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Pyroaerobiology: the aerosolization and transport of viable
microbial life by wildland fire
LEDA N. KOBZIAR ,
1,
MELISSA R. A. PINGREE,
1
HEATHER LARSON,
2
TYLER J. DREADEN,
3
SHELBY GREEN,
4
AND JASON A. SMITH
5
1
Department of Natural Resources and Society, University of Idaho, College of Natural Resources, 875 Perimeter Drive,
Moscow, Idaho 83844 USA
2
University of Florida, 53 Weaver View Circle #101, Weaverville, North Carolina 28787 USA
3
Forest Health Research and Education Center, USDA-Forest Service, Southern Research Station, 1405 Veterans Drive,
Lexington, Kentucky 40546 USA
4
Department of Environmental Science, University of Idaho, College of Natural Resources, 875 Perimeter Drive, Moscow, Idaho 83844 USA
5
School of Forest Resources and Conservation, University of Florida, 136 Newins-Ziegler Hall, Gainesville, Florida 32611 USA
Citation: Kobziar, L. N., M. R. A. Pingree, H. Larson, T. J. Dreaden, S. Green, and J. A. Smith. 2018. Pyroaerobiology: the
aerosolization and transport of viable microbial life by wildland fire. Ecosphere 9(11):e02507. 10.1002/ecs2.2507
Abstract. The field of aerobiology is expanding due to a recognition of the diversity of roles microbes play
in both terrestrial and atmospheric ecology. Smoke from global biomass burning has had significant and wide-
spread ecological and human health consequences, but the living component of smoke has received little
attention. Microbes aerosolized and transported by wildland fire may have profound effects on atmospheric
and environmental factors, acting as nuclei for ice condensation, transporting pathogens or symbionts, and
otherwise influencing ecosystems and human populations downwind. The potential for smoke to aerosolize
and transport viable microbes is a virtually blank piece of the microbial biogeography puzzle with far-
reaching implications. This study characterized the aerosolization of viable microbes via wildland fire smoke
from burns in contrasting coniferous forests. Seventy aerosolized microbial morphotypes were recovered, and
of these, a subset was identified using DNA analysis which revealed both pathogenic and non-pathogenic
fungal species. Overall microbial colony-forming units decreased with increasing distance from smoke source,
driven by bacterial abundance. Organisms were more abundant in smoke derived from mechanically treated
fuels than intact forest floors and were most abundant in smoke from a dry, biennially burned Pinus palustris
sandhill forest in Florida. Our findings of smoke-transported viable microbes have implications for ecosystem
restoration/conservation, global biodiversity, meteorology, and human health.
Key words: aerobiology; atmospheric biology; bioaerosols; emissions; fire ecology; forest pathogen; fungal dispersal;
microbial ecology; microbiology; prescribed fire; smoke; wildfire.
Received 3 October 2018; accepted 15 October 2018. Corresponding Editor: Michael F. Allen.
Copyright: ©2018 The Authors. This is an open access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
E-mail: lkobziar@uidaho.edu
INTRODUCTION
Long-distance transport of microbes has been
documented across continents and oceans (Brown
and Hovmoller 2002, Hara and Zhang 2012, Smith
et al. 2012, 2013), as well as before and after storm
winds and dust storms (Murata and Zhang 2014).
These large-scale changes in microbial distribution
demonstrate the likelihood for similar transport
mechanisms of viable microbial communities in a
differenttypeofatmosphericvector—wildland
fire smoke. Global biomass burning is responsible
for aerosolizing approximately 42.2 Tg of particu-
late matter (PM) annually (Andreae and Merlet
2001), yet the contribution of combustion-aeroso-
lized and viable microbial organisms has received
little scientific attention. Aerosolized microbes can
be pathogenic or beneficial to plant and human
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health (Roux and O’Brien 2001, Griffin 2007), or
may act to change microbial communities and
their roles in both atmospheric and terrestrial envi-
ronments (Morris et al. 2013, Golan and Pringle
2017). These diverse, abundant, and adaptable
organisms may be integral drivers of ecosphere
resilience and recovery, especially as natural and
human-induced changes to climate and distur-
bance regimes continue.
Although wildland fire smoke plume tempera-
tures can reach maximums well over the thresholds
for most life-forms (e.g., >290°Cat4.5mabovea
typical grassland fire; Clements 2010), the mixing
ofburnedandunburnedfuels,fluctuations in oxy-
gen availability, meteorological factors, and entrain-
ment of ambient air result in a mosaic of fire
intensities and temperatures across spatial and tem-
poral scales (Hiers et al. 2009). High-energy con-
vection columns carry a widerangeofparticlesizes
due to intense vertical air mixing (Clark et al. 1998,
Lynch et al. 2004) and can result in the aerosoliza-
tion and transport of organic matter and even min-
eral soils (Pisaric 2002, Bormann et al. 2008).
