RESEARCH ARTICLE
The abundance o heal h-associa ed bac e ia
is al e ed in PAH pollu ed soils—Implica ions
o heal h in u ban a eas?
Ani ud a Pa ajuli
1
, Mi a G o
¨n oos
1
, Sa i Kauppi
2
, Tomasz Płociniczak
3
, Ma ja I. Roslund
1
,
Polina Gali skaya
4
, Olli H. Lai inen
5
, Heikki Hyo
¨ y
5,6
, A i Jumpponen
7
, Rauni S o
¨mme
1
,
Ma in Roman schuk
1,4
, Nan Hui
1
*, Aki Sinkkonen
1,4
*
1Depa men o En i onmen al Sciences, Sec ion o En i onmen al Ecology, Uni e si y o Helsinki, Lah i,
Finland, 2Finnish En i onmen Ins i u e, SYKE, Cen e o Sus ainable Consump ion and P oduc ion,
Con aminan s, Helsinki, Finland, 3Depa men o Mic obiology, Uni e si y o Silesia, Ka owice, Poland,
4Kazan Fede al Uni e si y, Kazan, Russia, 5Depa men o Vi ology, School o Medicine, Uni e si y o
Tampe e, Tampe e, Finland, 6Fimlab Labo a o ies, Pi kanmaa Hospi al Dis ic , Tampe e, Finland,
7Di ision o Biology, Kansas S a e Uni e si y, Manha an, Kansas, Uni ed S a es o Ame ica
*aki.sinkkonen@helsinki. i (AS); [email p o ec ed] (NH)
Abs ac
Long- e m exposu e o polya oma ic hyd oca bons (PAHs) has been connec ed o ch onic
human heal h diso de s. I is also well-known ha i) PAH con amina ion al e s soil bac e ial
communi ies, ii) human mic obiome is associa ed wi h en i onmen al mic obiome, and iii)
al e a ion in he abundance o membe s in se e al bac e ial phyla is associa ed wi h ad e se
o bene icial human heal h e ec s. We hypo hesized ha soil pollu ion by PAHs al e ed soil
bac e ial communi ies ha had known associa ions wi h human heal h. The a ionale behind
ou s udy was o inc ease unde s anding and po en ially acili a e econside ing ac o s ha
lead o heal h diso de s in a eas cha ac e ized by PAH con amina ion. La ge con aine s
illed wi h ei he sp uce o es soil, pine o es soil, pea , o glacial sand we e le o incuba e
o con amina ed wi h c eoso e. Biological deg ada ion o PAHs was moni o ed using GC-
MS, and he bac e ial communi y composi ion was analyzed using 454 py osequencing.
P o eobac e ia had highe and Ac inobac e ia and Bac e oide es had lowe ela i e abun-
dance in c eoso e con amina ed soils han in non-con amina ed soils. Ea lie s udies ha e
demons a ed ha an inc ease in he abundance o P o eobac e ia and dec eased abun-
dance o he phyla Ac inobac e ia and Bac e oide es a e pa icula ly associa ed wi h
ad e se heal h ou comes and immunological diso de s. The e o e, we p opose ha pollu-
ion-induced shi s in na u al soil bac e ial communi y, like in PAH-pollu ed a eas, can con-
ibu e o he p e alence o ch onic diseases. We encou age s udies ha simul aneously
add ess he classic “ad e se oxin e ec ” pa adigm and ou no el “al e ed en i onmen al
mic obiome” hypo hesis.
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187852 No embe 16, 2017 1 / 18
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OPEN ACCESS
Ci a ion: Pa ajuli A, G o¨n oos M, Kauppi S,
Płociniczak T, Roslund MI, Gali skaya P, e al.
(2017) The abundance o heal h-associa ed
bac e ia is al e ed in PAH pollu ed soils—
Implica ions o heal h in u ban a eas? PLoS ONE
12(11): e0187852. h ps://doi.o g/10.1371/jou nal.
pone.0187852
Edi o : Ma ie-Joelle Vi olle, Uni e si e Pa is-Sud,
FRANCE
Recei ed: Augus 12, 2017
Accep ed: Oc obe 29, 2017
Published: No embe 16, 2017
Copy igh : ©2017 Pa ajuli e al. This is an open
access a icle dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License, which
pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal
au ho and sou ce a e c edi ed.
