bioe-09-642023 Ma ch 10, 2021 Time: 14:6 # 1
BRIEF RESEARCH REPORT
published: 16 Ma ch 2021
doi: 10.3389/ bioe.2021.642023
Edi ed by:
P asun Kuma ,
Chungbuk Na ional Uni e si y,
Sou h Ko ea
Re iewed by:
Suchada Chanp a eep
Napa ho n,
Chulalongko n Uni e si y, Thailand
Vic o ia Eugenia San os Mazo a,
Complu ense Uni e si y o Mad id,
Spain
*Co espondence:
Ignacio Poble e-Cas o
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Biop ocess Enginee ing,
a sec ion o he jou nal
F on ie s in Bioenginee ing and
Bio echnology
Recei ed: 15 Decembe 2020
Accep ed: 18 Feb ua y 2021
Published: 16 Ma ch 2021
Ci a ion:
Bo e o-de Acuña JM, Rohde M,
Saldias C and Poble e-Cas o I (2021)
Fed-Ba ch mcl-
Polyhyd oxyalkanoa es P oduc ion
in Pseudomonas pu ida KT2440
and 1phaZ Mu an on
Biodiesel-De i ed C ude Glyce ol.
F on . Bioeng. Bio echnol. 9:642023.
doi: 10.3389/ bioe.2021.642023
Fed-Ba ch mcl-
Polyhyd oxyalkanoa es P oduc ion in
Pseudomonas pu ida KT2440 and
1phaZ Mu an on Biodiesel-De i ed
C ude Glyce ol
José Manuel Bo e o-de Acuña1,2, Man ed Rohde3, Cesa Saldias4and
Ignacio Poble e-Cas o5*
1Ins i u e o Mic obiology, Technische Uni e si ä B aunschweig, B aunschweig, Ge many, 2B aunschweig In eg a ed
Cen e o Sys ems Biology (BRICS), Technische Uni e si ä B aunschweig, B aunschweig, Ge many, 3Cen al Facili y
o Mic oscopy, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many, 4Depa amen o de Química Física,
Facul ad de Química y Fa macia, Pon i icia Uni e sidad Ca ólica de Chile, Macul, Chile, 5Biosys ems Enginee ing Labo a o y,
Facul y o Li e Sciences, Cen e o Bioin o ma ics and In eg a i e Biology, Uni e sidad And es Bello, San iago, Chile
C ude glyce ol has eme ged as a sui able eeds ock o he bio echnological p oduc ion
o a ious indus ial chemicals gi en i s high su plus ca alyzed by he biodiesel indus y.
Pseudomonas bac e ia me abolize he polyol in o se e al biopolyme s, including
algina e and medium-chain-leng h poly(3-hyd oxyalkanoa es) (mcl-PHAs). Al hough
P. pu ida is a sui ed pla o m o de i e hese polyoxoes e s om c ude glyce ol, he
a ained concen a ions in ba ch and ed-ba ch cul u es a e s ill low. In his s udy, we
employed P. pu ida KT2440 and he hype -PHA p oduce 1phaZ mu an in wo di e en
ed-ba ch modes o syn hesize mcl-PHAs om aw glyce ol. Ini ially, he cells g ew in a
ba ch phase (µmax 0.21 h−1) o 22 h ollowed by a ca bon-limi ing exponen ial eeding,
whe e he speci ic g ow h a e was se a 0.1 (h−1), esul ing in a cell d y weigh (CDW) o
nea ly 50 (g L−1) a 40 h cul i a ion. Du ing he PHA p oduc ion s age, we supplied he
subs a e a a cons an a e o 50 (g h−1), whe e he KT2440 and he 1phaZ p oduced
9.7 and 12.7 gPHA L−1, espec i ely, a e 60 h cul i a ion. We nex e alua ed he PHA
p oduc ion abili y o he P. pu ida s ains using a DO-s a app oach unde ni ogen
deple ion. Ci ic acid was he main by-p oduc sec e ed by he cells, accumula ing in
he cul u e b o h up o 48 (g L−1) unde ni ogen limi a ion. The mu an 1phaZ amassed
38.9% o he CDW as mcl-PHA and exhibi ed a speci ic PHA olume ic p oduc i i y
o 0.34 (g L−1h−1), 48% highe han he pa en al KT2440 unde he same g ow h
condi ions. The biosyn hesized mcl-PHAs had a e age molecula weigh s anging om
460 o 505 KDa and a polydispe si y index (PDI) o 2.4–2.6. He e, we demons a ed
ha he DO-s a eeding app oach in high cell densi y cul u es enables he high yield
p oduc ion o mcl-PHA in P. pu ida s ains using he indus ial c ude glyce ol, whe e he
ed-ba ch p ocess selec ion is essen ial o exploi he supe io biopolyme p oduc ion
hallma ks o enginee ed bac e ial s ains.
Keywo ds: mcl-poly(3-hyd oxyalkanoa es), ed-ba ch, c ude glyce ol, pseudomonas pu ida, PHA depolyme ase
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INTRODUCTION
The wo ldwide manu ac u e o pe ochemical plas ics eaches
o e 359 million ons annually as hese ma e ials a e essen ial
in ou cu en li es yle (Poble e-Cas o e al., 2020a;Tou nie
e al., 2020). Un o una ely, syn he ic plas ics a e no p one
o biodeg ada ion and massi ely accumula e in he oceans
and soil ecosys ems (Ze le e al., 2013;Lau e al., 2020).
