Enginee ing he ou come o co e men a ion p ocesses by
1
al e ing he eeds ock suga - o-p o ein a io
2
R. Be ilacqua*, M. Mau icio-Iglesias, S. Balboa, J.M. Lema, M. Ca balla
3
CRETUS, Depa men o Chemical Enginee ing, Uni e sidade de San iago de Compos ela, 15782
4
San iago de Compos ela, Spain – icca do.be [email protected]
5
ABSTRACT
6
This wo k in es iga es he impac o he suga - o-p o ein (STP) a io on he ou come o hei
7
anae obic co e men a ion in e ms o subs a e con e sion and p oduc selec i i y. Fo his
8
pu pose, a con inuous s i ed ank eac o was ope a ed a pH 7 and ed wi h casein and glucose
9
a di e en STP a ios (0.25, 0.50, 0.75, 1.00 and 2.00 in COD basis). Casein con e sion was
10
una ec ed by glucose p esence as long as he a io was lowe o equal o 1. In his ange o STP
11
a io, n-bu y ic and n- ale ic acid p oduc ion was p omo ed due o he occu ence and p og essi e
12
in ensi ica ion o chain elonga ion p ocesses. Con e sely, STP a ios g ea e han 1 a e associa ed
13
wi h lowe amino acids consump ion, inhibi ion o he elonga ion me abolism and lowe ola ile
14
a y acids p oduc ion due o he o ma ion o al e na i e end p oduc s (e hanol, lac a e and
15
o ma e) and uniden i ied compounds. In e es ingly, hese nega i e e ec s a e e e sible, as
16
lowe ing he suga - o-p o ein a io allows o eco e p o ein acidi ica ion deg ee, p ocess
17
p oduc i i y and he chain elonga ion. O e all, his wo k success ully demons a es ha suga -
18
p o ein co e men a ion p ocesses can be s ee ed by adjus ing hei p opo ions in he eeds ock.
19
KEYWORDS: amino acids; bio e ine y; chain elonga ion; eeds ock composi ion; glucose; ola ile
20
a y acids
21
22
1 INTRODUCTION
23
Se e al s udies1–5 highligh ed he po en ial o mixing di e en subs a es o enhance he
24
p oduc ion o ola ile a y acids (VFAs) du ing mixed-cul u e e men a ion (MCF) p ocesses. The
25
posi i e e ec obse ed du ing he co e men a ion o p o eic s eams wi h hose ich in suga s is
26
gene ally associa ed wi h a be e balancing o mic onu ien s and ca bon/ni ogen p opo ions,
27
dilu ion o po en ially oxic o inhibi o y compounds, and/o an inc ease in hyd olysis a e due o
28
he highe biomass yields achie ed 6.
29
Howe e , mos li e a u e examples dealing wi h anae obic co e men a ion a e case s udies
30
in ol ing mix u es o speci ic was e and was ewa e s, as in his example s udying was e ac i a ed
31
sludge and po a o peel was e5. Thus, he applica ion o he esul ing knowledge is limi ed o hose
32
speci ic subs a es and he conclusions a e no alid o he con e sion o a gene ic mix u e o
33
p o eins and ca bohyd a es in o VFAs. Besides, all hese p e ious esul s appea no o be
34
conclusi e conce ning he in luence o mixing di e en o ganic ac ions. Fo example, B eu e e
35
al.9 obse ed ha he p esence o a suga (e.g. glucose) can pa ially inhibi he hyd olysis o
36
p o eins, and consequen ly hei con e sion in o VFAs, when he wo ac ions loading we e
37
simila , whe eas lowe suga loads did no show nega i e e ec s on p o ein e men a ion.
38
Con e sely, Ma e al.5 de e mined ha inc easing he ca bohyd a e ac ion in he eeds ock
39
a ou s he consump ion o p o eins, wi h his syne gis ic e ec being main ained e en when
40
ca bohyd a es we e dominan o e p o eins. In disag eemen wi h he wo p e ious esul s,
41
Tommaso e al.10 obse ed ha e en minimal glucose p esence induces a dec ease in he
42
deg ada ion a e o he chosen model p o ein, bo ine se um albumin.
