Ci a ion: Do ado, I.; Pineda, L.;
Ascencio-Gal án, M.L.; López-
Agudelo, V.A.; Caicedo, J.C.;
Gómez-Ríos, D.; Ramí ez-Malule, H.
Valo iza ion o Dex ose om Cassa a
S a ch and Suga cane Vinasse as
Polyhyd oxyalkanoa es by
Subme ged Cul u es o Cup ia idus
neca o : A Physicochemical–
Bio echnological App oach.
ChemEnginee ing 2024,8, 73.
h ps://doi.o g/10.3390/
chemenginee ing8040073
Academic Edi o : Ma ín Ramí ez
Recei ed: 12 Ma ch 2024
Re ised: 24 June 2024
Accep ed: 16 July 2024
Published: 23 July 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
chemenginee ing
A icle
Valo iza ion o Dex ose om Cassa a S a ch and Suga cane
Vinasse as Polyhyd oxyalkanoa es by Subme ged Cul u es o
Cup ia idus neca o : A Physicochemical–Bio echnological App oach
Isabel Do ado 1,†, Lau a Pineda 1,†, Ma ha L. Ascencio-Gal án1,† , Víc o A. López-Agudelo 2, Julio C. Caicedo 3,
Da id Gómez-Ríos 1and Howa d Ramí ez-Malule 1,*
1School o Chemical Enginee ing, Uni e sidad del Valle, Cali 760042, Colombia;
[email p o ec ed] (I.D.); lau a.c is ina.pineda@co eouni alle.edu.co (L.P.);
[email p o ec ed] (M.L.A.-G.); da id.and [email p o ec ed] (D.G.-R.)
2Ins i u e o Clinical Molecula Biology, Kiel Uni e si y and Uni e si y Medical Cen e Schleswig-Hols ein,
24105 Kiel, Ge many; [email p o ec ed]
3
T ibology, Polyme s, Powde Me allu gy and Solid Was e T ans o ma ions Resea ch G oup, Uni e sidad del
Valle, Cali 760042, Colombia; julio.cesa [email p o ec ed]
*Co espondence: howa [email p o ec ed]
†These au ho s con ibu ed equally o his wo k.
Abs ac : The p oduc ion o polyhyd oxyalkanoa es using subme ged cul u es o Cup ia idus neca o
DSM 428 was e alua ed using low-cos subs a es om ag oindus y: (i) dex ose om cassa a s a ch
and (ii) a mix u e o suga cane inasse om he bioe hanol indus y and dex ose om cassa a s a ch.
The e ec s o inasse composi ion (2.5, 5.0, 7.5, 25, 50, and 75% / ) and he use o aw and ac i a ed
ca bon-p e- ea ed inasse we e assessed. The esul s indica e ha cul i a ions using only cassa a
s a ch dex ose eached 4.33 g/L o biomass as he d y cell weigh and a poly(3-hyd oxybu y a e)
(PHB) p oduc ion o 47.1%. Raw inasse p opo ions o 25, 50, and 75% in he cul u e medium
esul ed in o al inhibi ion. Vinasse ea ed a he same a ios led o biomass p oduc ion in he ange
1.7–4.44 g/L. The highe PHB p oduc ion scena io was ob ained in a medium con aining dex ose and
ea ed inasse (7.5%), yielding 5.9 g/L o biomass and 51% o PHB accumula ion. The p oduced PHB
was cha ac e ized by XRD and FTIR o an analysis o c ys alline s uc u e and chemical unc ional
g oups, espec i ely. EDS was employed o a semi-quan i a i e analysis o he chemical composi ion,
and SEM was used o analyze he mo phology o he mic og anules. The esul s o DSC and TGA
analyses demons a ed he he mal s abili y o he ob ained PHB.
Keywo ds: polyhyd oxyalkanoa es; cassa a s a ch; suga cane inasse; Cup ia idus neca o ; low-cos
subs a es; mechanical es ing
1. In oduc ion
O e he las wo decades, an inc easing need has eme ged o look o al e na i es
o pe ochemical polyme s ha a e mo e en i onmen ally iendly. Cu en ly, he use o
con en ional plas ic is being eplaced by o he ma e ials such as glass, s eel, o silicone, and
biodeg adable bioplas ics such as polyhyd oxyalkanoa es (PHAs), polylac ic acid (PLA),
and polybu ylene succina e (PBS). PHA is he biopolyme ha has s ood ou he mos in
ecen yea s due o i s high biodeg adabili y, esis ance, and e sa ili y. The e o e, i is
es ima ed ha PHA could become a di ec compe i o agains pe ochemical plas ics [1].
Poly(3-hyd oxybu y a e) (PHB) is one o he mos well-known and s udied PHAs
and is he only homopolyme in his amily. PHB sha es simila physical p ope ies wi h
polyp opylene [
2
], including a simila mel ing poin , c ys allini y, molecula weigh , and
ensile s eng h. Conce ning mechanical p ope ies, PHB demons a es high mois u e
esis ance bu is a he b i le and igid. None heless, due o i s biodeg adabili y, hese
p ope ies may al e o e ime [
3
]. Fu he mo e, PHB is he mos en i onmen ally iendly
ChemEnginee ing 2024,8, 73. h ps://doi.o g/10.3390/chemenginee ing8040073 h ps://www.mdpi.com/jou nal/chemenginee ing
ChemEnginee ing 2024,8, 73 2 o 17
polyme , conside ing i s biocompa ibili y and biodeg adabili y. These cha ac e is ics en-
able i s use as an implan ma e ial in he human body and as a ca ie o he ex ended
elease o an ibio ics [
4
]. These p ope ies, combined wi h he homogeneous, dense, and
nanos uc u ed o ma ion achie ed h ough a bio echnology me hod, posi ion PHB as a
sui able biocompa ible ma e ial o me allic implan s.