Fac o s like empe a u e and adia ion decompose he polyme s
in small pa icles (<5 mm), en e ing now in o he ood
chain (Law and Thompson, 2014). This phenomenon is
de imen al o p ese ing na u al en i onmen s and human
heal h (Smi h e al., 2018), demanding ac ions o mo e owa d
a ci cula biop oduc ion economy. To his end, comme cial
subs i u es o pe ochemical plas ics a e he biodeg adable
poly(3-hyd oxyalkanoa es) (PHAs). These biopolyme s possess
physical and mechanical p ope ies simila o oil-based plas ics
displaying he mal malleabili y and elas ici y and ha ing
sui able b eaking poin s o he mo o ming (Laycock e al.,
2013). The indus ial sec o s ha exploi hese biopolyme s
include ood, ex ile, ag icul u e, biomedicine, and elec onics
(Raza e al., 2018).
PHAs a e na u ally occu ing inclusion bodies du ing nu ien
imbalance o med in mic obes’ cy osolic space and epo ed as
ene gy ese oi s (Madison and Huisman, 1999) and essen ial
elemen s o cope wi h di e en s ess agen s (Ob uca e al., 2017).
A mani old o companies cu en ly manu ac u es PHAs a a
la ge scale (Bo e o-de Acuña e al., 2017). Howe e , indus ial
p oduc ion o PHAs is cos ly, gi en high ope a ional expenses
because o he employed ca bon subs a e and downs eam
p ocessing (Chen e al., 2020). Hence, o o e come hese se backs,
was e ma e ials a ise as low-cos ca bon subs a es o sus ain
bac e ial g ow h and enewable polyes e p oduc ion (Cesá io
e al., 2014;Nielsen e al., 2017;Bo e o-de Acuña e al., 2019).
Combining enginee ed mic obial cell ac o ies wi h high cell
densi y e men a ions is a obus app oach o achie e ele a ed
PHA p oduc i i ies (Choi e al., 2020).
In he las decade, he biodiesel indus y has gene a ed la ge
quan i ies o c ude glyce ol, an ine i able by-p oduc esul ing
om he es e i ica ion p ocess o a y acids (Ga lapa i e al.,
2016). Gi en he high glyce ol su plus, he p oduc ma ke
p ice is con inually d opping (Zhang e al., 2020), making
i an a ac i e subs a e o de i e biochemicals (Kau e al.,
2020). Mic obial ca abolism o c ude glyce ol p esen s some
challenges since he biodiesel by-p oduc con ains me hanol,
aces o diesel, and hea y me als (Mo hes e al., 2007;Samul
e al., 2014). Rema kably, Pseudomonas s ains can endu e hese
oxic ma e ials (Poble e-Cas o e al., 2017) and uel glyce ol
me abolic p oduc s in o PHA biosyn he ic pa hways enabling
he syn hesis o medium-chain leng h (mcl-PHAs) (Kenny e al.,
2012;Pappala do e al., 2014;Fu e al., 2015;Liu e al., 2018)
and copolyme s o sho -co-medium-chain leng h polyes e s
(O ellana-Saez e al., 2019;Pacheco e al., 2019). Ba ch and ed-
ba ch p oduc ion o mcl-PHA is easible using c ude glyce ol
in Pseudomonas pu ida whe e he s ain KT2440 o med 34%
o he cell d y weigh (CDW) as polyes e wi h a inal p oduc
i e o 1.45 (g L−1) in 75 h (Poble e-Cas o e al., 2014a).
A newly isola ed soil s ain om Thailand, Pseudomonas sp.
ASC2, p o ed o syn hesize 3.02 (g L−1) o mcl-PHAs in lask
expe imen s. In a high cell densi y e men a ion, P. pu ida GO16
a ained 19 (g L−1) biomass using he was e polyol, showing a
speci ic PHA olume ic p oduc i i y o 0.13 (gPHA L−1h−1)
(Kenny e al., 2012).
While he polyme iza ion o PHAs in P. pu ida is well
cha ac e ized and elies on he PHA syn hase p o eins PhaC1 and
PhaC2, he no ye ully unde s ood depolyme iza ion p ocess is
a esul in pa o he enzyma ic ac ion o PHA depolyme ase
(PhaZ, PP_5004) (A ias e al., 2013). Di e en me abolic
s imuli go e n gene egula o y c oss alk, wi h he polyme iza ion
s. depolyme iza ion o PHA kine ics s ill unclea (Ka mann
e al., 2017;Velázquez-Sánchez e al., 2020). The accumula ing
e idence sugges s ha he polyme iza ion egula o y con ol
depends on he ype o ca bon subs a e (De Eugenio e al.,
2010). Inac i a ion o he phaZ gene in P. pu ida KT2442
boos ed mcl-PHA p oduc ion o cells g own on a y acids
(Cai e al., 2009). Con e sely, non- ela ed PHA ca bon sou ces,
glucose o glucona e, did no yield highe PHA i e s in his
mu an . Glyce ol-g own cells o a phaZ-lacking s ain o KT2440
p oduced 36% mo e biopolyme han he wild ype using c ude
glyce ol (Poble e-Cas o e al., 2014a). Using a y acids as
subs a es, a phaZ minus s ain o KT2440 o med mo e han 70
(g L−1) o unsa u a ed mcl-PHA in ed-ba ch cul u es (Vo e al.,
2015). These expe imen s laid he g oundwo k o challenging
he p oduc ion capaci ies o he phaZ-de icien mu an in high
cell densi y cul u es. He e, we assessed di e en eeding s a egies
o high i e PHA p oduc ion in P. pu ida s ains on indus ial
c ude glyce ol. The DO-s a ed-ba ch e men a ion is bes
sui ed o biocon e aw glyce ol in o he elas ome polyes e s
whe e he 1phaZ mu an and he pa en al KT2440 eached
mcl-PHA speci ic olume ic p oduc i i ies o 0.34 and 0.23 (g
L−1h−1), espec i ely.