Bioaerosols, or airborne particles with biological
origins, have the potential for long-range transport
and are likely to be associated with particulates, as
previous studies in continental dust transport have
shown (Hara and Zhang 2012). Wildland fire pro-
duces uniquely suitable substrates for organisms
that may not otherwise survive in smoke. For
example, pyrogenic C particles have been shown to
provide habitat for soil microbes (Pietik€
ainen et al.
2000), a role that may extend to PM within a smoke
column. A recent review of the long-distance trans-
port of fungi mentions the potential, yet unknown,
role for fire as a biogeographical dispersal vector
(Golan and Pringle 2017), because even prescribed
fire can evoke smoke plume rise to >1kmabove
ground level (Liu 2014). Yet wildland fire behavior
has received little attention for its singular potential
to aerosolize living microbes and transport them
via smoke plumes.
The viability of microorganisms in smoke
plumes is likely to be controlled by a combina-
tion of atmospheric and fire conditions, includ-
ing relative humidity; temperature; convective
forcing; degree of mixing; ultraviolet (UV) radia-
tion; and oxygen content. Of parallel importance
are the traits of the source microbial community
and the types of material aerosolized (e.g., fuel
source, pigmentation, high G +C nucleic acid
content, high DNA repair ability, and UV protec-
tion; Mohr 2007). However, these complex asso-
ciations have not been characterized in relation
to wildfire or prescribed fire smoke presence.
The composition of viable microbes transported
by smoke may have significant implications for
forest ecosystems and management. Understand-
ing the fate of specific pathogenic and beneficial
microbes can help direct broader restoration
efforts for the conservation of affected ecosys-
tems (Klopfenstein et al. 2010).
Of the numerous research publications pertain-
ing to wildland fire smoke or aerobiology, we
have only uncovered a single study that connects
the two disciplines, and no exploration of this
phenomenon in terms of microbiology, smoke
science, fire behavior, and fire ecology from an
interdisciplinary viewpoint (Fig. 1). Mims and
Mims (2004) found a strong correlation (r
2
=0.78)
between fungal spores and aerosolized PM
(assessed microscopically through visual counts
of particles) deposited in Texas, USA, by smoke
originating from wildfires on the Yucat
an Penin-
sula, M
exico. No assessment was conducted to
verify that smoke particles were physically or bio-
logically associated with the fungal spores. This
case study also incorporated biological samples
from a backyard experimental fire and measured
higher numbers of colony-forming units (CFUs)
on nutrient films exposed to smoke compared to
those in ambient air. However, statistical tests
were not conducted, and further study was not
pursued by the authors.
The transport of viable aerosolized microorgan-
isms via wildland fire smoke, hereafter referred to
as “pyroaerobiology”(PAB; Fig. 1), is an integra-
tion of micro- and aerobiology, smoke and atmo-
spheric sciences, fire behavior, and fire ecology in
a coherent effort to understand the ecological and
societal impacts of smoke-vectored microbes. The
objective of this study was to gain a foundational
understanding of the capacity of wildland fire to
aerosolize viable fungi and bacteria in smoke, and
how different combustion processes and sources
may affect the aerosolized communities. Altho-
ugh various microbiological methods could be
used to assess microbial composition and
abundance in air masses (Haig et al. 2016), a
commonly employed method for assessing the
likelihood that organisms would survive after
being aerosolized (i.e., capacity of microbes to
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KOBZIAR ET AL.
influence the environment where they land) is the
culturing of organisms (e.g., Yao and Mainelis
2007). This method is preferable because not only
does it assess microbial presence, but it also
allows for determination of post-fire viability.
Because prescribed fires consume more biomass
and typically burn more acreage than wildfires
across the United States on an annual basis (NIFC
2018), and because prescribed fire scenarios allow
for safe, direct access to the flaming front to con-
trol for differences in combustion type and fireline
intensity, we performed this initial experiment
using prescribed fires. To assess temperatures and
determine whether mass loss corresponded to cul-
turable microbial abundance, we conducted an
additional study using burns in a controlled com-
bustion laboratory using different fuel types. We
tested three hypotheses about smoke-transported
microbes during prescribed burns and laboratory
combustion experiments by culturing impacted
microbes, microscopic identification of morpho-
types, and genetic analyses: (1) Viable microbe
abundance as measured by CFUs will vary with
increasing distance from the smoke source and
will differ from ambient air; (2) viable species
abundance will differ with the type of combustion
(smoldering vs. flaming); and (3) viable species
abundance will differ by site, where historical fire
frequency, management, or fuel types differ.
