Jou nal o En i onmen al Chemical Enginee ing 10 (2022) 106943
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Chain elonga ion may occu in p o ein mixed-cul u e e men a ion wi hou
supplemen ing elec on dono compounds
R. Be ilacqua, A. Reguei a , M. Mau icio-Iglesias
*
, J.M. Lema, M. Ca balla
CRETUS, Depa men o Chemical Enginee ing, Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain
ARTICLE INFO
Keywo ds:
Resou ce eco e y
Amino acids
Bio e ine y
Ca boxyla e pla o m
Chain elonga ion
P o ein was e
ABSTRACT
This s udy ocuses on e i ying he occu ence o elonga ion p ocesses du ing p o ein mixed cul u e e men a-
ion, wi hou he supplemen a ion o elec on dono compounds. Du ing casein mixed-cul u e e men a ion a pH
5, i was obse ed ha longe chain ola ile a y acid p oduc ion inc eased, which could no be jus i ied by he
associa ed amino acid consump ion. Consequen ly, he occu ence o chain elonga ion p ocesses was hypo he-
sized. To e i y his hypo hesis, h ee casein ba ch es s, wi h and wi hou ace ic acid ini ial supplemen a ion,
we e pe o med a pH 5. The esul s sugges ha ace ic and p opionic acids a e indeed consumed o selec i ely
gene a e n- ale ic acid h ough he coupling wi h elec on dono amino acids, whose consump ion was u he
e i ied h ough he amino acid analysis. P olonged simul aneous a ailabili y o sui able amino acids and sho
chain ola ile a y acids and an acid equi alen concen a ion h eshold we e iden i ied as key pa ame e s o
he occu ence o chain elonga ion. The supplemen a ion o ace ic acid a he beginning o he es changed he
selec i i y o he elonga ion p ocess, p omo ing n-bu y ic and iso- ale ic p oduc ion. The associa ed mechanisms
we e p elimina y concep ualized, cons i u ing a i s s ep o u he s udies on he subjec . To he bes o ou
knowledge, his is he i s s udy demons a ing he easibili y o chain elonga ion p ocesses du ing p o ein mixed
cul u e e men a ion wi hou elec on dono supplemen a ion.
1. In oduc ion
Chain elonga ion (CE) is an appealing biop ocess which con e s
ola ile a y acids (VFA) and compa ible elec on dono compounds (e.
g. e hanol, lac a e) o longe chain ca boxylic acids in anae obic mi-
c obial cul u es [1]. Ca boxylic acids wi h ou o mo e ca bon a oms
a e gene ally mo e p o i able (≥2
€
/kg o C
4
+) and easie o sepa a e
om he e men a ion b o h han he sho e chain ones due o hei
g ea e hyd ophobici y [2], hus unde lining CE as an oppo uni y o
upg ade he p oduc s o mixed-cul u e e men a ions in a esou ce e-
co e y pe spec i e [3]. The accep ed CE mechanism, based on s udies
conduc ed mainly on C. kluy e i, is he e e se β-oxida ion [4,5]. This
cyclic eac ion elonga es sho chain VFAs by adding wo ca bon a oms
(ace yl-CoA molecule) pe cycle o he o iginal hyd oca bon backbone
while consuming educing powe . Bo h he ace yl-CoA and he educing
powe a e p o ided by he con e sion o sui able elec on dono com-
pounds, such as e hanol o lac a e. The end p oduc s p esen an e en o
an odd numbe o ca bon a oms depending on he p ecu so VFA,
espec i ely ace ic and bu y ic acid o p opionic and ale ic acid [6].
