Es ima ing he biodeg ada ion o PHB/PBAT ilms – An expe imen al
design app oach
Miguel Fe nandes
a,b,*
, And eia F. Sal ado
a,b
, Daniel A. Madalena
a,b
, An ´
onio A. Vicen e
a,b
a
Cen e o Biological Enginee ing, Uni e si y o Minho, Campus de Gual a , B aga 4710-057, Po ugal
b
LABBELS – Associa e Labo a o y, B aga, Guima ˜
aes, Po ugal
ARTICLE INFO
Keywo ds:
Cen al composi e o a ional design
Soil biodeg ada ion
Polyhyd oxybu y a e
Polybu ylene adipa e e eph hala e
P edic ion model
ABSTRACT
The massi e use o plas ics in a ious applica ions, pa icula ly packaging, gene a es eno mous amoun s o
plas ic was e ha can be ound almos e e ywhe e, including in soil. This ep esen s a se ious en i onmen al
pollu ion issue since he biodeg adabili y in soils can ake se e al yea s, depending on he mic obial composi ion,
he physical and chemical cha ac e is ics o plas ics, and o he ele an en i onmen al ac o s such as he soil
empe a u e, he soil mois u e holding capaci y (MHC), and he ca bon:ni ogen (C/N) a io. In his wo k, we
e alua ed he impo ance o hese soil physical-chemical pa ame e s on he biodeg ada ion o a plas ic ilm
composed o PHB/PBAT. A design-o -expe imen s me hodology, namely he Cen al Composi e Ro a ional
Design (CCRD) was used o de e mine he e ec s o hese pa ame e s in he biodeg ada ion o he ilms in soil.
The ca bon dioxide e olu ion was ollowed o 6 mon hs ollowing he guidelines o he ASTM D5988 (2018).
The esul s showed ha he mos impo an ac o was he empe a u e. Highe empe a u es (≥37 ◦C) accel-
e a ed biodeg ada ion while he adding o ni ogen (aqueous solu ion o ammonium chlo ide) had no impac on
his p ocess, p obably because he C/N a io p e-exis ing in he soil was sui able o gua an ee mic obial ac i i y.
Al hough a MHC be ween 80% and 100% is ecommended by in e na ional s anda ds, 60% MHC p oduced
simila esul s. Acco ding o he me hodology, he bes combina ion possible includes a empe a u e o 37 ◦C and
a MHC anging om 60 o 100%.
1. In oduc ion
Almos 50 % o plas ic is used in disposable applica ions, mainly
packaging such as ood packaging, bo les, pla es, cups, and clo hing [1].
Conce ning packaging, only a small po ion o he was e gene a ed,
abou 29.5 million ons (collec ed plas ic pos -consume was e: 34.6 %
ecycling, 42 % ene gy eco e y, 23.4 % land illing), is eco e ed, and
ecycled in Eu ope [2]. Recycling is a e y labo ious p ocess, ha in-
ol es he collec ion om consume s and manual so ing. The plas i-
cize s, addi i es, and colo ing elemen s, ha a e pa o plas ics
signi ican ly complica e he eco e y [3]. In he case o ood packaging,
he ecycling a e is much smalle due o con amina ion wi h o ganic
subs ances including oil and le o e oods [4]. Wi hou an economic
incen i e, mos plas ics collec ed a e sen o unde - egula ed coun ies
and des ined o he land ill [5]. In he las decades, soil has become one
o he en i onmen s mos hea ily pollu ed due o he disposal o se e al
ypes o was e, including haza dous was e, o ganic pollu an s, and
non-deg adable plas ics [6].
Beyond educing he applica ion o single-use plas ics, he eplace-
men o con en ional plas ics by biodeg adable al e na i es, ha
main ain he ideal cha ac e is ics o packaging, is belie ed o be a
aluable solu ion o educe plas ic pollu ion in he oceans and soils. This
way, soil pollu ion can be educed wi h he use and de elopmen o bio-
based biodeg adable plas ics. Biodeg adable plas ics ha e he ad an age
o being mo e suscep ible o mic oo ganisms, and can be mo e easily
ecycled (e.g., h ough compos ing), which should be hei main des iny
(G´
omez and Michel, 2013). None heless, biodeg adable plas ic may also
o e esis ance o biodeg ada ion in na u al en i onmen s, which is
highly dependen on he en i onmen al condi ions. I is o u mos
impo ance o unde s and he impac o se e al pa ame e s in he
biodeg ada ion p ocess in o de o e alua e he impac o plas ic was e
disposal in soil. The e ec o empe a u e is no mally conside ed o be
signi ican du ing biodeg ada ion o polyme s in soil, e en hough i s
impac is mo e signi ican o some polyme s han o he s. Nishide e al.
[7] ound ha 52 ◦C was an op imal empe a u e o he deg ada ion o
poly(bu ylene-succina e-co-adipa e) (PBSA) in soil, bu he same
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (M. Fe nandes).
Con en s lis s a ailable a ScienceDi ec
Polyme Deg ada ion and S abili y
jou nal homepage: www.jou nals.else ie .com/polyme -deg ada ion-and-s abili y
h ps://doi.o g/10.1016/j.polymdeg ads ab.2025.111182
Recei ed 22 No embe 2024; Recei ed in e ised o m 30 Decembe 2024; Accep ed 6 Janua y 2025
Polyme Deg ada ion and S abili y 233 (2025) 111182
A ailable online 8 Janua y 2025
0141-3910/© 2025 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license ( h p://c ea i ecommons.o g/licenses/by-
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empe a u e did no a ec Polycap olac one (PCL) and Polybu ylene
succina e (PBS) biodeg ada ion when compa ed o mesophilic empe -
a u es (30 ◦C). A lowe empe a u es, below 10 ◦C, i is challenging o
ha e signi ican deg ada ion [8]. Since he mesophilic mic oo ganisms
g ow h ange o empe a u e is 10 o 45 ◦C, alues ou side his ange
may in ac limi biodeg ada ion due o he low mic obial ac i i y [9].