MATERIALS AND METHODS
Study sites
Two distinct studies with different methodol-
ogy were conducted, based on limited available
resources. In the Florida-based study, we utilized
three 5- to 10-ha prescribed burns, while in
Idaho, we transported forest floor samples and
combusted them in a laboratory. The three burns
were conducted in humid sub-tropical forests at
the University of Florida’s Austin Cary Forest
approximately 18 km northeast of Gainesville,
Florida, USA. The first burn was conducted on 3
April 2015 in a mature (70–90 yr old) Pinus palus-
tris sandhill ecosystem (Myers and Ewel 1990)
maintained by a two-year prescribed fire return
interval since 2003 (Sandhill Biennial), while the
second burn was on 6 August 2015 in a mature
longleaf pine flatwoods (distinguished from
sandhills by a higher water table and sub-surface
spodic horizon) ecosystem that was burned
annually since ca. 1990 (Flatwoods Annual). The
third and final prescribed burn took place on 25
September 2015 in a hydric P. palustris and
Fig. 1. Pyroaerobiology integrates theory and methods from microbiology, smoke and atmospheric sciences,
and fire ecology, with a range of broader impacts and value added to each discipline.
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KOBZIAR ET AL.
Pinus elliottii flatwoods ecosystem that was pre-
viously (and incompletely) burned only once
since 1940 (in 2012), characterized by a heavy
buildup of surface and ground fuels (i.e., under-
story vegetation and organic soil horizons).
North Idaho forest floor sampling sites were
located in semi-arid steppe forests at the Univer-
sity of Idaho Experimental Forest (UIEF) on the
Palouse Range, about 20 km northeast of Mos-
cow, Idaho, USA. These mixed-conifer forest
stands consist of a diverse coniferous overstory
dominated by Pseudotsuga menziesii,Abies gran-
dis,Thuja plicata, and Pinus ponderosa var. pon-
derosa, and understory species dominated by
shrubs, with fuel reduction treatments as
described by Sparks et al. (2017).
Soil sample collection—mixed-conifer forest
in Idaho
Soil samples of the entire organic horizon (O
horizon) were collected from three forest stands
planted in 1982. Each stand had two treatments
including understory fuel reduction (all surface
fuels and small trees shredded and left on site—
masticated; see Sparks et al. 2017), and one left
untreated—control. Three soil samples were col-
lected to the entire depth of the organic soil hori-
zon (including O
i
,O
e
, and O
a
layers) using a
17 cm diameter ring at three randomly located
plots within both treatments in each stand
(n=18). Samples were kept cold for seven days
(2°C), air-dried for 48 h in the laboratory under a
sterilized closed laminar flow hood, and then
composited by treatment by stand prior to com-
bustion. Compositing was used to account for
high spatial variability within sub-sections, and
to achieve sufficient masses for continuous flam-
ing and residual smoldering combustion subse-
quently in the combustion laboratory (n=6).
Bioaerosol sampling—combustion laboratory
in Idaho
Because we sought to culture the microbes,
sampling durations were limited to 2 min in
order to reduce the potential for desiccation and
damage to the organisms (Mainelis and Tabayoy-
ong 2010). To assess background levels of aeroso-
lized microbes, ambient air samples were taken in
the field at each sampling location by exposing
one Petri dish with sterilized potato dextrose agar
medium at one meter above the ground surface,
quickly sealing it with Parafilm, and storing it in a
cooler for immediate transport to the laboratory
(n=18). A combustion laboratory (IFIRE Lab,
University of Idaho, Idaho, USA) was used for the
burn experiments. The laboratory is comprised of
a preparation and control room where data are
monitored, and a separate combustion chamber
within a climatically controlled room containing
an over-sized dedicated fume hood. O horizon
samples were transported to the combustion labo-
ratory, and using sterile techniques, ~100–200 g of
soil was transferred into sterilized metal pans
prior to ignition. Pan contents were burned on a
table scale to measure real-time mass loss rates
(per second, and as percent of initial mass). Three
type-K, 20-AWG fiberglass-sheathed thermocou-
ple wires (Omega Engineering, Stamford, Con-
necticut, USA) were positioned within the fuel
bed at 0, 15, and 60 cm above the fuel bed to
monitor temperatures during burning using a
datalogger in the preparation room. During flam-
ing and then smoldering combustion, three Petri
dishes were suspended approximately 50 cm to
1 m above the fuel source for a total of nine repli-
cate smoke samples for each stand 9fuel treat-
ment combination (total number of Petri dishes
subjected to smoke in laboratory =18, with nine
smoldering and nine flaming combustion sam-
ples). Two additional dishes were exposed for the
same time period prior to any ignitions to serve as
laboratory blanks. After exposing the dishes to
smoke and ambient laboratory air, dishes were
sealed, stored in a cooler, and transported to a
microbiology laboratory for incubation using ster-
ile handling techniques.