Many s udies highligh ha CE p ocesses o en equi e he supple-
men a ion o elec on dono compounds, such as e hanol o lac a e
[7–9]. Fo example, G oo schol en e al. [10] achie ed high n-cap oic
acid i e s, and o smalle ex en n-hep anoic and n-cap ylic acids, using
e hanol-supplemen ed municipal solid was e as subs a e. Liang and
Wan [11] obse ed ha e men ing b ewe s’ spen g ain wi h e hanol
p omo ed he p oduc ion o n- ale ic and n-cap oic acid, while lac a e
addi ion s imula ed n-bu y ic acid o ma ion as end p oduc . Howe e ,
o he s udies show ha he ex e nal elec on dono supplemen a ion can
be bypassed when hese compounds a e p oduced in-si u oge he wi h
he equi ed VFAs. Con e as-D´
a ila e al. [12] demons a ed he po-
en ial o pe o ming CE ia consecu i e lac a e o ma ion and con-
sump ion du ing ood was e sequen ial-ba ch e men a ion. Lac a e was
p og essi ely p oduced by Lac obacillus ssp. om he ca bohyd a es
ac ion o ood was e, and subsequen ly used by Cap oicip oducens ssp.
o elonga e n-bu y ic acid o n-cap oic acid. Simila ly, Ca ajal-A oyo
e al. [13] illus a ed he easibili y o high- a e n-cap oic acid p oduc-
ion using hin s illage wi hou ex e nal supplemen a ions, as he ca -
bohyd a es con ained in he eeds ock we e mainly being con e ed o
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (M. Mau icio-Iglesias).
Con en s lis s a ailable a ScienceDi ec
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Jou nal o En i onmen al Chemical Enginee ing 10 (2022) 106943
2
lac a e and subsequen ially used as elec on dono compound by he
elonga ion mic obiome.
Mos o he CE s udies a ailable in li e a u e ocus on ca bohyd a es
as he main subs a e, such as acid whey om he dai y indus y [14],
suga cane molasses [15], hin s illage [13] o ood was e [12]. On he
con a y, li le is known abou CE p ocesses du ing anae obic e men-
a ion o p o eins. Only wo s udies could be ound [16,17], which we e
pe o med wi h a pu e cul u e o Eubac e ium py u a i o ans, a bac e-
ium isola ed om bo ine umen. The au ho s obse ed ha , supplying
he amino-acid-en iched cul u e medium wi h ace ic, p opionic o
bu y ic acid, he VFAs we e consumed and elonga ed o n-bu y ic,
n- ale ic and n-cap oic acids, espec i ely, using some amino acids (AA)
as elec on dono compounds (e.g. alanine and leucine). Howe e , he e
is no in o ma ion abou CE elonga ion p ocesses du ing anae obic
mixed-cul u e e men a ion o p o eins. O he wen y mos equen
amino acids in p o eins, some can ake he ole o elec on dono s, ac-
cep o s o e en bo h [18]. The di e si y o edox oles sugges s ha
p o eins migh unde go simul aneous e men a ion and CE p ocesses
wi hou he need o elec on dono supplemen a ion.
In he amewo k o a p ojec on he alo iza ion o p o ein- ich
esidues, his s udy in es iga es he easibili y o CE elonga ion p o-
cesses du ing anae obic mixed cul u e e men a ion o p o eins wi hou
he supplemen a ion o elec on dono compounds. We ied o e i y
whe he hei occu ence could explain he inc eased p oduc ion o
longe chain VFA a low pH condi ions, which could no be comple ely
jus i ied by he associa ed amino acid consump ion.
2. Ma e ials and me hods
2.1. P e ious expe imen s
Two con inuous s i ed ank eac o s (CSTR) we e ope a ed wi h wo
di e en p o eins, casein and gela in, a di e en pH alues (5, 7 and 9)
o unde s and he e ec o subs a e composi ion [19] and pH [20] on
p o ein mixed cul u e e men a ion. The 1L (wo king olume) eac o s
we e con inuously spa ged wi h ni ogen (10 mL/min) and main ained
a 25 ◦C in a clima ised chambe . The o ganic loading a e and hyd aulic
e en ion ime we e 5.3 g COD/L⋅d and 1.5 d espec i ely.
2.2. Chain elonga ion ba ch expe imen s
Ba ch expe imen s we e conduc ed wi h casein as subs a e in o de
o: (i) con i m CE p esence du ing anae obic mixed-cul u e e men a ion
o p o eins, (ii) unde s and he ole o AA in he p ocess, and (iii) e i y
whe he he addi ion o sho e chain ca boxyla es (i.e. ace ic and p o-
pionic acid) can a ec CE p ocess ex ension and/o selec i i y.