Da a A ailabili y S a emen : The bac e ial
sequence da a a e a ailable in he Sequence Read
A chi e a NCBI unde accession numbe
SRR5229978. All o he ele an da a a e wi hin he
pape and i s Suppo ing In o ma ion iles.
Funding: The esea ch was unded by Finnish
Funding Agency o Technology and
Inno a ionTekes (decision numbe 40333/14),
h ps://www. ekes. i/en/. The unde s had no ole in
In oduc ion
Pollu an s, such as polya oma ic hyd oca bons (PAHs), can lead o shi s in mic obial commu-
ni ies [1–4]. PAH deg ada ion ypically las s o decades in pollu ed en i onmen s including
soil, wa e , ai , and sedimen s [5–7]. Di ec PAH oxici y, abio ic ans o ma ion, and mic o-
bial deg ada ion a o some mic obial axa, whe eas o he s become less p e alen in PAH-pol-
lu ed soils [8]. The bac e ial axa ha h i e unde PAH exposu e a y depending on he ypes
o soil and en i onmen al condi ions [9–12]. A sea ch o gene al ends in mic obial shi s in
PAH-pollu ed soils necessi a es concu en s udies o mic obial communi ies in di e en soil
ypes.
Exposu e o PAHs is associa ed wi h se e e human heal h de ici s and ecological impac s,
such as p ocess o PAH-DNA adduc o ma ion and ca cinogenesis [13], and he e o e hey
ha e been classi ied as p io i y en i onmen al pollu an s by he Uni ed S a es En i onmen al
P o ec ion Agency (US-EPA) and he En i onmen al Eu opean Agency (EEA) [14,15]. The
en i onmen al concen a ion o PAHs and he connec ion be ween di ec PAH exposu e and
human heal h ha e been well in es iga ed [16–23]. Almos 90% o he PAHs eleased in o he
a mosphe e accumula e in su ace soil laye s [14] whe e hey a e p ima ily deg aded by soil
bac e ia o bind o soil pa icles [22]. The heal h ou comes o PAH-exposu e ha e been a ib-
u ed o di ec and indi ec oxin e ec s on humans [23,24].
Se e al s udies ha e in es iga ed he ela ionship be ween human heal h and he mos
common bac e ial axa ound in he human mic obiome. A ich o dominan P o eobac e ial
and diminished Bac e oide es communi ies in he human gu can con ibu e o a isk o
immune sys em diso de s including ch onic obs uc i e pulmona y disease and as hma [25],
and such e ec s can be ansgene a ional. Fo example women whose in an s de eloped IgE-
associa ed eczema had lowe di e si y o Bac e oide es in hei gu du ing he p egnancy
[26]. Al e a ion in he P o eobac e ial abundance in a ious egions o human body is associ-
a ed wi h se e al heal h diso de s, bu i s e ec s a e wo old. On he posi i e side, di e se
P o eobac e ial communi y on skin is ela ed o a educed isk o a opy [27,28]. On he neg-
a i e side, o e g ow h o P o eobac e ia is connec ed o as hma and ch onic obs uc i e pul-
mona y disease [29]. Wi hin P o eobac e ia, Be ap o eobac e ia a e en iched in he gu
mic obiome o indi iduals wi h ype 2 diabe es [30]. Impo an ly, ecen indings indica e
ha P o eobac e ial communi y o indi idual’s skin depends on land use in hei li ing en i-
onmen and ha he en i onmen unes esponse o alle gens [27–31]. Soil is he majo es-
e oi o Ac inobac e ia, Bac e oide es, and P o eobac e ia [32,33], and hus he li ing
en i onmen likely unes indi idual’s esponses o alle gens, suga me abolism, and heal h
diso de s. Fo hese easons, i is possible ha ad e se heal h e ec s connec ed o li ing
in con amina ed en i onmen may pa ly be a ibu able o undamen al changes in he
en i onmen al mic obiome people a e exposed o, in addi ion o he di ec physiological
esponses caused by he oxins. Su p isingly, o he bes o ou knowledge, no s udies ha e
ocused on he po en ial connec ion be ween PAH pollu ion and soil communi y changes in
he abundance o heal h-associa ed bac e ia.