Bioaerosol sampling—prescribed burns in Florida
The three prescribed burns were conducted
within the late-spring-to-late-summer growing
season. All prescribed burns were ignited using a
combination of flanking and strip head fire igni-
tion patterns. Flame lengths (indicative of fire
intensity and ranging from 0.5 to 3 m) were esti-
mated by ocular comparison to a 1-m pole (Kreye
and Kobziar 2015) and ranged from an average
value of 0.9 m in the Flatwoods Annual site,
2.3 m in the Sandhill Biennial site, to 3.8 m in the
Long-Unburned site.
All samples were collected via passive impac-
tion onto malt extract agar (MEA) medium in
Petri dishes manually oriented into the wind.
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KOBZIAR ET AL.
Sampling locations at each burn were established
when consistent fire rate of spread was observed,
and outside of direct ignition zones. Samples
were collected at the origin (approximately
0.5 m) and increasing distances from the flaming
front relative to flame length. Sterilized Petri
dishes with MEA were suspended approximately
1–2 m above ground level on platforms attached
to extension poles facing upwind into smoke
plumes for two minutes, closed, sealed with Par-
afilm, and stored in a cooler for 1–2 h before
transportation to the laboratory. During the
burns, three to four ambient air samples per burn
were taken at a minimum of 9 m upwind and
away from any perceivable influence of smoke,
but within the same hour and site conditions.
Samples were also collected during smoldering
combustion at 0.5–1 m from the source of com-
bustion. No smoldering samples were collected
at the Flatwoods Annual site due to the insuffi-
cient source of smoldering combustion. The
number of samples taken was dictated by oppor-
tunities for safe entry into the burn zone, and the
total duration of each burn.
Microbial culture processing—both locations
Samples were transported to the laboratory
and kept at room temperature (~23°C) in the
dark until colonies had developed, 72 h and 7–
14 d for Florida and Idaho samples, respectively.
Plates were visually examined under a 409
microscope, classified into morphotypes, and
CFUs for fungi and yeasts were counted. After
approximately one week, a colony from each
morphotype from the Florida sites was subcul-
tured on acidified potato dextrose agar (APDA)
to enable targeted analysis of morphological
characteristics (e.g., spore production, colony fea-
tures). Of seventeen different fungal morpho-
types, eight randomly selected morphotypes
were subcultured on sterilized cellophane on
APDA for seven days, to facilitate growth and
harvesting of material for DNA extractions. Stan-
dard sterile technique and analysis within a lami-
nar air flow hood were used throughout with all
cultures maintained at ~23°C in the dark.
DNA extraction, amplification, and sequencing—
Florida samples
The randomly selected unique morphotypes
from the Florida smoke samples had DNA
extracted by harvesting a portion of the colony
from the cellophane, using the DNeasy Plant
Mini Kit (QIAGEN, Hilden, Germany), following
the manufacturer’s instructions. The nuclear ribo-
somal internal transcribed spacer (ITS) region
from fungal morphotypes was amplified by
polymerase chain reaction (PCR) with primers
ITS1F (50-CTTGGTCATTTAGAGGAAGTAA-30)
and ITS4 (50-TCCTCCGCTTATTGATATGC-30;
White et al. 1990) using PCR conditions from
Sena et al. (2018), Sanger-sequenced at the
University of Florida Genetics Institute in Gaines-
ville, Florida, USA, and compared to sequences in
GenBank. The Idaho samples were not sequenced
due to resource limitations.