In each assay, a 2L sealed glass essel (1L o wo king olume) was
used, and he ope a ional condi ions we e se o be simila o he
con inuous eac o s (25 ◦C, N
2
spa ging, pH 5). The inoculum was ob-
ained om he con inuous eac o and i s ini ial concen a ion was
main ained a 0.5 g VSS/L. Syn he ic hyd olyzed p o ein, pep one om
casein (A2208,0500 PanReac), was used as he sole ca bon sou ce. I s
composi ion was p e iously de e mined in a sepa a e s udy [19]. The
ini ial casein concen a ion was equal o 5 g COD/L o he i s es ,
while i was inc eased o 10 g COD/L o he ollowing wo es s, co -
esponding o a subs a e- o-inoculum a io (SIR) equal o 10 and 20 g
COD/g VSS, espec i ely. The hi d ba ch es was ini ially supple-
men ed wi h ace ic acid a an app oxima e concen a ion o 0.5 g/L.
Mac onu ien s we e also added in all ba ch es s a he ollowing con-
cen a ions (g/L): NaCl 0.292; KH
2
PO
4
0.780; NH
4
Cl 0.530, Na
2
SO
4
0.057; MgCl
2
.6 H
2
O 0.120.
10 mL samples we e aken a inc easing ime in e als (ini ially 2–3
h). Hal o he sample olume was cen i uged and il e ed o o al
ammonia ni ogen (TAN), VFA and al e na i e p oduc s (i.e. lac a e,
e hanol, o mic acid) de e mina ion. The emaining 5 mL we e cen i-
uged, and he biomass solid pelle s we e hen esuspended in 5 mL o a
0.7% w/w solu ion o NaCl and dis illed wa e o op ical densi y
de e mina ion. The supe na an was ozen o AA analysis. A he end o
each expe imen , VSS concen a ion o he e men a ion b o h was also
measu ed.
2.3. Analy ical me hods
Mos physicochemical pa ame e s we e de e mined acco ding o
S anda d Me hods [21]. Mixed liquo samples we e used o measu e
o al and ola ile suspended solids, while il e ed (0.45 µm) mixed liquo
samples we e used o de e mine TAN, VFA and amino acids concen-
a ion. All spec opho ome ic measu emen s we e pe o med wi h a
Shimazdu UV-1800.
VFAs om C2 o C7 we e measu ed h ough gas ch oma og aphy
(AGV-DB1 me hod), hough o ganic acids wi h mo e han i e ca bon
a oms we e ne e de ec ed. The gas ch oma og aphe was an Agilen
6850 wi h a lame ioniza ion de ec o , using a DB-Wax column (Agilen
Technologies, 30 m ×0.250 mm ×0.25 µm). The injec o and he de-
ec o we e se a a empe a u e o 200 ◦C and 300 ◦C espec i ely. The
ca ie gas was ni ogen. The samples we e il e ed (0.45 µm) and hen
acidi ied wi h 10 µM o concen a ed H
3
PO
4
(85%) be o ehand.
Seconda y me aboli es (e.g., lac a e) we e de e mined, albei ne e
de ec ed, using a Hewle -Packa d 1100 high pe o mance liquid ch o-
ma og aphe (GLEFG1 me hod) equipped wi h an in a ed Hewle -
Packa d 1047 A de ec o . The column used was an Aminex HPX-87 H
(Bio-Rad Labo a o ies, 300 ×7.8 mm) using H
2
SO
4
(5 mM) as eluen .
The column empe a u e was 30 ◦C while o he de ec o was 35 ◦C.
Fo o al AA de e mina ion, he AccQ-Tag me hod was used, con-
e ing p e en i ely hyd olyzed AAs (24 h wi h HCl 6N) in o s able
luo escen de i a i es, which we e hen analysed wi h a Wa e s 2695
high pe o mance liquid ch oma og aphe h ough a luo escence de-
ec o (Wa e s 2475).
Tu bidi y (i.e. op ical densi y) was de e mined using he OD600
me hod calib a ed wi h ac ual ola ile suspended solids measu emen s.
