wa e
A icle
De eloping a Bio echnological Tool o Moni o ing
Wa e Quali y: In Vi o Clone Cul u e o he Aqua ic
Moss Fon inalis An ipy e ica
So ía Debén * , Jesús Ramón Aboal, Pablo Gi áldez, Zulema Va ela and
Jose Ángel Fe nández
Ecology Uni , Depa men o Func ional Biology, Uni e sidade de San iago de Compos ela, Fac. Bioloxía,
Lope Gómez de Ma zoa s/n, San iago de Compos ela, 15702 A Co uña, Spain; [email p o ec ed] (J.R.A.);
[email p o ec ed] (P.G.); [email p o ec ed] (Z.V.); [email p o ec ed] (J.Á.F.)
*Co espondence: [email p o ec ed]; Tel.: +34-881-81-33-14
Recei ed: 18 Decembe 2018; Accep ed: 12 Janua y 2019; Published: 15 Janua y 2019
Abs ac :
One o he main ac o s limi ing ac i e biomoni o ing wi h aqua ic mosses is he lack o
su icien ma e ial. A labo a o y cul u e o he moss would sol e his p oblem and hus con e
he echnique in o a aluable bio echnological ool o moni o ing wa e quali y. Wi h his aim,
we i s es ablished small and la ge scale axenic
in i o
cul u e sys ems o he aqua ic moss
Fon inalis an ipy e ica. We hen a emp ed o enhance he g ow h a e o he cul u es by modi ying
empe a u e, pho ope iod and medium composi ion (N:P a io, P concen a ion, CO
2
supply,
NH
4
NO
3
supply and suc ose supply). None o hese modi ica ions g ea ly inc eased he
in i o
g ow h a e. Howe e , he g ow h a es we e su icien ly high ( ela i e o he ini ial weigh o he
cul u es) in bo h sys ems (45 and 6 mg
·
day
−1·
g
−1
o lasks and bio eac o s espec i ely) o enable
he p oduc ion o la ge amoun s o ma e ial. The abili y o cul u e he ma e ial will subs an ially
imp o e he applicabili y o he moss bag echnique.
Keywo ds: moss bag echnique; bio eac o s; b yophy es; Fon inalis p opagule; clonal p opaga ion
1. In oduc ion
I is beyond deba e ha managing wa e is essen ial o sus ainable de elopmen wo ldwide,
and moni o ing wa e quali y by means o adi ional me hods o by biomoni o ing should be
manda o y. The aqua ic moss Fon inalis an ipy ec ica Hewd. is he mos commonly used b yophy e o
biomoni o ing inland wa e quali y [
1
]. The wide ange o dis ibu ion o his species in empe a e
egions and i s ease o iden i ica ion and handling in he labo a o y make i ideal o his ype o
s udies [
2
]. I can be used bo h as a passi e (na i e specimens) and an ac i e biomoni o ing agen
( ansplan ed specimens). The ad an ages o ac i e biomoni o ing ha e ecen ly been desc ibed, and
a e summa ised as ollows: (i) he elimina ion o pheno ypic and geno ypic adap a ions; (ii) imp o ed
empo al in e p e a ion o he esul s as he du a ion o he exposu e pe iod is known; (iii) i enables he
assessmen o he magni ude o he pollu ion as he ini ial concen a ions o elemen s in he ansplan s
a e also known; and (i ) i emo es he need o he selec ed species o be p esen in he sampling
si es [
1
,
3
]. The me hod mos commonly used o expose he mosses in ac i e biomoni o ing su eys
(ca. 80% o he published pape s [
1
]) is he moss bag echnique, in which mosses a e ansplan ed inside
mesh bags om uncon amina ed si es o he s udy si es. The echnique has been used o de e mine
nume ous ypes o aqua ic pollu an s, including hea y me als and me alloids [
1
], pe sis en o ganic
pollu an s (such as PCBs (polychlo ina ed biphenyls) and PAHs (Polycyclic a oma ic hyd oca bons);
e.g., [
4
]), and e en some adioac i e iso opes (such as Cs
137
; e.g., [
5
]). Howe e , he me hod is limi ed
Wa e 2019,11, 145; doi:10.3390/w11010145 www.mdpi.com/jou nal/wa e
Wa e 2019,11, 145 2 o 10
by se e al ac o s: (i) he a ailabili y o unpollu ed si es whe e ma e ial can be collec ed; (ii) he
yea - ound p esence o su icien numbe s o indi iduals o he selec ed biomoni o ing species a
unpollu ed si es o enable collec ion wi hou h ea ening conse a ion o he na i e popula ion; and
(iii) he a ailabili y o homogeneous ma e ial, as he na u al a iabili y in he elemen al composi ion
o he moss may di e depending on an h opogenic and na u al ac o s (i.e., gene ic, pheno ypic,
mic o-si es, e c. [1]), which change o e ime.
