In e na ional Biode e io a ion & Biodeg ada ion 164 (2021) 105295
A ailable online 9 July 2021
0964-8305/© 2021 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license
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A labo a o y app oach on he combined e ec s o g ani e bio ecep i i y
and pa ame e s modi ied by clima e change on he de elopmen o
subae ial bio ilms on cul u al he i age
Elsa Fuen es
*
, Bea iz P ie o
Depa amen o Eda oloxía e Química ag ícola, Facul ad de Fa macia, Uni e sidade San iago de Compos ela, 15782, San iago de Compos ela, Spain
ARTICLE INFO
Keywo ds:
Clima e change
G ani e bio ecep i i y
Wa e a ailabili y
Tempe a u e
SABs
ABSTRACT
Conse a ion o cul u al he i age buildings and monumen s may be nega i ely a ec ed by he impac o clima e
change on subs a es and colonizing mic oo ganisms. In his s udy, ou ypes o comme cial g ani e, in which
he bio ecep i i y anged om e y low o e y high, we e inocula ed wi h a mul ispecies mic obial cul u e and
exposed o di e en empe a u es (18 and 24 ◦C) and le els o wa e a ailabili y (1 day, 3 days and 7 days o
wa e a ailabili y/week) o simula e di e en clima ic condi ions. The e ec s on bio ilm o ma ion o he in-
e ac ions be ween he subs a e bio ecep i i y and he en i onmen al pa ame e s we e in es iga ed. Bio ilm
g ow h and pho osyn he ic e iciency we e moni o ed o e 42 days by pulse-ampli ude modula ed (PAM) lu-
o ome y and spec opho ome ic colou measu emen . The p ima y bio ecep i i y o he g ani e was a de e -
mining ac o in he o ganisms’ capaci y o a ach o he subs a e and in e ac ed h ough he expe imen wi h
he clima ic condi ions by modi ying he de elopmen o he mic oo ganisms. Wa e a ailabili y was a limi ing
ac o o he colonizing mic oo ganisms, in e ms o bo h g ow h and pho osyn he ic e iciency. A he highe
empe a u e, me abolic eac ions we e enhanced unde wa e es ic ion (bu no unde maximum wa e
a ailabili y) and he mic obial ecology was al e ed, leading o a highe p opo ion o cyanobac e ia ela i e o
algae.
1. In oduc ion
Cul u al he i age is a legacy le by ou ances o s, wi h which we li e
a p esen and which we mus pass on o u u e gene a ions. Once los , i
is un eco e able, and conse a ion and p o ec ion o cul u al he i age
buildings and monumen s agains ha m ul agen s, such as apid en i-
onmen al changes, is he e o e o pa icula impo ance (UNESCO
Wo ld He i age Cen e, 2007). Since he Indus ial Re olu ion in he
18 h cen u y, he le els o g eenhouse gases o an h opogenic o igin
ha e been inc easing con inuously, leading o an inc ease in he a e age
empe a u e o be ween 0.6 and 4 ◦C. Global wa ming p oduces changes
in he clima e, wi h luc ua ions occu ing much as e han a any o he
ime in he Ea h’s his o y. One o he mos commonly s udied conse-
quences is he change in p ecipi a ion pa e ns and he dis ibu ion o
d ough s.
In his con ex , he e is g owing conce n abou he ex en o which
hese changes will a ec cul u al he i age, as he cons uc ion ma e ials
used a e usually agile o wea he ed as a consequence o hei long
exis ence. These ma e ials a e no designed o wi hs and he “new” cli-
ma ic condi ions, and despi e ha ing been exposed o en i onmen al
luc ua ions o many yea s, hey ha e ne e expe ienced he cu en
a e o change (Haugen e al., 2018). In ecen yea s, his ma e has
begun o be gi en special a en ion, wi h nume ous s udies p o iding
new insigh s in o he ela ionship be ween en i onmen al changes and
cul u al he i age (Sabbioni e al., 2009; B imblecombe e al., 2011;
Fa o i´
c and Seekamp, 2017; Sesana e al., 2020). In o ma ion abou he
e ec s can lead o an o ien ed p e en i e conse a ion, ocusing e-
sou ces on he he i age monumen s mos suscep ible o de e io a ion.
S one is one o he ma e ials mos commonly and widely used
h oughou his o y, and despi e i s s eng h and du abili y, i is a ec ed
by de e io a ion p ocesses. Clima e is one o he main causes o deg a-
da ion, due o bo h he di ec physical-chemical e ec s on he s one and
o he impac i has on he ac i i y o mic oo ganisms ha inhabi he
s one su ace. The complex ecology o s one-buil monumen s is s ongly
a ec ed by en i onmen al changes, as he ecological niche is exposed o
en i onmen s esso s. Bio ilms, which a e communi ies o algae, ungi,
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (E. Fuen es).