2.4. Calcula ions
Acidi ica ion deg ee was calcula ed o desc ibe he subs a e con-
e sion (in COD basis), exp essed as:
Acidi ica ion deg ee (%) = ƩCVFA−COD
Cp
×100 (1)
whe e C
VFA-COD
s ands o he o al concen a ion o he measu ed
alipha ic VFAs (in g COD-VFA/L) in he e men a ion liquo and C
p
o
he ini ial p o ein concen a ion (in g COD/L).
As CE leads o a highe ac ion o longe -chain VFA, i.e. mo e
educed VFA, he deg ee o educ ion, de ined as he a e age COD pe
g am o p oduced alipha ic VFAs, was he p oposed pa ame e o e al-
ua e he p esence o CE p ocesses:
Deg ee o educ ion (gCOD/gVFA) = ƩCVFA−COD
ƩCVFA
(2)
whe e C
VFA-COD
s ands o he o al COD concen a ion o he measu ed
VFAs (in g COD-VFA/L) and C
VFA
o he o al concen a ion o he
measu ed VFAs (in g VFA/L) in he e men a ion liquo . Fo VFA wi h
2–6 ca bon a oms, his index a ies be ween 1.07 g COD/g VFA co e-
sponding o pu e ace ic acid and 2.21 g COD/g VFA co esponding o
pu e cap oic acid.
To be e unde s and he mechanisms in ol ed in he AA-based CE
p ocess, balances be ween AA consump ion and VFA p oduc ion we e
es ablished. AA consump ion was calcula ed om he measu ed AA
mola concen a ions in he in luen and e luen o he eac o s. To link
AA consump ion and VFA p oduc ion, he s oichiome y o AA con e -
sion p oposed by Ramsay and Pullammanappallil [22] was used, wi h
due co ec ions in oduced by Reguei a e al. [23].
R. Be ilacqua e al.
Jou nal o En i onmen al Chemical Enginee ing 10 (2022) 106943
3
3. Resul s and discussion
3.1. pH and amino acids composi ion de e mine chain elonga ion
easibili y du ing p o ein mixed cul u e e men a ion
In a p e ious s udy [20], i was obse ed ha longe chain VFA
p oduc ion is p omo ed du ing p o ein con inuous e men a ion a acid
condi ions, esul ing in highe a e age deg ees o educ ion han a
neu al and alkaline pH alues (Table 1). Mo eo e , his inc eased
longe chain VFA p oduc ion did no co espond wi h he consump ion
Table 1
A e age deg ee o educ ion (exp essed as g COD/g VFA) achie ed du ing
con inuous mixed cul u e e men a ion o casein and gela in a di e en pH
alues.
P o ein pH 5 pH 7 pH 9
Casein 1.72 ±0.06 1.47 ±0.01 1.47 ±0.01
Gela in 1.40 ±0.02 1.36 ±0.02 1.29 ±0.02
Fig. 1. n-Vale ic acid mola balance in he con inuous casein (a) and gela in (b) eac o s a h ee di e en pH alues. P oline; n-Vale ic. P oline concen a ion is
exp essed in VFA equi alen s based on he associa ed s oichiome y.
Fig. 2. VFA p oduc ion du ing casein ba ch e men a ion a pH 5 and SIR 10 (a, b) and SIR 20 (c, d). Ace ic; P opionic; Iso-Bu y ic; x n-Bu y ic; Iso-Vale ic;
n-Vale ic.
R. Be ilacqua e al.
Jou nal o En i onmen al Chemical Enginee ing 10 (2022) 106943
4
o he p ecu so amino acids. Thus, i was hypo hesized ha CE p o-
cesses migh ha e been con ibu ing o he obse ed change o selec-
i i y. As his endency was only obse ed a acid condi ions and
especially du ing casein e men a ion, i was also hypo hesized ha pH
and p o ein composi ion could play a ole in p omo ing he elonga ion
p ocess.