Ano he ele an pa ame e is wa e , which is conside ed a c ucial
ac o o he de elopmen o mic oo ganisms. Ve y low wa e con en ,
i.e., 10 % esul s in no disin eg a ion o mo e han wo mon hs [10].
Ano he impo an ac o o be conside ed is he nu ien con en o he
soil. he mos impo an nu ien o biodeg ada ion is ni ogen since i is
equi ed o he g ow h o mic oo ganisms, due o i s exp essi e p es-
ence in nucleic acids and amino acids [10]. Hoshino e al. [11] showed
h ough a s udy conduc ed in se e al soil si es ha he deg ee o
deg ada ion o se e al polyme s was mo e co ela ed wi h he o al ni-
ogen con en in se e al sampling si es han wi h he soil ca bon con-
en . Acco ding o he au ho s, his may be ela ed o he plas ic
composi ion (ca bon, hyd ogen, oxygen), which lacks ni ogen.
Mos biodeg ada ion s udies usually e alua e he e ec s o a single
ac o a a ime by changing he alue o one a iable and keeping he
o he a iables cons an [9]. Howe e , in many a eas, expe imen al
design echniques ha e been employed in p oduc and p ocess op imi-
za ion aiming a minimizing cos s and sa ing ime, as well as maxi-
mizing p oduc i i y and p oduc quali y. Beyond educing he numbe
o ials, expe imen al design analyses ac o s simul aneously and allows
he op imiza ion o mo e han one esponse a a ime [12]. Fu he mo e,
he applica ion o expe imen al design s a egies (e.g., cen al composi e
o a ional design - CCRD) esul s in he ma hema ical modula ion o he
assessed scena io so ha es ima ions can be made. This app oach can be
used in esea ch, de elopmen and p oduc ion, o example o op imize
cul u e media [13].
In his wo k we e alua ed simul aneously he e ec o se e al
physical-chemical pa ame e s o he soil on he biodeg ada ion o PHB/
PBAT bilaye ilms, by applying he cen al composi e o a ional design
(CCRD). The pa ame e s es ed we e he empe a u e, soil mois u e-
holding capaci y (MHC), and ni ogen. The in e ac ions be ween he
pa ame e s we e in es iga ed. A p edic ion model was de eloped and
es ed, o es ima e he PHB/PBAT ilm biodeg adabili y in soil.
2. Ma e ial and me hods
2.1. Soil sampling
The soil was sampled om he Uni e si y o Minho (Campus o
Gual a , B aga, Po ugal), acco ding o ISO 18,400 (2018) and as
desc ibed in o he wo k [14]. Sampling was pe o med om he su ace
o he plowing dep h, and ege a ion, oo s, moss-co e ed li e laye , o
woody plan s we e emo ed o diminish he addi ion o esh o ganic
ca bon which could mask o al e he CO
2
p oduced om he es ed
ma e ial incuba ions.
In o al 19 ials we e conduc ed using combina ions o he 3 ac o s
es ed empe a u e (9 - 45 ◦C), soil mois u e-holding capaci y (60 −100
%), and he C/N a io (ni ogen added o he soil in ela ion o he
ca bon exis ing in he plas ic) (0 - 40), u he de ail can be ound in
Table 1 and S1 (supplemen a y me hods). In he end, a alida ion ial
was conduc ed using he bes combina ion possible. The wo samplings
occu ed in Oc obe 2020 and July o 2021. The sampling o he ali-
da ion ial occu ed in July 2022. No long pe iods o d ough , eezing,
o looding we e egis e ed be o e sampling. The soil was anspo ed in
a loosely ied black polye hylene bag as ecommended by ISO 18,400
(2018) wi h ee access o ai , o a oid exposu e o ligh ha could
p omo e he g ow h o algae on he su ace o he soil, physical
compac ion, and educe a ia ions in he soil wa e con en . The soil was
sie ed in he labo a o y o 1 mm pa icle diame e and hen s o ed in
ays wi h 2 cm o heigh o main ain he ae obic condi ion and in da k
a 4 ±1 ◦C.
2.2. Physicochemical cha ac e iza ion o soil samples
The physical-chemical cha ac e is ics o he soil we e de e mined by
using he ecommended guidelines in he ASTM D5988 [15] namely he
ASTM D1293 [16], ASTM D425 [17], and SM 2540 G [18] o pH,
mois u e-holding capaci y and he con en o o al ola ile and ixed
solids espec i ely, he ca bon o ni ogen a io was de e mined by
elemen al analysis wi h a LECO T uSpec CHN, and he alues a e p e-
sen ed in he Table S1.
2.3. Biodeg ada ion o PHB/PBAT ilms – an expe imen al design
app oach
2.3.1. Biodeg ada ion assays
Biodeg ada ion expe imen s we e pe o med using 38 eac o s (3 L
wide-mou h glass ja s), inocula ed wi h he soil (250 mg). PHB/PBAT
55/45 w % bilaye ilms (11.3 ×9 cm wi h 35 µm o hickness) we e
added o he eac o s (one ilm pe eac o ), o con ol assays we e se
up, in which he blend was eplaced by cellulose pape , Wha man no 1
(9 ×8.75 cm wi h 197 µm o hickness) as ecommended by he ASTM
D5988 [15]. The biodeg ada ion was e alua ed by espi ome ic es s,
ollowing he ASTM D5988 [15] es me hod using he measu emen o
CO
2
p oduc ion by i a ion.