A pa adigm shi has aken place in ecen yea s, and, ins ead o moss being collec ed in
he ield, selec ed species o moss can be cul u ed in he labo a o y, hus helping o minimize he
abo e-men ioned limi a ions. The abili y o cul u e he moss ma e ial should con e he echnique
in o a bio echnological ool which could be widely used by local, na ional and in e na ional bodies o
moni o wa e quali y. Unlimi ed a ailabili y o he ma e ial would emo e he need o he specialized
knowledge equi ed o species iden i ica ion and designing sampling s a egies, e c. Cul u ing he
moss would also ensu e a cons an supply o he ma e ial o biomoni o ing pu poses by p oducing
la ge amoun s o high-quali y ma e ial wi h he same ini ial ( e y low) concen a ions o elemen s [
6
].
This would also minimize he a iabili y in he esul s [
7
], as demons a ed o e es ial mosses [
8
].
Fu he mo e, i would p e en damage o he en i onmen associa ed wi h equen collec ion o
mosses a unpollu ed si es.
The i s a emp o cul u e an aqua ic moss (F. an ipy e ica) was ca ied ou by Rausch de
T aubenbe g and Ah-Peng [
7
]; howe e , he ma e ial was no cul u ed unde axenic condi ions and he
clone was no main ained o e y long. Un o una ely, he clone p oduced by hese au ho s has been
los and was ne e used as a biomoni o ing agen . Mo e ecen ly, a clone o Sphagnum pallus e was
success ully cul u ed unde axenic condi ions o use in a mosphe ic ac i e biomoni o ing su eys [
9
].
The cloning and use o his moss ha e been success ully s anda dized (e.g., in [
8
]). We a e awa e
o wo axenic cul u es o aqua ic moss clones ha a e cu en ly a ailable: Rinchos egium ipa ioides
(Ral Reski, pe s. comm. (pe sonal communica ion)) and he clone o F. an ipy e ica g own in ou
labo a o y, ob ained om he game ophy es s e ilized by A es e al. [10].
To ou knowledge, no p e ious a emp s ha e been made o cul u e hose aqua ic mosses unde
axenic condi ions in bio eac o s wi h he aim o p oducing ma e ial o use in ac i e biomoni o ing
su eys. On he o he hand, he g ow h a e o he clone mus be su icien ly high o make he
biomoni o ing echnique iable. Howe e , he g ow h condi ions ha e no ye been op imized o
his species. Thus, wi h he aim o cul u ing an aqua ic moss clone o he pu pose o biomoni o ing
wa e quali y, he s udy objec i es we e as ollows: (i) o cul u e F. an ipy e ica unde
in i o
axenic
condi ions in liquid medium, and (ii) o inc ease he g ow h a e o he cul u e by modi ying he
empe a u e, pho ope iod and medium composi ion (N:P a io, P concen a ion, CO
2
supply, NH
4
NO
3
supply and suc ose supply).
2. Ma e ial and Me hods
2.1. P opaga ion o Fon inalis An ipy e ica Game ophy es o In Vi o Cul u e
The F. an ipy e ica game ophy e used o ini ia e he cul u es was o iginally collec ed, s e ilised
and p ese ed on solid medium by A es e al. [
10
]. A single game ophy e om his collec ion was
isola ed, agmen ed and placed in Pe i dishes wi h modi ied Knop medium. The medium was
p epa ed as desc ibed by Hohe and Reski [
11
] (4.24 mM Ca(NO
3
)
2·
4H
2
O, 3.36 mM KCl, 1.84 mM
KH
2
PO
4
, 1.02 mM MgSO
4·
7H
2
O, 45
µ
M FeSO
4·
7H
2
O) and he pH was adjus ed o 6.5 wi h KOH
and HCl (be o e au ocla ing). The medium was also supplemen ed wi h 0.3% suc ose and he
mic oelemen s ecommended by Schween e al. [
12
] (i.e., 50
µ
M H
3
BO
4
, 15
µ
M ZnSO
4·
7H
2
O, 50
µ
M
MnSO
4·
1H
2
O, 2.5
µ
M KI, 500 nM Na
2
MoO
4·
2H
2
O, 50 nM Co(NO
3
)
2·
6H
2
O, 50 nM CuSO
4·
5H
2
O) and
was solidi ied wi h 1% (w/ ) Phy agel (Sigma-Ald ich, S . Louis, MO, USA). The Pe i dishes we e
sealed wi h Pa a ilm
®
and main ained a 15
◦
C wi h a day/ligh egime o 16/8 h and i adia ed by
luo escen ubes (OSRAM L36W/865 cool dayligh , OSRAM, Munich, Ge many). Once he asexual
Wa e 2019,11, 145 3 o 10
p opagules had o med and he apices had elonga ed, he elonga ed shoo s we e agmen ed as be o e
and he pieces we e ans e ed o new pla es wi h esh medium. This p ocess was epea ed un il
su icien ma e ial was a ailable o ini ia e he cul u es in liquid medium. The s e ili y condi ions
we e main ained du ing ans e o he cul u es and con olled wi h B medium (20 g/L LB b o h low
sal —Duche a Biochemie, Haa lem, The Ne he lands; and 1.2% Bac o
TM
Aga —Bec on, Dickinson and
Company, F anklin Lakes, NJ, USA) and wi h PDA medium (39 g
·
L
−1
Po a o Dex ose Aga —Pan eac
AppliChem, Da ms ad , Ge many), which we e held a oom empe a u e o a leas 4 weeks.