Con en s lis s a ailable a ScienceDi ec
In e na ional Biode e io a ion & Biodeg ada ion
jou nal homepage: www.else ie .com/loca e/ibiod
h ps://doi.o g/10.1016/j.ibiod.2021.105295
Recei ed 11 Feb ua y 2021; Recei ed in e ised o m 20 June 2021; Accep ed 5 July 2021
In e na ional Biode e io a ion & Biodeg ada ion 164 (2021) 105295
2
cyanobac e ia and bac e ia embedded in a ma ix o exopoly-
saccha ides, a e habi ual inhabi an s o s one walls in cul u al he i age
monumen s. Subae ial bio ilms occu a he in e ace be ween he a -
mosphe e and he subs a e, and any en i onmen al changes will in-
luence he mic oo ganisms, in u n, al e ing he s one (Viles and Cu le ,
2012; Fa e o-Longo and Viles, 2020). In his con ex , he concep o
bio ecep i i y, de ined by Guilli e (1995) as " he capaci y o a ma e ial
o be colonized by li ing o ganisms", is o pa icula impo ance. The
bio ecep i i y o ma e ials can be modula ed by clima ic ac o s, making
he ma e ials mo e suscep ible o coloniza ion; howe e , clima ic ac o s
can also a ec he mic oo ganisms and hei su i al. Al hough changes
in clima ic ac o s can inc ease he bio ecep i i y o he subs a e as i
deg ades, li le is known abou he impo ance o mic oo ganisms in
media ing hese changes. The complex ne wo k o in e ac ions makes i
di icul o de e mine he impac o en i onmen al changes as a esul o
he in e ac ion be ween he ini ial s a e o he s one and he ype o
mic oo ganisms colonizing he s one.
Among he pa ame e s a ec ed by clima e change, empe a u e and
wa e a ailabili y ha e he s onges impac on pho o ophic o ganisms,
while also a ec ing he in eg i y o he subs a e (A i˜
no e al., 2010).
Modi ica ions in po osi y and oughness can a ou wa e e en ion, and
wa e a ailabili y egula es he g ow h o mic oo ganisms (Cu le e al.,
2013a; Gladis-Schmacka e al., 2014) and hei physiology (H¨
aubne
e al., 2006; Ka s en and Holzinge , 2012; P ie o e al., 2020; Fuen es
and P ie o, 2021). Tempe a u e, on he o he hand, can al e he mi-
c obial composi ion o ecosys ems, causing displacemen o species and
modi ica ion o me abolic a es (Bea dall and Ra en, 2004; A i˜
no e al.,
2010; Hodkinson e al., 2011). Al hough he indi idual e ec s o em-
pe a u e and wa e a ailabili y on mic oo ganisms ha e been well
s udied, li le is known abou how he in e ac ion wi h each o he a ec s
he de elopmen o subae ial bio ilms, and e en less abou he di e -
ences linked o he p ima y bio ecep i i y o s ones.
A p esen , clima e condi ions may be changing as e han he
abili y o he en i onmen o adap . In his con ex , he s udy o he
e ec o di e en ac o s associa ed wi h clima e change in ela ion o
subs a e bio ecep i i y is o pa icula in e es . S udy o he in e ac ions
be ween mic obial g ow h and en i onmen al changes, aking in o ac-
coun he bio ecep i i y o he s one, is impo an as hese in e ac ions
can in luence he conse a ion o cul u al he i age (Go bushina, 2007;
Joh and Lee, 2012; Viles and Cu le , 2012; Wi e al., 2012). In his
pape , we epo a i s app oach o in es iga ing he combined e ec s o
empe a u e, wa e a ailabili y and s one p ima y bio ecep i i y on he
g ow h o pho o ophic o ganisms on g ani e. Wi h his aim, we moni-
o ed he de elopmen o bio ilms on g ani e samples di e ing in bio-
ecep i i y and subjec ed o di e en en i onmen al condi ions, by
using pulse-ampli ude modula ed (PAM) luo ome y and spec opho-
ome ic colo measu emen .
2. Ma e ials and me hods
2.1. P epa a ion o g ani e samples, cul u e inocula ion and expe imen al
design
Fou di e en ypes o comme cially a ailable g ani e, commonly
used as cons uc ion and o namen al ma e ial, we e used o e alua e he
de elopmen o mic obial coloniza ion unde di e en en i onmen al
condi ions. The o namen al g ani es we e selec ed aking in o accoun
hei bio ecep i i y index (p o ocol de eloped by V´
azquez-Nion e al.,
2018): wo wi h low bio ecep i i y (Neg o Suda ica (NS) and Sil es e B
(BLUE)) and wo wi h high bio ecep i i y (Sil es e AM (SAM) and Sil-
es e G (GOLDEN)) (Fig. 1). The cha ac e is ics o hese g ani es a e
lis ed in Table 1.
Eigh een 4 ×4 ×2 cm specimens o each g ani ic ock we e p e-
pa ed. Sawn su ace inish was applied o all o hem. A e au ocla ing,
a olume o 2 ml o a mul i-species cul u e cha ac e ized by
V´
azquez-Nion e al. (2016), in exponen ial g ow h phase, was inocu-
la ed on o he uppe su ace o each specimen using a mic opipe e. The
cul u e is mainly composed o he g een algae B ac eacoccus mino
(Schmidle ex Choda ) Pe o ´
a, S ichococcus bacilla is N¨
ageli and Chlo -
ella sp., and he cyanobac e ia Aphanocapsa sp. and Lep olyngbya
cebennensis (Gomon ) I.Umezaki and M.Wa anabe.