RESULTS AND DISCUSSION
Cons an -Feeding PHA Syn hesis Unde
Ni ogen Limi a ion
P. pu ida KT2440 and a phaZ-de icien mu an s ain
demons a ed in a p e ious s udy o syn hesize e icien ly
mcl-PHAs om c ude glyce ol in ba ch cul u es (Poble e-
Cas o e al., 2014a). We now challenged hese na u al polyes e
p oduce s in he p ocess o choice o he indus ial p oduc ion o
PHAs, he ed-ba ch cul u e. Applying di e en eeding s a egies
using indus ial c ude glyce ol wi hou any modi ica ion
(C eme Oleo, GmbH, Ge many), we de eloped a h ee-s age
e men a ion whe e he wo ini ial phases aimed o o m biomass
and he hi d s age mcl-PHA unde ni ogen deple ion. The ba ch
cul u es s a ed wi h a biomass concen a ion o 0.13 (g L−1)
in a 4 L wo king olume bio eac o . The cul u e b o h ini ially
con ained 20 (g L−1) c ude glyce ol and 1 (g L−1) o glucose o
p e en a cha ac e is ic ex ended lag phase (Escapa e al., 2012;
Becke s e al., 2016), usually aking mo e han 10 h o P. pu ida
cells g owing on glyce ol as he sole C sou ce (Poble e-Cas o
e al., 2020b). As he P. pu ida cells p opaga e, oam de eloped
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FIGURE 1 | Cons an - eeding ed-ba ch mcl-PHA p oduc ion. The e men a ion p ocess comp ised h ee phases: (i) ba ch, (ii) exponen ial eeding, and (iii) cons an
eeding o c ude glyce ol (50 g h−1) unde ni ogen deple ion. Time p o ile o (A) P. pu ida KT2440 and (B) phaZ-knockou mu an . The da a ep esen he mean
alues and s anda d de ia ion om wo independen expe imen s.
in he bio eac o (a 3 h), which dispe sed once we p o ided
an i oam (200 µL L−1). A e 22-h cul i a ion, P. pu ida KT2440
and 1phaZ mu an had simila maximum speci ic g ow h
a es o 0.21 h−1(Figu e 1A), eaching a CDW o 11.5 g L−1
(Figu es 1A,B). Then, we s a ed eeding glyce ol exponen ially,
se ing he speci ic g ow h a e a nea ly 50% o µmax (0.1 h−1)
(Figu e 1). Du ing his phase, no glyce ol accumula ed in he
cul u e b o h o he cells sec e ed by-p oduc s due o he coupled
ca abolism and anabolism unde ca bon limi ing condi ions
(Poble e-Cas o e al., 2012;González-Cabalei o e al., 2015).
A 40 h cul i a ion, we no longe p o ided glyce ol and
ammonium; we ins ead p o ided a subs a e pulse o a ain
10 (g L−1) o glyce ol wi hin he bio eac o . This p ocedu e
igge ed ni ogen limi a ion, and once glyce ol was unde he
de ec ion limi (a e 1 h), we ed he subs a e, bu his ime a
a cons an mass low a e o 50 (g h−1). Du ing he biopolyme
p oduc ion phase, we upheld he ai low a e, p o iding only
il e ai ins ead. The DO e olu ion showed a ma ked inc ease
as he cells accumula ed highe amoun s o PHAs h ough he
p ocess, showing a educed O2demand as he cells no longe
duplica e (Figu es 1A,B). The P. pu ida s ains sec e ed ci ic
acid as he main co-p oduc as glyce ol accumula ed in he
medium, showing a inal yield on he polyol o 0.18 ±0.01 (g
g−1) and 0.16 ±0.02, o he wild- ype and he mu an s ain,
espec i ely (Table 1 and Figu e 1). Indeed, ni ogen deple ion
o glyce ol-g own P. pu ida cells inhibi s he TCA cycle enzymes
like isoci a e dehyd ogenase (ICD), slowing he ca bon lux
h ough he oxida i e ou e (Becke s e al., 2016). The biomass
su e ed a sligh educ ion in KT2440 and he phaZ-knockou
mu an om ∼50 (g L−1) o 45.4 and 46.7 (g L−1), espec i ely,
a common end o cells endu ing o e low me abolism (Xu
e al., 1999;Poble e-Cas o e al., 2014b). A he end o he
e men a ion p ocess (60 h), he wild- ype KT2440 syn hesized
9.7 (gPHA L−1) wi h a biopolyme con en o 21.4% o he CDW
(Figu e 1A and Table 1), while he 1phaZ dele ion mu an
achie ed 12.7 (gPHA L−1), amassing 27.2% o he cell biomass
as polyes e (Figu e 1B and Table 1). Finally, he monome ic
composi ion o he gene a ed biopolyme s by cons an - eeding
s a egy was domina ed by 3-hyd oxydecanoa e wi h a 75.4
and 76.1% ela i e mola ac ion in he wild- ype and 1phaZ
mu an , espec i ely (Table 1). The es o he monome s had he
ollowing dec easing p opo ion wi hin he polyme ic chain in
bo h es ed s ains: 3-hyd oxyoc anoa e, 3-hyd oxydodecanoa e,
3-hyd oxy-5-cis-dodecanoa e, 3-hyd oxyhexanoa e, and
3-hyd oxy e adecanoa e.
