20
. While it has been generally accepted that
soil can serve as an intermediate home for pathogens before
they transfer to their hosts, it is not clear where and how the
microbes in the soil transfer from their original habitats to the
atmosphere
21–25
. Considering that most microbes prefer aqueous
environments to survive, it is still a mystery to explain how viable
soil-based microbes spread much further and faster than would
be expected through the air
1,26,27
. Futhermore, even though we
know that after a rainfall there is a rapid increase of bioaerosol
concentration in the air, we have not explained the wide spread of
microbes with the transfer modes discovered to date
9,28,29
.
In this work, aerosols represent small water droplets suspended
in the air; in particular, bioaerosols are defined as aerosols
containing microbes. Recently, we discovered a new mechanism
of aerosol generation by raindrops hitting soil
30
(Fig. 1a–d).
We demonstrated that when a raindrop hits soils, small bubbles
are formed inside the raindrop and then small droplets eject
when the bubbles burst at the air/raindrop interface. Depending
on soil wetting-properties and raindrop impact speed, the amount
of aerosols varies. Interestingly, for particular wetting conditions
and impact speeds, hundreds of aerosols are generated from a
single raindrop within a few microseconds (Supplementary
Movie 1). On soils with wetting properties similar to sandy-clay
and clay soils, most aerosols are generated when raindrops fall
at velocities corresponding to light and moderate rain
30
.
Furthermore, we have shown that fluorescent dyes permeated
in the soil can be dispersed by aerosols
30
. Based on our findings,
under actual field conditions, it was demonstrated that organic
materials can be transferred though aerosols generated by
raindrops
31
. Our previous work explained the physics of aerosol
generation (aerosolization) from soil; however, we still need to
understand if and how bacteria in soil are transferred through the
aerosolization process.
Here, we illuminate a previously unexplored mechanism that
transfers bacteria from soil to air through aerosols. We first
develop a visualization method for characterization of aerosols
containing bacteria. Using the method, we quantitatively examine
the effects of bacterial surface concentration, soil composition,
raindrop impact speed, and surface temperature to identify trends
in bacteria transfer from soil to air. We also verify that bacteria
can indeed survive after the aerosolization process. Finally,
we estimate the global transfer rate of soil bacteria by rainfall.
Results
Visualization of aerosols containing soil bacteria. In this work
we show that soil-borne bacteria can be transferred from soil to
Raindrop
Aerosols
Sampling plate
Bacteria
Bacteria
Bacteria
Soil
Aerosol
Aerosol
Raindrop
Falling hole
Aerosol
Raindrop
hitting on soil
Soil with
bacteria
t = 0 ms t = 31 ms t = 423 ms
ab c d
e f
g
h
Figure 1 | Bioaerosol generation by raindrops. (a–d) Aerosols generated by drop impingement on a reference surface, which maximized the aerosol
generation (a TLC plate (TLC-C) in Table 1). The TLC plates served as an ideal soil-like surface. The white lines are the trajectories of aerosols ejected from
the initial droplet after impact over a period of 400 ms. Due to air flow above the droplet, the trajectories of the ejected aerosols are curved. The scale bars
indicate 1 mm. For more details, see Supplementary Movie 1. (e) Schematic illustration of the experimental procedure for drop impingement on soil and
aerosol collection. (f) Confocal microscopy images of C. glutamicum on the surface of clay soil with the cell density of 250 cells mm
2
.(g,h) Fluorescent
microscopy images of aerosols generated by drop impingement on clay soil pre-permeated with C. glutamicum. The red circles and the yellow dots indicate
aerosols and C. glutamicum, respectively. The scale bars indicate 200, 50 and 25 mminf–h, respectively.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14668
2 NATURE COMMUNICATIONS | 8:14668 | DOI: 10.1038/ncomms14668 | www.nature.com/naturecommunications
air via aerosols generated by rain. Aerosols are collected on the
sampling plates positioned above the soil of interest (Fig. 1e).