2.2. In Vi o Cul i a ion Techniques
The F. an ipy e ica clones we e cul u ed a small and la ge scales in liquid medium o s udy he
e ec s o di e en cul u e condi ions. Fo he small-scale cul u e, E lenmeye lasks con aining
500 mL o medium we e held inside a g ow h chambe a 15
◦
C. Fo he la ge-scale cul u e,
pho obio eac o s (Bio Bundle 15M Applikon, Del , The Ne he lands) con aining 12 L o medium we e
used; he empe a u e o he bio eac o s was main ained by a double jacke sys em connec ed o a
cooling ba h (Iso emp—Fishe 9.5-14.5 L Ra12, Fishe Scien i ic SL, Mad id, Spain) and con olled by
so wa e in he bio eac o so wa e sys em (ezCon ol, Del , The Ne he lands). Al hough he cul u e
pH can also be con olled in hese bio eac o s, his op ion was disabled o all he expe imen s ca ied
ou (see below) and he pH was allowed o a y depending on he cul u e condi ions.
The cul u e medium used in bo h E lenmeye and bio eac o cul u es was he same as ha used
in he p opaga ion s ep, excep ha i was no solidi ied and was only modi ied when he aim o he
expe imen equi ed i (see below and Table 1). Be o e ans e o he lasks o bio eac o s, he moss
samples we e placed on a glass dish and weighed ( esh) on a balance Me le Toledo B502-S (Me le
Toledo, Columbus, OH, USA). Weighing and ans e we e ca ied ou in a lamina low cabine Tels a
AV-100 (Azbil Tels a Technologies Slu, Te assa, Ba celona, Spain) o p e en he con amina ion o
he cul u es.
Table 1.
Composi ion o he cul u e medium and a ia ion in he Fon inalis an ipy e ica cul u e
condi ions in he expe imen s (Exp.) ca ied ou in lasks (E.1–E.3) and bio eac o s (E.4–E.7).
A, B, ..., E
= di e en ea men s (T ea m.) o each expe imen (see ex o mo e de ails). n: numbe o eplica es;
T: empe a u e.
Type o
Cul u e Exp. T ea m. n Time
(days) T (◦C) Volume
(L)
Pho ope iod
(h ligh /da k)
Suc ose
(%)
N:P
Ra io
P
(mg·L−1)CO2NH4
(g·L−1)
Flasks
Con ol 13
57–76
15 0.5 16/8 0.3 2.1 250 - -
E.1 7 76 15 0.5 24/0 0.3 2.1 250 - -
E.2 A 3 70 15 0.5 16/8 2 2.1 250 - -
B 3 70 15 0.5 16/8 4 2.1 250 - -
E.3
A 3 57 15 0.5 16/8 0.3 23 250 - -
B 3 57 15 0.5 16/8 0.3 46 250 - -
C 3 57 15 0.5 16/8 0.3 2.1 5200 - -
D 3 57 15 0.5 16/8 0.3 23 500 - -
E 3 57 15 0.5 16/8 0.3 46 500 - -
Bio eac o s
Con ol 4
97–135
15 12 24/0 0.3 2.1 250 - -
E.4 A 1 113 10 12 24/0 0.3 2.1 250 - -
B 1 114 20 12 24/0 0.3 2.1 250 - -
E.5 1 97 15 + 20 4 + 8 24/0 0.3 2.1 250 - -
E.6 1 122 15 12 24/0 0.3 2.1 250 0.1 -
E.7 1 123 15 12 24/0 0.3 2.1 250 - 0.1
Di e en expe imen s we e conduc ed (see Table 1 o de ails) wi h he aim o inc easing he
g ow h a e o he moss by op imizing he ollowing a iables: (i) he pho ope iod o which he
cul u es a e subjec ed (Expe imen 1); (ii) he suc ose con en o he cul u e medium (Expe imen 2);
(iii) he N:P a io o he cul u e medium, and consequen ly he N and P con en (Expe imen 3);
(i ) he
empe a u e in con inuous cul u e (Expe imen 4); ( ) he cul u e empe a u e and eno a ion o
nu ien s by simula ing sp ing condi ions (i.e., cul u e du ing 1 mon h in 4 L o medium a 15
◦
C
ollowed by an inc ease in empe a u e o 20
◦
C and he addi ion o 8 L o esh cul u e medium)
Wa e 2019,11, 145 4 o 10
(Expe imen 5); ( i) he in e mi en addi ion o CO
2
(i.e., addi ion o 0.1 L pe day) (Expe imen 6);
and ( ii) he addi ion o NH4NO3(0.1 g·L−1) (Expe imen 7).