2.3.2. Measu emen o CO
2
The e ol ed CO
2
can be used o ollow he ae obic biodeg ada ion
since i is an indi ec measu e o O
2
consump ion, e lec ing he bio-
logical deg ada ion o he polyme . The Ti a ion me hod o measu e he
CO
2
was accomplished acco dingly o he ASTM D5988 [15] wi h he
ollowing modi ica ions: ja s we e used ins ead o desicca o s, and 0.5 M
po assium hyd oxide (KOH) solu ion (Labkem) was placed in a 150 ml
beake ins ead o a 100 ml beake . An au oma ic i a o Ti ando 888,
wi h he iamo™ 2.5 so wa e. was used o pe o m he i a ion wi h
hyd ochlo ic acid (HCL) solu ion 0.25 M ((Labbox AGR ISO). When
eplacing HCL, dis illed wa e was added o he soil o conse e he
ini ial mois u e-holding capaci y. B ie ly, he e ol ed CO
2
was cap u ed
by he KOH solu ion, c ea ing po assium bica bona e. Then he quan i y
o he CO
2
p oduced was de e mined by i a ing he KOH solu ion wi h
HCL. The ne CO
2
p oduced om he es ma e ial was calcula ed by
sub ac ing he a e age amoun o CO
2
p oduced in he soil con ol ja s
om he amoun o CO
2
p oduced in he es ma e ial ja s. The
biodeg ada ion pe cen ages we e calcula ed om he a io be ween he
ne CO
2
p oduc ion and he heo e ical CO
2
p oduc ion based on he
ca bon con en , o he es ed ma e ial.
2.3.3. PHB/PBAT ilms
The plas ic was a bilaye ilm o PHB/PBAT 45/55 h w %. The PHB
was an expe imen al PHB g ade (Biome P309) supplied by Biome
(K ailling, Ge many). The PBAT was a comme cial alipha ic-a oma ic
copolyes e -based polyme (Eco lexVR F blend C1200, a ilm-blowing
g ade). De ails abou he co-ex usion o he bilaye ilm can be ound
in he wo k o Teixei a e al., [19]. Di e en amoun s o he blend and
cellulose we e added o he bio eac o s o ob ain an equi alen mass o
o ganic ca bon, acco ding o Table S2.
Table 1
Va iables and le els used o he CCRD.
Independen Va iables −1.68 −1 0 1 1.68
C/N a io 0 6.1 15.0 23.9 30.0
Tempe a u e ( ◦C) 9.0 16.3 27.0 37.7 45.0
Soil Mois u e capaci y (%) 60.0 68.0 80.0 92.0 100.0
M. Fe nandes e al.
Polyme Deg ada ion and S abili y 233 (2025) 111182
2
2.3.4. Design o expe imen s
2.3.4.1. Expe imen s. A design o expe imen s (DoE) me hodology was
used o iden i y he e ec s o di e en pa ame e s and hei in e ac ions
on he biodeg ada ion p ocess: soil empe a u e (⁰C), soil mois u e-
holding capaci y (%), and he C/N a io (ni ogen added o he soil in
ela ion o he ca bon exis ing in he plas ic). The biodeg ada ion was
de e mined h ough he analysis o he accumula ed CO
2
(using he
same me hod desc ibed be o e and based on he ASTM D5988 [15]. A
h ee- a iable CCRD (2³) was used, wi h i e epe i ions o he cen al
poin (0) and six axial poin s. The addi ion o axial poin s (−1.68 and
+1.68) enables he adjus men o he da a o a second-o de model and,
consequen ly, o con i m he exis ence o cu a u e [12]. Va iables and
le els a e demons a ed in able 1.
In o al, 19 assays we e pe o med, as indica ed in Table S3. Since he
assays we e conduc ed in wo pa s, wi h wo soils, 5 assays o he
cen al poin we e included ins ead o 3 (i.e., 3 o he i s soil and 2 o
he second soil). The soils we e collec ed om he same si e, and p e-
sen ed simila p ope ies, al hough sligh di e ences may be possible,
o example a he mic obial communi y’s composi ion le el. The DoE
da a we e analysed using P o imiza Expe imen al Design So wa e
(B azil. h ps: //expe imen al-design.p o imiza.com.b /).
A alida ion ial was u he pe o med o con i m and assess he
quali y o he DoE. The alues o he alida ion ial, whe e he mos
signi ican o each pa ame e . In cases whe e he pa ame e s we e no
ele an , he cen al poin we e selec ed.
2.3.4.2. PHB/PBAT ilms biodeg ada ion p edic ion. The biodeg ada ion
o PHB/PBAT ilms was assessed o 6 mon hs, and a ma hema ical
modula ion o he PHB/PBAT ilms biodeg ada ion o each mon h o
he assay was made. Wi h his app oach, i is possible o co ela e he
physical-chemical cha ac e is ics o he es ed soil wi h he ilms’
biodeg ada ion. I is impo an o co ela e and es ima e he bioplas ics’
biodeg adabili y h ough ime by co ela ing he di e en physical-
chemical cha ac e is ics o he soil wi h he biodeg ada ion ime o he
biopolyme s. Fo his pu pose, a eg ession model (Eq. (1)) was adjus ed
using he expe imen al se up o he p e ious CCRD (Table S3)
y=
α
∗ln( ) + β(1)
Whe e y co esponds o he ilms’ biodeg adabili y and co esponds o
ime (in mon hs). I was also possible o obse e ha he wo coe icien s
o he eg ession model desc ibed in Eq. (1) a e ela ed o he physical-
chemical cha ac e is ics o he soil. As such, a simila CCRD was used o
de e mine he equa ions o calcula e he coe icien s
α
( esponsible o
he e ical s e ch o he cu e) and β ( esponsible o he ho izon al
displacemen o he cu e).
These equa ions can be used, o p edic he biodeg ada ion o he
ma e ial es ed in his wo k in simila soils, wi h a p e ious cha ac e -
iza ion o he soil ( empe a u e and mois u e holding capaci y). and he
sample (elemen al analysis o ca bon de e mina ion).