Fluorescent microscopy images are obtained by using soils pre-
permeated with a red-fluorescence dye (Rhodamine B) and green-
fluorescence (SYTO BC) bacteria (Fig. 1f). The soils were fully
dried under common laboratory environment after pre-treatment
with red fluorescent dye. In this work, we used three species of
soil bacteria: Corynebacterium glutamicum (C. glutamicum ATCC
13032), Bacillus subtilis (B. subtilis JMA222) and Pseudomonas
syringae (P. syringae ATCC 55389), and six kinds of soil: two
different types of clay, sandy-clay and sand (Table 1). In addition,
two different thin layer chromatography plates (TLC) were used
as reference porous surfaces (ideal soil-like surfaces). The aerosols
and bacteria appear as red circles (aerosols) and yellow dots
(bacteria; Fig. 1g,h). Depending on the species and strains of
bacteria, it is known that bacteria show different aerosolization
properties
32
. In this work, we used three representative bacteria
that are each abundant in soil and show long-term viability on
both soils and TLC plates. These strains allow us to evaluate the
effect of aerosolization on cell viability without considerable
numbers of dead cells on the surfaces. In Fig. 1g, the aerosol sizes
range from a few microns to hundreds of microns; but with the
higher magnification microscope-lens, we can observe submicron
droplets on the plates. The number of bacteria varies from zero to
several thousand in a single aerosol, mainly depending on the soil
types, the bacterial surface density, the surface temperature and
the raindrop impact speed (Fig. 1h).
Viability of bacteria dispersed by aerosols. All the bacteria used
in this work can be transferred from soil to air by aerosols while
remaining viable. The bioaerosols are collected on the sampling
plates and the plates are kept in a standard laboratory environ-
ment for a specific amount of time. Subsequently the aerosols on
the plates are transferred to agar plates to measure the number of
aerosols containing live bacteria. Depending upon the bacterial
species and the soil, different numbers of colonies on the agar
plates are observed (Fig. 2a,b). The most bioaerosols are
generated on the sandy-clay soils but no bioaerosol is observed
from the sand soils. This result can be explained by our previous
work showing that sandy-clay soils have the optimal wetting
properties for aerosolization, but sand soils absorb raindrops too
fast to generate aerosols
30
. The bacteria transferred to the aerosols
exhibit different viability depending on their residence time in the
air (Po0.05), but there is no significant difference in the soil
types tested and in the bacterial species (P40.05). As a result,
the three different soil bacteria remained alive even 1 h after
aerosolization. This result suggests two important conclusions;
(1) bacteria can survive aerosolization by drop impingement, and
(2) bacteria can be convectively transferred to distant locations
while remaining viable. The viability test used in this work may
not be applicable for other soil bacteria that may lose their
culturability when subjected to environmental stresses or an
inadequate cultivation environment. Indeed, it is well known that
a small percentage of airborne bacteria can be cultured
33,34
.In
fact most estimates suggest that the majority of bacteria on the
planet have evaded laboratory cultivation
35–38
. In this work, we
intentionally seeded culturable bacteria into the soil before
aerosolization to check if the soil bacteria can survive the
aerosolization process. We did not use bacteria directly recovered
from environmental samples transferred by aerosols. If the
aerosolization results in some bacteria remaining viable
but unculturable, the number of bacteria transferred through
aerosolization would be underestimated. However, this
underestimation does not undermine the main result of our
viability test. To minimize the limitations of viability testing, we
used direct visualization of aerosols containing bacteria to count
the number of bacteria transferred by aerosols; therefore, the
aerosolization efficiency reported is not distorted by bacteria
culturability.
Particle dispersion by bubble bursting. The number of bubbles
formed inside a raindrop is the key factor influencing bacterial
transfer (Fig. 3a and Supplementary Movie 2). With increasing
bubbles formed inside a raindrop, more bacteria can be trans-
ferred by bubble bursting. The proposed mechanism is most
relevant when the soil is not fully wet. After a couple of raindrops
impact the same location, the soil is fully wet and a thin water
film forms on the surface. In this case, we speculate that another
possible mechanism of bacteria transfer by splashing is more
relevant. Thoroddsen et al. have shown that the ejected sheet
brings liquid from the bottom of the film, and when it breaks
up into fine spray it could act just like the proposed bubble
mechanism
39
. If we consider this mechanism, it is possible that
even more bacteria can be dispersed by raindrop impact.
