Upcycling depolymerized PET waste into polyhydroxyalkanoates and triacylglycerols by a newly isolated Rhodococcus sp. strain.

Rebochoe al.
Bio echnology o he En i onmen (2025) 2:5
h ps://doi.o g/10.1186/s44314-025-00019-4
RESEARCH Open Access
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Bio echnology o
he En i onmen
Upcycling depolyme ized PET was e
in opolyhyd oxyalkanoa es and iacylglyce ols
byanewly isola ed Rhodococcus sp. s ain
Ana Te esa Rebocho1,2, C is iana A. V. To es1,2*, Helena Koninckx3, Lu ga S agie 3, Oli ia A. A allah4,
Ma ija Mojice ic4, Cuney E dinc Tas4, Ma ga e B ennan Fou ne 4, Ma ia A. Reis1,2 and Filomena F ei as1,2
Abs ac
The use o pos -consume polye hylene e eph hala e (PET) was es, which o en con ain a ious addi i es and con-
aminan s such as me als and pigmen s ha make mechanical ecycling and eusabili y di icul , as eeds ocks
o mic obial syn hesis o alue-added bio-based p oduc s is an eme ging sus ainable s a egy o managing such
was es. This s udy e alua ed he abili y o a s ain isola ed om a plas ic-con amina ed si e, Rhodococcus sp. isola e
A e7, o use e eph halic acid (TPA) ob ained by chemically depolyme izing PET was e, as sole eeds ock o cell
g ow h and p oduc ion o polyhyd oxyalkanoa es (PHAs) and iacylglyce ols (TAGs) as in acellula s o age com-
pounds. The ed-ba ch bio eac o cul i a ion esul ed in a cell d y weigh p oduc ion o 3.85 g/L, wi h PHA and TAG
con en s o 15.0 w .% and 15.4 w .%, espec i ely. O e all, he cul u e consumed 16.5 g/L TPA o e a pe iod o 73 h.
The p oduced PHA was mainly composed o 3-hyd oxy ale a e (3HV) monome s (> 90 w .%). The accumula ed TAGs
p esen ed a a y acids p o ile ich in oc adecenoic acid (C18:1; 52 w .%), hexadecanoic acid (C16:0; 32 w .%) and oc a-
decanoic acid (C18:0; 12 w .%). O e all, he s ain Rhodococcus sp. A e7 demons a ed a high capaci y o TPA emo al,
con e ing i in o cell biomass, PHA and TAGs, hus ende ing his biop ocess a p omising solu ion o educe he plas-
ic was e bu den, in a ci cula and sus ainable app oach.
Keywo ds Plas ic upcycling, Biodeg ada ion, Rhodococcus, Polye hylene e eph hala e, Polyhyd oxyalkanoa es,
Poly(3-hyd oxybu y a e-co-3-hyd oxy ale a e), T iacylglyce ols
In oduc ion
Polye hylene e eph hala e (PET) is a widely used he -
moplas ic, syn hesized h ough he condensa ion o e e-
ph halic acid (TPA) and e hylene glycol (EG) [1]. Gi en
i s simple syn hesis, low-cos p oduc ion, he mos abil-
i y and du abili y, make i ex ensi ely used in packaging
indus ies, namely plas ic bo les o so d inks, ood ja s,
clo hing, and plas ic ilms [2, 3]. Despi e accoun ing o
6.2% o he wo ldwide plas ics’ p oduc ion, only 25% o
PET is ecycled in Eu ope, wi h he majo i y disca ded in
he en i onmen [4, 5].
The inc easing demand and inadequa e disposal o
PET ha e led o se e e en i onmen al pollu ion, as la ge
olumes escape p ope was e managemen sys ems,
con amina ing oceans and ecosys ems. Due o i s esis -
ance o mic obial deg ada ion, PET pe sis o cen u ies,
in ensi ying plas ic was e accumula ion [6, 7]. Managing
PET was e emains a c i ical challenge, as con en ional
*Co espondence:
C is iana A. V. To es
c. o es@ c .unl.p
1 i4HB - Ins i u e o Heal h and Bioeconomy, NOVA School o Science
and Technology, NOVA Uni e si y Lisbon, Capa ica 2829-516, Po ugal
2 UCIBIO – Applied Molecula Biosciences Uni , Depa men o Chemis y,
NOVA School o Science and Technology, NOVA Uni e si y Lisbon,
Capa ica 2829-516, Po ugal
3 A ecom NV, Indus ieweg 122P, Wondelgem 9032, Belgium
4 LIFE Resea ch Ins i u e, Technological Uni e si y o he Shannon
Midlands Midwes , A hlone N37 HD68, I eland
Page 2 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
disposal me hods such as land illing and incine a ion
pose se e al disad an ages, including slow deg ada-
ion a es in land ills, limi ed spaces, long- e m isks o
con amina ion o soils and g oundwa e wi h leacha e
con aining oxic compounds (e.g. hea y me als, dioxins,
u ans and polychlo ina ed biphenyls), and he emission
o g eenhouse gases associa ed wi h incine a ion [2, 8].
Recycling is cu en ly ega ded as he mos sus ainable
PET was e managemen s a egy and is ca ego ized in o
ou ypes [9]. P ima y ecycling in ol es mechanical
e-ex usion o clean and single- ype PET in o ma e ials
wi h simila p ope ies [10], hough i is limi ed by con-
amina ion and ma e ial deg ada ion o e mul iple cycles
[11]. Seconda y ecycling p ocesses PET h ough clean-
ing, sh edding and emel ing in o lakes o pelle s, bu
i equi es high wa e consump ion o emo e con ami-
nan s (e.g. polyme s, di , and labels) and o en esul s in
downcycling due o he mal deg ada ion [10, 12, 13]. Fu -
he mo e, e ia y ecycling eco e s monome s, oligom-
e s o addi i es ia chemical depolyme iza ion me hods
such as hyd olysis, glycolysis, aminolysis and me hanoly-
sis [14, 15]. Al hough e ec i e o he e ogeneous PET
was e s eams, his p ocess is cos ly, ene gy-in ensi e,
suscep ible o equipmen co osion, and gene a es was e
sol en s [16, 17]. Las ly, qua e na y eco e y ha nesses
PET’s high calo i ic alue h ough combus ion, bu i is
o en associa ed wi h he elease o oxic umes [18].