43
Besides he con e sion e iciency, suga s and p o eins ea u e di e en VFA selec i i y. The
44
e men a ion o suga s (e.g. glucose) mainly yields ace ic, p opionic and bu y ic acid11 on
45
p opo ions ha can be s ee ed h ough pH adjus men s12, simila ly o lac ose e men a ion13.
46
Con e sely, p o eins selec i i y hea ily depends on hei composi ion,14 gi en he po en ial
47
combina ion occu ing om he mix o he 20 main amino acids (AAs). Ace ic acid ends o be he
48
main p oduc a neu al and alkaline condi ions whe eas low pH a ou s he con e sion o longe
49
chain VFAs15. Mo eo e , b anched chain VFAs and n- ale ic acid a e mos ly ob ained h ough he
50
e men a ion o speci ic AAs a he han om suga s16. This subs a e dependence sugges s ha
51
he VFA dis ibu ion o a co e men a ion p ocess could be s ee ed based on he eeding
52
p opo ions be ween he wo o ganic ac ions. Ye , he li e a u e conce ning his e ec is again
53
con adic o y. Fo example, supplemen ing gela in e men a ion wi h ei he glucose o lac ose in
54
equal p opo ions was associa ed o an inc eased p oduc ion o n-bu y ic acid and e hanol9.
55
Ins ead, Zhou e al.17 obse ed an inc ease in ace ic and p opionic concen a ions when
56
p og essi ely eeding g ea e p opo ions o ca bohyd a e- ich co n s aw o he sludge-deg ading
57
eac o . In ano he case s udy, limi ing he suga ac ion in he eeding mix u e seemed o a ou
58
he o ma ion o n- ale ic acid5.
59
The a o emen ioned in o ma ion poin s ou he need o a uni e sal pa ame e o be e
60
unde s and he in e ac ion be ween p o eins and ca bohyd a es in a co e men a ion p ocess in
61
o de o enginee he p ocess owa ds he desi ed ou come. The e o e, he p esen s udy
62
p oposes he suga - o-p o ein a io (STP), measu ed in chemical oxygen demand (COD) basis, as
63
he pa ame e o assess and unde s and such in e ac ion. The use o model p o ein and suga
64
compounds (i.e. casein and glucose, espec i ely) aims a acili a ing esul s in e p e a ion as well
65
as hei ex apola ion o a gene ic p o ein-ca bohyd a e co e men a ion p ocess.
66
2 MATERIALS AND METHODS
67
2.1 Feeds ock composi ion
68
Casein pep one (A2208,0500 PanReac) and D(+)-glucose anhyd ous (131341.1211 PanReac) we e
69
he model compounds used in his s udy. P o ein concen a ion was ixed a 7.50 g/L h oughou
70
he expe imen , while glucose concen a ion was p og essi ely inc eased om 1.87 g/L o 14.96
71
g/L. The eeds ock solu ion was supplemen ed wi h mac o- and mic o-nu ien s, as desc ibed in
72
Be ilacqua e al.18 , and i was main ained e ige a ed h oughou he expe imen (4°C).
73
2.2 Con inuous eac o s ope a ion
74
The con inuous s i ed ank eac o (CSTR) o 1 L used in he p esen s udy was he same as
75
desc ibed in Be ilacqua e al. 18 The pH was se a 7.0 o he whole du a ion o he expe imen ,
76
while he eac o was main ained a 25 ºC h ough a empe a u e-con olled oom. Being a CSTR,
77
he hyd aulic e en ion ime (HRT) and he solids e en ion ime we e bo h equal o 1.5 d. The
78
main di e ence be ween he wo s udies was glucose being included in he eeds ock a inc easing
79
concen a ions in o de o es se e al STP a ios (in COD basis): 0.25, 0.50, 0.75, 1.00, 2.00. Each
80
esul ing STP a io (Table 1) was main ained o a leas 40 days, in o de o e alua e i s impac on
81
he co e men a ion p ocess a e eaching a s eady-s a e ope a ion.
82
Table 1. Ope a ional condi ions o he di e en phases o he co e men a ion eac o . STP: suga -
83
o-p o ein a io (COD basis); OLR: o ganic loading a e (g COD/L·d).