In he p esen s udy, we sampled ou di e en su ace soils, con amina ed hem wi h
PAHs and ollowed popula ion shi s in bac e ial communi y in con amina ed and non-con-
amina ed con aine s unde con olled condi ions. Finally, based on p e ious empi ical e i-
dence as well as ou new indings and no el ideas, we p opose a new “al e ed en i onmen al
mic obiome” hypo hesis ha should be in es iga ed in pa allel wi h he adi ional “di ec
oxin e ec ” pa adigm as a po en ial explana ion o he complex ela ionship be ween human
heal h and en i onmen al pollu ion.
PAH pollu ion al e s he abundance o heal h-associa ed bac e ia
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187852 No embe 16, 2017 2 / 18
s udy design, da a collec ion and analysis, decision
o publish, o p epa a ion o he manusc ip .
Compe ing in e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
Ma e ials and me hods
PAH sou ce
We selec ed c eoso e as he con aminan because i is among he mos widely used wood p e-
se a i es wi h a his o y o mo e han a cen u y in wood imp egna ion indus y [5]. C eoso e
consis s o 85% PAHs which a e chemical compounds consis ing o a leas wo a oma ic ings
used oge he [34,35]. The emaining is less han 10% phenolic compounds and 5–10% he -
e ocyclic a oma ic compounds consis ing o oxygen, sulphu , and ni ogen. The e o e, c eo-
so e is a ele an compound o es en i onmen al changes caused by PAH-pollu ion since i
consis s mainly o PAHs and is a common p oblem in u ban a eas wi h a his o y o sawmills.
Soil collec ion
Su ace soil was collec ed a ou sepa a e si es in Finland. The i s si e (Pea in Table 1) was a
Haapasuo pea p oduc ion a ea in Lei onma¨ki, Finland [36] (61˚54’N 26˚4’E). The cu en
su ace laye was o med hund eds o yea s ago, and i ep esen s he ansi ion laye be ween
mine o ophic Ca ex and omb o ophic Sphagnum domina ed pea bogs. The o iginal bog
was d ained, and he wa e able has been kep 30–40 cm below he pea su ace o mo e han
h ee decades. The second si e, e e ed o as pine o es soil, is a bo eal pine (Pinus syl es is
L.) o es in Hollola, loca ed in sou he n Finland (61˚0’N 25˚29’E) mo aine idge ( he undis-
u bed ecosys em has been desc ibed be o e [36]). The si e is cha ac e ized by a hin o ganic
soil laye on op o mine al soil [37]. The hi d si e (Sp uce o es soil in Table 1) is a sp uce
o es in Vie uma¨ki, sou he n Finland (60˚52’7N 25˚41’E). Sp uce (Picea abies) is he domi-
nan ee species, and he ield laye consis ed o Sphagnum and Pleu ozium sch ebe i mosses
as well as dwa sh ubs, mainly Vaccinium species. The ou h si e (sand) was nex o (dis ance
50 m) he si e 3 (Sp uce o es soil), bu i consis ed o ba e glacial sand and sca e ed ude al
he bs as o iginal su ace soil was emo ed yea s ea lie when he si e became a s o age ield o
lumbe .
A all ou si es, soil was collec ed om h ee sepa a e (dis ance >5m) ca. 1 m
2
plo s as
desc ibed in ea lie wo k [38,39]. In sho , a each plo , li e ege a ion and plan deb is we e
emo ed and 15–20 L o su ace soil (dep h 2–15 cm) was collec ed and mixed ho oughly.
The soil was hen andomly di ided in o wo 10 L polye hylene con aine s, and he p ocedu e
was epea ed a each 1 m
2
plo . Filled con aine s we e co e ed wi h polye hylene lids wi h wo
5 mm diame e holes sealed loosely wi h co on wool o acili a e passi e ae a ion. Soil weigh
in con aine s a ied be ween 3–12 kg depending on soil ype, he hea ies being he mine al
soil- ypes.
Basic physicochemical analyses. Mois u e con en was measu ed by d ying samples in an
o en (+ 90˚C) o 24 hou s. O ganic ma e (OM) was de e mined as loss o igni ion a 550˚C
o 4h. Nu ien con en s o he di e en soil ypes we e de e mined wi h QuikChem 8000
low injec ion analysis sys em (LACHAT Ins umen s Inc., USA) [38]. To measu e he pH,
10g ( esh weigh ) o each soil ype was mixed in 50 mL o 1M CaCl
2
, shaken o 5 min and
allowed o se le o 2–24 hou s [38].