Acco ding o Mo lino e al. [
5
], Cup ia idus neca o , o me ly known as Rals onia
eu opha, is one o he mos s udied mic oo ganisms o PHB p oduc ion due o i s e sa ile
me abolic capaci y. C. neca o can g ow bo h as a chemoau o oph and a he e o oph in
ae obic and anae obic en i onmen s, wi h he abili y o u ilize a a ie y o ca bon sou ces
o PHB syn hesis. Cu en ly, PHA p oduc ion is limi ed by he high p oduc ion cos s,
which a e h ee o ou imes highe han hose o syn he ic polyme s (be ween 0.60 and
0.87 USD/lb), hinde ing i s indus ializa ion and comme cializa ion [
6
]. In he PHA
p oduc ion p ocess, he ca bon sou ce could ep esen up o 50% o he inal cos [7–10].
As e iewed by Wang e al. [
11
] and Ba hia e al. [
12
], he use o was e as a ca bon
sou ce in subme ged cul u es o C. neca o o PHA p oduc ion could be an al e na i e o
educe he inal p oduc ion cos s. Cu en ly, and o e he las decade, many wo ks a e
being ca ied ou on he biosyn hesis o PHA using a ious suga -con aining was es as
al e na i e subs a es ha allow o cos educ ions [11,13–16].
Bi e cassa a is one o he o emos p ospec i e c ops o added- alue p oduc gene a-
ion in he bioeconomy, due o i s high s a ch con en (76.7% a e age [
17
]), low equi emen s
o cul i a ion, and non-compe i ion wi h ood p oduc ion [
18
]. Speci ically, bi e cassa a
p oduc ion in Colombia has g own signi ican ly in ecen yea s, wi h a ie ies cha ac e -
ized by high yields (25 on/ha) becoming an impo an sou ce o income o local a me s
and con ibu ing o he coun y’s economic de elopmen [
19
]. Colombia p oduces abou
269,000 ons o bi e cassa a pe yea , used mainly in he ood indus y o p oduce glucose
and uc ose sy ups, lou , concen a ed ca le eed and bioe hanol [
18
,
20
]. The e o e, he
use o bi e cassa a o ob ain s a ch hyd olysa e (e.g., dex ose), a po en ial ca bon sou ce,
o p oduce high- alue compounds such as PHA has been p oposed.
Suga cane exploi a ion is an es ablished indus y in Colombia, p oducing mainly able
suga o di ec comme cializa ion and he ood indus y, sy ups o alcoholic e men a-
ion, and bagasse o pape p oduc ion and ene gy co-gene a ion. Bioe hanol dis ille ies
p oduce be ween 2.2 and 3.1 million li e s o inasse daily [
21
,
22
]. Vinasse is a esidual
s eam in dis ille ies cha ac e ized by a low pH, high chemical oxygen demand (COD) and
biochemical oxygen demand (BOD), high dissol ed o ganic ma e con en , as well as a con-
side able quan i y o ino ganic sal s composed o chlo ides, sul a es, phospha es, calcium,
magnesium, and po assium [
23
]. Vinasse is po en ially oxic because o i s bio- ecalci an
subs ance con en s, such as phenolic compounds and pigmen s like melanoidins, which
can inhibi he ac i i y o mic oo ganisms [
24
]. Since inasse p oduc ion anges be ween
11 and 15 li e s o each li e o e hanol dis illed [
22
], en i onmen al conce ns abou i s
ea men and disposal ha e mo i a ed he explo a ion o u ilizing al e na i es [23].
As a s a egy o ake ad an age o he p ope ies and mic onu ien s p o ided by bo h
subs a es, i is no un easonable o use a combina ion o dex ose om cassa a s a ch
and suga cane inasse as a ca bon sou ce o C. neca o cul i a ion. Thus, he aim o his
s udy was o e alua e di e en dex ose/ inasse a ios as a low-cos ca bon sou ce o
PHA p oduc ion by C. neca o , including no inasse supplemen a ion. To da e and based
on a de ailed e iew o he scien i ic li e a u e, his low-cos subs a e combina ion has no
been epo ed as a p ospec i e al e na i e o bioplas ic p oduc ion.
2. Ma e ials and Me hods
2.1. T ea men and Cha ac e iza ion o Raw Ma e ials Used as a Subs a e
The ca bon sou ces e alua ed in his s udy consis ed o solid dex ose om bi e
cassa a s a ch p oduced in he no he n egion o Colombia, and suga cane inasse om a
local dis ille y in he sou hwes e n egion. Bo h subs a es we e dona ed by local indus ies.
ChemEnginee ing 2024,8, 73 3 o 17
Raw and ea ed inasse we e es ed o e alua e he e ec o he o ganic load o
suga cane inasse on C. neca o g ow h and PHA p oduc ion. In bo h cases, he inasse
was cen i uged a 8000 pm o 7 min o emo e pa icula e ma e . To al polyphenols
we e de e mined using he Folin–Ciocal eu me hod in a UV–Vis Jasco V730 spec opho-
ome e a 760 nm [
25
]. The pH was de e mined po en iome ically and
◦
B ix was ob ained
ia e ac ome y.
Suga cane inasse was ea ed o emo e he o ganic componen , which can be oxic,
and/o inhibi he g ow h o mic oo ganisms [
26
], which a e di icul o biodeg ade [
27
].
An adso p ion sys em was designed based on p e ious wo k in ou labo a o y. Figu e 1
shows a schema ic o he expe imen al se up, which consis ed o a bo osilica e glass ba ch
adso p ion column (5 cm in e nal diame e and 30 cm leng h). A ixed bed (25 g) o g anula
ac i a ed ca bon (GAC) was placed a he bo om o he column and, o sus ain he ca bon
bed, a cylind ical suppo was cons uc ed wi h wo me al meshes a he op and bo om o
he GAC bed.
ChemEnginee ing 2024, 8, x FOR PEER REVIEW 3 o 18
based on a de ailed e iew o he scien i ic li e a u e, his low-cos subs a e combina ion
has no been epo ed as a p ospec i e al e na i e o bioplas ic p oduc ion.
2. Ma e ials and Me hods
2.1. T ea men and Cha ac e iza ion o Raw Ma e ials Used as a Subs a e
The ca bon sou ces e alua ed in his s udy consis ed o solid dex ose om bi e
cassa a s a ch p oduced in he no he n egion o Colombia, and suga cane inasse om
a local dis ille y in he sou hwes e n egion. Bo h subs a es we e dona ed by local
indus ies.