84
Phase
STP a io
Casein OLR
Glucose OLR
I
0.25
5.33
1.33
II
0.50
5.33
2.67
III
0.75
5.33
4.00
IV
1.00
5.33
5.33
V
2.00
5.33
10.7
85
The eac o pe o mance was moni o ed as desc ibed in Be ilacqua e al.18 . In b ie , he pH was
86
con olled a he 7.0 se poin ia a mul ipa ame ic analyse (CHEMITEC, I aly) and NaOH 3M
87
addi ions. VFA and To al Ammonia Ni ogen (TAN) concen a ions we e de e mined wice a week,
88
while COD ( o al and soluble) and solids concen a ions we e measu ed once a week. Amino acid
89
(AA) analysis was pe o med on samples speci ically selec ed om s eady s a e pe iods o
90
ope a ion.
91
2.3 Analy ical me hods
92
The analy ical me hods used a e p e iously desc ibed in Be ilacqua e al. 15,18. A summa y is
93
included in Supplemen a y In o ma ion.
94
2.4 Mic obial communi y analyses
95
A each suga - o-p o ein a io, h ee biomass samples we e aken, co esponding o h ee
96
consecu i e weeks o s able ope a ion. Genomic DNA om 1 mL homogenized samples was
97
ex ac ed by iplica e using he Nucleospin Mic obial DNA ex ac ion ki (Mache y-Nagel),
98
acco ding o he ins uc ions o he manu ac u e . The eplica om eachsample we e pooled
99
oge he a e quan i ica ion, ensu ing quali y con ol and no maliza ion wi h Nanod op and Qubi
100
luo ome e (The mo Fishe Scien i ic Wal ham, MA, USA). The V3-V4 hype a iable egion o
101
Bac e ia was ampli ied using Bak _341F (5’ CCT ACG GGN GGC WGC AG 3’) and Bak _805R (5’ GAC
102
TAC HVG GGT ATC TAA TCC 3’)19. DNA me aba coding analyses o he egion we e ca ied ou by
103
AllGene ics & Biology SL (www.allgene ics.eu) in an Illumina MiSeq pla o m.
104
Bioin o ma ic analyses we e pe o med using he Mic obial Genomics module ( e sion 21.1)
105
wo k low o he CLC Genomics wo kbench ( e sion 21.0.3). Raw sequences we e il e ed o
106
emo e low-quali y eads and hen clus e ed in o Ope a ional Taxonomic Uni s (OTUs) a 97%
107
cu o o sequence simila i y and classi ied agains he non- edundan e sion SILVA SSU
108
e e ence axonomy ( elease 132; h p://www.a b-sil a.de)20 . Only he mos abundan bac e ial
109
OTUs (abo e 1 % o he o al obse ed OTUs) we e conside ed o u he analysis.
110
Mic obial abundance om phyla o genus le el was analyzed, log- ans o med and he s a is ical
111
signi icance was de e mined o p < 0.05 by pe mu a ional mul i a ia e analysis o a iance
112

(PERMANOVA), including Bon e oni co ec ion. Alpha di e si y was es ima ed om he
113
a e ac ion analysis using he esul ing phylogene ic ee o OTUs gene a ed by he MUSCLE
114
algo i hm, wi h a maximum sampling dep h o 26,618 eads. Be a di e si y was measu ed by B ay-
115
Cu is dis ances be ween each pai o samples applying p incipal coo dina e analysis (PCoA) o he
116
dis ance ma ices. Signi icance was, likewise, assessed by PERMANOVA.
117
118
2.5 Calcula ions
119
Acidi ica ion deg ee was he pa ame e chosen o desc ibe subs a e con e sion (in COD basis),
120
while ammoni ica ion was also used as a p oxy o moni o p o ein con e sion o VFA, as amino
121
acid e men a ion is always ela ed o NH4+ elease. In addi ion, balances be ween AA
122
consump ion and VFA p oduc ion we e es ablished o e i y p o ein con e sion s oichiome y.
123
Mo e de ails can be ound in Supplemen a y In o ma ion.