2.3. S a is ical Analysis
The no mali y o he da a was checked wi h he Kolmogó o –Smi no es s o he da a o he
lask cul u es. As he da a ul illed he equi emen s o pa ame ic es s, he di e ences in g ow h
be ween di e en expe imen s (1 o 3) we e he e o e compa ed by one-way ANOVA ( he Analysis
o he Va iance). The analysis was ca ied ou using IBM SPSS s a is ical so wa e, e sion 24.0 (IBM,
New Yo k, NY, USA).
3. Resul s
The clone cul u ed in he o iginal cul u e medium (con ol in all expe imen s) g ew success ully
in bo h lasks and bio eac o s (Figu es 1and 2). The g ow h a e (mg
·
day
−1
), exp essed ela i e o he
ini ial weigh o he cul u es (g
−1
), was 45 and 6 mg
·
day
−1·
g
−1
o lasks and bio eac o s, espec i ely,
whe eas he daily g ow h a e was, espec i ely, 184 and 557 mg
·
day
−1
. The p opo ion o g ow h
ela i e o he o al weigh (ini ial and g ow h) o he lask cul u es a ied be ween 70% and 87%
(mean = 76%; n = 13). The g ow h o he cul u e ( ela i e o he con ol) was no signi ican ly a ec ed
(p< 0.05) by he a ia ions in ei he pho ope iod (E.1) o suc ose concen a ion (E.2). The modi ica ions
o he cul u e medium (E.3—N:P a io) nega i ely a ec ed he cul u es, leading o signi ican ly lowe
g ow h han in con ols. This di e ence inc eased as he concen a ion o P inc eased, independen ly
o he N:P a io.
Wa e 2018, 7, x FOR PEER REVIEW 4 o 10
The no mali y o he da a was checked wi h he Kolmogó o –Smi no es s o he da a o he
lask cul u es. As he da a ul illed he equi emen s o pa ame ic es s, he di e ences in g ow h
be ween di e en expe imen s (1 o 3) we e he e o e compa ed by one-way ANOVA ( he Analysis
o he Va iance). The analysis was ca ied ou using IBM SPSS s a is ical so wa e, e sion 24.0 (IBM,
New Yo k, NY, USA).
3. Resul s
The clone cul u ed in he o iginal cul u e medium (con ol in all expe imen s) g ew success ully
in bo h lasks and bio eac o s (Figu es 1 and 2). The g ow h a e (mg·day−1), exp essed ela i e o he
ini ial weigh o he cul u es (g−1), was 45 and 6 mg·day−1·g−1 o lasks and bio eac o s, espec i ely,
whe eas he daily g ow h a e was, espec i ely, 184 and 557 mg·day−1. The p opo ion o g ow h
ela i e o he o al weigh (ini ial and g ow h) o he lask cul u es a ied be ween 70% and 87%
(mean = 76%; n = 13). The g ow h o he cul u e ( ela i e o he con ol) was no signi ican ly a ec ed
(p < 0.05) by he a ia ions in ei he pho ope iod (E.1) o suc ose concen a ion (E.2). The
modi ica ions o he cul u e medium (E.3 – N:P a io) nega i ely a ec ed he cul u es, leading o
signi ican ly lowe g ow h han in con ols. This di e ence inc eased as he concen a ion o P
inc eased, independen ly o he N:P a io.
Figu e 1. F esh weigh ( .w.) o he moss clone Fon inalis an ipy e ica cul i a ed in he di e en
expe imen s ca ied ou in lasks (E.1 – E.3). A, B,…, E = di e en ea men s o each expe imen (see
ex o mo e de ails). G ow h a es (mg·day−1) a e shown in i alics, g ow h a es ela i e o he ini ial
weigh (mg·day−1·g−1) a e shown be ween b acke s and bold ype, and he p opo ion o g ow h
ela i e o he o al weigh (ini ial and g ow h) a e shown in egula ype.
Figu e 1.
F esh weigh ( .w.) o he moss clone Fon inalis an ipy e ica cul i a ed in he di e en
expe imen s ca ied ou in lasks (E.1–E.3). A, B,
. . .
, E = di e en ea men s o each expe imen
(see ex o mo e de ails). G ow h a es (mg
·
day
−1
) a e shown in i alics, g ow h a es ela i e o
he ini ial weigh (mg
·
day
−1·
g
−1
) a e shown be ween b acke s and bold ype, and he p opo ion o
g ow h ela i e o he o al weigh (ini ial and g ow h) a e shown in egula ype.
The pe cen age g ow h ela i e o he o al weigh o he con ols in he bio eac o s a ied be ween
37% and 39.4% (mean = 39%; n = 4). A sligh ly highe g ow h han in he con ols was only achie ed
in ea men B in E.4. (i.e., cul u e empe a u e, 20
◦
C). G ow h was lowe in he o he expe imen s
and was up o 4 imes lowe in he expe imen simula ing sp ing condi ions (E.5).