Statistical analysis
Data were explored and analyzed in the R
environment (R Core Team 2016). Total CFUs
from the Florida study were analyzed pooled
and separate for each site in order to evaluate the
relationships between colonized microbes and
fire or site factors, respectively. The total CFUs
were then analyzed by growth forms. To meet
statistical assumptions, total CFU data were
transformed by log(x+1) and then compared
with an ANOVA against combustion type and
distance from flaming front within each ecosys-
tem. When split into organism types, we used
Kruskal–Wallis tests as the data did not fit all
assumptions of ANOVA. Where applicable,
transformed CFU data were tested in a regres-
sion model against distance from the flaming
front. Total CFUs from the Idaho study were
transformed by log(x+1) to meet ANOVA
assumptions and tested against fuel type, organ-
ism types, and regressed against temperatures
and mass loss (%) during combustion.
RESULTS
Florida prescribed burn sites
Across the three Florida prescribed burn sites,
distance was negatively related to the average
number of CFUs for all organism types during
flaming combustion (n=36, r
2
=0.77, P<0.001;
Fig. 2). Although there were higher CFU counts
for filamentous fungi compared to bacteria and
yeast (n=123, 47, and 25, respectively), the rela-
tionships between CFUs and distance (Fig. 2)
were driven by bacteria. Overall, filamentous
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KOBZIAR ET AL.
fungal and yeast CFUs were not significantly
related to distance or distance divided by flame
length when analyzed independently (P>0.05).
Smoke samples from the Sandhill Biennial site
showed a significant (P<0.001) and negative
correlation (r
2
=0.40) between bacterial CFUs
and distance from the flaming front. The Flat-
woods Long-Unburned site showed significant
differences (P<0.01) in CFUs among distances
from the flaming front, which were lowest at the
origin and outside of the smoke (at 30 m) but
highest at 3- and 6-m (2–3 times the flame
lengths) collection points, suggesting convective
wind vortices may have aerosolized organisms
from the abundant and tall (>1 m) understory
vegetation unique to this site.
Samples taken during flaming combustion
yielded higher CFUs compared to ambient sam-
ples (P<0.05) but were not significantly differ-
ent from smoldering samples (ambient n=7,
flaming n=28, smoldering n=22; Fig. 3). Total
CFUs, regardless of combustion type, were high-
est in the driest (based on soil type) burned site
(Sandhill Biennial: 171, n=30) and the mesic
Flatwoods Annual (104, n=14) sites, compared
to the Flatwoods Long-Unburned site (69,
n=27). Colony-forming units were significantly
higher in the Sandhill Biennial site compared to
Flatwoods Long-Unburned site (P<0.001). In
the Sandhill Biennial site, CFUs were highest and
most variable when aerosolized by flaming com-
bustion; they were significantly lower in ambient
samples when compared to both types of com-
bustion (P<0.05; Fig. 3). Eight unique fungal
morphotypes isolated from smoke samples and
identified using ITS sequences show a diverse
group of fungi, representing several orders and
ranging from pathogens to non-pathogens with
diverse ecological roles (Table 1).
Smoke samples collected during laboratory
experiments on soils from Idaho bore unique
morphotypes in all treatments: These were high-
est in masticated fuel sites (ambient samples con-
tained 15 total morphotypes with five unique;
burned samples had 11 morphotypes with five
unique). The unique morphotype assemblages in
burned, unburned, and masticated fuel beds sug-
gest that combustion aerosolized microbes that
would not be found in ambient air in the condi-
tions and season in which we sampled.
Fig. 2. Number of colony-forming units averaged for each distance from the flaming front of experimental pre-
scribed fires in all north Florida sites (n=7, 8, 7, 2, 4, 2, 2, 2, 2, 2, from 0.5 to 30 m, respectively). Total number of
samples taken =38.
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KOBZIAR ET AL.
Fig. 3. Bacterial colony-forming units collected during ambient, flaming, and smoldering combustion in
Florida prescribed burns in Sandhill Biennial sites (n=3, 18, 9 for ambient, flaming, and smoldering combustion
types, respectively) and Long-Unburned Flatwoods (n=4, 10, 13 for ambient, flaming, and smoldering combus-
tion types, respectively). The average flame length at the Sandhill Biennial site was 2.3 m with a relative humid-
ity (RH) of 39%, and 3.8 m at the Long-Unburned Flatwoods sites with an RH of 57%. The Flatwoods Annual
site was not included because it lacked sufficient smoldering combustion.
Table 1. Fungal identifications for eight unique morphotypes from prescribed fire smoke samples in a biennially
burned Sandhill pine ecosystem and Long-Unburned Flatwoods pine ecosystems in north Florida.
Best BLAST GenBank no. Identities Ecological function
Trichoderma strigosum EU718074 601/604 Functions in nutrient and mineral uptake, genus important in
agricultural remediation
Dothideomycete sp. 1 EU680480 546/546 Often found as pathogens, endophytes, or epiphytes of living plants.