The complexi y o PET was e, o en mixed wi h munic-
ipal was e and con aining mul ilaye ed plas ics, ubbe ,
aluminium and unc ional addi i es, ha a e designed
o speci ic packaging unc ions, poses signi ican chal-
lenges o e ec i e ecycling [19]. To add ess hese chal-
lenges, upcycling s a egies combining depolyme iza ion
and biocon e sion a e being explo ed o enhance PET
was e alo isa ion [20, 21]. PET depolyme iza ion yields
in e media es, such as TPA, which can se e as mic obial
subs a es, sui able o biocon e sion by TPA-me abo-
lizing mic oo ganisms ia speci ic me abolic pa hways
[21]. This biological app oach enables he ans o ma-
ion o PET was e in o high- alue p oduc s wi h di e se
applica ions [22, 23]. Recen s udies ha e con i med he
mic obial me abolism o TPA, EG and o he PET deg a-
da ion p oduc s in o compounds, such as polyhyd oxyal-
kanoa es (PHA) [24–28], hyd oxyalkanoyloxy-alkanoa es
(HAAs) [27], bac e ial cellulose [29], muconic acid [30],
anillic acid [31], β-ke oadipic acid [32, 33], ca echol, gal-
lic acid and py ogallol [33, 34].
Among mic obial candida es o PET biocon e sion,
Rhodococcus has eme ged as a p omising genus due o
hei ema kable me abolic e sa ili y and en i onmen-
al esilience. Commonly ound in con amina ed si es,
Rhodococcus species a e known o deg ading a ious
ecalci an compounds [35]. Se e al s ains ha e been
iden i ied o hei abili y o me abolize pollu an s and
con e complex subs a es in o aluable compounds
ia di e se ca abolic pa hways [36]. Fo example, Rho-
dococcus jos ii RHA1 and Rhodococcus sp. SSM1 we e
epo ed o deg ade TPA [37, 38], while Rhodococcus
sp. DK17 and R. e y h opolis PR4 can deg ade a oma ic
and alicyclic ings, as well as alkanes, espec i ely [39].
Addi ionally, R. ae he i o ans IAR1 was epo ed o con-
e oluene in o PHA and iacylglyce ols (TAGs) [40],
while R. e yh h opolis MTCC3951 and R. py idini o ans
P23 exhibi bo h TPA deg ada ion and PHA p oduc ion
capabili ies [41, 42].
Fu he mo e, Rhodococcus sp. can syn hesize o he
aluable compounds wi h en i onmen al and indus ial
ele ance, including biosu ac an s and ca o enoids [35],
PHA [43], wax es e s (WEs) and TAGs [44, 45]. Thei
me abolic e sa ili y makes hem s ong candida es o
biocon e sion and bio emedia ion, wi h po en ial appli-
ca ions in b eaking down pe sis en plas ic pollu an s like
polye hylene [46] and p oducing sus ainable biop oduc s
such as PHA and TAGs [47]. This aligns wi h ci cula
economy p inciples, o e ing an eco- iendly al e na i e
o adi ional PET was e managemen [35].
In his s udy, Rhodococcus sp. isola e A e7 was cul i-
a ed in bio eac o using chemically depolyme ized pos -
consume PET was e, wi h TPA as he sole ca bon sou ce.
The s ain’s abili y o p oduce in acellula ese es o
PHA and TAGs was e alua ed unde di e en cul i a ion
modes, including ba ch and ed-ba ch p ocesses. Fol-
lowing cul i a ion, he biop oduc s we e ex ac ed and
cha ac e ized, including an analysis o hei composi ion,
as well as he molecula mass dis ibu ion and he mal
p ope ies o PHA.
Ma e ials andme hods
Feeds ock p ocessing andcha ac e iza ion
Chemical depolyme iza ion o pos ‑consume PET was e
Pos -consume PET was e (Fig.1(A)), con aining app ox-
ima ely 2–5% polye hylene (PE), 1–2% pigmen s, me al-
lic ing edien s and ca bon black addi i es, was supplied
by No elplas (I eland). This ma e ial was used as he
s anda d subs a e wi hou u he p ocessing. Depo-
lyme iza ion expe imen s ia eac i e ex usion (REX)
we e conduc ed using a bench- op P ismTM win-sc ew
ex ude (The mo Elec on GmbH, Ka ls uhe, Ge many)
ollowing a modi ied p ocedu e om Fou ne e al. [48].
The PET was e eeds ock was mixed wi h solid NaOH in
a 2:1 (w %/w %) a io. The well-mixed depolyme iza ion
eac ion mix u e was hen dispensed h ough he main
sha in o he ba el, which was main ained a a cons an
empe a u e o 250 °C, while he sc ew o a ional speed
was se a 20 pm. The esul ing REX p oduc , named
REX-PET, was subsequen ly p ocessed o ex ac and
Page 3 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
sepa a e i s e eph halic acid (TPA) con en , named eac-
i e ex usion- e eph halic acid (REX-TPA) (Fig.1(B)).
Cha ac e iza ion o depolyme ized PET was e
The depolyme ized PET was e was cha ac e ized in
e ms o pa icle size dis ibu ion, mois u e and ino -
ganic con en , elemen al analysis and FT-IR.
The pa icle size dis ibu ion o REX-PET was de e -
mined by sie ing a sample (~ 100 g) in a mechanical
sie ing shake , equipped wi h sie es o po e sizes ang-
ing om 106 o 2000 µm. The sample was agi a ed o
10 min, and he ma e ial collec ed on each sie e was
weighed sepa a ely o de e mine i s espec i e ac ion in
he sample.
Fo he mois u e con en de e mina ion, REX-PET
(~ 50 mg) was subjec ed o a empe a u e o 100 °C un il
a cons an weigh was achie ed. A e wa ds, he d ied
sample was placed a a empe a u e o 550 °C o 24 h,
and he ino ganic sal s con en was de e mined g a i-
me ically by weighing he esul ing ashes. The elemen al
analysis o REX-PET was pe o med using an elemen al
analyze (The mo Finnigan-CE Ins umen s, Flash EA
1112 CHNS se ies, I aly). A REX-PET sample was cha -
ac e ized ia Fou ie - ans o m in a ed spec oscopy
(FT-IR) wi h a spec um wo spec ome e (Pe kin-
Elme , Wal ham, MA, USA) equipped wi h he a enu-
a ed o al e lec ance (ATR) accesso y. The spec a we e
eco e ed based on eigh scans be ween a esolu ion o
4000 and 400 cm−1, a oom empe a u e.
Depolyme ized PET was e p ocessing
Fo he bac e ial cul i a ion expe imen s, REX-PET was
p ocessed in o an aqueous solu ion (named REX-TPA),
which was ob ained by p epa ing a 3.33% (w/ ) mix u e
o REX-PET (5 g) (Fig. 1(B)) in deionized wa e (150
mL), ollowed by homogeniza ion a 600 pm du ing 1
h. The mix u e was il e ed (using pape il e s wi h po e
size 20 µm) ollowed by pH 7 no maliza ion by HCl 5
M addi ion. The inal solu ion con ained app oxima ely
20 mg/mL o REX-TPA, ep esen ing a eco e y o 60%
(Fig.1(C)). The solu ion o REX-TPA was assessed o i s
pH, mois u e and ino ganic con en , ICP, o al ca bon
and TPA quan i ica ion.