Wa e 2019,11, 145 5 o 10
Wa e 2018, 7, x FOR PEER REVIEW 5 o 10
Figu e 2. F esh weigh o he moss clone Fon inalis an ipy e ica cul i a ed in he di e en expe imen s
ca ied ou in bio eac o s (E.4 – E.7). A, B = di e en ea men s o he expe imen (see ex o mo e
de ails). G ow h a es (mg·day−1) a e shown in i alics, g ow h a es ela i e o he ini ial weigh
(mg·day−1·g−1) a e shown be ween b acke s and bold ype, and he p opo ion o g ow h ela i e o
he o al weigh (ini ial and g ow h) a e shown in egula ype.
The pe cen age g ow h ela i e o he o al weigh o he con ols in he bio eac o s a ied
be ween 37% and 39.4% (mean = 39%; n = 4). A sligh ly highe g ow h han in he con ols was only
achie ed in ea men B in E.4. (i.e., cul u e empe a u e, 20 °C). G ow h was lowe in he o he
expe imen s and was up o 4 imes lowe in he expe imen simula ing sp ing condi ions (E.5).
The changes in pH eco ded by he bio eac o so wa e du ing E.4-Tª, E.5-Sp ing, E.6-CO2 and
E.7-Ammonium a e shown in Figu e 3. The pH o he medium in he con ol bio eac o s a ied
du ing he cul u e pe iod, om an ini ial alue o 5.62 (±0.09, n = 4) o 7.98 (±0.39). The a ia ions in
pH du ing E.6 e lec he in e mi en addi ion o CO2 du ing de elopmen o he expe imen . The
addi ion o NH4NO3 led o a dec ease in he cul u e pH o 3.43, which was accompanied by
physiological de e io a ion o he moss and a change in he colo o he apical bud (see Figu e 4A,B).
A e changing he cul u e medium o con ol condi ions (i.e., wi hou NH4NO3), he pH a ied as
usual, eaching a alue o 7.33. Cul i a ion o he moss a 20 °C led o he acidi ica ion o he medium
o pH 4.56; howe e , when shaking was no applied, he pH e u ned o 7.14.
Figu e 3. E olu ion in pH o he cul u e medium o he moss clone Fon inalis an ipy e ica o he
di e en expe imen s ca ied ou in bio eac o s (E.4 – E.7, see ex o mo e de ails) and eco ded by
he bio eac o so wa e. Black line and g ay shading: mean and in e al in con ols; blue line: E.4 A;
Figu e 2.
F esh weigh o he moss clone Fon inalis an ipy e ica cul i a ed in he di e en expe imen s
ca ied ou in bio eac o s (E.4–E.7). A, B = di e en ea men s o he expe imen (see ex o mo e
de ails). G ow h a es (mg
·
day
−1
) a e shown in i alics, g ow h a es ela i e o he ini ial weigh
(mg
·
day
−1·
g
−1
) a e shown be ween b acke s and bold ype, and he p opo ion o g ow h ela i e o
he o al weigh (ini ial and g ow h) a e shown in egula ype.
The changes in pH eco ded by he bio eac o so wa e du ing E.4-T
ª
, E.5-Sp ing, E.6-CO
2
and
E.7-Ammonium a e shown in Figu e 3. The pH o he medium in he con ol bio eac o s a ied du ing
he cul u e pe iod, om an ini ial alue o 5.62 (
±
0.09, n = 4) o 7.98 (
±
0.39). The a ia ions in pH
du ing E.6 e lec he in e mi en addi ion o CO
2
du ing de elopmen o he expe imen . The addi ion
o NH
4
NO
3
led o a dec ease in he cul u e pH o 3.43, which was accompanied by physiological
de e io a ion o he moss and a change in he colo o he apical bud (see Figu e 4A,B). A e changing
he cul u e medium o con ol condi ions (i.e., wi hou NH
4
NO
3
), he pH a ied as usual, eaching
a alue o 7.33. Cul i a ion o he moss a 20
◦
C led o he acidi ica ion o he medium o pH 4.56;
howe e , when shaking was no applied, he pH e u ned o 7.14.
Wa e 2018, 7, x FOR PEER REVIEW 5 o 10
Figu e 2. F esh weigh o he moss clone Fon inalis an ipy e ica cul i a ed in he di e en expe imen s
ca ied ou in bio eac o s (E.4 – E.7). A, B = di e en ea men s o he expe imen (see ex o mo e
de ails). G ow h a es (mg·day−1) a e shown in i alics, g ow h a es ela i e o he ini ial weigh
(mg·day−1·g−1) a e shown be ween b acke s and bold ype, and he p opo ion o g ow h ela i e o
he o al weigh (ini ial and g ow h) a e shown in egula ype.
The pe cen age g ow h ela i e o he o al weigh o he con ols in he bio eac o s a ied
be ween 37% and 39.4% (mean = 39%; n = 4). A sligh ly highe g ow h han in he con ols was only
achie ed in ea men B in E.4. (i.e., cul u e empe a u e, 20 °C). G ow h was lowe in he o he
expe imen s and was up o 4 imes lowe in he expe imen simula ing sp ing condi ions (E.5).