Table 1. The pH and o ganic ma e con en o each soil ype.
Pea Pine o es Sp uce o es Sand
pH 3.52 ±0.03
b
3.6 ±0.26
b
3.25 ±0.04
c
4.87 ±0.05
a
O ganic ma e (%) 97.75±0.41
a
63.42 ±8.15
b
35.19 ±12.41
c
1.21 ±0.17
d
Values a e mean ±1 SD. Le e s in he supe sc ip deno e s a is ical di e ences in Tukey’s es s among di e en soil ypes
h ps://doi.o g/10.1371/jou nal.pone.0187852. 001
PAH pollu ion al e s he abundance o heal h-associa ed bac e ia
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187852 No embe 16, 2017 3 / 18
PAH deg ada ion expe imen . Fo each soil ype, h ee pai s o 10L polye hene con ain-
e s we e included. In he beginning o he expe imen , soil was mixed ho oughly wi hin pai s
in la ge polye hene con aine s and he ea e di ided again in o wo 10L polye hene con ain-
e s. O each pai , one con aine was andomly chosen o be spiked wi h c eoso e, whe eas
ano he ecei ed 100g glacial sand (Lohja Rudus Oy, Lah i, Finland). Ini ial (day 0) samples o
basic chemical analyses we e acqui ed immedia ely a e spiking. C eoso e spiking was done as
desc ibed ea lie [38]. In sho , c eoso e (6–24 g) was mixed ho oughly wi h 100g glacial sand,
and he mix u e was inco po a ed in o soil in he con aine s selec ed o con amina ion. This
esul ed in he concen a ion o ~ 1% PAHs simila o hose in c eoso e con amina ed si es.
A e c eoso e addi ion, con aine s we e le o incuba e o ou weeks (28 days) a 16 ±1˚C
and sampled a days 28, 91, and 189 o chemical and bac e ial communi y analyses. Each sam-
ple ( o al weigh 10 g) consis ed o i e subsamples om ou dep hs (2, 5, 10, and 15 cm) col-
lec ed om each con aine . Communi y sequencing analyses we e pe o med on day 0 and
when he o al PAH concen a ion had dec eased by mo e han 20% om week ou (day 28)
alue. The e o e, week 13 (day 91) samples we e analyzed in mos cases. Howe e , week 27
(day 189) samples we e u ilized o each o con ol and con amina ed mine al soil con aine s
because hei PAH concen a ions a week 13 we e mo e han 95% o week 4 concen a ions.
To a oid he ans e o mic oo ganisms be ween con aine s, he sampling equipmen was
ca e ully lame s e ilized wi h 70% e hanol igh be o e aking each sample.
PAH analyses
PAH concen a ions we e de e mined using oluene ex ac ion as desc ibed in No dic Guide-
lines o Chemical Analysis o Con amina ed Soil Samples [40] and analyzed as desc ibed ea -
lie [41] wi h he excep ion ha aqueous sodium py ophospha e decahyd a e solu ion (0.05
M) ins ead o hexane was used. The ex ac s we e analyzed wi h Shimadzu GC–MS-QP5000
sys em equipped wi h AOC-20i au oinjec o and 30-m ZB-5MS column (0.25 mm i.d.,
0.25 μm ilm hickness). The o en p og am was se as ollows: 80˚C o 1 min, 10˚C/min o
250˚C, 7˚C/min o 280˚C, 20˚C/min o 320˚C wi h a hold o 10 min o a o al un ime o
34.29 min. PAH-mix 9 (16 PAHs included) was used o GC-MS and PAH-Mix 31 o i e deu-
e a ed PAHs o soil samples (D . Eh ens o e , GmbH Ge many) as s anda ds and An h a-
cene-D10 (D . Eh ens o e , GmbH Ge many) as a eco e y s anda d.