The inocula ed samples we e held in clima ic chambe s (SCLAB PGA-
1228/2 HR and AIR-FRIO AIR-1330-HRTLV) wi h 12 h ligh /da k cycles
(LED ubes, 6500 K, 1500 lm) and cons an ela i e humidi y (80%), a
wo di e en empe a u es and 3 di e en le els o wa e a ailabili y:
18 ◦C and 24 ◦C, and 7 days (H7, non-days o d ough ), 3 days (H3, wi h
4 days o d ough ) and 1 day (H1, wi h 6 days o d ough ) o wa e
a ailabili y pe week (Fig. 2). Fo he wa e a ailabili y expe imen , he
samples we e placed ho izon ally in ays wi h s e ile wa e up o he
sample su ace. Wa e was egula ly enewed o main ain s able le el.
The H3 and H1 d ough pe iods we e pe o med by emo ing samples
om wa e . These wa e -d y cycles we e epea ed weekly o 42 days.
2.2. Moni o ing bio ilm o ma ion
The chlo ophyll luo escence (p oxy o g ow h (Egge e al., 2006)
and physiological s a e (Gen y e al., 1989)) and he colou a ia ion
(p oxy o biomass and pigmen p oduc ion (P ie o e al., 2002)) we e
moni o ed immedia ely a e inocula ion o he samples and 7, 15, 28
and 42 days la e , o de e mine any e ec s on bio ilm de elopmen .
Fluo escence signals in he samples we e de e mined a 470, 645 and
665 nm by pulse-ampli ude modula ed (PAM) luo ome y, in a Phy o-
PAM sys em (Heinz Walz GmbH) equipped wi h a Phy o-EDF ib e op-
ics emi e -de ec o uni . The samples su aces we e hyd a ed wi h
wa e using a hand-held a omise and held in da kness o 20 min be o e
he da a we e eco ded, in o de o allow he oxido- educ ion s a e o he
PSII cen es. A o al o nine andom eadings we e made on he su ace
o each sample, o measu e he ollowing a iables: minimal luo es-
cence in he da k-adap ed s a e (F
0
, p oxy o biomass), i.e. wi h all he
PSII cen es open, and he maximal luo escence in he da k adap ed
s a e (F
M
), i.e. wi h all he PSII cen es closed. The alues o hese
Fig. 1. Pho og aph o he ou ypes o g ani es used in he expe imen ( om low o high bio ecep i i y index).
E. Fuen es and B. P ie o
In e na ional Biode e io a ion & Biodeg ada ion 164 (2021) 105295
3
Table 1
Desc ip ion o he li ho ype s udied.
Comme cial name Abb e ia ion Classi ica ion B.I.
a
Tex u e Size (cm) Open po osi y (%)
Neg o Sud´
a ica NS Gabb o 1.7 - Low Fine/medium g ained 4 ×4 ×2 0.53
Sil es e B Blue Monzog ani e 2.2 - Low Fine g ained 4 ×4 ×2 0.90
Sil es e G Golden Monzog ani e 8.3 - High Fine g ained 4 ×4 ×2 2.53
Sil es e AM SAM Monzog ani e 8.4 - High Medium g ained 4 ×4 ×2 3.66
a
Bio ecep i i y index (B.I.) was calcula ed ollowing he p o ocol de eloped by V´
azquez-Nion e al. (2018)
Fig. 2. Schema ic diag am o he expe imen al design applied o each g oup o samples (N =3) di ided in o he ou di e en g ani es (NS, BLUE, GOLDEN and
SAM), h ee le els o wa e a ailabili y (H1, H3 and H7) and wo empe a u es (18 and 24
◦C). ↓B.I indica es g ani es cha ac e ized by low bio ecep i i y and ↑ B.I
indica es g ani es cha ac e ized by high bio ecep i i y.
Fig. 3. F
0
(biomass p oxy) (a) and Y
MAX
(bio ilm pho osyn he ic e iciency) (b) alues o he di e en bio ilms o med on he g ani e samples (NS, SAM, BLUE and
GOLDEN) unde di e en condi ions o wa e a ailabili y (H1, H3 and H7) and empe a u e (18 and 24 ◦C) a 1, 7, 14, 28 and 42 days ( om da ke o ligh e ed,
espec i ely). Di e en lowe -case le e s indica e signi ican di e ences a he end o he expe imen be ween he h ee le els o wa e a ailabili y ( o each g oup
o med by g ani e ype and empe a u e), while he di e en capi al le e s indica e signi ican di e ences be ween he ou di e en ypes o g ani e ( o each g oup
o med by wa e a ailabili y and empe a u e) (Duncan pos -hoc es a
ρ
>0.01).