The changes in pH eco ded by he bio eac o so wa e du ing E.4-Tª, E.5-Sp ing, E.6-CO2 and
E.7-Ammonium a e shown in Figu e 3. The pH o he medium in he con ol bio eac o s a ied
du ing he cul u e pe iod, om an ini ial alue o 5.62 (±0.09, n = 4) o 7.98 (±0.39). The a ia ions in
pH du ing E.6 e lec he in e mi en addi ion o CO2 du ing de elopmen o he expe imen . The
addi ion o NH4NO3 led o a dec ease in he cul u e pH o 3.43, which was accompanied by
physiological de e io a ion o he moss and a change in he colo o he apical bud (see Figu e 4A,B).
A e changing he cul u e medium o con ol condi ions (i.e., wi hou NH4NO3), he pH a ied as
usual, eaching a alue o 7.33. Cul i a ion o he moss a 20 °C led o he acidi ica ion o he medium
o pH 4.56; howe e , when shaking was no applied, he pH e u ned o 7.14.
Figu e 3. E olu ion in pH o he cul u e medium o he moss clone Fon inalis an ipy e ica o he
di e en expe imen s ca ied ou in bio eac o s (E.4 – E.7, see ex o mo e de ails) and eco ded by
he bio eac o so wa e. Black line and g ay shading: mean and in e al in con ols; blue line: E.4 A;
Figu e 3.
E olu ion in pH o he cul u e medium o he moss clone Fon inalis an ipy e ica o he
di e en expe imen s ca ied ou in bio eac o s (E.4–E.7, see ex o mo e de ails) and eco ded by
he bio eac o so wa e. Black line and g ay shading: mean and in e al in con ols; blue line: E.4 A;
ed line: E.4 B; b own line: E.5; g een dashed line: E.6 and g een line: E.7. Reds poin s a e shown o
E.5 and E.7 when he cul u e condi ions we e changed.
Wa e 2019,11, 145 6 o 10
Wa e 2018, 7, x FOR PEER REVIEW 6 o 10
ed line: E.4 B; b own line: E.5; g een dashed line: E.6 and g een line: E.7. Reds poin s a e shown o
E.5 and E.7 when he cul u e condi ions we e changed.
Figu e 4. Appea ance o he moss clone Fon inalis an ipy e ica cul i a ed in bio eac o s: (A) in he
con ol cul u e medium and (B) physiological de e io a ion obse ed a e he addi ion o NH4NO3 in
he expe imen 7.
4. Discussion
Axenic cul u es o a clone o he aqua ic moss F. an ipy e ica we e success ully main ained. The
g ow h a es we e 3 imes g ea e in he bio eac o s han in lasks a e cul u e o 4 mon hs, al hough
he g ow h a e ela i e o he ini ial weigh was 7 imes highe in he lasks (see Figu es 1 and 2).
Despi e he high a e o g ow h o he clone, he inc ease in biomass was lowe han epo ed o
o he b yophy es (e.g., Sphagnum palus e) in which much g ea e inc eases in biomass we e achie ed
in only 4 weeks [9]. In o de o esol e his p oblem, we a emp ed o maximize he in i o g ow h
o he clone by modi ying bo h he cul u e medium and he di e en cul u e condi ions.
One o he echniques used o maximize he g ow h o S. palus e cul u es in bio eac o s was o
main ain cons an illumina ion [9]. Howe e , as F. an ipy e ica is a shade- ole an species, wi h
ela i ely low compensa ion and sa u a ion poin s [13,14], he low g ow h a es ela i e o he ini ial
weigh o he con ol cul u es in he bio eac o s (6 mg·day−1·g−1) and compa ed wi h he con ol
cul u es in he bio eac o s (45 mg·day−1·g−1) may indica e he pho oinhibi ion o g ow h as a esul o
he con inuous illumina ion. Howe e , he obse ed e ec s o he pho ope iod (E.1) did no indica e
any changes in g ow h wi h a pho ope iod o 16/8 o 24/0 h, and hus pho oinhibi ion can be uled
ou . The limi ing e ec o he low densi y o game ophy es pe olume o medium ela i e o hose
in he lasks is ano he possible explana ion o he low g ow h a e in he bio eac o s (see [9]) and
should be aken in o accoun in u u e s udies.
Figu e 4.
Appea ance o he moss clone Fon inalis an ipy e ica cul i a ed in bio eac o s: (
A
) in he
con ol cul u e medium and (
B
) physiological de e io a ion obse ed a e he addi ion o NH
4
NO
3
in
he expe imen 7.
4. Discussion
Axenic cul u es o a clone o he aqua ic moss F. an ipy e ica we e success ully main ained.
The g ow h a es we e 3 imes g ea e in he bio eac o s han in lasks a e cul u e o 4 mon hs,
al hough he g ow h a e ela i e o he ini ial weigh was 7 imes highe in he lasks (see
Figu es 1and 2
).