Raw and ea ed inasse we e es ed o e alua e he effec o he o ganic load o
suga cane inasse on C. neca o g ow h and PHA p oduc ion. In bo h cases, he inasse
was cen i uged a 8000 pm o 7 min o emo e pa icula e ma e . To al polyphenols
we e de e mined using he Folin–Ciocal eu me hod in a UV–Vis Jasco V730
spec opho ome e a 760 nm [25]. The pH was de e mined po en iome ically and °B ix
was ob ained ia e ac ome y.
Suga cane inasse was ea ed o emo e he o ganic componen , which can be oxic,
and/o inhibi he g ow h o mic oo ganisms [26], which a e difficul o biodeg ade [27].
An adso p ion sys em was designed based on p e ious wo k in ou labo a o y. Figu e 1
shows a schema ic o he expe imen al se up, which consis ed o a bo osilica e glass ba ch
adso p ion column (5 cm in e nal diame e and 30 cm leng h). A ixed bed (25 g) o
g anula ac i a ed ca bon (GAC) was placed a he bo om o he column and, o sus ain
he ca bon bed, a cylind ical suppo was cons uc ed wi h wo me al meshes a he op
and bo om o he GAC bed.
Figu e 1. Schema ic diag am o he expe imen al assembly used o he ea men o suga cane
inasse.
The GAC used in his s udy was p o ided by a local supplie o wa e ea men
solu ions. The GAC was o bi uminous o igin and p oduced unde s ic ly con olled
condi ions ia high- empe a u e s eam ac i a ion. This coal p o ides a la ge su ace a ea,
la ge po e olume, and an op imal po e s uc u e o adso p ion pu i ica ion ea men .
In acco dance wi h AWWA S anda d B 604-05 [28], he main physicochemical
cha ac e is ics o he GAC a e shown in Table 1.
Figu e 1. Schema ic diag am o he expe imen al assembly used o he ea men o suga cane inasse.
The GAC used in his s udy was p o ided by a local supplie o wa e ea men
solu ions. The GAC was o bi uminous o igin and p oduced unde s ic ly con olled
condi ions ia high- empe a u e s eam ac i a ion. This coal p o ides a la ge su ace a ea,
la ge po e olume, and an op imal po e s uc u e o adso p ion pu i ica ion ea men . In
acco dance wi h AWWA S anda d B 604-05 [
28
], he main physicochemical cha ac e is ics
o he GAC a e shown in Table 1.
Two ea men imes o suga cane inasse we e e alua ed: 1.0 and 3.0 h. Since he
polyphenols p esen in inasse a e one o he mos ele an and concen a ed inhibi o s
o biological ac i i y, he emo ion o polyphenols was e alua ed as an indica o o he
ea men e ec i i y, and each es was pe o med a a cons an up- low (2.7 L/min).
ChemEnginee ing 2024,8, 73 4 o 17
Table 1. Physicochemical cha ac e is ics o g anula ac i a ed ca bon used as an adso p ion agen o
he ea men o suga cane inasse.
Pa ame e Value
Iodine numbe 850 mg/g Min
Ash 15% Max
Humidi y 5% Max
Ha enes 90% Min
G anulome y Mesh 8 ×30
Mesh8 5% Max/2 mm
Mesh30 5% Max/2 mm
2.2. Mic oo ganism, Cul u e Media, and Cul i a ion Condi ions
C. neca o DSM 428 was ob ained om he Leibniz Ins i u e DSMZ collec ion (Ge man
Collec ion o Mic oo ganisms and Cell Cul u es GmbH) as a glass ampoule in a acuum-
packed double ial wi h a lyophilized able o a single s ain o mic oo ganisms. Fo i s
ac i a ion, he lyophilized cells om he ampoule we e ehyd a ed and g own in 5 mL o
liquid nu ien b o h (NB). NB medium (PanReac AppliChem, ITW Reagen s, Monza, I aly)
was composed as ollows: mea ex ac 3 g/L, mea pep one 5 g/L). The cul u es we e
incuba ed a 35
◦
C
±
2
◦
C o 24 o 48 h and he mic obial g ow h ob ained was asep ically
added o a liquid medium composed o 20% glyce ol and nu ien b o h in 1.5 mL ials o
subsequen s o age a −20 ◦C.
Ini ially, 1.5 mL o c yop ese ed cells we e eac i a ed in 50 mL o NB as seed medium,
disposed in 250 mL shake lasks a 35
◦
C and 150 pm o 24 h. Two p e-cul u e s ages we e
ca ied ou o s ain adap a ion p io o p oduc ion cul u es. Fi s p e-cul u es con ained
45 mL o modi ied mine al saline medium (MSM) and we e inocula ed wi h 5 mL o he
cul i a ed seed b o h. MSM medium con ained (pe li e ) dex ose om bi e cassa a
s a ch 20 g, Na
2
HPO
4
.7H
2
O 6.7 g, KH
2
PO
4
1.5 g, (NH
4
)
2
SO
4
1.0 g, MgSO
4
.7H
2
O 0.2 g, i on
and ammonium ci a e 60 mg, CaCl
2
.2H
2
O 10 mg, and elemen ace solu ion 1 ml. Elemen
ace solu ion (pe li e ): H
3
BO
3
0.3 g; CoCl
2
.6H
2
O 0.2 g; ZnSO
4
.7H
2
O 0.1 g; MnCL
2
.4H
2
O
30 mg; NaMoO4.2H2O 30 mg; NiCl2.6H2O 20 mg; CuSO4.5H2O 10 mg [29].
Then, he second p e-cul u es we e p epa ed iden ically and inocula ed wi h 5 mL o
b o h om i s p ecul u es. The PHB p oduc ion cul u es we e pe o med in modi ied
MSM medium, and using supplemen a ion wi h ea ed and un ea ed suga cane inasse.