Mois u e and ino ganic con en we e also de e -
mined o REX-TPA samples (~ 5 mL) as de e mined as
desc ibed in he p e ious sec ion. REX-TPA samples (~ 5
mL) we e il e ed (0.2 µm nylon, Wha man) and analysed
by induc i ely coupled plasma-a omic emission spec-
ome y (ICP-AES) (Ho iba Jobin–Y on, F ance, Ul ima,
equipped wi h a 40.68 MHz RF gene a o , Cze ny-Tu ne
monoch oma o wi h 1.00 m (sequen ial) and au osam-
ple AS500). The To al Ca bon (TC) was analysed in a
TOC-VCSH Analyse (Shimadzu) wi h a combus ion ca -
aly ic oxida ion a a empe a u e o 680 °C. High pu i y
ai se ed as ca ie gas a a low a e o 150 mL/min.
TPA concen a ion was de e mined by high pe o mance
liquid ch oma og aphy (HPLC) using an Agilen Eclipse
C18 250 × 4.6 mm, coupled o a UV de ec o . The analysis
was pe o med a 50 °C, wi h samples elu ed in isoc a ic
mode using me hanol (Fishe Chemical, HPLC g ade)
and 0.1% o mic acid (Sigma-Ald ich, HPLC g ade)
solu ion (1:1, / ). The low a e was se o1 mL/min,
and he injec ion olume 5 µL [49]. TPA de ec ion was
ob ained a 240 nm. A TPA s ock solu ion (1 g/L) (Me ck
Millipo e, 98%) was p epa ed in a phospha e bu e
(con aining pe li e : (NH4)2HPO4, 1.1 g (PanReac Appli-
Chem, 99%); K2HPO4, 5.8 g (PanReac AppliChem, 99%);
KH2PO4, 3.7 g (ChemLab, 99.5%)) and adjus ed o pH 7.
F om his s ock solu ion, TPA s anda ds we e p epa ed
by se ial dilu ion wi h a wa e –me hanol mix u e (10%
me hanol, Fishe Chemical, HPLC g ade) o achie e TPA
concen a ions anging om 4 o 400 mg/L. Simila ly, he
cell- ee supe na an samples we e dilu ed in he same
10% me hanol aqueous solu ion o ensu e consis ency o
Fig. 1 Pos -consume PET was e (A), REX-PET chemical depolyme ized sample ob ained om eac i e ex usion o pos -consume PET was e (B)
and REX-TPA solu ion used o bio eac o cul i a ions (C)
Page 4 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
he s anda ds and samples ma ix o he HPLC analysis.
All measu emen s we e done in iplica e.
Mic oo ganism
Rhodococcus sp. A e 7 was isola ed om land ill soil, by
se ially dilu ing and cul i a ing a sample on selec i e
pla es, con aining comme cial TPA (Me ck Millipo e,
98%) as ca bon sou ce. Single colonies we e isola ed
and used o he ampli ica ion o he 16S RNA gene ia
colony PCR using a T100 The mal Cycle (Bio-Rad). The
ampli ied 16S RNA genes we e hen sen o Mac ogen
Eu ope (The Ne he lands) o Sange sequencing.
Cul u e media
Mine al sal medium (MSM) con aining 8.86 g/L
K2HPO4 (PanReac AppliChem, 99%), 2.80 g/L KH2PO4
(ChemLab, 99.5%), 0.50 g/L NaCl (PanReac AppliChem,
99.5%), 0.10 g/L MgSO4·7H2O (Biochem Chemopha ma,
99.5%) and 0.10 g/L NH4Cl (Biochem Chemopha ma,
99.5%), was used o all expe imen s. The mic onu ien s’
solu ion was added o he medium a a concen a ion o
10 mL/L. The mic onu ien s solu ion con ained he ol-
lowing (pe li e ): FeSO4·7H2O, 1.83 g; MnSO4·1H2O,
0.56 g; ZnSO4·7H2O, 1.35 g; CaCl2·2H2O, 0.067 g; CoSO4
7H2O, 0.036 g; CuSO4 5H2O, 0.036 g; H3BO3, 0.65 g;
EDTA dissodium·2H2O, 1.104 g. This medium was used
o inocula p epa a ion o all assays. The medium was
supplemen ed wi h e eph halic acid (TPA) (Me ck Mil-
lipo e, 98%) as ca bon sou ce a a concen a ion o 0.5
g/L o inocula p epa a ion. Fo bio eac o assays, MSM
was supplemen ed wi h REX-TPA solu ion (p epa ed as
desc ibed abo e), o achie e a inal TPA concen a ion o
12 g/L. All media we e s e ilized by au ocla ing a 121 °C
and 1 ba , o 30 min.
Bio eac o cul i a ions
Th ee bio eac o expe imen s we e pe o med unde
di e en modes o cul i a ion: ba ch (Assay A) and ed-
ba ch wi h pulse eeding (Assay B) o con inuous eeding
(Assay C). The inocula o he bio eac o assays we e p e-
pa ed by inocula ing 1 mL o he c yop ese ed cul u e
in o 200 mL MSM, p epa ed as desc ibed abo e, in a 500
mL ba led shake lask. The lasks we e incuba ed in a
o a y shake (200 pm), a 30 °C, o 48 h. Fo he bio e-
ac o assays, MSM supplemen ed wi h REX-TPA (12 g/L)
and ammonia (0.3 g/L), yielding a C/N a io o 29.7 (gC/
gN), was p epa ed.
A e s e iliza ion in an au ocla e a 121 °C, 1 ba , o
30 min, he medium was inocula ed wi h 200 mL o he
p epa ed bac e ial cul u e o ini ia e he expe imen s.
Th oughou he assays, he pH was moni o ed bu no
con olled. The empe a u e was con olled a 30 ± 0.1 °C
in all assays. An ae a ion a e o 2 SLPM (s anda d li e s
pe minu e) was kep du ing he expe imen s. The dis-
sol ed oxygen (DO) concen a ion was con olled a 20%
o he ai sa u a ion, by au oma ically adjus ing he s i -
ing a e be ween 300 and 1000 pm. Foam o ma ion
was au oma ically supp essed by addi ion o An i oam
204 (Sigma-Ald ich).
Assay A, unde ba ch mode, was conduc ed in a 3 L
bio eac o (Jupi e 3, Sola is, I aly), wi h ini ial wo king
olume o 2 L. Assay B was pe o med in a 5 L bio eac o
(Jupi e 6.0, Sola is, I aly) wi h an ini ial wo king olume
o 2 L. A e ini ial TPA deple ion, signalled by an ab up
inc ease o DO concen a ion, REX-TPA pulse (1 L) con-
aining 20 g/L TPA was ed o he bio eac o . Assay C was
pe o med in a 3 L bio eac o (Bione F1, Spain), wi h an
ini ial wo king olume o 1.5 L. A con inuous eeding o a
REX-TPA solu ion con aining 20 g/L TPA and 0.01 g/L o
ammonia, was ed o he bio eac o a a 0.1 L/h low a e
o 15 h.