E. Fuen es and B. P ie o
In e na ional Biode e io a ion & Biodeg ada ion 164 (2021) 105295
4
a iables we e used o calcula e he maximum quan um e iciency o
PSII pho ochemis y (Y
MAX,
p oxy o physiological s a e), as (F
M
-F
0
)/F
M
(Logan, 2005). In addi ion, in o de o s udy he a ia ion in he ela i e
mic obial abundance, he F
0
470nm/F
0
645nm a io was calcula ed as
he signal a 470 nm, ela ed o chlo ophyll b (algae), and he signal a
645 nm, ela ed o allophycocyanin (cyanobac e ia). High alues o he
a io indica e a dominance o g een algae, while low alues indica e
dominance o cyanobac e ia (Fuen es and P ie o, 2021).
Colou a ia ions we e de e mined by he spec opho ome ic colou
echnique, using a po able spec opho ome e (Konica Minol a CM-
700d) wi h an 8 mm diame e a ge a ea and equipped wi h CM-
S100w so wa e (Spec a Magic TM NX). The analy ical condi ions
we e as ollows: D65 illuminan , 2◦obse e and SCI mode. Nine
andom measu emen s we e made di ec ly on he SABs su aces. The
da a we e analyzed using he CIELAB colou sys em (CIE, 1978), whe e
a* deno es he ed/g een alue (a* (+) = ed; a* (−) =g een) and b*
indica es he yellow/blue alue (b* (+) =yellow, b* (−) =blue). These
pa ame e s we e ep esen ed as a ia ions in he alues ela i e o he
o iginal colou o he s one, hus yielding Δa* and Δb*. To analyse da a,
colo a ia ion o 2 CIELAB uni s was used as indica i e o di e ences as
his is he h eshold o pe cep ible changes no iced by expe ienced ob-
se e s (Mok zycki and Ta ol, 2011).
2.3. S a is ical analysis
The chlo ophyll luo escence de i ed da a (F
0
, Y
MAX
and F
0
470nm/
F
0
645nm a io) ob ained jus a e inocula ion and a he end o he
expe imen we e subjec ed o a Th ee-Way ANOVA wi h a pos -hoc
Duncan’s es (p- alue≤0.01). Fo his pu pose, SPSS s a is ical p o-
g am ( e sion 23.0) was used.
3. Resul s
The minimum luo escence (F
0
) o he bio ilm (biomass p oxy) o e
ime is ep esen ed in Fig. 3a, o each g oup o samples o g ani e ocks
subjec ed o di e en empe a u es (18 and 24) and di e en humidi y
le els (H7, H3 and H1). Fo NS, he luo escence was e y low unde all
o condi ions and dec eased o e ime. This was obse ed in he samples
main ained a 18 ◦C and a 24 ◦C. In he case o BLUE ock, F
0
was e y
low, eaching sligh ly highe alues a he end o he expe imen a
18 ◦C, bu only o maximum wa e a ailabili y (H7). The alues we e
low in he samples held a 24 ◦C, ega dless o he le el o wa e a ail-
abili y, and ended o dec ease o e ime. The F
0
alues o he GOLDEN
samples inc eased o e ime in he samples subjec ed o maximum wa e
a ailabili y (H7) a bo h 18 and 24 ◦C, bu in he la e case lowe
maximum alues o F
0
we e ob ained. When wa e a ailabili y was
educed, he luo escence was much lowe , dec easing o e ime, bo h a
18 and 24 ◦C. Fo he SAM g ani e, he highes F
0
alues a e ound,
especially a maximum wa e a ailabili y (H7). In his scena io (H7), he
bio ilm luo escence inc eased o e ime, bo h a 18 and 24 ◦C. How-
e e , he F
0
alues o he bio ilms subjec ed o 3 and 1 days o wa e
a ailabili y (H3 and H1) a ied wi h he empe a u e. A 18 ◦C he
alues dec eased o e ime, while a 24 ◦C his pa ame e ended o
inc ease, no eaching alues as high as wi h maximum wa e
a ailabili y.