Saprobes degrade cellulose and other complex carbohydrates in dead
or partially digested plant matter
Dothideomycete sp. 2 HQ631008 584/595 As above
Pestalotiopsis sp. KX757719 546/546 Parasitic fungus that targets ants, also plant pathogens
Epicoccum nigrum MF687186 541/541 Endophyte and plant pathogen, produces anti-fungal and anti-bacterial
compounds
Neopestalotiopsis australis KY398730 547/547 Endophytic fungus capable of breaking down and digesting
polyurethane, can metabolize under anaerobic conditions
Hypocreales sp. KP306921 437/451 Diverse functions by species
Penicillium lagena NR_153223 549/549 Non-pathogenic fungi present around mycorrhizal roots
Notes: Identities are based on best BLAST matches to the NCBI database using the internal transcribed spacer region.
Identities are nucleotide matches between the morphotype and GenBank accession. Ecological function is based on brief
literature review of the best BLAST match.
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KOBZIAR ET AL.
Unlike trends in the Florida samples, Idaho for-
est microbial CFUs in smoke derived from flam-
ing combustion did not differ significantly
between combustion types nor when compared
with the ambient samples. The composition of
morphotypes, however, differed between the
ambient and the combusted samples, with eight
unique morphotypes occurring only in smoke
samples. In addition, the masticated fuel type
aerosolized more CFUs compared to untreated
fuel, but neither had significantly different CFU
numbers from the ambient samples (P<0.01).
While the quality of the fuel source (i.e., masti-
cated or not) of burned organic matter had a sig-
nificant influence on the number of CFUs in
smoke, the quantity burned (% mass loss or total
mass loss [g]) did not correlate with total CFUs
(n=18, r
2
=0.30, P=0.26). Idaho smoke samples
showed higher mean CFUs per sample of fungi
than bacteria (13 and 4, respectively). Tempera-
tures were not measurably correlated with mor-
photype composition or number of CFUs;
however, the maximum temperatures of flaming
vs. smoldering combustion from the fuel bed ther-
mocouples were higher (528°C vs. 395°C, respec-
tively), and temperatures at 60 cm height, where
samples were taken, at times exceeded 60°Cdur-
ing both flaming and smoldering combustion.
DISCUSSION
Pyroaerobiology, a term we introduce in this
study, represents an interdisciplinary and little-
researched line of inquiry, integrating terrestrial
ecology, aerobiology, smoke science, microbiol-
ogy, fire behavior, and fire ecology in a coherent
effort to understand the impacts of aerosolized
live pyrogenic material. Because this line of
inquiry is a new application of aerobiology, our
exploratory study was designed to provide evi-
dence for the potential for smoke to aerosolize
and transport viable microorganisms and to test
some basic hypotheses. Our study was inher-
ently limited by the specific source/fuels sam-
pled, fire behavior characteristics, sampling
duration and methods, and processing methods,
so that specific results should not be extrapolated
to other fires. We used culture-based methods to
capture and grow viable microbes from smoke
using a single medium in each study. It is well
established that the media used will affect the
microbes recovered and that most microbes are
unculturable. We used a general growth medium
able to grow many fungi and bacteria but pre-
sumably only cultured a small portion of the
potentially viable microbes in the smoke. In
addition, different sampling durations would
likely lead to different results (Mainelis and
Tabayoyong 2010).
These initial studies using prescribed burns
and laboratory experiments show that fire aero-
solizes and smoke transports a variety of viable,
culturable microbes, and these assemblages are
dissimilar in composition and abundance from
the communities aerosolized by background
aerosolization drivers (e.g., wind, gravity, spore
propulsion) in paired samples. While our study
addressed forest stand-level transport of organ-
isms, longer-distance transport and its implica-
tions would depend on fire behavior and sources
of the microbes (e.g., the microbial community),
season (which affects sporulation, activity, and
probably survival), environmental conditions
(recent rain events, winds affect background
levels of aerosolized organisms), and the physio-
logical hardiness and growth potential of the
organisms or propagules aerosolized (e.g., fungal
hyphae, spores, and their dispersal mechanisms;
Golan and Pringle 2017).