Samples (10–20 mL) we e pe iodically aken om
he bio eac o o quan i ica ion o he cell d y weigh
(CDW), TPA, ammonium, PHA and TAG.
Analy ical me hods
CDW quan i ica ion
Fo de e mina ion o he CDW, cul u e b o h samples (5
mL) we e cen i uged (20 min a 18,516 g, 4 °C). The cell
pelle was washed wi h deionized wa e (5 mL) wice and
lyophilized o 48 h. The CDW was de e mined g a i-
me ically by weighing he d ied cell pelle s. All measu e-
men s we e done in iplica e.
Quan i ica ion o TPA
TPA concen a ion was de e mined using HPLC, as
desc ibed abo e, wi h an Agilen Eclipse C18 column
(250 × 4.6 mm) and UV de ec ion a 240 nm. The analysis
was conduc ed a 50 °C in isoc a ic mode wi h a mobile
phase o me hanol and 0.1% o mic acid (1:1, / ), a a
low a e o 1 mL/min and an injec ion olume o 5 µL.
A TPA s ock solu ion (1 g/L) was p epa ed in phospha e
bu e (pH 7), and TPA s anda ds we e p epa ed h ough
se ial dilu ion o ob ain concen a ions anging om 4
o 400 mg/L. Cell- ee supe na an samples we e dilu ed
wi h a 10% me hanol–wa e solu ion o ma ch he ma ix
o he s anda ds. All measu emen s we e pe o med in
iplica e.
Ammonium quan i ica ion
Ammonia concen a ion was de e mined by colo im-
e y using a low segmen ed analyse (Skala 5100, Skala
Analy ical, Ne he lands). NH4Cl (Biochem Chemop-
ha ma, 99.5%) samples a concen a ions anging om
2 o 20 mg/L we e used as s anda ds. All measu emen s
we e done in iplica e.
Page 5 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
PHA andTAGs quan i ica ion
S o age compounds con en in he biomass, namely
PHA and TAGs, and hei composi ion we e de e mined
by gas ch oma og aphy (GC) a e acidic me hanoly-
sis o eeze-d ied cells’ samples. F eeze d ied samples
(3 o 5 mg) we e mixed wi h 2 mL 20% ( / ) sulphu ic
acid (Honeywell Fluka, HPLC g ade) in me hanol (Fishe
Chemical, HPLC g ade) and 2 mL benzoic acid in chlo o-
o m (0.5 g/L) (Fishe Chemical, HPLC g ade) and hea ed
a 100 °C, o 4 h. Benzoic acid (Sigma-Ald ich, ≥ 99.5%)
ac ed as in e nal s anda d. The calib a ion cu e o PHA
quan i ica ion was p epa ed using a s anda d solu ion
o P(HB-co-HV) (Sigma-Ald ich) composed o 14 mol%
3-hyd oxy ale a e (3HV). Fo TAGs quan i ica ion, a
mix u e o a y acid me hyl es e s (FAME) composed o
C14-C22 (Sigma-Ald ich) a concen a ions anging om
0.1 o 1.0 g/L was used. The me hyl es e s ob ained om
he me hanolysis, de i ed simul aneously om bo h
PHA and TAGs, we e analysed in a single un using a
T ace 1300 GC appa a us (The mo Fishe Scien i ic, US)
equipped wi h a lame ioniza ion de ec o (FID) (The mo
Fishe Scien i ic, US) and a Res ek column (C ossbond,
S abilwax). The sys em ope a ed a cons an p essu e (96
kPa) using helium as ca ie gas. The o en empe a u e
p og am was he ollowing: 20 °C/min un il 100 °C; 3 °C/
min un il 155 °C and, inally, 20 °C/min un il 230 °C wi h
a holding ime o 30 min. All measu emen s we e done in
iplica e.
Glycogen analysis
Glycogen accumula ion was analysed ollowing he p o-
ocol desc ibed by [50]. The p e iously weighed eeze-
d ied biomass was ea ed wi h 2 mL o a dilu e solu ion
o HCl. The ubes we e incuba ed a 100 °C o 3 h. The
samples we e il e ed ( il e wi h 0.2 µm po e size, Wha -
man) and analysed by high-pe o mance liquid ch oma-
og aphy (HPLC) using a ch oma og aph equipped wi h
an Aminex HPX-87H HPLC column (Bio-Rad, USA). A
solu ion o 0.01 N o H2SO4 was used as a mobile phase
wi h a low a e o 0.5 mL/min and a 30 °C ope a ing em-
pe a u e. The de ec ion wa eleng h was se a 210 nm.
Glucose (Scha lau, Ba celona, Spain) was used as s and-
a d anging om 0.06 o 1 g/L. Samples we e analysed in
iplica e.
Polyphospha e s aining me hod
Fo s aining polyphospha e inclusions, samples we e
ixed wi h gen le hea on glass mic oscopic slides and
exposed o Loe le ’s me hylene-blue s aining in each
case wi h ligh washing in dis illed wa e [51].
Calcula ions
The maximum speci ic cell g ow h a e (μmax, h−1) was
calcula ed by de e mining he linea eg ession slope
o he exponen ial phase o Ln X /X0 e sus ime cu e,
whe e X /X0 (g/L) is he ac i e cell biomass concen a-
ion a ime (h) and a he beginning o he un ( 0),
espec i ely. The ac i e cell biomass (X, g/L) (wi hou
PHA and TAG) used o yield calcula ions, a ime , was
de e mined by Eq. (1).
whe e CDW (g/L), PHA (g/L) and TAG
(g/L) ep e-
sen he CDW and he concen a ions o PHA and ia-
cylglyce ol (TAG) a ime (h), espec i ely.
The o e all olume ic p oduc i i y ( P, g/ L.(day)),
whe e P isindica i e o PHA o TAG, we e de e mined
by Eq. (2):
whe e ΔP (g/L) is he p oduc (PHA o TAG) p oduced
in ime in e al Δ (h).
The yields o ac i e biomass (YX/S, gX/gTPA) and he
p oduc s (P) on subs a e basis (YP/TPA, gP/gTPA) we e
de e mined by Eqs. (3) and (4):
whe e ΔX and ΔP a e he ac i e biomass and he PHA
and/o TAG p oduced (g/L), espec i ely, and ΔS (g/L)
is he concen a ion o TPA om REX-TPA esidue con-
sumed du ing he same ime ange o he cul i a ion un.