2.4. Analy ical me hods
To e alua e he e ec o he biodeg ada ion on ilm p ope ies.in he
alida ion assay, he PHB/PBAT ilms we e analyzed be o e and a e
he incuba ions o e i y he e ec s on he unc ional g oups using
A enua ed To al Re lec ance-Fou ie T ans o m In a ed Spec oscopy
(ATR-FTIR) and Di e en ial Scanning Calo ime y (DSC) o e alua e he
changes in he he mal p ope ies.
2.4.1. A enua ed o al e lec ance- ou ie ans o m in a ed spec oscopy
(ATR-FTIR)
To assess he e ec s o he PHB/PBAT biodeg ada ion on he
chemical s uc u e o he ilms, an ATR-FTIR analyses an ALPHA II-
B uke spec ome e (E lingen. Ge many) was used acco ding o he
p ocedu e desc ibed by Sil a e al., [20].
2.4.2. Di e en ial scanning calo ime y (DSC)
A Pe kin Elme DSC 4000 Di e en ial Scanning Calo ime e (Pe kin
Elme . Wal ham. MA. USA). An assay based on he wo k o Sil a e al.
[21] was used o he assessmen o he ilms’ he mal p ope ies.
Howe e , only po ions o he ilm (8 mg each) we e used, and he
p o ocol was se o: hea ing a 10 ⁰C/min om 5 ⁰C o 190 ªC o e ase he
he mal his o y; cooled down o −25 ⁰C a −10 ⁰C/min; hea ing o 190
⁰C a 10 ⁰C/min. F om he inal hea ing un he mel ing (Tm) and
c ys alliza ion empe a u es (Tc) we e de e mined. The onse empe a-
u es (Tm Onse ) and mel ing en halpies (ΔH) we e calcula ed using
Py is so wa e e sion 12.1 (Pe kin Elme . Wal ham. MA. USA). The
c ys allini y (Xc) o he samples was de e mined acco dingly wi h Bebe
e al., [22], by he a io o he mel ing en halpy (ΔH0) o 100 % c ys-
alline PHB o PBAT, which is 146 J/g [23] and 114 J/g espec i ely
[24]. The absolu e c ys allini y can be calcula ed acco ding o Eq. (2):
Xc =ΔH
ΔH0 x100 (2)
2.5. S a is ical analysis
All he DSC alues we e exp essed as mean ±s anda d de ia ion
(SD) o he ini ial and inal PHB/PBAT ilms shee s be o e and a e
deg ada ion. The da a was assessed using one-way analysis o a iance
(ANOVA) ollowed by Tukey´s es using O iginPRO 2019b s a is ical
p og am and he s a is ical signi icance was accep ed as p <0.05 (95 %
signi icance). The p incipal componen analysis (PCA) conduc ed on he
expe imen al ials o he CCRD was also pe o med using he same
so wa e.
3. Resul s
3.1. Design o expe imen s o he biodeg ada ion assay
The physicochemical p ope ies o he soil we e wi hin he ecom-
mended anges by he ASTM D5988 [15], namely he pH was in he
ange 6 - 8 in all ials as indica ed in Table S1. Mo e impo an ly, he
di e ences among he pH o he di e en soils we e qui e small, which
con ibu ed o he maximum homogenei y possible be ween he ials.
The alues o biodeg ada ion ob ained, a ied be ween 28.65 % and
60.73 %, which co esponded o he condi ions o ial 11 (lowes
empe a u e) and 8, espec i ely (Table 2). The 19 ials pe o med o
he DoE demons a ed ha he inc eased empe a u e esul ed in an
inc eased biodeg ada ion. The PCA analysis (Fig. 1) also suppo ed his
conclusion. In he ials whe e 37.7 ◦C o highe empe a u es we e used
( ials 3, 4, 7, 8 and 12) he biodeg ada ion eached >51 % a e 6
mon hs, while wi h he lowe empe a u es es ed he maximum
biodeg ada ion ob ained was o 48. 2 % ( ial 15). Wi h he wo lowes
empe a u es es ed (9 and 16.3 ◦C), he biodeg ada ion ne e passed
he 41 % ba ie ( ial 5). The mos signi ican ca bon mine aliza ion
cu es a e ep esen ed in Figu e S1.
All he pa ame e s which we e no signi ican we e elimina ed om
he CCRDs equa ions. The esul s o he CCRDs demons a ed ha he
empe a u e was he only pa ame e es ed ha was signi ican in all he
CCRDs pe o med each mon h (Table 3). The humidi y was jus signi -
ican in he six h mon h, and in in e ac ion wi h empe a u e in he hi d
and ou h mon hs. The C:N a io was only signi ican in in e ac ion wi h
he empe a u e in all mon hs, excep o he i s one (Table 3).
3.2. A mul i a ia e pe spec i e
In he PCA analysis (Fig. 1) he p incipal componen 1 (PC1) and
p incipal componen 2 (PC2) explain 71.52 % and 11.55 % o he da a
a iabili y, espec i ely. Consequen ly, i is impo an o men ion ha
M. Fe nandes e al.
Polyme Deg ada ion and S abili y 233 (2025) 111182
3
he ho izon al dis ance be ween he di e en poin s is signi ican ly mo e
impo an han hei e ical displacemen . As such, i is possible o see
in Fig. 1 he ca ego iza ion o he expe imen al ials acco ding o he
expe imen al empe a u e, wi h he condi ions wi h he highe em-
pe a u es on he igh (45 and 37 ⁰C), he middle empe a u e (27 ⁰C) on
he cen e and he lowe empe a u es on he le (9 and 16 ⁰C). The PCA
clea ly shows ha he empe a u e was he mos impo an ac o since
i is mainly ep esen ed by he PC1. Mo eo e , i is also possible o
con i m ha in ac , highe empe a u es p omo e a highe deg ada ion
o he PHB/PBAT ilms since he loadings indica e a co ela ion be ween
he expe imen al empe a u e and he biodeg adion on each mon h. I
was also possible o con i m ha bo h humidi y and C/N did no
signi ican ly impac he biodeg ada ion o he polyme s due o hei
o hogonal placemen when compa ed o he empe a u e and biodeg-
ada ion on each mon h.