P oduc ion cul u es we e inocula ed a 10% / o second p e-cul u es. and he cells we e
g own o 48 h a 35
◦
C and 150 pm. The pH was adjus ed o 6.8 wi h NaOH 2M. Cul u es
we e pe o med in iplica e. The composi ion o suga cane inasse a ied be ween 2.5
and 75% / in each case. This a ia ion aimed o e alua e he inhibi ing e ec s o a wide
ange o suga cane inasse composi ions in subme ged cul u es o C. neca o .
2.3. Biomass and Suga s Quan i ica ion
Biomass concen a ion was quan i ied as cell d y weigh in 1.0 mL samples. Samples
we e cen i uged a 5000 pm o 10 min. Supe na an s we e used o suga de e mina ion
and we cells we e d ied a 70 ◦C o 24 h o u he weigh de e mina ion.
Reducing suga s we e de e mined using he 3,5-Dini osalicylic acid (DNS) me hod [
30
].
2.4. PHA De ec ion and Ex ac ion
The ela i e in acellula o ma ion o he polyme was de e mined by lipophilic
s aining wi h Sudan black B, whe e he o ma ion o PHA is de ec ed along wi h he
p esence o black g anules inside he cells. Fo he PHB ex ac ion, he emaining cul u es
we e cen i uged a 5000 pm o 10 min. The ob ained pelle s we e dissol ed in 5 mL o
sodium hypochlo i e and 5 mL o chlo o o m. Then, cells we e agi a ed in a o ex and kep
a oom empe a u e o 20 h in agi a ion, be o e being cen i uged a he a o emen ioned
condi ions. As a esul , h ee sepa a e phases we e ob ained: an ino ganic phase (uppe )
co esponding o he sodium hypochlo i e, he b oken cellula ma e ial (middle), and he
ChemEnginee ing 2024,8, 73 5 o 17
o ganic phase o chlo o o m wi h he dissol ed biopolyme (lowe ). The PHA p esen in
each sample was p ecipi a ed by he addi ion o isop opyl alcohol. The p ecipi a e was
d ied a 70 ◦C o 24 h and PHB d y weigh was quan i ied [31].
2.5. Ma e ial Cha ac e iza ion
The c ys allog aphic analysis o he PHB was pe o med using a panaly ical X- ay
di ac ome e wi h Cu-K
α
adia ion (wa eleng h
λ
= 1.5405 Å) ope a ing a 45 kV and
40 mA. A pa allel beam op ical sys em was implemen ed, comp ising a pa abolic mi o
in he inciden beam, a 0.18
◦
pa allel pla e collima o , and a la g aphi e monoch oma o
in he di ac ed beam. X- ay scanning was conduc ed in he ange o 5 o 90 deg ees
2
θ
, in s ep scanning mode, wi h inc emen s o 0.03
◦
(2
θ
) and a coun ing ime o 2 s. The
unc ional g oups and moie ies p esen in he PHB powde samples we e de e mined using
Fou ie ans o m in a ed Spec oscopy (FTIR) (IR A ini y-1, Shimadzu Scien i ic Ins u-
men s, Columbia, MD, USA). The FTIR spec a we e eco ded in he ange om 4000 o
500 cm
−1
. Fo chemical composi ion e alua ion, an ene gy-dispe si e X- ay (EDS) analysis
was pe o med using a Philips XL 30 FEG wi h a high-pu i y Ge EDS de ec o (Philips
N.V, Eindho en, The Ne he lands). ZAF co ec ion was applied o he s oichiome ic
analyses due o he low eliabili y o EDS unde ni ogen concen a ions. The s uc u al
analysis o he PHB was ca ied ou ia scanning elec on mic oscopy (SEM) using a Philips
XL
30 FEG
, ope a ing a 15 keV wi h a backsca e ed elec on de ec o (EDAX-EDS). The
he mal s abili y o he ex ac ed PHB, along wi h s anda d samples, we e analyzed ia
he mog a ime ic analysis (TGA). The empe a u e ange was om 30
◦
C o 600
◦
C, a a
hea ing a e o 10
◦
C/min in a ni ogen a mosphe e (N
2
low a e = 40 mL/min). The deg a-
da ion a e o PHB samples was pe o med using empe a u e da a o T5%, T10%, and T50%
ob ained om he TGA analysis. The mel ing poin (Tm) and glass ansi ion empe a u e
(Tg) o he PHB samples we e de e mined ia di e en ial scanning calo ime y (DSC) anal-
ysis (DSC-1 se ies, Me le -Toledo, Columbus, OH, USA) wi h a hea ing and cooling a e o
10
◦
C/min in a N
2
en i onmen wi h a gas low o 20 mL/min. Fo DSC analysis, 3.5 mg o
he PHB sample was loaded in an aluminum pan and hea ed in he empe a u e ange o
−
10
◦
C o 200
◦
C a a hea ing a e o 10
◦
C/min. The poin o in lec ion in he DSC cu e
be ween onse and o se empe a u es co esponds o he glass ansi ion empe a u e and
he mel ing poin , measu ed as he peak empe a u e o an endo he mic e en .
3. Resul s and Discussion
3.1. T ea men and Cha ac e iza ion o Suga cane Vinasse
The aw suga cane inasse exhibi ed high concen a ions o polyphenolic compounds,
in addi ion o high acidi y (Table 2). The physicochemical pa ame e s e alua ed in he
inasse exceeded he a e age alues epo ed in he li e a u e [
26
,
27
], which may be ela ed
o he concen a ion p ocess used in dis ille ies.
Table 2. Physicochemical cha ac e iza ion o aw suga cane inasse and polyphenolic compounds’
emo al in ea ed suga cane inasse.
Pa ame e Resul
T ea ed Vinasse
1 h 3 h
To al polyphenolic
compounds (g/L) 18.39 18.39 15.31
Ashes (g/L) 93.19
pH 4.88 ±0.01
◦B ix 30.0 ±0.1
Table 2also shows a compa ison o he suga cane inasse ea men using di e en
adso p ion imes. As can be obse ed, o a ime o 3 h, a educ ion o 16.75% o he
polyphenolic compounds p esen in he inasse was achie ed.