S a is ical analysis
The s a is ical di e ences o he mean and s anda d
de ia ion o he kine ic and s oichiome ic pa ame e s
ob ained om he h ee assays pe o med by Rhodo-
coccus sp. A e7 using REX-TPA we e assessed using
one-way ANOVA ollowed by Bon e oni’s mul iple com-
pa ison es s in G aphPad P ism 5 wi h he c i e ia o
s a is ical signi icance se a p < 0.05.
Biop oduc s ex ac ion andcha ac e iza ion
The cul i a ion b o h was cen i uged (10,350 g, 20 min,
4 °C) and he ob ained cell pelle s we e eeze-d ied and
milled. The biop oduc s we e ex ac ed om he eeze-
d ied biomass by Soxhle ex ac ion wi h chlo o o m
(Fishe Chemical, HPLC g ade), a 80 °C, o 48 h. La e ,
(1)
X
=
CDW
−
(PHA
+
TAG )
(2)
p=
P

(3)
Y
X/S=
�X
�S
(4)
Y
P/S=
�P
�S

Page 6 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
he PHA was p ecipi a ed in ice-cold e hanol (1:10, / ),
unde igo ous s i ing, and d ied in a ume hood a
oom empe a u e [52].
The e hanol used o PHA p ecipi a ion was collec ed
and allowed o e apo a e a oom empe a u e in a ume
hood, o eco e he p oduced TAGs.
Fo u he pu i ying he PHA, he sample was mixed
wi h 1-bu anol (≥ 99.5%, PanReac AppliChem) a a con-
cen a ion o 0.3% (w/ ), and hea ed o 75 °C o 2 h,
unde cons an s i ing, o dissol e he TAGs ac ion.
The sol en was emo ed while s ill ho and he insoluble
PHA was eco e ed and le o d y a oom empe a u e
in a ume hood.
FT‑IR
FT-IR analysis we e conduc ed o he eco e ed TAGs
and PHA samples in a Pe kin-Elme Spec um wo spec-
ome e coupled wi h ATR accesso y. The polyme was
di ec ly analysed on he FTIR cells. The spec a we e
eco ded be ween 400 and 4000 cm−1 esolu ion and 10
scans we e conduc ed a oom empe a u e.
The mal p ope ies
Di e en ial scanning calo ime y (DSC) was ca ied ou
wi h a DSC Q2000 ins umen (TA Ins umen s, New
Cas le, FL, USA). He me ic aluminium pans we e used o
place he samples, and he analysis was pe o med wi h a
hea ing and cooling a e o 10 °C/min o e a empe a u e
ange o − 90 °C o 180 °C, h ough h ee hea ing cycles.
The endo he m peak’s empe a u e and a ea o he i s
hea ing cycle we e used o de e mine mel ing empe a-
u es (Tm) and mel ing en halpies (∆Hm), espec i ely,
while he glass ansi ion empe a u e (Tg, °C) was aken
as he midpoin o he hea lux s ep. The c ys allini y
(XC, %) was es ima ed as he a io be ween he ob ained
mel ing en halpy and he mel ing en halpy o 100% c ys-
alline PHB, es ima ed as 146 J/g [53].
The mog a ime ic analysis (TGA) was pe o med
using a he mog a ime ic Analyze Labsys EVO (Se a-
am, F ance), wi h weighing p ecision o + / − 0.01%. Sam-
ples we e placed in aluminium c ucibles and analysed in
a gon a mosphe e wi h empe a u e ange be ween 25
and 800 °C, a a a e o 10 °C/min. The maximum he mal
deg ada ion empe a u e (Tdeg, °C) co esponds o he
empe a u e alue ob ained o he maximum dec easing
peak o he sample mass.
Molecula mass dis ibu ion
Size-exclusion ch oma og aphy (SE-HPLC) was pe -
o med o de e mine he numbe a e age molecula
weigh (Mn), weigh a e age molecula weigh (Mw) and
polydispe si y index (Mw/Mn) o he PHA. Monodis-
pe se polys y ene s anda ds (370–2,520,000 Da) and he
biopolyme we e p epa ed a a concen a ion o 0.2%
(w/ ) in chlo o o m. Analysis was conduc ed using a
KNAUER Sma line SE-HPLC sys em (Be lin, Ge many)
equipped wi h a Phenomenex Phenogel Linea Liquid
Ch oma og aphic Column (300 × 7.8 mm; To ance, CA,
USA), ope a ed a 30 °C wi h a 1 mL/min chlo o o m
low a e as he mobile phase, using a Wa e s2414 e ac-
i e index de ec o (RID) (Mil o d, CT, USA).
Resul s anddiscussion
Feeds ock cha ac e iza ion
The esidue ob ained om he depolyme iza ion o PET
was e, named REX-PET, was a uni o m da k powde
(Fig.1(B)). The majo i y o he ma e ial comp ised pa -
icles in he 1000–2000 µm size ange (46 ± 2.0% w/w),
ollowed by pa icles la ge han 2000 µm (28 ± 5.7%
w/w) and hose a 500–1000 µm (16 ± 1.4% w/w) (Fig.2).
Smalle pa icles (< 500 µm) collec i ely accoun ed o
less han 12 ± 1.1% w/w. This pa icle size dis ibu ion
indica es ha he ma e ial is p edominan ly g anula ,
wi h la ge size ac ions domina ing he sample.
The mois u e and ino ganic con en o REX-PET is
shown in Table1, and i p esen s a high ino ganic sal
con en o REX-PET (46.46 ± 4.69 w .%), since NaOH
was used as a ca alys o he depolyme iza ion o he
PET was e, o ming a sodium sal o TPA [54]. The ele-
men al analysis (Table 1) e ealed ha REX-PET was
mainly composed o ca bon (44.21 ± 2.4%), wi h a mino
hyd ogen con en (2.97 ± 0.39%) o ace o ni ogen
(0.04 ± 0.02%), while no sulphu was de ec ed.