The maximum yield (Y
MAX
), which indica es he pho osyn he ic e -
iciency, is ep esen ed in Fig. 3b. Fo he NS samples, he mic oo -
ganism e iciency gene ally inc eased o e ime. The highes
pho osyn he ic e iciency (45%) was eached on day 28 a 18 ◦C wi h
maximum wa e a ailabili y (H7). A he end o he expe imen , he
e iciency was abou 30% o samples exposed o maximum wa e
a ailabili y, and below his alue o he lowe le els o wa e a ail-
abili y (H3 and H1), bo h a 18 and 24 ◦C. An inc ease in pho osyn he ic
e iciency o e ime a bo h 18 and 24 ◦C was obse ed o he BLUE
samples. A 18 ◦C he highes pho osyn he ic e iciency o hose sam-
ples (55%) was obse ed a he end o he expe imen o he bio ilms
wi h maximum wa e a ailabili y (H7) bu when he a ailabili y was
dec eased o 1 day (H1), he maximum pho osyn he ic e iciency was
g ea ly educed (<10%). This did no occu when samples we e held a
24 ◦C, as he maximum pho osyn he ic e iciency a he end o he
expe imen wi h maximum wa e a ailabili y was lowe (37%) and
when he wa e a ailabili y dec eased, he pho osyn he ic e iciency did
no a y g ea ly (>20%). Fo GOLDEN samples, he pho osyn he ic e -
iciency inc eased o e ime o maximum wa e a ailabili y (H7) and
18 ◦C, exceeding 50% by he end o he expe imen . Howe e , when he
wa e a ailabili y was dec eased, al hough he end con inued up-
wa ds, he alues we e below 40%. When bio ilms we e exposed o
24 ◦C, he mic obial abundance inc eased o e ime (be ween 44 and
63%) o all le els o wa e a ailabili y, eaching 63% o maximum
wa e a ailabili y (H7). Finally, SAM pho osyn he ic e iciency was high
when bio ilms we e exposed o maximum wa e a ailabili y and 18 ◦C,
eaching an e iciency o 60% by he end o he expe imen . In he
samples exposed o 24 ◦C, he pho osyn he ic e iciency eached 55%.
Howe e , o he mic oo ganisms exposed o es ic ed wa e le els
(bo h H3 and H1) a 18 ◦C, he e iciency dec eased o e ime, eaching
lowe alues han o maximum wa e a ailabili y (H7). Howe e , a
24 ◦C he e iciency emained cons an o e ime o H3 and H1
(exceeding 40% e iciency) and sca cely di e ing om he alues ob-
ained o H7.
Fig. 4 ep esen s di e ences in he F
0
470 nm/F
0
645 nm a io
( ela i e o he ini ial alue) a 7, 14, 28 and 42 days. This a io p o ides
in o ma ion abou he p opo ion o algae and cyanobac e ia in he
bio ilms. As he signal a 470 nm is ela ed o chl b (algae) and he signal
a 645 nm, o allophycocyanin (cyanobac e ia), high alues indica e a
p edominance o g een algae while low alues indica e a p edominance
o cyanobac e ia. When he samples we e held a 18 ◦C, he a io
inc eased o e ime o all le els o wa e a ailabili y, ela i e o he
alue a he beginning o he expe imen o NS, BLUE and GOLDEN
g ani es. Fo he SAM samples, he e was no g ea a ia ion a he end o
he expe imen ela i e o he ini ial alue in he case o samples in
which wa e a ailabili y was es ic ed (H1 and H3) al hough an in-
c ease was no ed in he samples subjec ed o maximum wa e a ail-
abili y (H7). In he samples held a 24 ◦C, his end was somewha
modi ied. Fo he mic oo ganisms g owing on he g ani es wi h highes
le els o bio ecep i i y (SAM and GOLDEN) and in which wa e a ail-
abili y was maximum (H7) he index dec eased. This was also obse ed
o NS unde maximum wa e a ailabili y un il day 28 bu hen e e sed
owa ds he end o he expe imen .
The colou a ia ions o he bio ilms (sub ac ing he alues o he
uninocula ed subs a e) a he end o he expe imen wi h espec o he
alues ob ained jus a e inocula ion a e shown in Fig. 5, wi h Δa*
plo ed agains Δb* bo h o 18 and 24 ◦C (le and igh espec i ely).
Conside ing ha he cen al poin implies no di e ence o he jus
inocula ed samples, o NS and BLUE g ani es bo h pa ame e s sca cely
a ied o e ime (below he h eshold o 2 CIELAB uni s) o any o he
condi ions s udied. In GOLDEN samples a ma ked end owa ds
g eening (Δa* dec eases) and yellowing (Δb* inc eases) was obse ed
o maximum wa e a ailably (H7) and 18 ◦C. Howe e , he colou o
he same samples unde he same wa e condi ions bu kep a 24 ◦C,
end o g eening bu no yellowing. In he case o SAM g ani e, bo h Δa*
and Δb* alues o bio ilms subjec ed o maximum wa e a ailably (H7)
and bo h empe a u es we e o e he 2 CIELAB uni s’ h eshold. In
summa y, mo e no able colou changes owa ds g eening and yellowing
we e obse ed in SAM and GOLDEN g ani es, especially o maximum
wa e a ailabili y, while he colou o he o he bio ilms did no a y
du ing he expe imen . Fu he mo e, o all g ani es, he di e ences in
colou a ia ion among he h ee di e en le els o wa e a ailabili y
(H1, H3 and H7) a e g ea e a 18 han a 24 ◦C.