ChemEnginee ing 2024,8, 73 6 o 17
Some s udies ha e been epo ed ha hei p esence can be oxic and/o inhibi he
g ow h o ce ain mic oo ganisms. Howe e , he e a e epo s indica ing ha no inhibi o y
e ec s a e obse ed when inasse is used as a ca bon sou ce [
32
]. Addi ionally, he high
suga con en p esen in he inasse makes i a iable subs a e o he g ow h o C. neca o .
In gene al, he chemical composi ion o inasse is qui e a iable, depending on he
quali y o he juice, p o enance, ha es ing condi ions, e men a ion, and dis illa ion
p ocess used. All hese condi ions ep esen challenges o he use o inasse as a ca bon
sou ce o p oduce PHA h ough mic obial e men a ion.
3.2. Kine ic E alua ion o C. neca o in Dex ose Medium and PHB P oduc ion in Shake
Flask Cul u es
Figu e 2shows he e olu ion o glucose consump ion, biomass, and PHB p oduc ion
o e ime in shake lask cul i a ions o C. neca o using dex ose om cassa a s a ch as
a low-cos ca bon sou ce. The lag phase ended 12 h a e cul u e s a ed. Subsequen ly,
an exponen ial phase was obse ed, anging om 12 o 48 h o cul i a ion, ollowed by
a s a iona y phase o 12 h. The maximum biomass concen a ion achie ed was 5.17 g/L
a 48 h. A he end o he cul i a ions, he glucose concen a ion was 10.75 g/L, which
co esponds o 53.7% o he ini ial concen a ion. PHB p oduc ion and accumula ion was
e alua ed a he end o he cul u es (72 h). PHB concen a ion was 2.01
±
0.10 g/L, which
co esponded o a PHB con en o 47.1%. The accumula ions o PHB con en we e isually
con i med h ough a mic oscopy u ilizing he Sudan black s aining me hod (see Figu e 3),
which was moni o ed a e 24, 48, and 72 h o cul i a ion. This quali a i e analysis allows
o he obse a ion o he p og essi e inc ease in polyme in acellula accumula ion as
cul i a ion ad ances.
ChemEnginee ing 2024, 8, x FOR PEER REVIEW 7 o 18
Figu e 2. Glucose consump ion, biomass, and PHB p oduc ion o e ime in shake lask cul i a ions
o C. neca o using dex ose om cassa a s a ch hyd olysa e as a ca bon sou ce.
(a) (b) (c)
Figu e 3. De ec ion o PHB by 100× mic oscopy using Sudan black s aining: (a) 24 h, (b) 48 h, and
(c) 72 h.
Fu he mo e, he p oduc ion o PHB h ough he mic obial cul i a ion o C. neca o
has been ex ensi ely s udied, and o he wo ks epo ed he u iliza ion o simila ca bon
sou ces o biopolyme p oduc ion [15,34,35]. In his p ocess, he PHB con en is highe
han ha epo ed by [36] o C. neca o a e 58 h in cul u e in a s udy u ilizing b oken
ice as a ca bon sou ce (38% PHB). Simila ly, Oli ei a e al. [37] epo ed a PHB
accumula ion o 33.3% when employing soybean and 2.5% molasses in solid-s a e
e men a ion wi h C. neca o . Likewise, a s udy epo ed an accumula ion o 42.2% a 31 h
o cul i a ion o C. neca o DSM 545 using uc ose as he ca bon sou ce [38]. In con as ,
highe accumula ions (84.3 and 92%) we e epo ed a 72 h o cul i a ion o s ains C.
neca o IBP/SFU-1 and C. neca o B-10646, espec i ely, using glucose as he ca bon sou ce
[16,39]. The e o e, glucose is a p omising ca bon sou ce o PHB p oduc ion using C.
neca o . E en hough uc ose is he only suga capable o being me abolized by hyd ogen-
oxidizing bac e ia, hey can easily mu a e, enabling hem o me abolize glucose h ough
he En ne –Doudo off pa hway o o m py u a e, which is ans o med by
dehyd ogenase in o Ace yl-CoA, one o he p ecu so s o PHA, as demons a ed in his
s udy using C. neca o DSM 428 [16,40].
3.3. E alua ion o C. neca o G ow h and PHB P oduc ion wi h Dex ose om Cassa a S a ch
and Suga cane Vinasse as a Supplemen
The esul s o biomass and biopolyme accumula ion ob ained using inasse
supplemen a ion and dex ose om cassa a s a ch in diffe en p opo ions a e shown in
Figu e 2. Glucose consump ion, biomass, and PHB p oduc ion o e ime in shake lask cul i a ions
o C. neca o using dex ose om cassa a s a ch hyd olysa e as a ca bon sou ce.
In his cul u e, he g ow h o C. neca o in ca bon sou ces ich in glucose, such as
dex ose om cassa a s a ch, was demons a ed. Addi ionally, i was obse ed ha he e
was no deple ion o he ca bon sou ce by he end o he cul u e. This beha io mi o s
ha epo ed by [
28
] when employing he same s ain (C. neca o DSM 428) in cul u es
wi h concen a ions anging om 5 o 20 g/L o glucose, whe e subs an ial amoun s we e
epo ed a he end o he cul u e (be ween 1.4 g/L and 17.4 g/L). The same au ho epo s
ha only wi h 2 g/L glucose was he g ow h phase limi ed by he o al consump ion o
he ca bon sou ce. O he au ho s [
33
] epo ed a dec ease in biomass quan i y when he
glucose concen a ion was 20 g/L, a ibu ing his concen a ion o he inhibi ion o g ow h
in C. neca o DSM 545. Fu he mo e, hey indica e ha wi h glucose concen a ions highe
han 10 g/L, comple e glucose deple ion does no occu a he end o he cul i a ion. In ou
s udy, glucose concen a ions o 20 g/L we e u ilized; howe e , he biomass p oduc ion a
48 h exceeded he da a epo ed by [
33
]. Despi e he absence o glucose deple ion in he
ChemEnginee ing 2024,8, 73 7 o 17
medium, i is likely ha ni ogen sou ce deple ion (ammonium sul a e) occu ed, enabling
PHB accumula ion.