DNA ex ac ion, ampli ica ion, and sequencing
To al DNA was ex ac ed om each soil sample using Fas DNA SPIN Ki o Soil (MP Biome-
dicals, Illki ch, F ance) acco ding o he manu ac u e ’s s anda d p o ocol. The highly hype -
a iable egion V3 o bac e ial 16S RNA gene was ampli ied using p ime cons uc s ha
inco po a ed he py osequencing adap e s (A), sample-speci ic DNA ags, and MF341 5’
CTA CGG GAG GCA GCA G 3’ o R518 50ATT ACC GCG GCT GCT GG 30[42]. The
PCR we e conduc ed unde he ollowing condi ions: 200 nM o each o wa d and e e se
p ime s, 5 ng empla e DNA, 200 μM o each dNTP, 2.5 mM MgCl
2
, 1 U GoTaq Ho S a
DNA polyme ase (P omega, Madison, WI), and 2.5 μl PCR bu e . The PCR cycle pa ame e s
consis ed o an ini ial dena u a ion a 94˚C o 3 min, hen 25 cycles o dena u a ion a 94˚C
o 1 min, annealing a 54˚C o 1 min, and ex ension a 72˚C o 2 min, ollowed by a inal
ex ension s ep a 72˚C o 10 min. The PCR p oduc s we e pu i ied using Ampu e XP Mag-
ne ic Clean-up (Agencou Bioscience Co po a ion, Be e ly, MA, USA), and quan i ied using
Nanod op (The mo Scien i ic, Rock o d, IL, USA) and Bioanalyze 2100 wi h DNA 1000
chips (Agilen Technologies Inc., San a Cla a, CA, USA). The sequencing was pe o med
using he 454 GS FLX p o ocol and he GS FLX Ti anium Rapid Lib a y P epa a ion Ki (454
PAH pollu ion al e s he abundance o heal h-associa ed bac e ia
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187852 No embe 16, 2017 4 / 18
Li e Sciences, Roche Diagnos ics, CT, USA). The bac e ial sequence da a a e a ailable in he
Sequence Read A chi e a NCBI unde accession numbe SRR5229978.
Sequence analysis. The sequence da a we e analyzed using MOTHUR ( 1.35.0, 64-bi o
Linux) acco ding o he s anda d ope a ing p o ocol as desc ibed ea lie [43]. B ie ly, he aw
sequence da a we e quali y con olled, and eads wi h ambiguous bases o homopolyme s lon-
ge han 8 bp (722 sequences) we e emo ed. The UCHIME algo i hm [44] iden i ied 943
sequences as chime ic, and hese we e subsequen ly omi ed. Sequences we e assigned in o
Ope a ional Taxonomic Uni s (OTU) a 97% simila i y and OTUs assigned o axon a ini ies
using Naï e Bayesian Classi ie [45] agains he RDP aining se ( e sion 10). Ra e OTUs
occu ing h ee o ewe imes ac oss all samples we e omi ed o a oid p oblems caused by
unce ain y in o igin.
S a is ical analyses. The ela i e abundance was calcula ed as he numbe o sequences in
a axon di ided by o al numbe o sequences in a sample. To compa e con amina ed and p is-
ine ea men s, - es on ela i e abundances o bac e ial phyla was conduc ed. Signi ican
esul s a e indica ed by as e isks (p<0.05). False De ec ion Ra e (FDR) was used o he p- al-
ues co ec ion in - es s. The di e ence in he pH and he o ganic ma e con en ac oss he
ou soil ypes was calcula ed using ANOVA in JMP ( .11.0 64-bi ; SAS Ins i u e, Ca y, No h
Ca olina). To isualize bac e ial communi y composi ions o whole bac e ial communi ies as
well as he majo bac e ial phyla, non-me ic mul idimensional scaling (NMDS) analyses was
pe o med based on ela i e abundance o OTUs using egan package in R ( 3.2.2, R De elop-
men Co e Team 2015). The B ay-Cu is dis ance was chosen in he NMDS analysis since
he e we e null alues be ween samples in he da a [46]. We pe o med he NMDS analyses a
99, 97, 95, 93, 91% OTU simila i y le els bu only p esen he da a a 97% because OTU h esh-
old had no impac on he o e all conclusions.
Di e ences in he bac e ial communi y composi ion be ween c eoso e con amina ed and
con ol g oups we e es ed using pe mu a i e analysis o a ia ion (PERMANOVA, unc ion
adonis in R-package egan and B ay-Cu is me ic). The di e ence in he ela i e abundances
o bac e ial phyla be ween c eoso e con amina ed and con ol soils was calcula ed using he T-
es in JMP.