3.3. Analy ical analysis o he alida ion ial
The analy ical echniques e ealed ha biodeg ada ion du ing he
alida ion ial (C/N a io o 20, 37.7 ⁰C empe a u e and 80 % o soil
holding capaci y), was mainly a ibu ed o he PHB laye . In Figu e S2 is
possible o see he di e ence be ween he ini ial ilm and he inal es-
idues o he alida ion ial. The ATR-FTIR demons a ed ha he PHB
laye was he main con ibu o o he biodeg ada ion since no PHB
spec a we e p esen on he esidues a he end o he assay (Figu e S3).
The PBAT laye p esen ed clea signs o se ious biodeg ada ion wi h a
ma ked educ ion in all peaks, such as he a oma ic (1267 cm
-1
) and he
ca bonyl abso bance peak (1709 cm
-1
) Figu e S3) [25]. Se e al small
new peaks de eloped (e.g., 3937 3872, 3745, 3694, 3617, 3289 cm
-1
)
some o hem ela ed o he o ma ion o ee O
–
H and hyd oxyl and
ca boxylic, linked o he scission in he main chain esul ing om he
hyd olysis o he ac ion o enzymes [26]. The DSC analysis also e ealed
ha he PHB disappea ed om he sample and all he pa ame e s we e
signi ican ly a ec ed (p <0.05), excep o he empe a u e mel ing
onse (Table S4). The inc eased c ys allini y (Table S4) demons a ed
he p e e ence o biodeg ada ion o he amo phous a eas o he PBAT
laye [27]. The PBAT mel ing empe a u e inc eased signi ican ly,
indica ing he biodeg ada ion and hyd olysis o he alipha ic pa which
lead o an imp o ed c ys alline s uc u e wi h ewe de ec s [28]. I is
also no ewo hy ha in he ials wi h he empe a u e in he ange
ecommended by he s anda d me hod (20–28 ⁰C) he con ol biodeg-
ada ion (cellulose), was equal o supe io o he 70 % <necessa y o
he assay o be conside ed alid. The same was obse ed a 16.3 ⁰C and
45 ⁰C, howe e a 9 ⁰C and 37.7 ⁰C he condi ion was no achie ed
(Table S5). This may happen, because some empe a u es a ou he
cellulose-deg ading mic oo ganisms o he e a e some di e ences in he
Table 2
Pe cen age o biodeg ada ion o he PHB/PBAT ilms du ing 6 mon hs o each DoE ial. The alida ion was pe o med in iplica e. SD - S anda d de ia ion.
T ials C/N a io T ( ◦C) MHC (%) Biodeg ada ion (%)
Mon hs
123456
1 8.1 (−1) 16.3 (−1) 68.1 (−1) 5.31 14.68 18.35 27.31 33.23 38.44
2 31.9 (1) 16.3 (−1) 68.1 (−1) 1.89 6.36 14.67 22.77 27.19 32.93
3 8.1 (−1) 37.7 (1) 68.1 (−1) 34.36 41.31 42.12 46.12 48.89 51.53
4 31.9 (1) 37.7 (1) 68.1 (−1) 32.18 42.55 46.51 49.01 54.36 59.30
5 8.1 (−1) 16.3 (−1) 91.9 (1) 4.50 19.71 26.35 31.93 35.45 40.58
6 31.9 (1) 16.3 (−1) 91.9 (1) 2.79 9.69 18.47 31.22 34.02 39.53
7 8.1 (−1) 37.7 (1) 91.9 (1) 31.05 36.51 38.22 43.85 52.52 57.63
8 31.9 (1) 37.7 (1) 91.9 (1) 27.32 43.65 45.32 47.68 53.50 60.73
9 0.0 (−1.68) 27.0 (0) 80.0 (0) 6.49 23.13 27.33 32.42 40.70 42.64
10 40.0 (1.68) 27.0 (0) 80.0 (0) 9.01 26.97 31.83 36.73 42.42 47.99
11 20.0 (0) 9.0 (−1.68) 80.0 (0) 5.32 14.76 18.06 21.45 25.12 28.65
12 20.0 (0) 45.0 (1.68) 80.0 (0) 12.41 38.36 51.70 55.45 57.06 58.53
13 20.0 (0) 27.0 (0) 60.0 (−1.68) 16.79 25.06 30.84 37.79 43.30 45.06
14 20.0 (0) 27.0 (0) 100.0 (1.68) 14.52 27.12 35.31 37.82 40.22 44.87
15 20.0 (0) 27.0 (0) 80.0 (0) 11.80 23.24 30.70 36.40 45.28 48.23
16 20.0 (0) 27.0 (0) 80.0 (0) 12.45 25.24 34.58 33.70 39.96 43.67
17 20.0 (0) 27.0 (0) 80.0 (0) 14.57 26.30 29.64 33.24 40.97 46.80
18 20.0 (0) 27.0 (0) 80.0 (0) 14.73 25.33 32.74 37.38 43.34 47.64
19 20.0 (0) 27.0 (0) 80.0 (0) 15.32 24.23 28.91 34.03 40.43 45.97
Valida ion (A e age ±SD) 20 37.7 80 22.79 ±1.58 36.54 ±1.14 42.0 ±2.31 45.97 ±1.85 51.09 ±2.28 55.28 ±1.72
Fig. 1. P incipal componen analysis o he biodeg ada ion expe imen al ials
( ep esen ed by T1-T14, and TC o he cen al poin s) whe e H ep esen s he
soil’s humidi y, R co esponds o he C/N a io, T co esponds o he empe -
a u e and M(1–6) o he mon h. 919.