PHA P oduc ion Using a
Dissol ed-Oxygen-S a Feeding
(DO-S a ) Unde Ni ogen Limi a ion
Despi e he sound PHA olume ic concen a ion ob ained in
his s udy ope a ing he cons an - eeding scheme, he P. pu ida
s ains did no amass he PHA con en p e iously epo ed
in ba ch cul u es (Poble e-Cas o e al., 2014a). Thus, o ully
ha ness he biopolyme p oduc ion capaci ies o he 1phaZ
mu an and he wild ype, a dissol ed oxygen-s a (DO-s a )
eeding app oach was applied. This well-con olled s a egy has
p o en o yield highe PHA amoun and con en in enginee ed
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TABLE 1 | Medium-chain leng h (mcl-PHA) p oduc ion in Pseudomonas s ains unde a ious e men a ion modes u ilizing c ude glyce ol.
S ain P oduc ion
mode
Biomass PHA PHA
con en
YCIT/Gly
Ci a e
yield
YPHA/Gly
PHA yield
Speci ic PHA
olume ic
p oduc i i y
Monome ic composi ion (%) Re e ences
(g L−1) (g L−1) (%w ) (g g−1) (g g−1) (g L−1h−1) C6 C8 C10 C12 C12:1 C14
P. pu ida
KT2440
Cons an
eeding
45.3 9.7 21.4 0.18 0.08 0.16 1.2 15.3 75.4 5.7 2.4 0.3 This s udy
1phaZ Cons an
eeding
46.7 12.7 27.2 0.16 0.10 0.21 1.3 16.1 76.1 5.9 0.6 N.D. This s udy
P. pu ida
KT2440
DO-s a 49.5 13.8 27.9 0.36 0.11 0.23 0.8 15.8 73.3 5.6 4.5 0.4 This s udy
1phaZ DO-s a 52.4 20.4 38.9 0.32 0.13 0.34 1.2 16.7 76.2 5.4 0.5 N.D. This s udy
P. pu ida GO16 Fed ba ch 19.0 6.3 33.2 N.D. N.S 0.13 3 18 35 13 15 7 Kenny e al., 2012
Pseudomonas
sp. ASC2
Ba ch 10.7 3.0 28.2 N.D N.S. 0.04 N.D. 4.8 N.D. N.D. 95.2 N.D. Muangwong e al., 2016
N.D., No de ec ed; N.S., No shown.
P. pu ida s ains on glucose and a oma ics (Poble e-Cas o e al.,
2014b;Bo e o-de Acuña e al., 2020). Fo biomass o ma ion,
we epea ed he same g ow h condi ions as hose employed
du ing he cons an - eeding app oach, whe e a mix u e o
pu e oxygen and ai was p o ided o a oid oxygen limi a ion.
Ni ogen was always su icien a his s age, eaching bo h s ains
biomass p oduc ions o nea ly 50 (g L−1) (Figu e 2). A his
poin , we p o ided a pulse o glyce ol (10 g L−1) o enable
he cells o consume he emaining ammonium, and o ge
a be e DO esponse, we p o ided only il e ai as ca ied
ou in he cons an - eeding cul i a ions. Likewise, ci a e began
o accumula e in he cul u e b o h due o he me abolic shi
p o oked by ni ogen limi a ion (Figu e 2). As a esponse o
glyce ol exhaus ion, he DO sa u a ion inc eased; hus, e e y ime
he dissol ed oxygen exceeded a alue o 70%, an au oma ed
addi ion o he subs a e (20 g L−1glyce ol) occu ed in an
in e al o 15 min (Figu e 2). This eeding ope a ion did
no educe he o med biomass, and he in acellula polyes e
syn hesis boos ed, main aining a apid pace o accumula ion
un il he e mina ion o he p ocess (Figu e 2). A e 20 h o
eeding d i en by he DO esponse, he wild- ype s ain a ained
a biomass i e o 49.5 (gCDW L−1), o which 13.8 (g L−1)
consis ed o mcl-PHA (Figu e 2A and Table 1). Simila ly, he
depolyme ase-de icien mu an achie ed a biomass yield o 52.4
(gCDW L−1) bu comp ised an enhanced biopolyme yield o
20.4 (g L−1)—o e all PHA con en 38.9%w (Figu e 2B). The
speci ic PHA olume ic p oduc i i y (0.34 g L−1h−1) eached
by he 1phaZ mu an is he highes epo ed oday o de i e
mcl-PHA using indus ial c ude glyce ol (Table 1). P. pu ida
KT2440 and i s phaZ knockou mu an s ain sec e ed 48 (g
L−1) o ci ic acid in he DO-s a s age (Figu e 2). Howe e ,
ci a e yields on glyce ol ob ained in he ed-ba ch cul i a ions
we e lowe (Table 1) han he alues p e iously epo ed (0.5 g
g−1) in ba ch cul u es (Poble e-Cas o e al., 2014a). The co-
p oduc ion o ci a e no only is de imen al in e ms o ca bon
loss o biopolyme syn hesis bu also impac s nega i ely o he
PHA p oduc ion p ocess as p oduc ion cos s boos , and he
addi ion o he base solu ion o main ain he op imal pH exe s
a dilu ion e ec lowe ing he PHA olume ic p oduc i i ies
(Wa necke and Gill, 2005).