4. Discussion
The pa ame e s de i ed om chlo ophyll luo escence and colou
E. Fuen es and B. P ie o
In e na ional Biode e io a ion & Biodeg ada ion 164 (2021) 105295
5
quan i ica ion p o ide es ima es o he g ow h and he physiological
s a e o he bio ilms (Egge e al., 2006; V´
azquez-Nion e al., 2016;
Geno a e al., 2020). Bo h echniques e ealed di e ences in g ow h in
ela ion o bio ecep i i y and he en i onmen al pa ame e s, wi h he
highes g ow h and pho osyn he ic e iciency in he bio ilms on samples
supplied wi h maximum wa e a ailabili y o 7 days (H7). Tempe a u e
did no appea o ha e a signi ican e ec on g ow h, bu signi ican ly
a ec ed he pho osyn he ic e iciency (Table 2). Depending on he ype
o g ani e, he clima ic ac o s, empe a u e and wa e a ailabili y, had
an e ec on he biological coloniza ion as well as on he bio ilm
pho osyn he ic e iciency.
Biocoloniza ion o ma e ials depends on bo h he p e ailing clima ic
condi ions and he bio ecep i i y o he ma e ial (Guilli e, 1995).
Al hough many s udies ha e in es iga ed he bio ecep i i y o s one
(P ie o and Sil a, 2005; Mille e al., 2012; V´
azquez-Nion e al., 2018;
Fa e o-Longo and Viles, 2020; Sanma ín e al., 2021) and he e ec o
en i onmen al condi ions on mic obial coloniza ion (A i˜
no e al., 2010;
Cu le e al., 2013b; Gladis-Schmacka e al., 2014; Gayla de and
Au ho Anonymous, 2020) no s udies ha e add essed bo h ac o s a he
same ime. The bio ecep i i y o a ma e ial, speci ically o g ani e, de-
pends on he su ace oughness, po osi y and chemical composi ion
(Guilli e, 1995; P ie o and Sil a, 2005; Mille e al., 2012). In his s udy,
he biomass emaining on he su ace o he sample 24 h a e inocula-
ion was analyzed by de e mining he minimum luo escence pa ame e .
The luo escence signals (F
0
) and maximum yield (Y
MAX
) di e ed
signi ican ly be ween he di e en ypes o g ani e (be ween g oups)
(Table 2), bu no in he 18 samples o he same ype o g ani e (wi hin
g oup). This indica es ha he se lemen a ied on di e en subs a es.
This di e en esponse is consis en wi h he bio ecep i i y index (BI) o
each ype o g ani e conside ed, as he mean F
0
alues inc eased wi h
hose o he bio ecep i i y index. The subs an ial di e ence in he
abundance o o ganisms on he g ani e samples wi h di e en le els o
bio ecep i i y, only 24 h a e inocula ion, may indica e ha he p op-
e ies o he s one ha de e mine bio ecep i i y a e impo an du ing
Fig. 4. Changes in he F
0
470/F
0
645 a io o e ime (42 days) o he di e en bio ilms o med on he g ani e samples (NS, SAM, BLUE and GOLDEN) a 18 and 24 ◦C.
Fig. 5. Plo o Δb* agains Δa* colou changes in he bio ilms a he end o he expe imen (day 42) wi h espec o jus a e inocula ion, a wo di e en em-
pe a u es (18 and 24
◦C). Rock subs a es a e indica ed in di e en colou s (black, NS; yellow, SAM; blue, BLUE and ed, GOLDEN) and wa e a ailabili y is ep-
esen ed by di e en shapes (ci cle, H1; iangle, H2 and squa e H3). The dashed g een line ma ks he h eshold o 2 CIELAB uni s, a e which colou changes a e
pe cei ed by an expe ienced obse e .
E. Fuen es and B. P ie o
In e na ional Biode e io a ion & Biodeg ada ion 164 (2021) 105295
6
he ini ial s eps o coloniza ion. Al hough he e ec o su ace oughness
was educed in his s udy by placing he samples ho izon ally, o he
p ope ies such as open po osi y, bulk densi y and capilla y wa e
(P ie o and Sil a, 2005) appea o be key ac o s in de e mining es ab-
lishmen o he mic oo ganisms on he su ace. This is consis en wi h
he indings epo ed by D’O azio e al. (2014), who in es iga ed he
e ec o chemical composi ion, po osi y and wa e e en ion on di e en
ypes o b icks and obse ed ha su ace oughness and o al po osi y
a e he mos impo an physical p ope ies de e mining he adhesion o
algal cells o he subs a e. In he p esen s udy, he e ec o g ani e
bio ecep i i y on bio ilm a achmen was obse ed a e inocula ion.
Mo eo e , i was also ound a signi ica i e in e ac ion e ec o bio-
ecep i i y and clima ic ac o s in he bio ilm de elopmen a he end o
he expe imen . Thus, in hose cases whe e he subs a e a o s he
ancho age o he o ganisms, a o able en i onmen al condi ions such as
high-wa e a ailabili y (H7) o inc ease in empe a u e led o a highe
g ow h o he bio ilm (Table 2). This is a e y impo an inding ha
mus be aken in o accoun in labo a o y expe imen s in which di e en
ypes o s one a e es ed, in o de o a oid a ibu ing he obse ed e -
ec s o en i onmen al pa ame e s a he han o he subs a e bio-
ecep i i y. Howe e , Manso e al. (2015), who s udied mic obial
coloniza ion on di e en cemen mix u es, ound ha he main di e -
ences in bio ouling we e caused by changes in humidi y, while ough-
ness and po osi y did no ha e subs an ial e ec s. Thus, hese esul s
lead o s udy he ole o bio ecep i i y on he ini ial s ages o
biocoloniza ion independen ly o clima ic condi ions, as well as o he
s udy o main pa ame e s ( oughness, po osi y, capilla i y, e c.) de e -
mining he ini ial adhesion o o ganisms and he e o e he inal
coloniza ion.