Table 3
DoE equa ions o each mon h. x
1
– C/N a io, x
2
– empe a u e, x
3
– mois u e
holding capaci y.
Mon hs DoE Equa ions
1 Y₁ =14.38 +8.98 x₂R² =52.86 %
2 Y₂ =26.18 +11.07 x₂ +2.93 x₁ x₂R² =89.24 %
3 Y₃ =31.66 +11.07 x₂ +2.88 x₁ x₂ - 2.09 x₂ x₃R² =94.67 %
4 Y₄ =36.15 +9.56 x₂ +0.99 x₂² +1.50 x₁ x₂ - 2.08 x₂ x₃R² =95.89 %
5 Y₅ =41.99 +9.75 x₂ +1.74 x₁ x₂R² =95.45 %
6 Y₆ =46.51 +9.37 x₂ +1.17 x₃ +2.19 x₁ x₂R² =92.90 %
M. Fe nandes e al.
Polyme Deg ada ion and S abili y 233 (2025) 111182
4
communi ies in each eac o , and hus di e en biodeg ada ion po en-
ial. Fu he mo e, he highe biomass build-up and consequen ca bon
e en ion, may happen a supe io empe a u es in his case 37.7 ⁰C [29].
This could explain he in e io biodeg ada ion obse ed in some ials
using his empe a u e.
3.4. Valida ion and es ima ion he biodeg ada ion o PHB/PBAT ilms
Es ima ing he biodeg ada ion o PHB/PBAT ilms could be a e y
use ul s a egy o de e mine he biodeg ada ion o PHB/PBAT ilms in
soils. Fo his pu pose, a alida ion assay was used, wi hin he es ed
pa ame e s’ ange, wi h a di e en combina ion o expe imen al con-
di ions, i.e., a C/N a ion o 20, a 37.7 ⁰C and a soil mois u e capaci y o
80 %. The esul s o he alida ion ial a e depic ed in Fig. 2.
Fig. 2 shows ha he es ima ed and expe imen ally ob ained
biodeg ada ion alues in he alida ion ial ha e a 0.98 co ela ion
coe icien . A loga i hmic eg ession model was u he used o es ima e
he biodeg ada ion o PHB/PBAT ilms by co ela ing he physico-
chemical cha ac e is ics o he soil wi h he biodeg ada ion a a speci ic
ime. Thus, he equa ions o calcula e he model’s coe icien s we e
ob ained using a CCRD and he esul s a e exp essed in Eq. (3) and Eq.
(4).
α
=17.34 +x2+0.13x2
2+0.37x3+0.07x1x2+0.42x2x3(3)
β=13.80 +x1+9.97x2+0.02x2
2−0.21x3+1.43x1x2+0.79x2x3(4)
Whe e x
1
co esponds o C/N a ion, x
2
co esponds o he soil em-
pe a u e and x
3
co esponds o he mois u e-holding capaci y. This way,
he loga i hmic model (Eq. (1)) can be u he used o es ima e he
biodeg ada ion o PHB/PBAT ilms h ough ime and he esul s a e
depic ed in Fig. 3.
I is possible o obse e in Fig. 3 ha he p edic ion model (Eq. (1))
can p edic wi h high accu acy he biodeg ada ion o PHB/PBAT ilms
since a co ela ion be ween he es ima ed and expe imen al esul s is ca.
0.99 which can be conside ed a high co ela ion, conside ing ha his
p ocess is mainly media ed by he p esence o mic oo ganisms which
can p esen a high a iabili y. The co ela ion is 0.93 be ween all he
expe imen al alues indica ed in able 2 and he es ima ions o he
model unde he same condi ions.
4. Discussion
Conside ing he alues ecommended by he s anda d me hod [15]
o he C:N a io (ni ogen added o he soil in ela ion o he ca bon in
he es specimen, in his case in he o m o ammonium chlo ide),
al hough 0 and 40:1 we e used, his pa ame e had no clea impac on
he inal biodeg ada ion, no being signi ican in any equa ion (Table 3).
Kijcha engkul e al. [27] ound ha compos wi h a highe C:N a io
han he ecommended, esul ed in less polyme biodeg ada ion. I has
been obse ed in o he wo k ha he ca bon inpu p esen ed by he
PHBV deg ada ion may cause he mic obial immobiliza ion o ni ogen
sou ces o sus ain mic obial g ow h, since he PHBV deg ada ion
inc eased by 45 % he mic obial biomass ni ogen and educed he
dissol ed o ganic ni ogen by 66 %. [30]. In his wo k, he addi ion o
ni ogen (ammonium chlo ide) did no ha e a signi ican impac on he
biodeg ada ion o he PHB/PBAT ilms, p obably because he ca bon o
he polyme and ni ogen added we e no su icien o signi ican ly shi
he C:N a io exis ing in he soil, which explains he simila biodeg a-
da ion be ween all he C:N a io condi ions es ed. In hese biodeg a-
da ion es s, ni ogen was no limi ing as shown by he close
biodeg ada ion alues be ween he in e media e (20) C:N a io (43.7 % -
48.2 %, ials 14 o 19) and he lowes (0) and highes (40) C:N a io
es ed, 42.6 % ( ial 9) and 48 % ( ial 10) espec i ely.
Rega ding humidi y, i is ecommended an 80 o 100 % MHC [15].