As shown in Fig.3, he spec al peaks o all REX-PET
ba ches exhibi ed high simila i y among he ba ch sam-
ples, al hough hey di e om hose epo ed o com-
me cial TPA. The ca boxylic g oup (-OH) s e ching peak
appea s a ound 3000–2800 cm−1, bu i is less in ense in
he REX-PET samples, sugges ing a lowe concen a ion
o ee ca boxylic acid g oups compa ed o pu e TPA
[55]. The in ense zone o peaks be ween 1718 and 1270
cm−1 co espond o he C = O and C = C bonds o he
benzene ing in TPA, wi h in ense peaks a 1557 and 1391
cm−1 indica i e o he acidic ca bonyl g oup (-C = O) and
a oma ic ing ib a ions [55–57]. No ably, his egion
shows conside able di e ences om comme cial TPA, as
he peaks co espond o he o ma ion o TPA disodium
sal , a p oduc o he depolyme iza ion p ocess [58]. This
is e idenced by he absence o -O–H bending bond in
REX-PET, a 940 cm−1 a ibu ed o he p esence o dis-
odium e eph hala e [59], and he disappea ance o he
ca boxylic acid g oups (-COOH) a 1625 and 1423 cm−1
[60]. Mo eo e , peaks displayed a 1088 and 1023 cm−1
can be a ibu ed o he = C-H bending ib a ions o he
a oma ic ing [56]. The FT-IR spec a o he h ee REX-
PET samples in Fig.3 show s ong simila i y among he
Page 7 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
ba ches, indica ing consis en chemical s uc u es wi h
no signi ican a ia ions in peaks o in ensi ies, e lec ing
a s able and ep oducible depolyme iza ion p ocess.
The REX-TPA solu ion ob ained om REX-PET p e-
sen ed a da k, anslucid wi hou isible suspended pa -
icles (a e il a ion) as can be obse ed in Fig.1(C).
The pH o he REX-TPA solu ion was ound o be 11.05
(Table2), which co ela es wi h he d y REX depolyme i-
za ion p ocess ha ollows he hyd olysis me hod unde
alkaline condi ions p o ided by adding NaOH [43]. This
pH alue is compa able o ha ob ained o a solu ion
con aining PET depolyme ized upon ela able hyd oly-
sis [61]. The REX-TPA solu ion had a o al ca bon (TC)
con en o 12.06 ± 0.24 g/L (as de e mined by he TOC-
VCSH Analysis) and a TPA concen a ion o 19.69 ± 0.09
g/L (as de e mined by he HPLC analysis). This TPA con-
cen a ion accoun s o a ca bon con en o 11.39 g/L
which shows ha he solu ion p edominan ly comp ised
TPA, wi h only a mino con en o o he ca bonaceous
compounds.
The mois u e and ino ganic con en (Table 2) o
REX-TPA was o 96.20 ± 0.43 w .% and 2.21 ± 0.13 w .%,
espec i ely. As expec ed, REX-TPA showed a high con-
en in Na (461.12 ± 71.71 mg/L) (Table2), which can be
a ibu ed o he depolyme iza ion p ocedu e conduc ed
unde alkaline condi ions. Thus, HCl was used o neu-
alize he aqueous solu ion [62]. The main ad an age
o using his ype o depolyme iza ion condi ions is i s
sui abili y o complex PET was e s eams, which o en
con ain signi ican amoun s o me als, pigmen s o o he
ypes o plas ic [9, 63]. O he elemen s ound in REX-
TPA we e Fe (8.84 ± 2.45 mg/L), Ti (4.16 ± 1.48 mg/L), Sb
(3.82 ± 1.23 mg/L), Si (2.12 ± 0.84 mg/L) and aces o C ,
Al, Zn, W and Mo (< 0.5 mg/L) (Table2). This wide ange
o componen s ound in REX-TPA e eals he high he -
e ogenei y o addi i es ha can be ound in mixed plas ic
li e samples [64].
Ba ch bio eac o cul i a ion
Rhodococcus sp. A e7 was cul i a ed unde ba ch mode
using an ini ial TPA concen a ion o 12 g/L as sole ca -
bon sou ce, unde a con olled empe a u e o 30 °C and
an ini ial pH o 7. The ini ial concen a ions o TPA (12
g/L) and ammonium (0.3 g/L) in he bio eac o medium
we e de e mined based on p io indings om an RSM
s udy (TableS1 and Fig. S1, Supplemen a y Ma e ial).
This op imiza ion s udy iden i ied hese pa ame e s as
mos sui able o enhancing cell g ow h and he accumu-
la ion o in acellula s o age compounds, namely PHA
and TAGs.
A e a 10-h lag phase, Rhodococcus sp. A e7 en e ed
an exponen ial phase, eaching a maximum speci ic cell
g ow h a e o 0.18 ± 0.05 h−1 and a CDW o 1.78 ± 0.08
g/L a 20 h o cul i a ion, when ammonia was exhaus ed
Fig. 2 Pa icle size dis ibu ion o REX-PET
Table 1 Cha ac e iza ion o REX-PET eeds ock
n.d. no de ec ed
Pa ame e REX-PET
Mois u e (w .%) 1.07 ± 0.41
O ganic con en (w .%) 52.47 ± 4.92
Ino ganic sal s (w .%) 46.46 ± 4.69
Elemen al analysis (%):
C 44.21 ± 2.4
H 2.97 ± 0.39
N 0.04 ± 0.02
S n.d
Page 8 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
(Fig.4(A1)). A inal CDW o 2.67 ± 0.06 g/L was eached
a he end o he assay. This alue is sligh ly highe han
he 2.3 g/L o CDW epo ed o Pseudomonas umson-
gensis G016 KS3 g ew in a ba ch eac o , wi h simila
du a ion, using as ca bon sou ce TPA and EG monome s
ob ained by enzyma ic PET hyd olysis [27].
PHA accumula ion s a ed du ing he exponen ial
cell g ow h phase, a 13 h o cul i a ion, and con inued
(Fig.4(A2)) un il he end o he assay, eaching a maxi-
mum PHA con en in he biomass o 4.22 ± 0.03 w .%,
co esponding o a PHA concen a ion o 0.11 ± 0.02
g/L (Table3). Sligh ly highe alues we e epo ed o P.
umsongensis G016 KS3 (7 w .%), co esponding o 0.15
g/L o PHA [27].