Two of our hypotheses were supported by our
data, including that abundance (CFUs) varied
with distance from the smoke sources and that
species abundance and composition would differ
by site/site conditions (e.g., mastication). Our
results suggest that the more frequently burned
sites have higher numbers of viable aerosolized
organisms in smoke overall, which may reflect
fire history and associated microbial fire adapta-
tions (Glassman et al. 2016), or differences in the
types of fuels combusted. Grasses and pine litter
drove fire behavior in the frequently burned sites
in contrast to shrubs, grasses, and even small trees
in the Long-Unburned site. Comparisons of the
source microbial populations among sites would
be needed to draw conclusions about whether the
differences in aerosolized communities are a func-
tion of source, fire behavior, or even sampling
protocols (including culture medium used). These
results suggest that future PAB research should
includeanassessmentofsmokesourcecommuni-
ties in order to derive predictions for the potential
impact on atmospheric and downwind terrestrial
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KOBZIAR ET AL.
communities. Other improvements would include
larger sampling sizes, methods parameterized for
expected aerosol densities, and employing
metagenomic analyses to reduce bias against non-
culturable species.
The Idaho mixed-conifer forest samples
demonstrated that masticated fuels produced
more CFUs and unique microbial communities
than non-treated stands, as indicated by distinct
morphotypes, and that both differed from ambi-
ent communities. Mastication changes individual
fuel (soil O horizon) surface areas, fuel packing
ratios, moisture content, and depth (Kreye et al.
2013), hence changing the microhabitat. As such,
fuels treatments may have an impact on micro-
bial communities that then extends to aeroso-
lized and dispersed microbes when and if fire
occurs. Mixing of the soil organic horizons due
to heavy equipment used for fuel reduction treat-
ments may expose organisms whose habitats
would otherwise be unavailable for aerosoliza-
tion via combustion.
Our second hypothesis that species abundance
differed with combustion phase was only sup-
ported in the Florida sites. Flaming combustion
burned smaller diameter woody litter, soil
organic horizons, and surface fuels including
shrubs, grasses, and herbaceous vegetation. In
contrast, smoldering combustion samples were
necessarily obtained from residual fuel, often lar-
ger woody debris after the initial passage of the
fire front. The source of the microbial materials
was therefore different between the two phases
of combustion; we cannot isolate the effect of the
fuel source from that of the phase of combustion.
That the different phases of combustion did not
produce significant results in the laboratory
burns using Idaho mixed-conifer O horizon sam-
ples, along with the lack of a temperature effect,
implies that the energetic differences between the
combustion phases were not significant for the
microbes we were able to culture.
Heating from wildfire and prescribed fire
events have poorly understood physiotemporal
effects on soil microbial populations (Pingree and
Kobziar 2019). In forests devoid of regular fire dis-
turbances, prescribed burning employed as a
restoration effort may negatively impact ecosys-
tem processes. For example, soil heating may be
substantially increased where organic soil hori-
zons are deeper, as in the Long-Unburned Florida
site, leading to prolonged heating and increased
temperatures (Varner et al. 2005), and increased
potential for greater numbers of microbes to be
aerosolized. Indirectly, the exclusion of frequent,
low-severity fires may favor the proliferation of a
soil microbial community with lower temperature
thresholds and disturbance adaptability com-
pared to a frequently burned forest soil commu-
nity (Hart et al. 2005). These altered microbial
communities may also be transported and relo-
cated via aerosolization or particle-mediated
transport in smoke with unknown consequences
for adjacent ecosystems. Efforts to measure and
characterize wildland fire effects on microbial spe-
cies can help to improve management of sensitive
and rare ecosystems where recurrent fire and
adapted microbial species are closely coupled
with ecosystem function (Glassman et al. 2016).
Societal impacts of smoke-transported living
microbes could be both indirect (e.g., ecosystem
services) and direct (human health). Microorgan-
isms provide integral functions in forest ecosys-
tems including decomposition and C cycling,
nutrient cycling, production and consumption of
greenhouse gases, development of soil structure
and maintenance, and effects on other soil biota.
Understanding the fate of specific pathogenic and
beneficial microbes could help direct broader
restoration efforts for the conservation of affected
ecosystems (Klopfenstein et al. 2010). Theoreti-
cally, managers could retard spreading of detri-
mental pathogens and promote dissemination of
beneficial mycorrhizae or nitrogen-fixing bacteria,
or other microbes that would benefitsociety.