Despi e he high a e o g ow h o he clone, he inc ease in biomass was lowe han epo ed o o he
b yophy es (e.g., Sphagnum palus e) in which much g ea e inc eases in biomass we e achie ed in only
4 weeks [
9
]. In o de o esol e his p oblem, we a emp ed o maximize he
in i o
g ow h o he
clone by modi ying bo h he cul u e medium and he di e en cul u e condi ions.
One o he echniques used o maximize he g ow h o S. palus e cul u es in bio eac o s was o
main ain cons an illumina ion [
9
]. Howe e , as F. an ipy e ica is a shade- ole an species, wi h ela i ely
low compensa ion and sa u a ion poin s [
13
,
14
], he low g ow h a es ela i e o he ini ial weigh
o he con ol cul u es in he bio eac o s (6 mg
·
day
−1·
g
−1
) and compa ed wi h he con ol cul u es
in he bio eac o s (45 mg
·
day
−1·
g
−1
) may indica e he pho oinhibi ion o g ow h as a esul o he
con inuous illumina ion. Howe e , he obse ed e ec s o he pho ope iod (E.1) did no indica e any
changes in g ow h wi h a pho ope iod o 16/8 o 24/0 h, and hus pho oinhibi ion can be uled ou .
The limi ing e ec o he low densi y o game ophy es pe olume o medium ela i e o hose in he
lasks is ano he possible explana ion o he low g ow h a e in he bio eac o s (see [
9
]) and should be
aken in o accoun in u u e s udies.
Acco ding o o he au ho s, he p o ision o an addi ional sou ce o C has also been shown o be
e ec i e o inc easing he
in i o
g ow h o b yophy es (e.g., [
9
,
15
,
16
]). These au ho s demons a ed
Wa e 2019,11, 145 7 o 10
ha he addi ion o C in he o m o suc ose o glucose inc eased he g ow h o S. palus e hanks
o he abili y o pea mosses o assimila e exogenous C in he o m o suga s (mixo ophy) in he
same way ha he addi ion o CO
2
enhanced he g ow h o F. an ipy e ica. Howe e , he indings
o he p esen s udy show ha nei he he addi ion o suc ose o o CO
2
inc eased he g ow h o
his moss ela i e o he con ols (71% in E.2 A, 70% in E.2 B ela i e o 76% in he con ol lask
cul u es in and 32% in E.6 ela i e o 39% in he bio eac o con ols; Figu es 1and 2). This appea s o
indica e ha F. an ipy e ica is no u ilizing exogenous sou ces o C o maximize biomass p oduc ion.
In he case o CO
2
, a high concen a ion o his gas and/o i s in e ac ion wi h o he a iables such
as empe a u e and pho ope iod may limi he g ow h o F. an ipy e ica as he pho osyn he ic ac i i y
would be inc eased and he espi a ion dec eased due o he induc ion o s oma al closu e, as sugges ed
by Mabe ly [
17
,
18
]. This would also explain he pH luc ua ions obse ed in E.6, as he addi ion o
CO
2
leads o he acidi ica ion o he medium due o he addi ion o ca bona es. Howe e , once he
CO2is consumed du ing pho osyn hesis, he pH will again inc ease (Figu e 3).
Likewise, we in es iga ed whe he he inc ease in P concen a ion, he a ia ion in he N:P a io
(E.3) and he addi ion o NH
4
NO
3
as an addi ional sou ce o N (E.7) would enhance he g ow h o
he moss clones. N and P a e o en g ow h-limi ing nu ien s in aqua ic en i onmen s [
19
], and a
highe a ailabili y o N will cause a g ea e imbalance be ween N and P (possibly causing P o be
a limi ing nu ien o moss de elopmen [
20
,
21
]). Howe e , i is no known how hese elemen s
a ec he g ow h and de elopmen o moss in cul u e. Some au ho s ha e epo ed ha he addi ion
o N and P p omo es g ow h o b yophy es in cul u e (e.g., [
22
–
24
]) while o he s ha e obse ed a
dec ease in biomass a e he addi ion o hese nu ien s [
25
]. The esul s o expe imen s E.3 (N:P
a io) and E.7 (ammonium) a e consis en wi h he la e indings. In hese expe imen s, he g ow h o
F. an ipy e ica was no a ec ed by he inc eased concen a ion o NH
4
NO
3
(22% compa ed wi h 39%,
Figu e 2), and al hough he moss showed some de e io a ion and he apices u ned b ownish in colou ,
he appea ance imp o ed once he samples we e ans e ed o cul u e medium wi hou NH
4
NO
3
(wi h he subsequen e u n o highe pH) (Figu e 4). Con e sely, we ound ha g ow h dec eased on
he addi ion o g ea e amoun s o P (independen ly o he N:P a io used) (57–25% compa ed wi h
76%, Figu e 1).