Resul s
Soil chemical cha ac e is ics
The ou soil ypes used in his s udy di e ed in pH (F = 164.4, d = 3, 20, p<0.001) and
o ganic ma e con en (F = 183.2, d = 3, 20, p<0.001). Sandy soil had he highes and sp uce
o es soil he lowes pH (Table 1). O ganic ma e con en dec eased in he ollowing o de :
pea >pine o es soil >sp uce o es soil >sandy soil (Table 1). Thus, he ou soil ypes ep-
esen a ange o habi a s wi h highly a iable bac e ial communi ies.
Cha ac e is ics o bac e ial ope a ional uni s (OTUs) and ela i e
abundances o bac e ial phyla
The py osequenced bac e ial 16S RNA gene agmen da ase s we e analysed om 24 soil
samples aken on days 0, 91, and 189. Day 0 samples o mic obiological analyses we e aken
be o e con amina ion. Then he ime poin a which 20% o mo e o PAH concen a ion on
day 28 had disappea ed was de e mined. In sand, 20% deg ada ion was eached on day 189
and in o he soils on day 91 (S1 Table). Day 28 was used as a e e ence day because he educ-
ion in PAH concen a ion un il day 28 is ypically caused by he e apo a ion o naph halene
and o he low molecula weigh compounds [47,48].
PAH pollu ion al e s he abundance o heal h-associa ed bac e ia
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187852 No embe 16, 2017 5 / 18

In o al 3626 OTUs we e ob ained, which ep esen ed mo e han 20 bac e ial phyla om
di e en soil ypes wi h and wi hou PAH con amina ion. Bac e ial OTUs ep esen ing P o-
eobac e ia we e he mos abundan g oup accoun ing o 50.9%, 31.5%, and 35.8% o he o al
sequences in con amina ed, p is ine, and day 0 samples, espec i ely (Table 2). Likewise, P o-
eobac e ia was he p edominan phylum when compa ed ac oss he di e en soil ypes wi h
46.20% (pea soil), 37.7% (pine o es soil), 36.0% (sand), and 37.8% (sp uce o es soil) o all
sequences. O he dominan phyla in all soils we e Acidobac e ia, Ac inobac e ia, Ve ucomi-
c obia, and Bac e oide es (Tables 2and 3).
The ela i e abundances o di e en phyla be ween con amina ed and p is ine ea men s
we e compa ed. P o eobac e ia (d = 1, p= 0.046) we e mo e abundan in con amina ed soils,
whe eas he ela i e abundances o Ac inobac e ia (d = 1, p= 0.043) and Bac e oide es (d = 1,
p= 0.005) declined in con amina ed soils (Table 2). In addi ion, i was obse ed ha he ela-
i e abundances o Pa cubac e ia and Candidus Saccha ibac e ia we e lowe in he con ami-
na ed soils, al hough he ela i e abundances o hese wo we e e y low (Table 2). As axa
belonging o P o eobac e ia ep esen ed mo e han 35% o mic obial communi y in all soil
ypes, we compa ed he ela i e abundances o he classes unde he phylum P o eobac e ia
Table 2. Rela i e abundances o bac e ia phyla (classes) on day 0 and when 20% o PAH con amina ion had disappea ed, i.e. days 91 and 189 in
sand and o he soils (bo h p is ine and con amina ed), espec i ely.
Week 0 No c eoso e C eoso e added
Phylum Mean 1SD Mean 1SD Mean 1SD
Acidobac e ia 0.240 0.110 0.269 0.089 0.268 0.170
Ac inobac e ia 0.150 0.060 0.092*0.041 0.049*0.016
A ma imonade es 0.001 0.001 0.002 0.002 0.000 0.000
Bac e oide es 0.048 0.026 0.069*0.034 0.010*0.013
Candida e_di ision_WPS-1 0.000 0.000 0.001 0.002 0.000 0.000
Candida us_Saccha ibac e ia 0.014 0.010 0.013*0.006 0.002*0.003
Chlamydiae 0.001 0.001 0.004 0.007 0.001 0.002
Chlo o lexi 0.002 0.003 0.002 0.004 0.001 0.001
Fib obac e es 0.001 0.003 0.002 0.003 0.000 0.000
Fi micu es 0.008 0.010 0.015 0.015 0.031 0.062
Gemma imonade es 0.030 0.060 0.007 0.013 0.001 0.003
Ni ospi ae 0.001 0.004 0.002 0.003 0.001 0.002
Pa cubac e ia 0.001 0.001 0.003*0.003 0.001*0.000
Planc omyce es 0.004 0.005 0.005 0.005 0.004 0.006
P o eobac e ia 0.358 0.077 0.315*0.071 0.509*0.166
Alphap o eobac e ia 0.100 0.026 0.120 0.033 0.127 0.075
Be ap o eobac e ia 0.165 0.035 0.082*0.032 0.234*0.057
Gammap o eobac e ia 0.043 0.021 0.054 0.019 0.076 0.018
Del ap o eobac e ia 0.047 0.015 0.057 0.041 0.061 0.022
Unclassi ied p o eobac e ia 0.004 0.002 0.003 0.001 0.010 0.003
Spi ochae es 0.003 0.008 0.009 0.022 0.000 0.001
Unclassi ied bac e ia 0.081 0.070 0.130 0.107 0.049 0.061
Ve ucomic obia 0.040 0.030 0.060 0.035 0.074 0.054
To compa e con amina ed and p is ine ea men s, we conduc ed - es be ween c eoso e added and no c eoso e.