124
3. RESULTS AND DISCUSSION
125
3.1 Co e men a ion eac o ope a ion
126
The co e men a ion eac o was con inuously ope a ed o 344 days (Fig.1a). The i s 56 days
127
we e join ly conside ed as a phase o s a -up and acclima ion o glucose p esence (1.33 g
128
COD/L·d), gi en ha he inoculum was used o deg ade only p o eins du ing a p e ious
129
expe imen 18. To inhibi me hanogenesis, which began o occu a day 42, sodium 2-
130
b omoe hanesulphona e (BES, 137502, SigmaAld ich) was added o he eac o eeds ock a a
131
concen a ion o 0.5 g/L s a ing om day 45. A day 344, he eac o was s opped due o Co id-
132
19 lockdown and es ic ions on esea ch ac i i y and i s con en was s o ed a 4°C. The ope a ion
133
was hen esumed a e wo mon hs (Fig. 1b) by acclima ing he s o ed biomass a he o iginal
134
condi ions o pH, empe a u e and ni ogen spa ging. The eac o was ope a ed in ba ch mode o
135
he i s 10 days by adding a dilu ed eeds ock pulse o he essel, in o de o sa ely eac i a e he
136
biomass ac i i y. A e ha ing de ec ed he occu ence o VFA p oduc ion (Fig. 1d), con inuous
137
eeding s a ed a an hyd aulic e en ion ime (HRT) equal o 3 d (STP 1.00), o a oid po en ial
138
washou o he biomass. A e one week i was lowe ed o 2 d, and inally se a he o iginal alue
139
o 1.5 d a day 24. On day 45, glucose concen a ion was inc eased o achie e he highes STP
140
alue (2.00). The eac o ope a ion was hen inalised a day 88.
141
Biomass concen a ion apidly g ew om 0.6 o 1.0 g VSS/L when exposed a he lowes glucose
142
loading (STP 0.25), compa ibly wi h he highe yields associa ed wi h suga subs a es21. Inc easing
143
he STP a io u he a ou ed biomass g ow h, eaching 1.4 g VSS/L and 2.8 g VSS/L a STP a ios
144
o 1.00 and 2.00, espec i ely.
145
Me hanisa ion was success ully inhibi ed om day 50 on, since no di e ence was de ec ed
146
be ween he o al COD concen a ions in he eac o in luen and e luen (Fig. 1a and b). The
147
di e ence be ween o al and soluble COD in he e luen s ma ched he biomass concen a ions
148
achie ed in he eac o . The o e all concen a ion o VFA (COD basis) inc eased p og essi ely wi h
149
he applica ion o highe STP a ios, peaking a app oxima ely 10 g COD/L (STP 1.00). VFA
150
p oduc ion was 20% lowe (8 g COD/L) a e he eac o ope a ion was esumed a he same
151
condi ions (Fig. 1b), sugges ing ha he in e up ion and subsequen s o age migh ha e a ec ed
152
he mic obial popula ion. Soluble COD concen a ion was sys ema ically highe han he VFA-COD
153
concen a ion, sugges ing he p esence o non-con e ed subs a e, al e na i e end p oduc s (e.g.
154
e hanol) and/o uniden i ied p oduc s. As glucose could no be de ec ed in he eac o e luen s,
155
only p o ein can accoun o he non-con e ed subs a e.
156
As expec ed, global VFA p oduc ion inc eased a highe STP a ios. Howe e , he e ec o STP a io
157
on indi idual VFA p oduc ion was acid-dependan (Fig. 1c and d). Ace ic, n-bu y ic and n- ale ic
158
acids we e he main p oduc s o mos o he eac o o iginal ope a ion (≥750 mg/L), p og essi ely
159
inc easing wi h he STP a io. In e es ingly, n- ale ic acid p oduc ion peaked a 1500 mg/L when
160
applying an STP alue o 0.75, becoming he VFA wi h he highes concen a ion. Ace ic acid
161
eplaced i a STP 1.00, eaching 2000 mg/L. In compa ison, n-bu y ic acid concen a ion g ew
162
mo e s eadily, s abilising a a inal concen a ion o 1500 mg/L a STP 1.00. Con e sely, iso-bu y ic
163
and iso- ale ic acid p oduc ion dec eased om 330 o 250 mg/L and om 650 o 500 mg/L
164
espec i ely when applying an STP alue g ea e han 0.25. n-Cap oic acid was only de ec ed o a
165
limi ed amoun o ime (STP 0.50) and only in small concen a ions (≤ 150 mg/L). Du ing he
166
esumed ope a ion, he inc ease in STP a io especially a ou ed ace ic and p opionic p oduc ion
167
(≥2200 mg/L) in de imen o all he o he VFAs, whose concen a ions we e equal o lowe han
168
500 mg/L. Lac a e, o ma e and e hanol p oduc ion was no obse ed du ing he o iginal
169
expe imen and a a iable concen a ions du ing he esumed ope a ion (da a no shown).