I is clea ha u he me abolic enginee ing e o s mus
a oid he en y o he ca bon lux in o he TCA cycle du ing
he biopolyme p oduc ion phase o diminish o ganic acid
o ma ion in P. pu ida KT2440. The e a e se e al ou es o
achie e his, beginning wi h he o e exp ession o ace yl-CoA
ca boxylase (ACC), which con e s ace yl-CoA in o malonyl-
CoA, whe e he la e is he main p ecu so o PHA syn hesis
o subs a es me abolized h ough he cen al ca bon me abolism
(Rehm e al., 1998;Poble e-Cas o e al., 2013). Ano he pa h
is o block he en y o ace yl-CoA in o he K ebs cycle by
inac i a ing in i o he ci a e syn hase enzyme using RNA
in e e ence o an isense RNA (Desai and Papou sakis, 1999;Ko
e al., 2020), as he gene ic sys ems can be ac i a ed du ing he
ni ogen-limi ing phase, which may ha e no nega i e impac s on
biomass o ma ion in he ini ial nu ien -su icien phase. Finally,
inspec ion o he monome composi ion o he biosyn hesized
PHA highly esembled he p e ious eeding egime’s alues. The
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FIGURE 2 | DO-s a ed-ba ch mcl-PHA p oduc ion. The cul u e comp ised h ee phases: (i) ba ch, (ii) exponen ial eeding, and (iii) DO-s a esponse o subs a e
eeding unde ni ogen limi a ion. Time p o ile o (A) P. pu ida KT2440 and (B) phaZ-knockou mu an . The da a ep esen he mean alues and s anda d de ia ion
om wo independen expe imen s.
mos abundan hyd oxy acid encoun e ed in bo h s ains was
he C10 (Table 1). This monome ’s ela i e mola ac ion in he
pa en al s ain was 73.3%, whe eas he 1phaZ knockou mu an
p esen ed 76.2% (Table 1).
Visualiza ion o he Biosyn hesized
mcl-PHA and Physical P ope ies
C ude glyce ol con ains ha m ul compounds, including
me hanol, sal s, and hea y me als (Mo hes e al., 2007;Samul
e al., 2014). These elemen s impai cell g ow h and e oke
s ess esponses a he p o eome and ansc ip ome le els
(Mana a e al., 2012;Fu e al., 2015;Bojano iˇ
c e al., 2017).
This oxic eeds ock migh also in luence cell mo phology and
he polyme iza ion p ocess o he polyes e s. Figu e 3 depic s
mic og aphs acqui ed by ansmission elec on mic oscopy
aken a he maximum PHA o ma ion poin (60 h) in he
cons an eeding (3A, KT2440 and 3B, 1phaZ mu an ) and
DO-s a e men a ions (3C, KT2440 and 3D, 1phaZ mu an ).
Nei he he pa en al s ain no he phaZ-dis up ed mu an
displays signi ican cellula mo phological a iance o al e ed
PHA inclusion bodies (Figu e 3). Con e sely o a p e ious
s udy on c ude glyce ol, he cells showed no agg ega ion
(Poble e-Cas o e al., 2014a), indica ing obus g ow h and
p ope mixing. Impu i ies p esen in he subs a e sou ce
as black do s we e dis inc i e in bo h g owing P. pu ida
s ains. No ably, in acellula PHA s uc u es had no appa en
al e a ion by dele ing he polyme disassembling enzyme PhaZ
(Figu es 3B,D). As p e iously no ed, P. pu ida s ains g own on
c ude glyce ol p esen ed la ge numbe s o in acellula PHA
g anules han hose cul i a ed on pu e glyce ol (Poble e-Cas o
e al., 2014a), which a e mo e une enly shaped (Figu es 3A–D).
The PHA mo phology depends on a ious ac o s, including he
le el o exp ession o g anula associa e p o eins like phasins
and PHA polyme ase and depolyme ase (Jend ossek, 2009),
which in u n ely on he cul u e condi ions and he g ow h
subs a e (De Eugenio e al., 2010). Unde s ess, PHA-p oducing
bac e ia al e he PHA con en and i s mo phology as a esponse
mechanism imp o ing he su i al a e when cells h i e unde
high sal concen a ions and low and ele a ed empe a u es o
encoun e ing oxic compounds (Ob uca e al., 2020). As aw
glyce ol con ains me hanol and hea y me al, his could explain
he in acellula g anule’s obse ed al e a ions.
We also un eiled he he mal p ope ies o he biosyn hesized
mcl-PHA ia di e en ial scanning calo ime y (DSC) o pu i ied
biopolyme sampled a 60-h cul i a ion om he bio eac o s.