TAGs syn hesis was ini ia ed la e , a a ound 21 h o
cul i a ion (Fig.4(A2)), eaching an in acellula con en
o 13.45 ± 0.69 w .% and a concen a ion o 0.15 g/L by 21
h o cul i a ion (Table3). This co esponds o an o e -
all olume ic p oduc i i y o 0.305 g/(L day). Du ing he
i s 21 h, he bac e ial s ain consumed 7.74 g o TPA,
o bo h cellula g ow h and PHA accumula ion, esul -
ing in g ow h yield o 0.19 gX/gTPA. A e ammonium
deple ion, he cul u e used he a ailable TPA o PHA
and TAG accumula ion. The PHA yield was 0.011 ± 0.00
gPHA/gTPA, while a highe yield was eached o TAGs
(0.031 ± 0.002 gTAG
/gTPA). An o e all consump ion o
10.7 g/L o TPA was achie ed o e 28 h. Simila alues
Fig. 3 Fou ie - ans o m in a ed (FT-IR) spec a o h ee ba ches o REX-PET samples de i ed om PET was e depolyme iza ion unde simila
condi ions, and o comme cial TPA (Me ck Millipo e, 98%)
Table 2 Cha ac e iza ion o REX-TPA solu ion used in bio eac o
media cul i a ion as eeds ock
Pa ame e REX-TPA
pH 11.05
Mois u e (w .%) 96.20 ± 0.43
O ganic con en (w .%) 1.58 ± 0.55
Ino ganic sal s (w .%) 2.21 ± 0.13
To al ca bon (g/L) 12.06 ± 0.24
[TPA] (g/L) 19.69 ± 0.09
Elemen (mg/L)
Na 461.12 ± 71.71
K 9.30 ± 3.91
Fe 8.84 ± 2.45
Ti 4.16 ± 1.48
Sb 3.82 ± 1.23
P 3.74 ± 0.98
Mg 3.11 ± 0.86
Ca 2.15 ± 0.36
Si 2.12 ± 0.84
C 0.51 ± 0.28
Cu 0.29 ± 0.03
Al 0.27 ± 0.09
Zn 0.25 ± 0.18
W 0.25 ± 0.06
Mo 0.13 ± 0.06
Page 9 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
we e ob ained o P. umsongensis GO16 KS3 cul i a ed in
TPA and EG ob ained by enzyma ic PET hyd olysis, (0.21
gCDW/gsubs a e and 0.014 gPHA/gsubs a e, espec i ely) [27].
Fed-ba ch cul i a ion wi hpulse eeding
In Assay B (Fig.4(B1)), Rhodococcus sp. A e7 en e ed he
exponen ial g ow h phase a e 9 h o cul i a ion, p e-
sen ing a maximum speci ic g ow h a e o 0.18 ± 0.02
h−1, which was no signi ican ly (p > 0.05) di e en om
Assay A (0.18 ± 0.05 h−1). Ammonia deple ion was
obse ed a e 17 h o cul i a ion, esul ing in a CDW
o 2.18 ± 0.13 g/L. By 24 h, he cul u e achie ed a maxi-
mum CDW o 3.17 ± 0.03 g/L (Fig.4(B1)), wi h PHA and
TAGs con en s o 2.77 ± 0.01 w .% and 11.03 ± 0.36 w .%,
espec i ely. A his momen , dissol ed oxygen concen-
a ion s a ed o inc ease (da a no shown), indica ing
deple ion o he ca bon sou ce. The e o e, a 1 L REX-TPA
pulse (con aining 20 g/L TPA) was ed o he cul u e, is-
ing he TPA concen a ion o 6.76 ± 0.87 g/L.
Du ing he i s 24 h, Rhodococcus sp. A e7 p oduced
0.09 ± 0.00 g/L o PHA (Fig.4(B2)). Despi e he subse-
quen eeding o a TPA pulse, no u he inc ease in
Fig. 4 Cul i a ion p o iles o ba ch (A), ed-ba ch wi h pulse eed ( he dashed line deno es he ime he REX-TPA pulse was gi en) (B) and ed-ba ch
wi h con inuous eed ( he g ey a ea deno es he ime REX-TPA was ed o he bio eac o , a a a e o 0.1 L/h) (C) o Rhodococcus sp. A e7 using
REX-TPA as eeds ock. E o ba s co espond o iplica e measu emen s
Page 16 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
con en (58 o 98 mol%) [85]. Mo eo e , he Mw and PDI
ob ained was also compa able o he PHBV, 260 kDa and
PDI o 1.9, p oduced by mixed mic obial cul u es wi h 82
mol% 3HV [84]. Ne e heless, he Mw a ained o Rho-
dococcus sp. A e7 PHBV was low compa ed o he p o-
duced by R. py idini o ans P23 (Mw o 600 kDa) using
TPA, bu s ill wi hin he same o de o magni ude [42].
The PDI o he PHBV a ained in assay C sugges s a
good polyme uni o mi y, which may acili a e i s p o-
cessing and biodeg ada ion a e [95, 96].
The mal p ope ies
The biopolyme p esen ed a mel ing empe a u e (Tm)
o 95.1 °C ha is wi hin he ange o se e al PHBV wi h
3HV con en s a ying be ween 58 and 98 mol% (89.9–
109.4 °C) [85], and simila o he Tm (101 °C) epo ed
o PHBV p oduced wi h TPA as eeds ock [42]. The high
Fig. 7 FT-IR spec a o (a) PHBV p oduced by Rhodococcus sp. A e7 du ing ed-ba ch wi h con inuous eed condi ions and (b) PHBV wi h 11% 3HV
con en (adap ed om [94])
Table 6 Physical- chemical and he mal p ope ies o PHBV
p oduced in Assay C om Rhodococcussp. A e7
Mw molecula weigh , PDI polydispe si y index, Tm mel ing empe a u e, Tg
glass ansi ion empe a u e, Tdeg deg ada ion empe a u e, ΔHm mel ing
en halpy, Xc c ys allini y ac ion
Cha ac e iza ion Value
Mw (kDa) 277
PDI 1.5
Tm (°C) 95.1
∆Hm (J/g) 26.3
Xc (%) 18.0
Tg (°C) − 21.1
Tdeg (°C) 270.0
395.5
503.6

Page 17 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
3HV con en in he copolyme lowe s he Tm signi i-
can ly, hus b oadening i s p ocessabili y window, which
acili a es he polyme p ocessing in compa ison o he
homopolyme composed by 3HB, cha ac e ized by a Tm
e y close o he deg ada ion empe a u e [97].
The PHBV also exhibi ed a glass ansi ion empe a u e
(Tg) o − 21.1 °C (Table6), which is signi ican ly lowe
han he ypical alues epo ed o PHBV wi h 3HV con-
en s o 82 mol% (− 13.2 °C) [84]. The low Tg obse ed o
PHBV may esul om TAGs s ill p esen in he polyme
ma ix a e 1-bu anol p ecipi a ion, po en ially ac ing as
plas icize s ha inc ease he ee olume be ween PHBV
chains and enhance hei mobili y a lowe empe a u es,
as epo ed o PHBV and plas icize s blends [89, 98].
The polyme p esen ed a c ys allini y o 18.0%
(Table6), indica ing i was mo e amo phous han o he
3HV- ich PHBV (40–50 mol % o 3HV) ha p esen
c ys allini ies wi hin 50% [85]. Fo copolyme s whe e
3HV con en was highe , a highe c ys allini y was
expec ed since i would mainly ake he c ys al s uc u e
o he P(3HV) homopolyme la ice [99]. This dec ease
in c ys allini y may be ela ed o he p esence o em-
nan s o TAGs ha we e no comple ely emo ed om
he sample [100], as p e iously de ec ed in he biopoly-
me FT-IR spec um. Ne e heless, he biopolyme ’s low
c ys allini y may p o ide mo e lexibili y compa ed o
o he PHBV blends wi h lowe 3HV con en , making i
sui able o applica ions ha equi e so e , mo e lexible
ma e ials [84].