I is gene ally es ablished ha aqua ic b yophy es do no ole a e p olonged exposu e o
empe a u es abo e 20
◦
C [
26
,
27
] and ha op imal g ow h occu s in he ange 5–15
◦
C [
28
,
29
]; mo e
speci ically a 10
◦
C in F. an ipy e ica [
27
]. The limi ed g ow h a high empe a u es may be a ibu ed
o he ac ha he apid a e o espi a ion exceeds he a e o pho osyn hesis and p oduces a dec ease
in pH ha in luences he g ow h [
30
]. Howe e , he esul s o he p esen s udy show ha he g ow h
o he cul u e a 10
◦
C was hal ha o he con ol (i.e., 24% compa ed wi h 39%; E.4 A), whe eas a
20
◦
C, he g ow h was sligh ly highe han ha o he con ol (i.e., 47% compa ed wi h 39%; E.4 B)
e en a he lowes pH in all o he expe imen s (Figu e 3). Al hough he F. an ipy e ica cul u e was
de i ed om axenic cul u es inocula ed in he bio eac o s, s e ili y checks usually yielded posi i e
esul s o bac e ial con amina ion. Tempe a u es close o 20
◦
C may a ou he g ow h o his bac e ial
communi y, which would ac as a bio e ilize , hus a ou ing he g ow h o he cul u e (e.g., [
10
,
31
]).
I is possible ha some species o Fon inalis may be mo e p oduc i e a he low empe a u es ha
occu du ing win e . Wa e low is g ea e a his ime o yea , and nu ien s a e con inually enewed,
especially in d ough -associa ed habi a s du ing pe iods o high empe a u e [
27
]. Howe e , i is
also possible ha low empe a u es may limi p oduc ion [
32
]. Conside ing he high a e o g ow h
obse ed a 20
◦
C (i.e., E.4 B), as well as he possible e ec o nu ien enewal and he beha iou o
his species unde na u al condi ions, in which we ha e obse ed a g ow h spu in he ield du ing
sp ing (pe sonal obse a ion), we simula ed sp ing condi ions (E.5) in o de o de e mine whe he he
change in empe a u e and enewal o nu ien s would a ou g ow h o he clone. The esul s we e
unexpec ed as he g ow h was almos 4 imes lowe han ha o he con ols (11% compa ed wi h 39%;
E.5) and was lowe han in any o he o he expe imen s (Figu e 2).
Wa e 2019,11, 145 8 o 10
In ligh o he abo e indings, and on he basis o ou obse a ions o he species unde na u al
condi ions, we suspec ha he e may be some as-ye unde ec ed way o maximizing he p oduc ion o
F. an ipy e ica in he bio eac o s. As commen ed abo e, he low densi y o game ophy es in he olume
o medium used in he bio eac o s ela i e o he lasks may be one o he ac o s limi ing p oduc ion [
9
].
The ac o s ha may ha e led o he low p oduc ion in he bio eac o s we e he ae a ion (con inual
in he bio eac o s bu absen in he lasks) and he ype and in ensi y o ligh used in each cul u e.
None heless, despi e he obse ed limi a ions, he
in i o
cul u e o moss in he liquid medium con ol
(see Table 1) p o ides a iable al e na i e o ield collec ion o ma e ial. This pa icula ly applies o
lask cul u e, as in addi ion o he p e iously men ioned limi a ions, we mus also conside he high
cos and main enance o he bio eac o sys ems.
5. Conclusions
None o he modi ica ions o he cul u e condi ions g ea ly inc eased he
in i o
g ow h o he
F. an ipy e ica clones wi h espec o he con ol. Howe e , he g ow h a e o he clone was su icien ly
high o enable he p oduc ion o la ge amoun s o ma e ial o he pu pose o biomoni o ing wa e
quali y. The abili y o clone he ma e ial will subs an ially imp o e he applicabili y o he moss
bag echnique.
Au ho Con ibu ions:
Concep ualiza ion, S.D., J.R.A. and J.Á.F.; Fo mal analysis, S.D., Z.V. and P.G.; Funding
acquisi ion, J.R.A. and J.Á.F.; In es iga ion, S.D., Z.V., P.G., J.R.A. and J.Á.F.; Me hodology, J.R.A. and J.Á.F.;
Resou ces, J.R.A. and J.Á.F.; Valida ion, S.D.; Visualiza ion, S.D.; W i ing—o iginal d a , S.D. and Z.V.;
W i ing— e iew & edi ing, J.R.A. and J.Á.F.
Funding:
This wo k was unded by MINECO p ojec CTM2015-70578-P. The au ho s belong o he Galician
Compe i i e Resea ch G oup GRC/GPC2016-002 and o he CRETUS S a egic Pa ne ship (AGRUP2015/02).
All o hese p og ammes a e co- unded by FEDER (EU).
Acknowledgmen s:
The au ho s a e e y g a e ul o Ve onica Fe nández González o aluable assis ance in
he labo a o y.
Con lic s o In e es :
The au ho s decla e ha he e a e no con lic s o in e es ega ding he publica ion o
his pape .
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