Howe e , lowe alues we e used in his s udy and his pa ame e had
no signi ican impac on biodeg ada ion in he es ed ange ( om 60 o
100 %) only di e ing by 0.2 % ( ials 13 and 14). Wa e abso p ion can
in e ac wi h he polyme s uc u e causing hyd oly ic deg ada ion
(abio ic p ocess), esul ing in he clea age o es e bonds [31]. In he
case o PBAT, he abso p ion o wa e in he amo phous egions o he
a oma ic polyes e can cause swelling and s ess esul ing in mic o-
ca i a ion [32]. The deg ada ion o PHB is also highe in soils wi h mo e
wa e con en han in soils wi h less wa e [33]. The mois u e con en
can hus in luence he deg ada ion. P oduc s wi h se e al ma e ials,
such as PHAs can e en p esen a supe io wa e abso p ion pe cen age,
o example, PHBHHx/KF p esen ed a highe wa e abso p ion pe -
cen age and a highe pe cen age o weigh loss du ing a soil deg ada ion
s udy when compa ed o he PHBHHx [34]. B iassoulis and Mis io is
[8] used he same s anda d me hods, o es lub ican s, and ecom-
mended also a ange o 60 % o 100 % o he soil wa e holding capaci y
and an ideal alue o 80 % o he g ea es possible disin eg a ion a e.
Chinaglia e al., [35] indica ed di e ences in he biodeg ada ion o
Ma e -bi (a comme cial, biodeg adable plas ic) be ween soils wi h 55 %
and 88 % MHC using he same s anda d me hod, al hough wi h bo h
soils, he plas ic ma e ial has exceeded he biodeg adabili y h eshold o
90 % de ined by he s anda d EN 17,033. These esul s may explain why
he soil’s mois u e in he p esen biodeg ada ion assay did no p esen
signi ican di e ences in he ange used, i.e., i was al eady wi hin he
op imal ange.
I is expec ed ha en i onmen al pa ame e s ha e an immedia e
e ec on biodeg adable plas ic build-up a e elease (acciden al o
in en ional) since hey di ec ly in luence he plas ic biodeg ada ion
a es. Fo modeling pollu an deg ada ion hal -li es in chemical a e
assessmen and exposu e models used o isk assessmen pu poses, he
empe a u e is used as a p edic o a iable [36]. The ange o empe -
a u es ecommended by he s anda d me hods is be ween 20 ◦C and 28
◦C. The inc eased empe a u e es ed, namely 37.7 and 45 ◦C, inc eased
he biodeg ada ion by 10.8 % and 11.5 % espec i ely. This obse a ion
could be due o he abio ic deg ada ion o /and he enhanced ac i i y o
some mic oo ganisms in hese condi ions. Kim e al. [37] demons a ed
ha he deg ada ion o PHB was highe in di e en soils a 37 ◦C han a
28 ◦C. These di e ences we e caused by he ac i i y o he soil mic obial
communi y, which is s ongly in luenced by he empe a u e o he
en i onmen . PBAT deg ada ion is also as e a compos ing empe a-
u es (i.e., 55 ◦C) [38,27]. I was expec ed ha highe empe a u es
should inc ease o a leas accele a e he biodeg ada ion p ocess. This
was con i med by he PCA analysis (Fig. 1). Tempe a u e can e ec i ely
dis u b chemical and biochemical eac ions and in luence he axonomic
s uc u e and me abolic ac i i ies o he communi ies [39]. Usually, he
biodeg ada ion p ocess inc eases wi h empe a u e inc ease un il i
eaches a pla eau (i.e., whe e i is included in he he mal op imum) and
inally, as he empe a u e inc eases mo e, he biodeg ada ion de-
c eases, because enzymes s a o dena u e, and he biodeg ada ion
Fig. 2. Compa a ion be ween he expe imen al alida ion ial and he p e-
dic ion model using mon hly equa ions.
M. Fe nandes e al.
Polyme Deg ada ion and S abili y 233 (2025) 111182
5
ou come becomes ine ec i e [40]. This e ec explains he small di -
e ence in he biodeg ada ion be ween he 37.7 ◦C and 45 ◦C condi ions,
which was only 1.3 % mo e, o he highe empe a u e.
Fo his speci ic bilaye ilm, an inc ease in empe a u e was bene-
icial unde he es ed soil cha ac e is ics. Al hough wi h 45 ◦C he
biodeg ada ion was sligh ly be e , a empe a u e a ound 37 ◦C should
be used o maximize soil biodeg ada ion since i equi es less ene gy o
be main ained. Fu he mo e, unde his empe a u e condi ion (i.e., 37
◦C) he maximum empe a u e o 45 ◦C o he g ow h o mesophilic
mic oo ganisms is a oided, and he mic obiome is p obably no signi -
ican ly shi ed. The mic obiome is no mally e icien in he deg ada ion
o nea PHAs in empe a u es be ween 20 - 30 ◦C. PBAT is compos able
and se e al he mophilic o ganisms ha e been iden i ied, wi h highe
empe a u es bene i ing he deg ada ion o his polyme [41]. The
abio ic e ec a hese empe a u es may be also highe [41]. This e ec
in combina ion (o no ) wi h he mo e e ec i e ac ion o he PBAT
deg ading mic oo ganisms caused he enhanced deg ada ion in his
wo k. The analysis o he communi ies in di e en condi ions can be
e y in e es ing o es in u u e wo ks.
The esul s indica e ha he PHB ac ion was mo e biodeg aded in
he alida ion ial (Table S4 and Figu e S3). The PHB polyme was no
longe de ec ed a e he assays in he ilm by he ATR-FTIR and DSC
analyses. The PBAT was less deg aded bu also con ibu ed o he CO
2
e olu ion, which was con i med by he de elopmen o small peaks
ela ed o he scission in he main chain, and he PBAT inc eased c ys-
allini y ela ed o he deg ada ion o he amo phous egions.