The biopolyme was he mally s able un il 270 °C
(Table6), in concomi an wi h deg ada ion empe a u e
(Tdeg) (279 °C) o o he 3HV- ich PHBV, whe e i su -
e ed a weigh loss o 46% [101]. Abo e his empe a u e,
a second weigh loss o 15.5% was a ained a 395.5 °C ol-
lowed by a hi d obse ed a 503.6 °C o 33.8% o weigh
loss. These la e s ages o deg ada ion a e a ibu ed o
he deg ada ion o a y acids, con i ming he decomposi-
ion o TAGs monounsa u a ed and sa u a ed a y acids,
mainly composed by C18:1, C16:0 and C18:0 [102].
The p esence o a y acids signi ican ly a ec ed he
he mal p ope ies o he sample. The a y acids wi hin
he polyme ma ix exhibi ed highe Tdeg han he indi-
idual oleic, s ea ic and palmi ic me hyl es e s (210–229
°C o 5% weigh loss) o hei e hylene glycol es e s
(228–248 °C) es ed as po en ial PHBV plas icize s [89].
This shows ha he p esence o TAGs-de i ed a y acids
in he polyme p o ide a highe window o he mal s a-
bili y compa ed o p e iously PHBV composi es wi h
simila a y acids. This demons a es ha PHBV ma e-
ials wi h high 3HV con en can be ailo ed by adding
na u al a y acids as plas icize s, enhancing he biopoly-
me ’s pe o mance, pa icula ly in he he mal s abili y
o end-use applica ions [89].
Conclusions
This wo k alida ed Rhodococcus sp. A e7 as a p omis-
ing mic oo ganism o he bio emedia ion o PET was e,
gi en i s high capaci y o TPA biocon e sion. The cul-
u e e icien ly upcycled depolyme ized PET was e in o
biomass, PHA and TAGs. The p oduced co-polyes e
PHBV, wi h a 3HV con en up o 90 w .%, has po en-
ial o being used in PHBV copolyme blends. The bio-
syn hesized TAGs, on he o he hand, we e en iched
in oc adecenoic and hexadecanoic acids, which migh
be o in e es o pai wi h he exis ing p oduc ion om
ege able oils sou ces. O e all, his s udy demons a ed
he po en ial o Rhodococcus sp. A e7 o e ec i e bio-
deg ada ion o chemically depolyme ized PET was e
in o alue-added bio-based p oduc s, hus con ibu ing
o educe he impac o PET was e and alo ising i in o
alue-added p oduc s, wi hin he ci cula economy con-
cep . Th ough he biocon e sion o PET was e in o sus-
ainable al e na i es o pe oleum-de i ed plas ics and
oils, his esea ch suppo s e o s o educe plas ic was e
and dependence on ossil-based esou ces, unde lin-
ing i s ole in ad ancing sus ainable was e managemen
solu ions and ein o cing i s en i onmen al and socie al
ele ance.
Abb e ia ions
PET Polye hylene e eph hala e
TPA Te eph halic acid
REX Reac i e ex usion
REX-TPA Reac i e ex usion- e eph halic acid
PHAs Polyhyd oxyalkanoa es
TAGs T iacylglyce ols
PHBV Poly(3-hyd oxybu y a e-co-3-hyd oxy ale a e)
3HV 3-Hyd oxy ale a e
C18:1 Oc adecenoic acid
C16:0 Hexadecanoic acid
Supplemen a y In o ma ion
The online e sion con ains supplemen a y ma e ial a ailable a h ps:// doi.
o g/ 10. 1186/ s44314- 025- 00019-4.
Supplemen a y Ma e ial 1. Table S1 Ma ix o he Response Su ace
Me hodology (RSM), composed o ele en expe imen s: ou ac o ial
design poin s; ou expe imen s o axial le el; and a cen al poin wi h
h ee eplicas. The wo independen a iable, X1 (REX-TPA concen a ion,
g/L) and X2 (Ammonium concen a ion, g/L) and he esponse Y1 (CDW,
g/L), Y2 (PHA w .%) and Y3 (TAG, w .%) o Rhodococcus sp. A e7. Table S2
One-way ANOVA esul s o kine ic and s oichiome ic pa ame e s o he
h ee assays pe o med by Rhodococcus sp. A e7 using REX-TPA. Fig. S1
Th ee-dimensional esponse su ace and con ou plo s show he in e ac-
i e e ec s o di e en concen a ions o ammonium and REX-TPA on
CDW (g/L) (a), PHA (w .%) (b) and TAG (w .%) (c) o Rhodococcus sp. A e7.
Acknowledgemen s
No applicable.
Au ho s’ con ibu ions
Concep ualiza ion: ATR, CT, and FF; o mal analysis: ATR, CT and FF; in es iga-
ion: ATR; me hodology: ATR; HK, LS, OA; w i ing—o iginal d a : ATR; w i ing—
e iew and edi ing: MF, CT, CET, MM, MR and FF. All au ho s ha e ead and
ag eed o he published e sion o he manusc ip .
Page 18 o 21
Rebochoe al. Bio echnology o he En i onmen (2025) 2:5
Funding
This wo k was inanced by he Eu opean Union’s Ho izon 2020 esea ch and
inno a ion p og am h ough P ojec Bio Inno a ion o a Ci cula Economy
o Plas ics (BioICEP), unde g an ag eemen No. 870292, suppo ed by he
Na ional Na u al Science Founda ion o China (g an numbe s: Ins i u e o
Mic obiology, Chinese Academy o Sciences: 31961133016; Beijing Ins i u e
o Technology: 31961133015; Shandong Uni e si y: 31961133014). Eu opean
Union’s Ho izon Eu ope EIC Pa h inde p og amme, Eco con e sion o lowe
g ade PET and mixed ecalci an PET plas ic was e in o high pe o ming
biopolyme s (EcoPlas ic), unde g an ag eemen No 101046758. By na ional
unds om FCT—Fundação pa a a Ciência e a Tecnologia, I.P., in he scope o
he p ojec s UIDP/04378/2020 and UIDB/04378/2020 o he Resea ch Uni on
Applied Molecula Biosciences—UCIBIO and he p ojec LA/P/0140/2020 o
he Associa e Labo a o y Ins i u e o Heal h and Bioeconomy—i4HB. A.T. R.
acknowledges FCT I.P. o he Ph.D. g an 2020.06470.BD.
Da a a ailabili y
No da ase s we e gene a ed o analysed du ing he cu en s udy.
Decla a ions
E hics app o al consen o pa icipa e
No applicable.
Consen o publica ion
No applicable.
Compe ing in e es s
The au ho s decla e no compe ing in e es s.
Recei ed: 8 July 2024 Accep ed: 3 Ma ch 2025
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