ChemEnginee ing 2024, 8, x FOR PEER REVIEW 7 o 18
Figu e 2. Glucose consump ion, biomass, and PHB p oduc ion o e ime in shake lask cul i a ions
o C. neca o using dex ose om cassa a s a ch hyd olysa e as a ca bon sou ce.
(a) (b) (c)
Figu e 3. De ec ion o PHB by 100× mic oscopy using Sudan black s aining: (a) 24 h, (b) 48 h, and
(c) 72 h.
Fu he mo e, he p oduc ion o PHB h ough he mic obial cul i a ion o C. neca o
has been ex ensi ely s udied, and o he wo ks epo ed he u iliza ion o simila ca bon
sou ces o biopolyme p oduc ion [15,34,35]. In his p ocess, he PHB con en is highe
han ha epo ed by [36] o C. neca o a e 58 h in cul u e in a s udy u ilizing b oken
ice as a ca bon sou ce (38% PHB). Simila ly, Oli ei a e al. [37] epo ed a PHB
accumula ion o 33.3% when employing soybean and 2.5% molasses in solid-s a e
e men a ion wi h C. neca o . Likewise, a s udy epo ed an accumula ion o 42.2% a 31 h
o cul i a ion o C. neca o DSM 545 using uc ose as he ca bon sou ce [38]. In con as ,
highe accumula ions (84.3 and 92%) we e epo ed a 72 h o cul i a ion o s ains C.
neca o IBP/SFU-1 and C. neca o B-10646, espec i ely, using glucose as he ca bon sou ce
[16,39]. The e o e, glucose is a p omising ca bon sou ce o PHB p oduc ion using C.
neca o . E en hough uc ose is he only suga capable o being me abolized by hyd ogen-
oxidizing bac e ia, hey can easily mu a e, enabling hem o me abolize glucose h ough
he En ne –Doudo off pa hway o o m py u a e, which is ans o med by
dehyd ogenase in o Ace yl-CoA, one o he p ecu so s o PHA, as demons a ed in his
s udy using C. neca o DSM 428 [16,40].
3.3. E alua ion o C. neca o G ow h and PHB P oduc ion wi h Dex ose om Cassa a S a ch
and Suga cane Vinasse as a Supplemen
The esul s o biomass and biopolyme accumula ion ob ained using inasse
supplemen a ion and dex ose om cassa a s a ch in diffe en p opo ions a e shown in
Figu e 3. De ec ion o PHB by 100
×
mic oscopy using Sudan black s aining: (a) 24 h, (b) 48 h, and
(c) 72 h.
Fu he mo e, he p oduc ion o PHB h ough he mic obial cul i a ion o C. neca o
has been ex ensi ely s udied, and o he wo ks epo ed he u iliza ion o simila ca bon
sou ces o biopolyme p oduc ion [
15
,
34
,
35
]. In his p ocess, he PHB con en is highe
han ha epo ed by [
36
] o C. neca o a e 58 h in cul u e in a s udy u ilizing b oken ice
as a ca bon sou ce (38% PHB). Simila ly, Oli ei a e al. [
37
] epo ed a PHB accumula ion
o 33.3% when employing soybean and 2.5% molasses in solid-s a e e men a ion wi h C.
neca o . Likewise, a s udy epo ed an accumula ion o 42.2% a 31 h o cul i a ion o C.
neca o DSM 545 using uc ose as he ca bon sou ce [
38
]. In con as , highe accumula ions
(84.3 and 92%) we e epo ed a 72 h o cul i a ion o s ains C. neca o IBP/SFU-1 and C.
neca o B-10646, espec i ely, using glucose as he ca bon sou ce [
16
,
39
]. The e o e, glucose
is a p omising ca bon sou ce o PHB p oduc ion using C. neca o . E en hough uc ose
is he only suga capable o being me abolized by hyd ogen-oxidizing bac e ia, hey can
easily mu a e, enabling hem o me abolize glucose h ough he En ne –Doudo o pa hway
o o m py u a e, which is ans o med by dehyd ogenase in o Ace yl-CoA, one o he
p ecu so s o PHA, as demons a ed in his s udy using C. neca o DSM 428 [16,40].
3.3. E alua ion o C. neca o G ow h and PHB P oduc ion wi h Dex ose om Cassa a S a ch and
Suga cane Vinasse as a Supplemen
The esul s o biomass and biopolyme accumula ion ob ained using inasse supple-
men a ion and dex ose om cassa a s a ch in di e en p opo ions a e shown in Table 3.
In addi ion, a he end o he p oduc ion ime (48 h), he p esence o PHB g anules in he
cells was con i med by s aining wi h Sudan Black B and h ough mic oscopic obse a ion,
as displayed in Figu e 4.
As obse ed in Table 3, he use o a medium wi h a aw inasse composi ion o 25%
/ (and highe ) o ally inhibi ed C. neca o g ow h. On he con a y, in media wi h a aw
inasse composi ion be ween 2.5 and 7.5% / , bac e ial g ow h was obse ed.
A clea end is obse ed in cul u es wi h a aw and ea ed inasse composi ion
be ween 2.5 and 7.5% / : as he p opo ion o inasse in he medium inc eases, bac e ial
g ow h inc eases, e idencing he nu i ional e ec o he o ganic assimilable compounds
in he mix u e, mainly ca bohyd a es. Despi e no signi ican changes in he accumula ion
o he biopolyme being obse ed in his ange o concen a ions (2.5–7.5% / ), mo e
biomass was p oduced in each case, yielding a highe inal PHA concen a ion. Mo eo e ,
o he mos concen a ed media wi h he ea ed inasse (25, 50, 75% / ), a lowe PHB
accumula ion was a ained.
ChemEnginee ing 2024,8, 73 8 o 17
As inc easing alues o ni ogen a e p o ided o he cul u e by inc easing he inasse
concen a ion, he addi ion o bo h nu ien s, ca bon and ni ogen a o cell g ow h bu no
he PHB accumula ion, as shown in he esul s o Table 4.