The viability and composition of microbes
transported by smoke may have significant impli-
cations for forest health. For example, the fungal
pathogen Cronartium ribicola (J.C. Fisch.), which
causes white pine blister rust and threatens the
endangered whitebark pine (Pinus albicaulis
Engelm.), was spread to new hosts in the western
United States via long-distance dispersal by atmo-
spheric transport (Frank et al. 2008). If this patho-
gen is viable in smoke, disease spread may be
vectored by smoke as well. It is currently
unknown what role smoke and wildfire play in
the transport of forest pathogens. These conse-
quences may in fact be an undesired impact of
management practices. For example, a recom-
mended practice to dispose of biomass infected
with plant pathogens (e.g., Phytophthora ramorum,
❖www.esajournals.org 9November 2018 ❖Volume 9(11) ❖Article e02507
KOBZIAR ET AL.
which causes Sudden Oak Death) is to burn the
material (Agrios 2005). Such attempts at pathogen
control may actually disperse pathogens depend-
ing on environmental conditions (e.g., as has been
shown in a study of wheat field burning; Roux
and O’Brien 2001). With additional knowledge,
managers could plan burns when conditions are
unlikely to transport pathogens present in a stand
to uninfected areas.
Relationships between microbial transport and
smoke composition may thereby help guide
smoke management decisions with significant
consequences (Bowman and Johnston 2005).
Smoke plumes from wildland fires impact natu-
ral resource management decision-making, pub-
lic opinion, and public safety and have catalyzed
immense planning and coordination efforts by
multiple stakeholders (Hardy et al. 2001). Future
investigation into targeted species of special con-
cern to human health impacts is also warranted,
because aerosolized microbes are well known to
aggravate patients with asthma and even cause
illness in otherwise healthy individuals (Griffin
2007).
CONCLUSIONS
The addition of viable organisms to the atmo-
sphere may alter bioaerosol species composition,
activity, and growth, with effects on biogeochem-
ical cycling, atmospheric cloud development,
and weather (Morris et al. 2013, Krumins et al.
2014). Our study provides an initial foundation
for a broad spectrum of future inquiries. Under
what conditions does wildland fire smoke trans-
port and deposit active plant pathogens to adja-
cent or distant locations, and what are the
potential consequences? Can human pathogens
and allergens be transported in smoke to such an
extent that they affect sensitive populations, and
more immediately, wildland fire personnel? With
these and other questions in mind, the establish-
ment of appropriate conceptual and methodolog-
ical guidelines for this line of inquiry is needed.
The approach should be grounded in the estab-
lished principles and theory of aerobiology,
which emphasize the importance of addressing
the complete aerobiological pathway. Ultimately,
pyroaerobiology should take into consideration
the source, characteristics of aerosolization (i.e.,
launch), atmospheric transport, deposition, and
direct and indirect subsequent ecological impacts
(Edmonds 1979).
We suggest the following considerations be
applied to future studies:
1. PAB sampling systems should sample smoke
plumes from multiple fire types at increasing
heights and distances from the source using
mobile platforms in smoke columns; these
would enable smoke communities to be sam-
pled specifically, the influence of ambient air
entrainment to be characterized, and the
degree of transport to be quantified;
2. PAB sampling should incorporate a wide
range of microbiological diversity assessments
including community sequencing, various
media, and baiting. Phylogenetic analyses cou-
pled with physiological/morphological exami-
nations of the species identified may shed
light on the evolution of the pyroaerobiome.
3. PAB sampling strategies should integrate
environmental and aero-habitat conditions
(e.g., PM levels, relative humidity, tempera-
ture, UV exposure) concurrently with sam-
ple extraction in order to characterize and
compare aero-habitats;
4. A variety of sampling techniques (e.g.,
impaction, filtering, impingement) and
durations need to be laboratory- and field-
tested for maximum capture of all viable
organisms to determine appropriate sam-
pling duration and volume for the unique
habitat of wildland fire smoke;
5. To link these effects with predictive models
of smoke transport and effects, an under-
standing of fire behavior, source substrates,
and how they interact to aerosolize microbes
is needed. PAB must include all sub-disci-
plines to address the questions of relevance
to ecological systems and processes, as well
as potential human health impacts.
ACKNOWLEDGMENTS
We wish to acknowledge the personnel of the
University of Florida’s Austin Cary Forest for assis-
tance with prescribed burning operations, and the con-
tributions of University of Idaho’s IFIRE combustion
laboratory personnel. The INBRE program supported
one of the study’s authors through a summer intern-
ship associated with the Environmental Science
❖www.esajournals.org 10 November 2018 ❖Volume 9(11) ❖Article e02507
KOBZIAR ET AL.
program in the University of Idaho’s College of Natu-
ral Resources. This work was supported in part by
grant no. IDAZ-MS-0115/project accession no. 1009933
from the USDA National Institute of Food and Agri-
culture. Thanks to Dr. Jennifer Andrew for initial con-
tributions. Publication of this article was funded by the
University of Idaho Open Access Publishing Fund.
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