The en i onmen al e ec s, in e ms o g ow h and physiological
s a e, began o be obse ed 14 days a e he inocula ion. A he end o
he expe imen , bio ilm g ow h inc eased unde maximum wa e
a ailabili y, and he g ow h slowed down when he wa e a ailabili y
was in e up ed by pe iods o d ough . D ough pe iods o 4–6 days
educed s a is ically he e iciency and g ow h o pho o ophic bio ilms
(Table 2). The majo e ec o wa e in con olling g ow h o he bio ilms
(mo e impo an han empe a u e) is consis en wi h he indings o
o he s udies, iden i ying wa e a ailabili y as one o he mos impo an
ac o s in he de elopmen o pho o ophic o ganisms (H¨
aubne e al.,
2006; Gladis and Schumann, 2011; Fuen es and P ie o, 2021). Mo e-
o e , bio ilm g ow h was no co ela ed wi h o al p ecipi a ion, bu
a he wi h he equency o ainy days, i.e. longe pe iods o exposu e o
high humidi y a ou g ow h o mic oo ganisms (Viles and Cu le , 2012;
Gladis-Schmacka e al., 2014). Thus, in he con ex o di e en clima e
change scena ios in which an inc ease in he du a ion o d ough s is
p edic ed (Hewi son e al., 2014), a educ ion in he de elopmen o
subae ial bio ilms may be expec ed, especially du ing he summe
pe iod.
The e ec o wa e a ailabili y on g ow h and physiological s a e o
bio ilms was also e lec ed by colou a ia ion da a especially o bio-
ilms de eloped on SAM and GOLDEN g ani es, whe e colou di e ences
g ea e han 2 CIELAB uni s, o bo h Δa* and Δb* (mo e yellow and
g een), indica e a bio ilm de elopmen pe cep ible o he human eye.
This is a e y impo an subjec o be aken in o accoun when bio-
colonisa ion o cul u al he i age is in ol ed as pe cep ion o bio ilm
de elopmen do means aes he ical biode e io a ion. To his espec ,
beyond he chemical and physical changes ha o ganisms can cause in
he subs a e, colou modi ica ions such as hose epo ed in his wo k
ha e been poin ed as cause o aes he ic de e io a ion o monumen s
(P ie o e al., 2006; Smi h e al., 2011; Sanma ín e al., 2012).
The g ow h o o ganisms also depends on empe a u e and nu ien
a ailabili y. In gene al, inc eased empe a u e, up o an op imal le el,
ends o lead o accele a ion o me abolic (Cal in cycle) eac ions (Fal-
kowski, 1980). The op imal empe a u e o g ow h o algae and cya-
nobac e ia is species speci ic (Baume and Pe zod , 2008; F anz e al.,
2012). We obse ed ha empe a u e s a is ically a ec s pho osyn he ic
e iciency (Y
MAX
pa ame e ), which was highe a 24 ◦C han a 18 ◦C.
Al hough in one p e ious s udy, pho osyn he ic e iciency did no a y,
di e ences in g ow h we e obse ed and despi e he wide ange o
empe a u e anges a which g ow h is iable, he bes esul s we e
ob ained be ween 21 and 24 ◦C (H¨
aubne e al., 2006). In he p esen
s udy, al hough no di ec e ec o empe a u e was obse ed in e ms o
bio ilm g ow h, bo h he di ec e ec o empe a u e and he in e ac ion
wi h wa e clea ly a ec ed he pho osyn he ic e iciency (Table 2). A
18 ◦C, he pho osyn he ic e iciency o he bio ilm was signi ican ly
highe a maximum wa e a ailabili y han wi h es ic ed wa e a ail-
abili y. Howe e , a 24 ◦C, al hough he pho osyn he ic e iciency o he
bio ilms ended o be highe wi h maximum wa e a ailabili y, he
di e ence was no s a is ically signi ican . The pho osyn he ic e iciency
o he bio ilms wi h es ic ed wa e a ailabili y (H3 and H1) was highe
a 24 ◦C han a 18 ◦C, while a maximum wa e a ailabili y (H7) he e
we e no di e ences in his pa ame e a he di e en empe a u es. This
di e en beha io o he bio ilm’s de elopmen unde changing em-
pe a u e condi ions is also e iden by colou da a as colou di e ences
among he 3 le els o wa e a ailabili y we e g ea e in bio ilms kep a
18◦ han on hose a 24◦. These esul s, oge he wi h hose ob ained
om chlo ophyll luo escence could indica e a bu e ing e ec o wa e
(Ga e and G isham, 2013), which would explain why he pho osyn-
he ic e iciency o he mic oo ganisms only inc eased in esponse o an
inc ease in empe a u e when he a ailabili y o wa e was es ic ed.