Table 3. Expe imen al esul s o biomass p oduced and PHB accumula ion unde di e en p opo -
ions o suga cane inasse ( aw and ea ed) in subme ged cul u es o C. neca o .
Vinasse
Vinasse
P opo ion in
Medium ( / )
Biomass
Concen a ion
(g/L)
Polyme
Concen a ion
(g/L)
Polyme
Accumula ion
(%)
Raw
inasse
2.5% 2.97 ±0.13 1.43 ±0.06 48%
5.0% 3.72 ±0.13 1.77 ±0.07 48%
7.5% 5.50 ±0.13 2.66 ±0.11 48%
25.0% No g ow h No p oduc ion 0%
50.0%
75.0%
T ea ed
inasse
2.5% 3.30 ±0.14 1.57 ±0.06 48%
5.0% 3.87 ±0.14 1.96 ±0.08 51%
7.5% 5.90 ±0.14 2.98 ±0.12 51%
25.0% 1.70 ±0.13 0.69 ±0.03 41%
50.0% 3.25 ±0.13 1.43 ±0.06 44%
75.0% 4.44 ±0.13 1.82 ±0.08 41%
ChemEnginee ing 2024, 8, x FOR PEER REVIEW 8 o 18
Table 3. In addi ion, a he end o he p oduc ion ime (48 h), he p esence o PHB g anules
in he cells was con i med by s aining wi h Sudan Black B and h ough mic oscopic
obse a ion, as displayed in Figu e 4.
Table 3. Expe imen al esul s o biomass p oduced and PHB accumula ion unde diffe en
p opo ions o suga cane inasse ( aw and ea ed) in subme ged cul u es o C. neca o .
Vinasse
Vinasse
P opo ion in
Medium ( / )
Biomass
Concen a ion
(g/L)
Polyme
Concen a ion
(g/L)
Polyme
Accumula ion
(%)
Raw
inasse
2.5% 2.97 ± 0.13 1.43 ± 0.06 48%
5.0% 3.72 ± 0.13 1.77 ± 0.07 48%
7.5% 5.50 ± 0.13 2.66 ± 0.11 48%
25.0%
No g ow h No p oduc ion 0% 50.0%
75.0%
T ea ed
inasse
2.5% 3.30 ± 0.14 1.57 ± 0.06 48%
5.0% 3.87 ± 0.14 1.96 ± 0.08 51%
7.5% 5.90 ± 0.14 2.98 ± 0.12 51%
25.0% 1.70 ± 0.13 0.69 ± 0.03 41%
50.0% 3.25 ± 0.13 1.43 ± 0.06 44%
75.0% 4.44 ± 0.13 1.82 ± 0.08 41%
Figu e 4. In acellula accumula ion o PHB in he bac e ium C. neca o in medium wi h ea ed
suga cane inasse 7.5% / ; (a) iden i ica ion o PHB by s aining wi h Black Sudan B (magni ica ion
100×); (b) scanning elec on mic og aphs (SEM), magni ica ion 5000×.
As obse ed in Table 3, he use o a medium wi h a aw inasse composi ion o 25%
/ (and highe ) o ally inhibi ed C. neca o g ow h. On he con a y, in media wi h a aw
inasse composi ion be ween 2.5 and 7.5% / , bac e ial g ow h was obse ed.
A clea end is obse ed in cul u es wi h a aw and ea ed inasse composi ion
be ween 2.5 and 7.5% / : as he p opo ion o inasse in he medium inc eases, bac e ial
g ow h inc eases, e idencing he nu i ional effec o he o ganic assimilable compounds
in he mix u e, mainly ca bohyd a es. Despi e no signi ican changes in he accumula ion
o he biopolyme being obse ed in his ange o concen a ions (2.5–7.5% / ), mo e
biomass was p oduced in each case, yielding a highe inal PHA concen a ion. Mo eo e ,
o he mos concen a ed media wi h he ea ed inasse (25, 50, 75% / ), a lowe PHB
accumula ion was a ained.
As inc easing alues o ni ogen a e p o ided o he cul u e by inc easing he inasse
concen a ion, he addi ion o bo h nu ien s, ca bon and ni ogen a o cell g ow h bu
no he PHB accumula ion, as shown in he esul s o Table 4.
(a) (b)
Figu e 4. In acellula accumula ion o PHB in he bac e ium C. neca o in medium wi h ea ed
suga cane inasse 7.5% / ; (a) iden i ica ion o PHB by s aining wi h Black Sudan B (magni ica ion
100×); (b) scanning elec on mic og aphs (SEM), magni ica ion 5000×.
Table 4. Chemical composi ion o he PHB p oduced by C. neca o using dex ose om cassa a s a ch
as a ca bon sou ce.
Spec um In S a s. C O Na Cl To al
A e age Yes 50.74 38.77 4.61 5.88 100.00
Addi ionally, when he ea ed inasse p opo ion in he medium is 25% / , a con-
side able dec ease in he amoun o biomass ob ained is seen. Ne e heless, when he
p opo ion inc eases again be ween 50 and 75% / , biomass p oduc ion ends o inc ease
again, which can be jus i ied because al hough he concen a ion o polyphenols in he
medium inc eases, suga s a e mo e a ailable. The e o e, he inc ease in inasse in he
medium leads o an inc ease in compounds ha ha e nega i e and posi i e e ec s ha
coun e ac each o he .
Ne e heless, i is impo an o highligh he case in which ea ed inasse was used
in a p opo ion be ween 50 and 75%. Al hough a highe inasse concen a ion does no
imply ha highe a ios o PHB accumula ion will be achie ed, he esul s a e p omising.
ChemEnginee ing 2024,8, 73 9 o 17
Signi ican cell g ow h was ob ained wi h a high amoun o inasse using a ea men o a
small emo ion o inhibi o s, educing he need o wa e in he medium.
The e o e, in e ms o esou ce u iliza ion, he use o he medium wi h he highes
amoun o ea ed inasse (75%) could be he bes scena io om he indus y s andpoin .