Signi ican di e ences indica ed by as e isk (p<0.05).
P- alues co ec ed by FDR.
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and ound ha he ela i e abundance o Be ap o eobac e ia was highe (d = 1, p= 0.013) in
con amina ed soils.
Bac e ial communi y composi ion
The o al bac e ial communi y composi ion did no di e be ween c eoso e con amina ed and
p is ine soils a he OTU le el as e ealed by he nonme ic mul idimensional scaling (NMDS)
o dina ion (Fig 1a) and pe mu a ional mul i a ia e analysis o a iance PERMANOVA
(Table 3). The NMDS a he genus le el, howe e , e ealed dis inc ly di e en bac e ial com-
muni ies in he c eoso e con amina ed and p is ine soils (Fig 1b). PERMANOVA indica ed
ha con amina ion and soil ype had dis inc main e ec s on mic obial communi y a he
genus le el and wi hou a s ong in e ac ion e ec (Table 3). These indings unde line ha
Table 3. The e ec s o soil ype and c eoso e con amina ion on bac e ial communi y composi ion a genus and OTU le els in PERMANOVA.
Genus le el D Sum o squa es Mean squa e F Model R
2
P alue
Soil ype (1) 3 0.09 0.03 4.81 0.38 0.001
C eoso e addi ion (2) 1 0.06 0.06 9.08 0.24 0.001
1:2 3 0.04 0.01 2.16 0.17 0.014
Residuals 8 0.05 0.01 0.21
To al 15 0.24 1.00
OTU le el
Soil ype (1) 3 2.02 0.67 2.74 0.37 0.001
C eoso e addi ion (2) 1 0.35 0.35 1.41 0.06 0.078
1:2 3 1.07 0.36 1.45 0.20 0.028
Residuals 8 1.98 0.25 0.36
To al 15 5.41 1.00
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Fig 1. NMDS o dina ion (B ay-Cu is dis ance) o soil bac e ial communi ies in he c eoso e con amina ed and con ol soil samples. (a) The
bac e ial communi y composi ion in he wo ea men g oups o e lap a he OTU le el. (b) The communi ies a e dis inc i ely di e en a he genus
le el. Fo pea (Pea in he igu e), pine o es soil (Pine.F) and sp uce o es soil (Sp uce.F), samples aken on week 31 (day91) we e u ilized, whe eas
week 27 (day 189) samples we e used in he case o mine al soil (Sand).
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c eoso e con amina ion changed mic obial communi y a he genus le el ega dless o he soil
ype.
Communi y composi ion analysis o majo bac e ial phyla a OTU le el
The majo bac e ial phyla de ec ed om he soil samples we e examined o hei di e ences
in communi y composi ion a he OTU le el be ween he c eoso e con amina ed and p is ine
soil samples. Since OTUs ep esen ing Bac e oide es we e no de ec ed in wo o he c eoso e
con amina ed samples, communi y composi ion analysis was no pe o med o Bac e oide es.
Consequen ly, he analyses we e pe o med only o P o eobac e ia and Ac inobac e ia.
P o eobac e ia. P o eobac e ial communi ies we e dis inc ly di e en in con amina ed
han in con ol soils in NMDS (Fig 2). PERMANOVA e ealed ha he soil ype and c eoso e
con amina ion had s ong indi idual e ec s on he P o eobac e ial communi y composi ion,
while he signi icance o he in e ac ion e m was en imes lowe (Table 4).