The ob ained glass ansi ion poin s (Tg) we e e y simila
among he p oduced mcl-PHAs (−10.5 o −11.9◦C) showing
a single Tg cha ac e is ic o a copolyme o medium-chain
leng h polyes e s (Cheema e al., 2012;Pacheco e al., 2019;
Figu e 3E). As dis inc i e elas ome s, he mel ing endo he ms
we e in he ange o 56.7–59.3◦C (Chan Sin e al., 2010;
Gumel e al., 2012). Mos impo an ly, he molecula weigh
o he pu i ied PHA was highe han 400 KDa (Figu e 3E),
a p e equisi e o p ocessing hese kinds o mac omolecules
indus ially (Mo hes e al., 2007), especially as so ene ma e ials
(Muangwong e al., 2016;Liu e al., 2018). Toge he , he
PHA he mal and physical p ope ies did no a y signi ican ly
be ween he polyes e s syn hesized by wild- ype and he phaZ
minus mu an (Figu e 3E). A Pseudomonas esino o ans s ain
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FIGURE 3 | T ansmission elec on mic og aphs o P. pu ida cells in ed-ba ch e men a ion aken a 60 h e men a ion. (A) P. pu ida KT2440 cons an eeding,
(B) phaZ-knockou mu an cons an eeding, (C) P. pu ida KT2440 DO-s a , and (D) phaZ-knockou mu an DO-s a . (E) The mal p ope ies o he biosyn hesized
mcl-PHA: T ansi ion glass empe a u e (Tg), mel ing empe a u e (Tm), and c ys alliza ion empe a u e (Tc). Molecula weigh (Mw), a e age-numbe molecula weigh
(Mn), and polydispe si y index (PDI). The alues ep esen mean and s anda d de ia ion om wo eplica es.
lacking he phaZ gene p esen ed he same molecula weigh s and
he mal p ope ies o he accumula ed PHA as he pa en al s ain
(Solaiman e al., 2003). Fu he , gi en he high polydispe si y
index alues (PDI >2), he syn hesized mcl-PHA on c ude
glyce ol would also pe o m app op ia ely in ex ude s as
he elas ome s exhibi na ow a e age-numbe mola mass
dis ibu ion (Mn, 190 KDa), whe e hese alues a e ela i ely
close o comme cial polyes e s (Debuissy e al., 2017). While
ex ac ing he PHA om he cells, we could app ecia e he
elas ome ilm ha emained a e chlo o o m e apo a ion in a
Pe i dish o pu i ica ion. Film ma e ials a e pa amoun in he
packaging and ag oindus y sec o s. A ilmable mcl-PHA was
syn hesized by Pseudomonas medi e anea using aw glyce ol
(Pappala do e al., 2014). The ob ained mcl-PHA p esen ed nea ly
he same p opo ion o C10 in he monome ic chain han he
ilmable biopolyme , and abo e all, bo h elas ome s display Tm
alues highe han 50◦C and Mw o 400 KDa.
CONCLUSION
In his s udy, we demons a ed ha he applied eeding s a egy
du ing mcl-PHA syn hesis in P. pu ida s ains using he c ude
glyce ol as subs a e in luences he amoun o o med polyes e
bu no hei physical p ope ies. We p o ed ha a DO-s a
ed-ba ch p ocess is a mo e sui able eeding s a egy han he
cons an - eeding app oach o me abolize he oxic c ude glyce ol
esul ing in nea ly 50% mo e biopolyme a he end o he
e men a ion in he phaZ-de icien mu an and he wild- ype
KT2440. The a ained speci ic PHA olume ic p oduc i i y
by 1phaZ knockou mu an (0.34 g L−1h−1) is a s ep
u he in he ques o de i e mcl-PHA om he polyol o he
biodiesel indus y in a mo e cos -e ec i e ashion. The e is s ill
oom o imp o emen conce ning PHA p oduc ion since he
P. pu ida cells sec e ed high ci ic acid le els. Fu he me abolic
enginee ing endea o s mus educe he ca bon was age and
edi ec ca bon lux owa d polyes e biosyn he ic pa hways o
mining he cell ac o y’s PHA p oduc ion pe o mance.
METHODS
P. pu ida S ains
The wild- ype P. pu ida KT2440 (DSM 6125) was ob ained om
he DSMZ collec ion, Ge many, and he 1phaZ mu an was
cons uc ed in a p e ious s udy (Poble e-Cas o e al., 2014a).
These s ains we e used o he di e en ed-ba ch e men a ions.
Cul u e Condi ions
S ains we e s o ed in 25% glyce ol a −80◦C as glyce ol s ocks.
Cells we e ou inely s eaked on o Lu ia–Be ani (LB) aga pla es
and g own o e nigh o isola e single colonies. Fo any shake
lask cul i a ion, P. pu ida s ains we e ae obically g own a
180 pm and 30◦C. Single colonies we e picked om he pla e and
ans e ed in o a 50 mL shake lask con aining 10 mL o LB liquid
medium. De ined minimal medium (M9) con aining 6 (g L−1)
c ude glyce ol (C eme Oleo, GmbH, Hambu g, Ge many) was
employed o subsequen p e-cul u e cul i a ion. The indus ial
glyce ol con ains 80% glyce ol, 0.5% me hanol, 10% ash, 3%
o ganic ma e , and 6.5% wa e . The M9 medium composi ion
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consis ed (pe li e ) o 12.8 g Na2HPO4·7H2O, 3 g KH2PO4,
4.7 g (NH4)2SO4, and 0.5 g NaCl. A e au ocla e s e iliza ion,
he medium was supplemen ed wi h il e ed ace elemen s [6.0
FeSO4·7H2O, 2.7 CaCO3, 2.0 ZnSO4·H2O, 1.16 MnSO4·H2O,
0.37 CoSO4·7H2O, 0.33 CuSO4·5H2O, and 0.08 H3BO3(mg
L−1)] and 0.12 g o MgSO4·7H2O. Ten millili e s o M9 medium
in a 50 mL shake lask was inocula ed wi h he o e nigh LB-
g own cul u e a an ini ial OD a 600 nm o 0.2 and incuba ed
o e nigh . A second p e-cul u e was ini ia ed by ans e ing
a p ede e mined olume o he p e ious one in o 300 mL o
M9 medium con aining 10 g L−1c ude glyce ol in 1 L ba led
E lenmeye lasks and cul i a ed o e nigh .