However, in the present work, we solely deal with the
dispersion of bacteria by aerosolization
39
. We investigated the
effect of bubble bursting on the dispersion of particles for two
different initial conditions (Fig. 3b); first, with different particle
concentrations in raindrops and clean surfaces (Case 1), and
second, with different particle densities on the surfaces and pure
raindrops (Case 2). Interestingly, for the wide range of surface
particle density and raindrop particle concentration, the number
Table 1 | Surface properties related to bioaerosol generation by raindrops impinging on soils and TLC plates.
Media Media hydraulic
diffusivity
Critical surface
temp.
Critical impact
speed
Aerosolization efficiency: e (%)
Name D
cap
(mm
2
s
1
) T
c
(°C) V
c
(m s
1
)1lm latex bead 10 lm latex bead C. glutamicum P. syringae B. subtilis
TLC
A
9.5 30 1.4 0.16 2.34 0.093 NA NA
TLC
C
22.4 30 1.33 2.24 2.68 0.16 0.034 0.0074
Clay
A
2.6 30 1.53 0.15 0.87 0.004 0.0066 0.0021
Clay
B
1.4 40 1.33 0.15 3.56 0.02 0.0392 0.0088
Sandy
clay
A
12 20 1.47 0.83 NA 0.013 0.0103 0.0034
Sandy
clay
B
4.8 50 1.33 1.24 NA 0.0060 0.0072 0.0102
Sand
A
127.6 30 1.53 0.01 0.05 NA NA NA
Sand
B
252.8 NA NA NA NA NA NA NA
D
cap
, T
c
and V
c
are hydraulic diffusivity
54
, critical temperature and critical impact velocity of the surfaces, respectively. In the case of Sand
A
and Sand
B
, the dispersion of bacteria was not observed due to
the low aerosolization efficiency; especially any particles and bacteria were not transferred by raindrops on Sand
B
.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14668 ARTICLE
NATURE COMMUNICATIONS | 8:14668 | DOI: 10.1038/ncomms14668 | www.nature.com/naturecommunications 3
of dispersed particles shows opposite tendencies for the two cases
with respect to surface temperature (Fig. 3c). At the surface
temperature of 30 °C, the least particles are dispersed for Case 1
(Po0.05); however, the most particles are dispersed at 30 °C for
Case 2 (Po0.05). Further, the trends of both cases are opposite
one another.
To further illuminate this result, we estimated the number of
bubbles formed inside a droplet as a function of surface
temperature. We can estimate the number of bubbles formed
inside a raindrop from the aerosol size distribution (Fig. 4a).
Here, when the bubbles are smaller than 1 mm, the number
of aerosols exponentially decreases with decreasing bubble
radius
40–43
; therefore, the number of aerosols can be expressed
as N
aerosols
¼ Aexp( 3
1
d
bubble
), where N
aerosols
is the number
of aerosols, A is a constant value (AB7.5) and d
bubble
is the
bubble diameter
40,41
. The bubble diameter is ten times greater
than the mean diameter of the aerosols
44,45
; thus, the number of
aerosols can be expressed as N
aerosols
¼ N
o
exp( 30
1
d
aerosol
),
where N
o
is a constant and d
aerosol
is the aerosol diameter. Under
the assumption that the bubbles have comparable diameters, the
total number of bubbles (N
bubbles
) formed inside a raindrop can
be estimated as N
bubbles
¼ N
o
A
1
; therefore, we can estimate the
number of bubbles formed inside the raindrop from the aerosol
size distribution.
The number of bubbles was estimated by the curve fitting
method. As we expected, the number of aerosols decreases
exponentially with respect to aerosol droplet size (Fig. 4a).