The 0.98 co ela ion coe icien (Fig. 2) ob ained o he es ima ed
and expe imen ally biodeg ada ion alues (Valida ion ial) indica es
ha he DoE app oach can be used, no only o op imize a p e-
de e mined p ocess ( he mos common applica ion o DoE) bu also used
o modula e he biodeg ada ion o bioplas ics using ewe ials. The
quali y o he p edic ion model (Fig. 3) enables he possibili y o p edic
he soil biodeg ada ion o hese ilms a any gi en empe a u e, MHC
and C:N a ios, wi hin he es ed ange. Fo example, in B aga ( he a ea
whe e he soil was sampled) he annual empe a u e o soils a a plough
dep h in 2021 was be ween 1.3 ◦C and 26.2 ◦C (IPMA - Ins i u o
Po uguˆ
es do Ma e da A mos e a (pe sonal communica ion)), being he
a e age soil empe a u e o 14.4 ◦C. Conside ing he MHC o he soil is
no in e io o he h eshold es ed (60 %) and no ni ogen is added o
he soil in he case o he elease o he ilms o he en i onmen , and he
ype o soil is simila o he one used o p oduce he model, he model
es ima es ha he o al deg ada ion will each in 6 mon hs, 35.74 %.
Ins ead o he empe a u e annual a e age, he alue o any pa ame e
can be changed o e ime, du ing he p edic ion, using he eal alues
measu ed and ollowing he seasonal changes. Using he same model
beyond he 6 mon hs es ed (Fig. 3), i is possible o conside ha he
bilaye ilm would each almos 70 % biodeg ada ion in 12 mon hs.
Howe e , hese ypes o ex apola ions beyond 6 mon hs should only be
conside ed indica i e since he model was buil using es ed (expe i-
men al) alues o 6 mon hs.
This model is a ool ha conside s se e al a iables, none heless, i
has se e al simpli ica ions and ep esen s an app oxima ion o he
biodeg ada ion and no an exac p edic ion. Fo example, UV ligh can
in e ac wi h polyme s. The pho odeg ada ion can cause c osslinking
wi hin he ilm due o he ecombina ion o he p oduced ee adicals
om No ish I. Kijcha engkul e al. [32] obse ed he pho odeg ada ion
o PBAT ilm in Cos a Rica soils, wi h he o ma ion o ee adicals.
Howe e , Kijcha engkul e al. [42] disco e ed ha he c osslinking
caused a dec ease in he biodeg ada ion o PBAT ilms in compos ing.
This and o he ac o s, including nu ien s and ype o soil could be
conside ed o enhance he quali y o he model. Ano he aspec ha
should be add essed is he cha ac e is ics o he polyme s being es ed.
Phase ansi ion empe a u es (glass ansi ion o mel ing empe a u e)
o he polyme s in his ange (20–37 ◦C) can change he biodeg ada ion
p ocess. Fo example, o PLA, abo e he glass ansi ion empe a u e,
he biodeg ada ion is imp o ed, because he chains in he amo phous
egions become lexible [43]. These ypes o ques ions need o be aken
in o conside a ion when es ing o he polyme s.
5. Conclusion
The key pa ame e o he biodeg ada ion e iciency o his bilaye
ilm in soil was he empe a u e, wi hin he ange be ween 37.7 and 45
◦C. In his case, o accele a e he biodeg ada ion in soil, a alue o 37 ◦C
was ound as he bes , since i equi es less ene gy, han he highe
empe a u es es ed. The ange o MHC (60 - 100 %) es ed, esul ed in
simila ou comes indica ing ha any alue wi hin his ange can be used
o p ope ly es biodeg ada ion o plas ics. The addi ion o ni ogen in
he o m o ammonium chlo ide didn’ in luence biodeg ada ion, which
indica es ha he soil con ained he necessa y ni ogen and an adequa e
C:N a io (wi hin he ange ecommended by he s anda ds). The esul s
o his wo k could also help o sa e ime on biodeg ada ion plas ic
es ing, i he bes condi ions a e applied.
CRediT au ho ship con ibu ion s a emen
Miguel Fe nandes: W i ing – e iew & edi ing, W i ing – o iginal
d a , So wa e, Resou ces, Concep ualiza ion. And eia F. Sal ado :
Fig. 3. Compa ison be ween he expe imen al alida ion ial (do s) and he p edic ion model using he inal equa ion (line) o 6 mon hs and p edic ion using he
alida ion pa ame e s beyond he 6 mon hs es ed (dashed line).
M. Fe nandes e al.
Polyme Deg ada ion and S abili y 233 (2025) 111182
6
W i ing – e iew & edi ing. Daniel A. Madalena: W i ing – e iew &
edi ing, Me hodology, Concep ualiza ion. An ´
onio A. Vicen e: Me h-
odology, Funding acquisi ion.
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 suppo ed by he Po uguese Founda ion o Science
and Technology (FCT) unde he scope o he s a egic unding o UIDB/
04469/2020 uni , and by LABBELS – Associa e Labo a o y in Bio ech-
nology, Bioenginee ing and Mic oelec omechanical Sys ems, LA/P/
0029/2020. Fu he mo e, his s udy was also suppo ed by BioTecNo e
ope a ion (NORTE-01–0145- FEDER-000004) unded by he Eu opean
Regional De elopmen Fund unde he scope o No e2020 - P og ama
Ope acional Regional do No e. The ellowship is suppo ed by a
Doc o al ad anced aining (call NORTE-69–2015–15) unded by he
Eu opean Social Fund unde he scope o No e2020 - P og ama Ope -
acional Regional do No e. Finally, by he doc o al g an PD/BD/
146195/2019 and he g an COVID/BD/153308/2023.
Supplemen a y ma e ials
Supplemen a y ma e ial associa ed wi h his a icle can be ound, in
he online e sion, a doi:10.1016/j.polymdeg ads ab.2025.111182.
Da a a ailabili y
Da a will be made a ailable on eques .
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