Al hough i canno be comple ely excluded, he o ma ion ia elon-
ga ion o iso-bu y ic [24,25], n-bu y ic [26] and iso- ale ic [27,28]
acids was disca ded due o hei p oduc ion being gene ally consis en
wi h he associa ed AA consump ion. Con e sely, n- ale ic acid o ma-
ion ia elonga ion can be mo e easily e i ied since p oline is he only
p ecu so AA (1 mmol P o =0.5 mmol n-Val [29]). In e es ingly, p o-
line consump ion alone could no jus i y n- ale ic p oduc ion du ing
casein e men a ion a acid condi ions (Fig. 1a), s eng hening he hy-
po hesis ha low pH alues a e equi ed o he p o ein-based CE o
occu . Howe e , no excess o n- ale ic acid was de ec ed du ing gela in
eac o ope a ion, ega dless o he ope a ional pH (Fig. 1b).
These esul s sugges ha he bac e ial communi ies could be pe -
o ming CE no only as a sink s a egy o NADH su pluses [1], bu also
as a mean o educe he numbe o acid equi alen s in he eac o bulk.
Indeed, a low pH he e is a la ge ac ion o undissocia ed VFA ha can
di use back h ough he cell memb ane o he in acellula space. The
highe con e sion o casein con e sion o VFAs a pH 5 (30%) han
gela in (20%) could explain why his de oxi ica ion s a egy was no
obse ed o bo h p o eins. Mo eo e , casein e men a ion gene a es an
excess o educing powe [19] which could be consumed by elonga ing
sho chain VFAs, whe eas gela in composi ion ea u es a highe pe -
cen age o elec on accep o AAs (58.6%) han casein (26.4%), limi ing
he need o CE p ocesses o main ain he edox balance.
3.2. Subs a e- o-inoculum a io a ec s he ex en o he elonga ion
p ocess
To e i y he abo emen ioned hypo heses, casein ba ch es s we e
conduc ed a wo di e en SIR alues: 10 and 20 g COD/g VS. A SIR 10,
a inal acidi ica ion and deg ee o educ ion o 43.5% and 1.76 we e
espec i ely a ained. The majo p oduc s we e n-bu y ic and iso- ale ic
acids, wi h concen a ions g ea e han 250 mg/L (Fig. 2b). The con-
cen a ions o ace ic and p opionic quickly ose du ing he i s 24 h,
hen s agna ed o ew hou s o con inue u he inc easing la e
(Fig. 2a), while he o he ca boxylic acids kep being s eadily p oduced.
No ably, n- ale ic acid p oduc ion showed a lag phase o app oxima ely
12 h (Fig. 2b). A compa able inal acidi ica ion and deg ee o educ ion
we e achie ed a SIR 20 (45.3% and 1.80, espec i ely), al hough he
du a ion o he ba ch es was inc eased o 384 h (Fig. 2c and d), hus
uling ou he occu ence o p oduc s inhibi ion. Once again, n-bu y ic
and iso- ale ic acids we e he majo p oduc s oge he wi h n- ale ic
acid, he h ee VFA eaching concen a ions highe han 550 mg/L.
Simila ly o he p e ious es , ace ic and p opionic acid concen a ion
inc eased du ing he i s 24 h and hen dec eased e en mo e ma kedly
han he end obse ed o he SIR 10 es (Fig. 2c). These s agna ion
pe iods migh co espond o in-si u consump ion o sho chain VFAs
associa ed wi h elonga ion p ocesses [28].
The VFA concen a ions o he SIR 20 es we e expec ed o double
hose o he SIR 10 one, as simila acidi ica ion deg ees we e achie ed,
hus uling ou p oduc inhibi ion. Howe e , his pa e n was only
e i ied o h ee VFAs (Fig. 3): iso-bu y ic, n-bu y ic and iso- ale ic
acids. The a io o ace ic and p opionic acid was below 2 while n-
ale ic acid one was highe han 3.0 (Fig. 3), con i ming he occu ence
o CE p ocesses. Al hough he elonga ion o ace ic and p opionic acid o
n- ale ic acid a SIR 10 canno be excluded, n- ale ic p oduc ion
(1.80 mmol/L) i ed qui e well wi h he maximum p oduc ion
Fig. 3. Ra ios be ween he inal VFA mola concen a ions ob ained du ing
casein ba ch e men a ion a SIR 20 and SIR 10, espec i ely. The ho izon al
line ep esen s he p opo ionali y h eshold.