Different constants of the exponential functions, N
o
, are
obtained for different surface temperatures. From the power
law functions; we can obtain the estimated number of bubbles,
N
bubble
¼ N
o
A
1
. The maximum number of bubbles is obtained
at the surface temperature of 30–40 °C for two reference surfaces
(TLC-A and TLC-C in Table 1), respectively. When we compared
the number of bubbles obtained from the curve fitting method
and by counting with the digital high-speed images, we find
reasonable agreement (Fig. 4b). The results obtained with two
different TLC plates show that the maximum number of bubbles
is formed at the surface temperature of around 30 °C. When we
calculated the total volume of aerosols, the least volumes were
obtained at the surface temperature of 30 °C. As a result, the total
volume of aerosols governs the number of dispersed particles
when the particles are dissolved in the raindrop (Case 1), but the
number of bubbles mainly governs the number of dispersed
particles when the particles are placed on the surface (Case 2;
Fig. 4c).
Aerosolization efficiency . We employ aerosolization efficiency
46
(e) to characterize how many particles or bacteria can be
transferred by a single raindrop from soil to air. Figure 5a
shows the key parameters related to aerosolization efficiency.
Aerosolization efficiency is the ratio of the number of particles on
the surface (N
particles.surface
) to the number of particles dispersed
from the surface (N
particles.aerosols
) on the surface area same as the
cross-sectional area of the raindrop. To obtain aerosolization
efficiency, fluorescent microspheres, with average diameter of
1 mm, were placed on the surfaces with particle densities ranging
from 10–10
4
particles per mm
2
(Fig. 5b). After drop impingement
on the surfaces, we quantified the number of microspheres in the
aerosols collected on the sampling plate (Fig. 5c). To verify the
mechanism of particle dispersion, we first characterized the size
distribution of aerosols generated from the reference surfaces
(TLC-A and TLC-B) and the soils (clay-A and sandy clay-A). The
size of aerosol ranges from a few microns to a few hundred
Corynebacterium
glutamicum
C. glutamicum
P. syringe
B. subtilis
100
10
Initial number of colonies
1
0.1
TLC-C
Clay-A
Sandy clay-A
Sandy clay-B
Clay-B
Pseudomonas
syringae
Bacillus
subtilis
10
1
0.1
0.01
0 102030405060
TLC-C, C. glutamicum
Clay-A, C. glutamicum
Clay-B, C. glutamicum
Sandy clay-A, C. glutamicu
m
Sandy clay-B, C. glutamicu
m
TLC-C, B. subtilis
Clay-A, B. subtilis
Clay-B, B. subtilis
Clay-A, P. syringae
Clay-B, P. syringae
Sandy clay-A, P. syringae
Sandy clay-B, P. syringae
Sandy clay-A, B. subtilis
Sandy clay-B, B. subtilis
TLC-C, P. syringae
Time (min)
Viability
0 min 15 min 30 min
90 min
60 min
45 min
ab
cd
Figure 2 | Viability of bacteria transferred by aerosols. (a) Colonies of three kinds of soil bacteria, C. glutamicum, P. syringae and B. subtilis, cultured on
agar plates for 2 days after they were aerosolized by raindrops on sandy-clay soil (Sandy clay-A in Table 1). The inner black circles indicate the location
where raindrops hit on the soil. The yellow dots indicate the colonies where bacteria grew. The scale bars represent 10 mm. (b) Viability test with respect to
the duration of drying the aerosols collected on the sampling plates. The time, displayed in the images, indicate the drying duration. Aerosols were
generated from TLC plates (TLC-C in Table 1) pre-permeated with C. glutamicum. The colonies were cultured on agar plates for 2 days after the
aerosolization. The scale bars indicate 10 mm. (c) Average number of colony-forming units from a single raindrop when the aerosols, collected on the
sampling plates, were transferred to the agar plates immediately after aerosolization. The error bars represent
±
1 s.d. resulting from nine drop
impingements. The impact velocity was 1.4 m s
1
, the drop diameter of 2.8 mm, and the surface temperature 20 °C for all cases. (d) Viability of bacteria
with respect to time after aerosolization. The viability is the ratio of the number of colonies on the agar plate to the number of aerosols containing bacteria
collected on the sampling plate. For more details, see ‘Methods’ section.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14668
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microns and the number of aerosols decreases exponentially with
increasing aerosol size regardless of the surfaces used (Fig. 5d).