170
To assess he impac o STP a io on casein-glucose co e men a ion, se e al s eady-s a e pe iods
171
we e iden i ied: day 56 – 119, day 142 – 232, day 249 – 295 and day 312 – 344 o STP a ios o
172
0.25, 0.50, 0.75 and 1.00, espec i ely. Fo he esumed ope a ion, he selec ed s able pe iods
173
we e day 24 – 45 and day 52 – 88 o STP a ios o 1.00 and 2.00, espec i ely.
174
175
Figu e 1. COD balance (a, o iginal ope a ion; b, esumed ope a ion: ▲ In luen o al COD; ●
176
E luen o al COD; □ E luen soluble COD; ◇ VFAs COD) and indi idual VFA concen a ions in he
177
co e men a ion eac o (c, o iginal ope a ion; d, esumed ope a ion: ● Ace ic; ◆ P opionic; ▲ Iso-
178
Bu y ic; x n-Bu y ic; ⁕ Iso-Vale ic; ■ n-Vale ic). The e ical black lines indica e he change in he
179
STP a io.
180
3.2 The in luence o STP a io on p o ein con e sion and amino acid consump ion
181
Glucose consump ion was comple e ega dless o he STP a io, while casein consump ion was
182
abo e 60% based on he ammoni ica ion pa ame e , excep o he STP a io o 2.00 (Fig. 2). Gi en
183
3.4 The in luence o STP on he mic obial communi y s uc u e
275
A o al o 296,998 eads we e ob ained a e imming and quali y il e ing, anging om 46,105 o
276
32,006 wi h an a e age o 36,512 eads pe sample, iden i ying 466 di e en OTUs (Table S1). In
277
addi ion, he a e ac ion cu es ob ained by he no maliza ion o OTUs coun o indi idual
278
biological eplica es eached pla eau (Fig. S1), poin ing ou an adequa e sample sequencing dep h
279
(26,618 eads).
280
Only OTUs wi h a minimum combined abundance o 1% we e used o u he analysis, esul ing in
281
132 OTUs dis ibu ed in 16 classes among 10 phyla (Fig. 6). Fi micu es and Ac inobac e ia we e he
282
dominan phyla in all he samples (abo e 65%), pa icula ly a STP a io o 1.0 (abo e 97%). Bo h
283
phyla, oge he wi h P o eobac e ia and Bac e oide es, a e obliga ed o acul a i ely anae obic
284
bac e ia well known by hei abili y o decompose polysaccha ides and p o einaceous subs a es
285
o p oduce VFAs27.
286
287
Figu e 6. Bac e ial classes wi h a o al abundance highe han 1% a di e en STP a ios. The
288
e ical black lines indica e he change in he STP a io. Class abundances a e colo ed acco ding o
289
he phyla hey belong o.
290

Howe e , a mo e clea in luence o inc easing glucose loads was obse ed a class le el (Fig. 6).
291
O e all, Bac e oidia abundance shows a dec easing end, while he p esence o Ac inobac e ia
292
and E ysipelo ichia is a o ed. In e es ingly, he mic obial communi y composi ion was simila up
293
o STP alues o 0.75, bu signi ican changes occu ed when his a io was inc eased o 1 and
294
u he o 2. This pa e n was also con i med by Be a di e si y analysis (Fig. 7), whe e all samples
295
belonging o STP a ios below 1.0 clus e ed oge he (mo eo e , PERMANOVA analyses showed no
296
signi ican di e ences among STP a ios o 0.25, 0.50 and 0.75 (pseudo- s a is ic 1.99, 6.90, 1.3, p-
297
alues > 0.1)) and sepa a ely om hose belonging o STP a ios o 1 and 2, espec i ely.
298
299
Figu e 7. P incipal componen analysis (PCoA) showing he di e ences on he communi y
300
composi ion ela ed o inc easing glucose loads. Poin s ep esen each sample and a e colou ed
301
acco ding o he STP alue: 0.25 (ligh g een), 0.50 (medium g een), 0.75 (da k g een), 1.0 (blue)
302
and 2.0 ( ed).