Fig. 4. n-Vale ic (a: P oline; n-Vale ic), iso- ale ic (b: Isoleucine; Leucine; Iso-Vale ic acid) and n-bu y ic acid (c: Glu amic acid; His idine;
Th eonine; Lysine; n-Bu y ic acid) balances in he casein ba ch es a SIR20 and pH 5. AA concen a ions a e exp essed in VFA equi alen s based on he
associa ed s oichiome y.
R. Be ilacqua e al.
Jou nal o En i onmen al Chemical Enginee ing 10 (2022) 106943
5
es ima ed assuming comple e con e sion o p oline (1.60 mmol/L), he
only AA assumed o yield n- ale ic acid [29]. Con e sely, he same
es ima ion applied o he SIR 20 es unequi ocally con i ms he
occu ence o CE (Fig. 4a). In ac , he SIR 20 esul s sugges ha almos
50% o he n- ale ic p oduc ion could ha e been p oduced h ough he
elonga ion pa hway. The CE p ocess appea s o be s ongly selec i e
owa ds n- ale ic o ma ion, as he iso- ale ic (Fig. 4b) and he
n-bu y ic (Fig. 4c) balances close qui e well.
Based on hese esul s, apa om he pH and p o ein composi ion,
he p olonged simul aneous a ailabili y o sui able elec on dono AA
and sho chain VFAs seems o play an impo an ole in de e mining he
ex en o he CE p ocess. In ac , highe ini ial subs a e concen a ions
in he ba ch es allowed he p oduc ion o he sho chain VFAs which
we e hen elonga ed wi hou deple ing he AAs equi ed by he CE
p ocess. In a con inuous e men a ion p ocess, he con inuous eeding
seems o bypass his AA limi a ion, since CE con ibu ion o n- ale ic
o ma ion was g ea e du ing CSTR ope a ion (68.2%, Fig. 1a) han in
he SIR 20 ba ch es (53.2%, Fig. 4a).
3.3. Unde s anding he ole o ace ic and p opionic acid du ing p o ein-
based chain elonga ion
A hi d ba ch es was pe o med wi h casein a SIR 20 and wi h he
ini ial supplemen a ion o ace ic acid a a concen a ion o app oxi-
ma ely 500 mg/L o unde s and whe he he supplemen a ion o sho
chain VFAs can p omo e CE p ocesses du ing p o ein e men a ion, hus
a oiding he associa ed subs a e limi a ions desc ibed in Sec ion 3.2.
Assuming ha ace ic acid p oduc ion and consump ion occu s
simul aneously, h ee pe iods can be di e en ia ed (Fig. 5): (i) simila
consump ion and p oduc ion a e du ing he i s 32 h (i.e., ace ic acid
concen a ion emains mainly unchanged); (ii) highe consump ion a e
han p oduc ion du ing he subsequen 40 h, and, (iii) highe p oduc ion
a e han consump ion om 72 h on. Excep o p opionic acid, he o he
VFAs s a ed o be p oduced only a e 24 h, sugges ing ha he ace ic
acid supplemen a ion led o an adap a ion phase du ing which he
biomass was no able o e icien ly con e he subs a e. In e es ingly,
n- ale ic acid o ma ion began only a e ace ic acid concen a ion
s a ed o dec ease isibly. n-Bu y ic and iso- ale ic acids we e
con i med o be he main p oduc s o he e men a ion (app oxima ely
800 mg/L). P opionic acid did no show any sign o s agna ion and i s
inal concen a ion was 300 mg/L, while iso-bu y ic and n- ale ic le els
we e sligh ly highe (400 mg/L). A he end o he es , he ace ic acid
consump ion almos balanced i s p oduc ion as he inal concen a ion is
compa able o he ini ial one. The acidi ica ion deg ee (excluding he
ini ially added ace ic acid) and he deg ee o educ ion we e 50.9% and
1.86 g COD/g VFA, espec i ely.
The supplemen a ion o ace ic acid a o s he p oduc ion o n-bu y ic
and iso- ale ic acids bu limi s he o ma ion o n- ale ic acid (Fig. 6).