Fig 2. NMDS o dina ion o P o eobac e ial OTUs in c eoso e con amina ed and p is ine (con ol) soil samples. The communi y
composi ion is no iceably di e en in he wo ea men g oups. The o dina ion is based on B ay-Cu is dissimila i y me ic.
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Ac inobac e ia. Al hough he ela i e abundance o Ac inobac e ia dec eased in c eoso e
con amina ed soil samples, i was obse ed ha i s communi y composi ion did no di e
be ween he c eoso e con amina ed and p is ine soil samples (Fig 3). Ins ead, he soil ype
a ec ed he Ac inobac e ial communi y composi ion ound in ha soil ype (Table 5).
Tempo al a ia ion. The o al bac e ial, P o eobac e ial and Ac inobac e ial communi ies
sampled on day 0 we e compa ed wi h hose sampled la e . Day 0 samples we e compa ed
wi h bo h c eoso e con amina ed and p is ine samples o de e mine he empo al communi y
dynamics. Only P o eobac e ial communi y on day 0 dis inc ly di e ed om he communi ies
sampled a he la e ime poin s (Fig 4a). Nei he o al bac e ial communi y no Ac inobac e -
ial communi y changed du ing he expe imen signi ican ly (Fig 4b and 4c).
Discussion
In he p esen s udy, we compa ed he esponses o bac e ial communi y o c eoso e con ami-
na ion in ou di e en ypes o soil. Ou da a indica es shi s in bac e ial communi y composi-
ion and al e a ions in he ela i e abundances o h ee majo bac e ial phyla as a esponse o
c eoso e con amina ion. The pollu ion-induced shi was a ibu able o an inc ease in he ela-
i e abundance o P o eobac e ia, pa icula ly Be ap o eobac e ia, and a dec ease in he ela-
i e abundance o Ac inobac e ia and Bac e oide es. These phyla a e he majo bac e ial phyla
p esen in soil as well as on human skin and o he biological samples [49–51]. Because hey
ha e been ecen ly linked o human immune unc ion and ch onic diseases [25–27,52], a
majo change in hei communi y composi ion in he en i onmen can po en ially a ec
human exposu e o hem and, e en ually, lead o modula ed human immune sys em unc ions
[27,31]. As a as we know, his idea ha we call he al e ed en i onmen al mic obiome
hypo hesis has ne e been p oposed no es ed. The i s s ep o e alua e he hypo hesis is
he e o e o pe o m a li e a u e e iew on he po en ial connec ions be ween heal h-associ-
a ed and pollu ion-induced changes.
While se e al s udies ha e in es iga ed and poin ed ou he al e a ion o he en i e bac e ial
communi y as a esul o PAH pollu ion, only a hand ul o s udies ha e ocused on pollu ion-
induced changes in P o eobac e ial, Bac e oide es, and Ac inobac e ial communi ies in su ace
soil [1,12,53–58]. Those s udies ha e e ealed ha P o eobac e ia we e mo e abundan in
PAH pollu ed soils compa ed o p is ine soils. Phylum Bac e oide es, in con as , ei he
dec eased in abundance [55] o was no s udied. The ela i e abundance o Ac inobac e ia was
usually lowe in con amina ed soil han in non-con amina ed soil [55], bu i s abundance is
also dependen on he PAHs in ol ed[57] (Table 6). The inc ease in he abundance o P o eo-
bac e ia and he dec ease in Ac inobac e ia and Bac e oide es a e consis en wi h ou indings.
As he s udies lis ed in Table 6 co e a wide ange o geog aphic a eas and land use his o ies, i
seems plausible o assume ha shi s in he abundance o P o eobac e ia, Ac inobac e ia, and
Bac e oide es a e a ypical consequence o PAH pollu ion in soil.
Table 4. The e ec s o soil ype and c eoso e con amina ion on P o eobac e ial communi y composi ion a he OTU le el in PERMANOVA.
D Sum o squa es Mean squa e F Model R
2
P alue
Soil ype (1) 3 1.50 0.50 2.13 0.28 0.001
C eoso e addi ion (2) 1 0.82 0.82 3.50 0.15 0.001
1:2 3 1.06 0.35 1.51 0.20 0.010
Residuals 8 1.87 0.23 0.36
To al 15 5.26 1.00
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