Fed-Ba ch Cul i a ions
The ed ba ches we e seeded wi h he second p e-cul u e
o a ain an ini ial cell densi y o (OD600 0.26). The ed-
ba ch eac o con ained M9 medium supplemen ed wi h 20 g
L−1c ude glyce ol, 0.12 g L−1MgSO4·7H2O, and 8 ml o
he ace elemen solu ion. O e all, 4 L o wo king olume
was se up in a 15 L essel (B10 s i ed ank bio eac o ,
Biologische Ve ah ens echnik, Basel, Swi ze land) o conduc
he e men a ion p ocesses. The ai low a e was main ained a
10 L min−1(ai - o-pu e oxygen a io was se o 10:1) du ing he
biomass p oduc ion phase. In he PHA p oduc ion phase, he
ai low a e was kep , bu pu e oxygen was no longe needed,
and he eac o was spa ged only wi h comp essed ai , p o iding
be e DO esponse when he ca bon subs a e was supplied.
The empe a u e was se a 30◦C and 12.5 (w/ ) o NH4OH
was added as equi ed o s abilize pH o 6.8 ±0.1 du ing he
cou se o he biomass o ma ion phase and as ni ogen supply
o a oid N limi a ion. Then, in he PHA p oduc ion phase,
he base was eplaced by NaOH 10% (w/ ). When equi ed,
Tego An i oam (E onik, Ge many) was supplemen ed o p e en
oaming (200 µL L−1). The agi a ion speed was au oma ically
adjus ed o 800 pm in o de o keep he dissol ed oxygen le el
abo e 20% ai sa u a ion.
The eeding solu ion con ained pe li e : 780 g c ude glyce ol
and 12 g MgSO4·7H2O. An exponen ial eeding s a egy was
applied du ing he biomass p oduc ion phase, ollowing an
exponen ial unc ion (Eq. 1).
F( )=µse (V0.X0)e(µse . )(S0.Yxs)−1(1)
He eby, Fis he eed a e (L h−1), µse is he desi ed speci ic
g ow h a e (se o 0.1 h−1), S0is he subs a e concen a ion o
he eed medium (780 g L−1c ude glyce ol), is he ime a e
eed s a (h), Yxs is he biomass yield on c ude glyce ol aken
om Poble e-Cas o e al. (2014a),V0is he ini ial olume o he
cul u e (L), and X0is he ini ial biomass le el (g cells L−1).
Biomass Quan i ica ion and Analy ical
P ocedu es
The op ical densi y (OD600 nm) was egis e ed o e ime in a
spec opho ome e (Ul aspec 2000; Hi achi, Japan) o de e mine
cell g ow h. Ten millili e s o cells was ha es ed a 9,000×g o
10 min a 4◦C and washed once wi h dis illed wa e p io o CDW
g a ime ic quan i ica ion in p e-weighed ubes. The cell pelle
was d ied o cons an weigh a 100◦C. A pho ome ic es (LCK
303 ki , Hach Lange, Danahe , Uni ed S a es) se ed o measu e
o line he ammonium le els in he supe na an . Supe na an
samples we e wi hd awn and acco dingly dilu ed o analyze he
c ude glyce ol and o ganic acid (ci a e, isoci a e, succina e,
uma a e, mala e, py u a e, and oxaloace a e) concen a ions by
HPLC Agilen 1260 (Agilen , K e eld, Ge many). The HPLC
sys em was equipped wi h an 8-mm Rezex ROA-o ganic acid
H column (Phenomenex, Uni ed S a es), which was ope a ed
a 65◦C. The mobile phase consis ed o 0.013 N H2SO4a
a 0.5 ml min−1 low coupled wi h a RID de ec o sys em
(Agilen se ie1260).