The inal concen a ion o ace ic acid (600 mg/L) is lowe han he sum
o he supplemen a ion (500 mg/L) plus he p oduc ion du ing he SIR
20 es (300 mg/L), indica ing a lowe o ma ion o his VFA du ing he
e men a ion o casein o a highe consump ion du ing CE p ocesses.
The inc ease in p opionic and iso-bu y ic acid p oduc ion is p obably
no signi ican , as i was qui e limi ed (<90 mg/L) in bo h cases, and
consequen ly a ibu ed o expe imen al a iabili y.
These esul s sugges ha he ini ial a ailabili y o ace ic acid can
al e he selec i i y o he CE p ocess, especially p omo ing i s elonga-
ion o n-bu y ic acid. Al hough i does no comple ely jus i y he in-
c ease in n-bu y ic acid inal concen a ion (200 mg/L), he di e ence
be ween expec ed and measu ed ace ic acid concen a ion (200 mg/L)
seems o be closely ela ed. Mo eo e , he mola balance o his VFA
(Fig. 7a) e idenced ha i s ou p ecu so AAs we e no con e ed
du ing he i s 54 h, s eng hening he hypo hesis o he supplemen ed
ace ic acid being consumed o p oduce n-bu y ic acid [30] h ough he
coupling wi h ace yl-CoA om a sui able elec on dono AA such as
alanine [17].
Fig. 5. VFA p oduc ion du ing casein ba ch e men a ion a SIR 20 and pH 5, wi h ini ial ace ic acid addi ion (500 mg/L). a: Ace ic; P opionic; b: Iso-Bu y ic;
x n-Bu y ic; Iso-Vale ic; n-Vale ic.
Fig. 6. Compa ison be ween he VFA inal concen a ions ob ained du ing he
ba ch es s a SIR 20 and pH 5 wi h (■) and wi hou (▨) ace ic acid supple-
men a ion (500 mg/L).
R. Be ilacqua e al.

Jou nal o En i onmen al Chemical Enginee ing 10 (2022) 106943
6
The inc ease o iso- ale ic acid in he supplemen ed es co esponds
o he dec ease o n- ale ic acid. This can be e i ied as he sum o iso
and n- ale ic acid in each es is app oxima ely 1200 mg/L (Fig. 6),
sugges ing ha he ace ic acid supplemen a ion migh ha e di e ed he
CE p ocess om he p oduc ion o he linea o m o he b anched-chain
one. The global ale ic acid balance (Fig. 7b) unequi ocally con i ms he
con ibu ion o CE o he p oduc ion o bo h acid o ms, as hei
concen a ions exceed he consump ion o he pa en AA. In e es ingly,
CE was ini ially he only esponsible o n- ale ic acid gene a ion (54 h),
e en be o e p oline s a ed being consumed.
Fig. 7. n-Bu y ic acid (a: Glu amic acid; His idine; Th eonine; Lysine; n-Bu y ic acid) and global ale ic (b: P oline; n-Vale ic; Isoleucine;
Leucine; Iso-Vale ic acid) balances in he casein ba ch es a SIR20 and pH 5 wi h ini ial ace ic acid addi ion. AA concen a ions a e exp essed in VFA equi alen s
based on he associa ed s oichiome y.
Fig. 8. Concep ual p o ein-based chain elonga ion mechanisms based on sho chain VFA and AA consump ion (a: ini ial lack o ace ic and p opionic acids; b: ini ial
a ailabili y o ace ic acid). sho chain VFA; supplemen ed sho chain VFA; AA; in e media e me aboli e; Elonga ed p oduc . The wide a ow ep esen s
he a o ed elonga ion eac ions, whe eas he aded colo s indica e absen o mino ou es.
R. Be ilacqua e al.
Jou nal o En i onmen al Chemical Enginee ing 10 (2022) 106943
7
3.4. Highligh ing he unde lying mechanisms o p o ein-based chain
elonga ion
F om he abo emen ioned esul s, i can be concluded ha casein-
based CE is indeed easible and occu s by using ace ic o p opionic
acid as elec on accep o compound (Fig. 8). Sui able elec on dono
AAs will supply educing powe and p opionyl-CoA o CE wi h ace ic
acid, o ace il-CoA o CE wi h ei he p opionic o ace ic acid [28].