303
Changing STP a io om 0.75 o 1.0 lead o a e y signi ican inc ease o E ysipelo ichia
304
abundance (Fig. 6) in de imen o Bac e oidia and Nega i icu es (Fig. S2). In addi ion, a dec ease
305
in Alpha di e si y was obse ed (Fig. 8). Inc easing u he he STP a io om 1.0 o 2.0 a o ed
306
he p esence o Ac inobac e ia, Bac e oidia and Clos idia in de imen o Co iobac e iia (Fig. S2),
307
and also he o e all di e si y o he mic obial communi y inc eases (Fig. 8). Combining hese
308
esul s wi h he di e en p oduc selec i i ies obse ed du ing he eac o ope a ion (Fig. 4), we
309
could specula e he posi i e link be ween Ac inobac e ia and p opionic acid p oduc ion as well as
310
he link be ween Co iobac e iia and he p oduc ion o longe chain VFA (bu y ic and ale ic acids).
311
312
Figu e 8. Summa y o alpha di e si y s a is ics shown as boxplo . A) Shannon en opy index; B)
313
Simpson index. S a is ical signi icance was measu ed by K uskal-Wallis es .
314
3.5 CE can be eco e ed by lowe ing he STP a io
315
To e i y whe he he CE p ocess could be eco e ed by lowe ing he glucose loading, a pa allel
316
co e men a ion eac o was inocula ed wi h biomass aken om he main eac o on day 45 o he
317
esumed ope a ion (STP 1.00) and ope a ed a an STP a io o 0.50 (Fig. 9).
318
319
320
Figu e 9. Ope a ion o he pa allel eac o a an STP a io o 0.50 o assess CE p ocess eco e y (a,
321
COD balance: ▲ In luen o al COD; ● E luen o al COD; □ E luen soluble COD; ◇ VFAs COD; b,
322
VFA concen a ions: ● Ace ic; ◆ P opionic; ▲ Iso-Bu y ic; x n-Bu y ic; ⁕ Iso-Vale ic; ■ n-Vale ic).
323
The e ical black lines sepa a e he acclima ion phase om he s eady-s a e ope a ion.
324
Bo h he o al and he soluble COD o he eac o e luen dec eased compa ibly wi h he lowe
325
STP applied o he eac o (Fig. 9a). Based on he VFA p oduc ion (COD basis), i was possible o
326
iden i y wo ope a ional pe iods: om he s a up o day 30 (acclima ion s age) and om day 30
327
o 43 (s eady-s a e ope a ion). In e es ingly, he alues o all COD pa ame e s we e simila o
328
hose p e iously ob ained a STP 0.50 (Fig. 1a), p o iding he i s p oo conce ning he
329
e e sibili y o excessi e suga supplemen a ion.
330
In e ms o VFA p oduc ion (Fig. 9b), he acclima ion pe iod was associa ed wi h a dec ease in
331
ace ic and p opionic acid concen a ions, whe eas he o he VFAs emained mos ly s able. In
332
con as , excep o p opionic and iso-bu y ic acid, VFAs p oduc ion inc eased be ween day 30 and
333
43. In pa icula , n-bu y ic, n- ale ic acid and iso- ale ic gene a ion showed a wo- old inc ease
334
which, coupled wi h he absence o lac a e and e hanol in he eac o e luen s, u he con i ms
335
he e e sibili y o he e ec s caused by STP a ios g ea e han 1.00. Also, he balance o ale ic
336
acids (Fig. 10) indica es ha CE p ocess was eco e ed du ing he s eady-s a e pe iod, as p oline
337
consump ion alone is no able o jus i y n- ale ic acid p oduc ion. This balance also highligh s he
338
occu ence o isome isa ion om he iso o he n- o m du ing he acclima ion s ep.