PHA Cha ac e iza ion and Quan i ica ion
The polyes e s we e i s ly me hanolized in o de o de e mine
he PHA monome ic composi ions and he in acellula PHA
con en . Fo his, 10 ml o cul u e was ans e ed o a alcon
ube and cells we e ha es ed a 9,000 ×g o 10 min a 4◦C
(Eppendo 5810 R, Hambu g, Ge many). Pelle s we e washed
once wi h dis illed wa e . The supe na an s we e disca ded,
and he pelle ed cells we e s o ed a −20◦C un il needed. The
me hanolysis p ocedu e was pe o med as p e iously speci ied
(Bo e o-de Acuña e al., 2014). Gas ch oma og aphy (GC)
coupled wi h mass spec ome y (MS) was used o analyze he
me hyl es e s o monome s. One millili e o he o ganic phase
was injec ed in o a Va ian GCMS sys em 450GC/240MS ion
ap mass spec ome e (Va ian Inc., Agilen Technologies) a
a spli a io o 1:10. The so wa e employed o p ocess he
esul ing da a was he MS Wo ks a ion 6.9.3 (Va ian Inc., Agilen
Technologies). The di e en compounds, i.e., he me hyl es e s o
3-hyd oxyexanoa e, 3-hyd oxyoc anoa e, 3-hyd oxydecanoa e,
3-hyd oxydodecanoa e, 3-hyd oxy-5-cis-dodecanoa e, and 3-
hyd oxy e adecanoa e, we e spli by using a Fac o Fou VF-
5ms capilla y column (30 m ×0.25 mm i.d. ×0.25 mm
ilm hickness), including calib a ion wi h comme cial PHB
(Sigma−Ald ich, MI, Uni ed S a es) and pu i ied mcl−PHA
om a p e ious wo k (Oli a-A ancibia e al., 2017). The ca ie
gas helium was se o a low a e o 0.9 ml min−1. The
empe a u es o he injec o and ans e line we e es ablished a
275 and 300◦C, espec i ely. The o en empe a u e was s epwise
p og ammed as ollows: 40◦C o 2 min, ising p og essi ely om
40 o 150◦C a a a e o 5◦C min−1and ul ima ely inc easing o
280◦C a a cons an a e o 10◦C min−1. To cap u e posi i e ions,
an elec on ioniza ion a 70 eV was se led, while he mass spec a
we e egis e ed by scanning ions o m/z 50 o m/z 650. The
PHA concen a ion was de e mined by he me hod desc ibed by
Lage een e al. (1988). The pe cen age o biopolyme in ela ion
wi h he CDW in wo biological eplica es was a e aged ou o
asce ain PHA con en (w %).
T ansmission Elec on Mic oscopy
P io o ixa ion, he bac e ia we e cooled down o 4◦C. Nex ,
2% o glu a aldehyde and 5% o o maldehyde (5%) we e added.
Cells we e subsequen ly washed wi h cacodyla e bu e (0.01 mol
L−1cacodyla e, 0.01 mol L−1CaCl2, 0.01 mol L−1MgCl2
6H2O, and 0.09 mol L−1suc ose, pH 6/9) and s ained o
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1 h a oom empe a u e wi h 1% aqueous osmium solu ion.
Dehyd a ion was achie ed by adding ace one a inc easing
concen a ions (10, 30, 50, 70, 90, and 100%) and incuba ing
he samples o 30 min a each ime. Solely he dehyd a ion
wi h 70% ace one con aining 2% u anyl ace a e was allowed
o e nigh . The Spu o mula o ha d esin was applied o
in il a e samples wi h an epoxy esin. A diamond kni e was
used o slice he samples in o ul a- hin sec ions, which we e
u he coun e s ained wi h a mix u e o u anyl ace a e and
lead ci a e. A TEM910 ansmission elec on mic oscope (Ca l
Zeiss, Obe kochen, Ge many) was ope a ed a an accele a ion
ol age o 80 kV o acqui e images. Digi al imaging o ul a-
hin sec ions was acqui ed wi h a Slow-Scan CCD-Came a
(P oScan, 1,024 ×1,024, Scheu ing, Ge many) wi h ITEM-
So wa e (Olympus So Imaging Solu ions, Muns e , Ge many).
Di e en ial Scanning Calo ime y
Analysis
The glass ansi ion, c ys alliza ion, and mel ing empe a u es
(Tg,Tc, and Tm, espec i ely) o each sample we e de e mined
by a Me le –Toledo DSC 821e. The ollowing i e cycles we e
pe o med: (i) a i s hea ing om −40 o 200◦C a 10◦C min−1,
(ii) an iso he m o 3 min, (iii) a cooling om 200 o −40◦C a
10◦C min−1, (i ) an iso he m o 3 min, and ( ) a second hea ing
om −40 o 200◦C a 10◦C min−1.
Size Exclusion Ch oma og aphy
Measu emen s
The weigh a e age molecula weigh s (Mw) and he espec i e
polydispe si y indices o he samples we e de e mined by
size exclusion ch oma og aphy (SEC) along wi h a s a ic
ligh sca e ing Dawn EOS in line wi h an Op ilab DSP
in e e ome ic e ac ome e (bo h we e ob ained om Wya
Technology) using CHCl3as he mobile phase and a calib a ion
cu e cons uc ed using polys y ene s anda d samples. The
SEC measu emen was pe o med on a Dionex P590A liquid
ch oma og aphy pump equipped wi h a gua d column and wo
PLgel 5-mm Mixed C (300 ×7.5 mm) columns in se ies wi h
a Visco ek di e en ial e ac ome e . The eluen was CHCl3a a
low a e o 1.0 mL min−1a 25◦C. Polys y ene s anda ds wi h a
molecula weigh ange o 1,020–1,944,000 we e used o gene a e
a uni e sal calib a ion cu e.
DATA AVAILABILITY STATEMENT
The o iginal con ibu ions p esen ed in he s udy a e included
in he a icle/supplemen a y ma e ial, u he inqui ies can be
di ec ed o he co esponding au ho /s.
AUTHOR CONTRIBUTIONS
IP-C concei ed and pe o med he ed-ba ch e men a ion and
analy ics. CS pe o med he he mal analysis. MR ca ied ou he
TEM s udies. JB-dA and IP-C in e p e ed he da a and w o e he
manusc ip . All au ho s con ibu ed o he a icle and app o ed
he submi ed e sion.
FUNDING
IP-C acknowledges inancial suppo om he p ojec s
INACH RG_17_19 and ANID-PIA-Anillo INACH ACT192057.
We acknowledge he Ge man Resea ch Founda ion and
he Open Access Publica ion Funds o he Technische
Uni e si ä B aunschweig.
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