Se e al AAs (alanine, a ginine, aspa ic acid, cys eine, glu amic acid,
his idine, se ine) can supply ei he p opionyl-CoA o ace yl-CoA as hey
ha e py u a e as in e media e p oduc o hei con e sion [29].
Me hionine and lysine a e associa ed o only p opionyl-CoA o only
ace yl-CoA, espec i ely. Th eonine can p o ide bo h hese compounds
ia di e en pa hways [29]. O e all, he ac ha no ex e nal elec on
dono supplemen a ions a e equi ed makes his kind o CE pa icula ly
appealing in a bio e ine y amewo k. In ac , he ope a ional cos s and
he chemical use should be lowe han o e hanol and/o lac a e-based
CE [1], mo i a ing u he in es iga ion on his aspec o he p ocess.
In p o ein-based CE, he p ocess selec i i y seems highly dependen
on he ini ial a ailabili y o sho chain VFA. I he p ocess is ini ially
limi ed by he a ailabili y o sho chain VFAs, i becomes pa icula ly
selec i e owa ds n- ale ic acid (Fig. 8a), albei he elonga ion o he
o he VFAs canno be comple ely excluded (Sec ion 3.2). Con e sely, he
ini ial supplemen a ion o ace ic acid clea ly al e s he selec i i y o he
p ocess (Fig. 8b), p omo ing he elonga ion o his sho chain VFA o n-
bu y ic acid while p io i izing he o ma ion o iso- ale ic acid o e he
n- ale ic (Sec ion 3.3).
4. Conclusions
To he bes o ou knowledge, his s udy iden i ied and a ge ed o
he i s ime he occu ence o chain elonga ion p ocess du ing p o ein
mixed-cul u e e men a ion, wi h he ollowing main conclusions:
•Acid condi ions a e hypo hesized o be p omo ing p o ein-based CE,
since his p ocess educes he o e all acid equi alen s yielded om
he subs a e, consequen ly a oiding po en ial oxici y cons ain s.
•The easibili y o CE p ocesses du ing p o ein e men a ion is
signi ican ly in luenced by p o ein composi ion, wi h due p e e ence
o hose ich in elec on dono AAs.
•P o ein-based CE does no equi e ex e nal elec on dono supple-
men a ions o occu , po en ially making his kind o p ocess mo e
sus ainable han he con en ional ones.
•The occu ence o CE s ongly depends on he p olonged simul a-
neous a ailabili y o sho chain VFAs and elec on dono AAs.
•The selec i i y o he CE p ocess depends on he speci ic a ailabili y
o ace ic and p opionic acid.
The knowledge gene a ed h ough his wo k cons i u es a s a ing
poin o u he s udies on p o ein-based CE aiming a an op imal
in eg a ion o p o ein- ich (was e)wa e s in bio e ine y amewo ks,
cu en ly en isioned o ca bohyd a e- ich s eams only.
CRediT au ho ship con ibu ion s a emen
Ricca do Be ilacqua: Me hodology, In es iga ion, Sample p o-
cessing, Da a analysis, W i ing – o iginal d a , W i ing – e iew &
edi ing. Albe e Reguei a: Me hodology, Da a analysis, W i ing –
o iginal d a , W i ing – e iew & edi ing. Miguel Mau icio-Iglesias:
Me hodology, Concep ualiza ion, Supe ision, Da a Analysis, W i ing –
o iginal d a , W i ing – e iew & edi ing, Juan M. Lema: Me hodology,
Concep ualiza ion, Supe ision; W i ing – o iginal d a . Ma a Ca -
balla: Me hodology, Concep ualiza ion, Funding acquisi ion; Supe i-
sion, Da a Analysis, W i ing – o iginal d a , W i ing – e iew & edi ing.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgmen s
This p ojec has ecei ed unding om he Spanish Go e nmen
h ough BIOCHEM p ojec (PCIN-2016-102, ERA-IB-2 7 h call, ERA-IB-
16-052) . The au ho s belong o a Galician Compe i i e Resea ch G oup
(ED431C-2021/37), co- unded by ERDF (UE).
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