On the clay and sandy clay soils, we observed more than
one hundred aerosols smaller than 10 mm from a single drop
impingement. This result indicates that micro bubbles are formed
inside the drops that impact the soils and the particles are
dispersed when bubbles burst at the surface of droplets.
We observe a linear relationship between the surface particle
density (S
surface
), which is the number of particles (N
particles.surface
)
per unit area and the total number of particles (N
particles.aerosols
)
dispersed by aerosols generated by a single raindrop (Fig. 5e).
To evaluate the aerosolization efficiency of particle transfer, we
introduce the dispersed particle density (S
aerosols
), which can be
expressed as S
aerosols
¼ N
particles.aerosols
A
raindrop
1
, where A
raindrop
t = 0 t = 5 ms t = 25 ms t = 50 ms
Case 1: drops with bacteria
Case 2: soil with bacteria
1,000
1,00
10
1
0.1
0.01
010
Case 1:
Case 2:
20 nl
–1
2 nl
–1
0.2 nl
–1
620 mm
–2
72 mm
–2
7 mm
–2
20 30 40 50 60
Surface temperature (°C)
Number of particles
a
b
c
Figure 3 | Particle transfer by bubble bursting inside raindrops.
(a) Bubble formation at the interface of surface and raindrop. The red boxes
indicate the regions magnified in the images below. The scale bars
represent 1 mm. (b) Schematic illustrations of the two cases of bacteria
existence. Bacteria can exist inside the raindrop (Case 1) or on the surface
(Case 2). (c) The number of particles dispersed by aerosols generated on a
TLC (TLC-C) plate with respect to surface temperature. Drop impingements
were conducted with two different initial conditions: first, particles are in
the raindrops (Case 1) and second, particles are on the surfaces (Case 2).
In Case 1 and Case 2, different particle concentrations and densities were
used; Case 1: 20 particles per nl, 2 particles per nl, and 0.2 particles per nl;
Case 2: 620 particles per mm
2
, 72 particles per mm
2
, and 7 particles per
mm
2
. For both cases, 1 mm diameter yellow-green fluorescent microspheres
were used. The red symbols and the white symbols indicate the drop
impingements of Case 1 and Case 2, respectively. The error bars
represent
±
1 s.d. resulting from nine drop impingements.
1,000
100
10
5 °C
10 °C
20 °C
30 °C
40 °C
50 °C
1
0.1
0.001 0.01
0.1
1
Aerosol droplet size (mm)
0
10
20
30
40
50
60
Surface temperature (°C)
Number of droplets
1,000
100
160
140
120
100
80
60
40
20
0
160
140
120
100
80
60
40
20
0
0
10
20
30
40 50
60
Surface temperature (°C)
10
TLC-C: estimated number
TLC-A: measured number
TLC-C: measured number
TLC-C: volume of aerosols
TLC-A: volume of aerosols
TLC-C: number of bubbles
TLC-A: number of bubbles
TLC-A: estimated number
1
Number of bubbles
Number of daerosols (nl)
Number of bubbles
a
b
c
Figure 4 | Relationship between aerosol generation and bubbles.
(a) The number of aerosols as a function of aerosol diameter. From the
curves, we can estimate the number of bubbles formed inside the raindrop
as a function of surface temperature. The impact velocity was 1.4 m s
1
with the raindrop diameter of 2.8 mm for all the surface temperatures.
(b) The number of bubbles estimated by the theory (the red symbols) and
counted using high-speed images (the white symbols). The theoretical data
were estimated by curve fittings and an empirical equation reported
40,41
.
(c) The number of bubbles created inside a droplet (the white symbols) and
the total volume of aerosols (the red symbols) with respect to surface
temperature.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14668 ARTICLE
NATURE COMMUNICATIONS | 8:14668 | DOI: 10.1038/ncomms14668 | www.nature.com/naturecommunications 5