Howe e , in e ms o p oduc ion, he esul s indica e ha using ea ed inasse a a p opo -
ion o 7.5% is he bes op ion, which leads o a PHB p oduc ion o 2.98
±
0.12 g/L, which
sligh ly exceeds he concen a ion epo ed in o he s udies wi h simila subs a es [
26
],
and a PHB accumula ion o 51%. The abo e is a p omising esul ha demons a es ha
he ea men o suga cane inasse con ibu es signi ican ly o he inc ease in PHB p o-
duc ion, as in es iga ions using he same mic oo ganism and ope a ing condi ions, using
aw inasse wi hou any ea men , led o lowe biopolyme accumula ions (be ween
26 and 33%) [
32
]. Howe e , he esul s ob ained may be be e i he cul u e medium is
supplemen ed wi h ano he ca bon sou ce, such as molasses, which is also a esidue om
e hanol dis illa ion. Resea che s e alua ing di e en p opo ions o inasse/molasses in a
mine al medium epo ed accumula ions om 56 [
41
,
42
] o 97% [
26
]. Likewise, accumula-
ions o up o 76% a e eco ded when inc easing he glucose concen a ion in he medium
(50 g/L) [43].
The e o e, conside ing ha one o he main disad an ages o he comme cializa ion
o PHA is i s high p oduc ion cos , up o h ee imes highe han ha o con en ional
plas ics [
44
], he use o low-cos ca bon sou ces, such as hose s udied in his wo k, could
educe his gap; i is epo ed ha he use o inasse could achie e a dec ease in p oduc ion
cos o up o 22.2% [6].
Figu e 5shows he biomass and suga ime-cou ses o C. neca o o he p oduc ion
medium supplemen ed wi h ea ed inasse 7.5% / and he p oduc ion medium wi h
only dex ose.
ChemEnginee ing 2024, 8, x FOR PEER REVIEW 10 o 18
Figu e 5. Mean alues o biomass g ow h and glucose consump ion by C. neca o in cul u e media
wi h dex ose and dex ose- ea ed suga cane inasse a concen a ion o 7.5%.
As can be obse ed in Figu e 5, o cul u e supplemen ed wi h suga cane inasse, he
exponen ial phase s a s a 12 h and g ow h las s up o abou 36 h. Then, he s a iona y
phase can be obse ed. The biomass accumula ion was 5.80 ± 0.12 g/L a 48 h o cul u e.
The emanen glucose a he end o he cul u e was 12.10 g/L, wi h a PHB accumula ion
o 51%. Simila ends we e obse ed in he case o cul u es wi h only dex ose, wi h a
maximum biomass concen a ion o 5.17 ± 0.027 g/L. The glucose concen a ion a he end
o he cul u e was 10.75 g/L, wi h a PHB accumula ion o 47.1%.
Acco ding o he esul s ob ained, he addi ion o inasse o he medium had a
posi i e impac on cell g ow h due o i s supply o suga s (glucose, uc ose, suc ose) and
o he nu ien s, such as o ganic acids, as epo ed in he li e a u e [45]. This con ibu ed
o a biomass accumula ion du ing he exponen ial phase ha was highe han ha
ob ained using ca bon sou ces like dex ose [41]. Howe e , compa ing he pe cen ages o
PHB accumula ion in bo h cul u es, dex ose can be conside ed o be sui able as he sole
ca bon sou ce, achie ing a good pe cen age o his bioplas ic.
Now, conside ing he p ocesses in ol ed in using inasse as a ca bon sou ce, and o
minimize p oduc ion cos s, he PHB ob ained om dex ose was chosen o be physically
and s uc u ally cha ac e ized.
3.4. Biopolyme cha ac e iza ion
3.4.1. XRD Analysis
Figu e 6 p esen s he XRD pa e n o he PHB ob ained ia he mic obial cul i a ion
o C. neca o using dex ose om cassa a s a ch. F om hese esul s, an O ho hombic
phase wi h a 19-P212121 space g oup belonging o PHB was e idenced by he e lec ions
o he c ys allog aphic planes: (020), (110), (130), (202), and (410), loca ed a angles 2θ =
13.52°, 16.91°, 27.28°, 31.62°, and 45.341°, espec i ely, indexed h ough he e e ence ile
JCPDF 00-001-0182. Simila XRD pa e ns o PHB we e epo ed by [46,47]. The in ense
(202) peak indica es he c ys alline na u e and i has been p oposed ha he polyme
ma ix adop s a egula helicoidal con o ma ion wi h wo an ipa allel chains in he
O ho hombic uni cell wi hin he c ys alline domain. In his sense, i is possible o obse e
he PHB ob ained by C. neca o wi h a uni cell, which consis s o an o ho hombic
c ys alline s uc u e sys em [47,48].
Figu e 5. Mean alues o biomass g ow h and glucose consump ion by C. neca o in cul u e media
wi h dex ose and dex ose- ea ed suga cane inasse a concen a ion o 7.5%.
As can be obse ed in Figu e 5, o cul u e supplemen ed wi h suga cane inasse, he
exponen ial phase s a s a 12 h and g ow h las s up o abou 36 h. Then, he s a iona y
phase can be obse ed. The biomass accumula ion was 5.80
±
0.12 g/L a 48 h o cul u e.
The emanen glucose a he end o he cul u e was 12.10 g/L, wi h a PHB accumula ion
o 51%. Simila ends we e obse ed in he case o cul u es wi h only dex ose, wi h a
maximum biomass concen a ion o 5.17
±
0.027 g/L. The glucose concen a ion a he end
o he cul u e was 10.75 g/L, wi h a PHB accumula ion o 47.1%.
Acco ding o he esul s ob ained, he addi ion o inasse o he medium had a posi i e
impac on cell g ow h due o i s supply o suga s (glucose, uc ose, suc ose) and o he
nu ien s, such as o ganic acids, as epo ed in he li e a u e [
45
]. This con ibu ed o a
biomass accumula ion du ing he exponen ial phase ha was highe han ha ob ained
ChemEnginee ing 2024,8, 73 16 o 17
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