Taking in o accoun he speci ic esponses o di e en ypes o
Table 2
Resul s o h ee-way ANOVA o Phy o-PAM measu emen s jus a e inocula ion
and a he end o he expe imen , conside ing ype o g ani e, wa e a ailabili y
and empe a u e as ac o s. p- alues <0.01 a e indica ed in bold ype.
Jus a e
inocula ion
End o he
expe imen
Fac o s F p-
alue
a
F p-
alue
a
F
0
TEMPERATURE 1.965 0.167 2.852 0.098
WATER 0.818 0.448 42.792 0.000
GRANITE B.I. 186.867 0.000
b
39.543 0.000
TEMPERATURE *
WATER
2.463 0.096 3.457 0.040
TEMPERATURE *
GRANITE B.I.
2.254 0.094 5.156 0.004
WATER * GRANITE B.I. 0.953 0.467 9.066 0.000
TEMPERATURE *
WATER * GRANITE B.I.
1.424 0.225 1.364 0.248
Y
MAX
TEMPERATURE 4.828 0.033 19.304 0.000
WATER 1.239 0.299 23.669 0.000
GRANITE B.I. 103.370 0.000
b
51.018 0.000
TEMPERATURE *
WATER
1.723 0.189 1.339 0.000
TEMPERATURE *
GRANITE B.I.
1.363 0.265 9.507 0.273
WATER * GRANITE B.I. 0.869 0.524 0.723 0.633
TEMPERATURE *
WATER * GRANITE B.I.
0.847 0.540 1.014 0.427
ΔF
0(470
nm)
/F
0
(645)
TEMPERATURE - - 4.585 0.037
WATER - - 0.287 0.752
GRANITE B.I. - - 22.126 0.000
TEMPERATURE *
WATER
- - 3.507 0.038
TEMPERATURE *
GRANITE B.I.
- - 5.569 0.002
WATER * GRANITE B.I. - - 1.194 0.325
TEMPERATURE *
WATER * GRANITE B.I.
- - 5.989 0.000
a
p- alue is signi ican when is <0.01.
b
Fo bo h, F
0
and Y
MAX
, he Duncan pos -hoc es (
ρ
>0.01) e ealed sig-
ni ican di e ences jus a e inocula ion be ween all he ypes o g ani es
ollowing he nex o de : NS <BLUE <GOLDEN <SAM.
E. Fuen es and B. P ie o
In e na ional Biode e io a ion & Biodeg ada ion 164 (2021) 105295
7
mic oo ganisms o he en i onmen al condi ions and he ac ha bio-
ilms a e complex communi ies o o ganisms, compe i ion o esou ces
will lead o some species being selec ed o e o he s. In esponse o
changes in en i onmen al condi ions, cyanobac e ia and algae will
compe e wi h each o he , la gely in esponse o an inc ease in empe -
a u e whe e he o me is a o ed (C ispim e al., 2003; Gayla de and
Gayla de, 2005). In he p esen s udy, he F
0
470nm/F
0
645 nm a io
p o ided in o ma ion abou he p opo ion o algae and cyanobac e ia
in he bio ilms. In he bio ilms in which he physiological s a e was
op imal (SAM and GOLDEN samples a H7) he p opo ion o cyano-
bac e ia inc eased when he samples we e main ained a 24 ◦C (nega i e
alues o ΔF
0
470nm/F
0
645 nm a io), bu no a 18 ◦C. Tha is, unde
ideal g ow h condi ions, i.e., a highly bio ecep i e subs a e and
high-wa e a ailabili y, empe a u e is he ac o con olling di e si y in
bio ilms as a ise in empe a u e a o s he de elopmen o cyanobac-
e ia e sus algae (Table 2). Thus, he p esen indings indica e ha in
he con ex o he expec ed global wa ming, cyanobac e ia will be p e-
dominan in a eas whe e an inc ease in he equency o ain is
p edic ed.
5. Conclusions
The e ec o he combina ion o p ima y bio ecep i i y oge he wi h
clima ic pa ame e s was o he i s ime e alua ed. The esul s show an
ini ial e ec o bio ecep i i y which is modula ed by empe a u e and
wa e a ailabili y along he expe imen . Once o ganisms a e s ablished,
a educ ion in wa e a ailabili y educes bio ilm de elopmen , while
empe a u e ac s as a me abolic ac i a o , imp o ing he e iciency o
he o ganisms e en unde un a o able wa e condi ions. In u n, when
condi ions a e op imal, i.e., highly bio ecep i e subs a e and o al
wa e a ailabili y, an inc ease in empe a u e a o s he de elopmen o
cyanobac e ia e sus algae.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgmen s
This s udy was pa ly inanced h ough p ojec CGL2016-79778-R
(AEI/FEDER, UE) and Xun a de Galicia (ED431 2018/32). E. Fuen es
was inancially suppo ed by a PhD Fellowship-Con ac MICINN-FPI
(BES-2017-079927).
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