339
Compa ing hese esul s wi h hose desc ibed in he p e ious sec ions, i was hypo hesised ha
340
he inc eased a ailabili y o glucose associa ed wi h highe STP a ios migh be making u he
341
con e sion o e hanol and lac a e in o VFAs less appealing o he mic obial communi y due o
342
kine ic limi a ions associa ed wi h high OLRs (16 g COD/L·d a STP a io 2.00). Besides, he absence
343
o subs a e limi a ions migh be making specialised me abolic pa hways, such as CE, less
344
appealing om a bioene ge ics poin o iew. S ill, he dis up i e e ec caused by he ope a ion
345
in e up ion canno be comple ely disca ded, as i migh ha e accele a ed he disappea ance o
346
he CE p ocess a he highes STP a ios by al e ing he mic obial communi y in i s place. Besides,
347
he VFA concen a ions we e no s ic ly he same as du ing he o iginal expe imen a STP 0.50
348
(Fig. 1c), sugges ing ha longe ope a ion ime migh be equi ed o ully eco e he p e iously
349
ob ained s eady s a e.
350
Figu e 10. Iso and n- ale ic acid balance in he pa allel eac o a an STP a io o 0.50: ■ P oline; ■
351
n- ale ic acid; ■ Isoleucine; ■ Leucine; ■ Iso- ale ic acid. AA concen a ions a e exp essed as
352
VFA equi alen s acco ding o he s oichiome y desc ibed by Reguei a e al.16
353
4. CONCLUSIONS
354
This s udy success ully in es iga ed he in e ac ions be ween amino acids and glucose du ing hei
355
co e men a ion in o de o unde s and he impac o he STP a io on subs a e consump ion,
356
acidi ica ion deg ee, p oduc selec i i y and mic obial communi y s uc u e. In pa icula , he main
357
indings a e:
358
• STP a ios equal o lowe han 1.00 do no a ec he ex en o p o ein con e sion, bu
359
excessi e suga loading hinde s AA consump ion and a ou s he p oduc ion o al e na i e
360
end p oduc s.
361
• The p oduc s dis ibu ion can be s ee ed owa ds he p oduc ion o n-bu y ic and n- ale ic
362
acid by inc easing he suga p opo ion up o he op imal STP a io o 1.00, which p omo es
363
he occu ence o CE p ocesses.
364

• The inc easing load o glucose a ec ed mic obial communi y composi ion, especially a he
365
highes STP a io es ed (2.0). O e all, he p esence o Ac inobac e ia and E ysipelo ichia
366
was a o ed in de imen o Bac e oidia abundance.
367
• The changes p oduced by excessi e suga loadings a e e e sible, as lowe ing he STP a io
368
allows o eco e he longe chain VFA p oduc ion o a ce ain ex en .
369
CONFLICTS OF INTEREST
370
The e a e no con lic s o in e es o decla e.
371
ACKNOWLEDGEMENTS
372
This p ojec has ecei ed unding om he Eu opean Union’s ERA-IB p og amme unde g an
373
ag eemen numbe PCIN-2016-102 (BIOCHEM p ojec ). The au ho s belong o a Galician
374
Compe i i e Resea ch G oup (GRC), co- unded by ERDF (UE).
375
REFERENCES
376
1 H. Rughoonundun, R. Mohee and M. T. Hol zapple, In luence o ca bon- o-ni ogen a io on
377
he mixed-acid e men a ion o was ewa e sludge and p e ea ed bagasse, Bio esou .
378
Technol., 2012, 112, 91–97.
379
2 Á. Val Del Río, T. Palmei o-Sanchez, M. Figue oa, A. Mosque a-Co al, J. L. Campos and R.
380
Méndez, Anae obic diges ion o ae obic g anula biomass: e ec s o he mal p e- ea men
381
and addi ion o p ima y sludge, J. Chem. Technol. Bio echnol., 2014, 89, 690–697.
382
3 A. F. Duque, C. S. S. Oli ei a, I. T. D. Ca mo, A. R. Gou eia, F. Pa delha, A. M. Ramos and M.
383
A. M. Reis, Response o a h ee-s age p ocess o PHA p oduc ion by mixed mic obial
384
cul u es o eeds ock shi : impac on polyme composi ion, N. Bio echnol., 2014, 31, 276–
385
288.
386
4 Z. Guo, A. Zhou, C. Yang, B. Liang, T. Sangee ha, Z. He, L. Wang, W. Cai, A. Wang and W. Liu,
387
Enhanced sho chain a y acids p oduc ion om was e ac i a ed sludge condi ioning wi h
388
ypical ag icul u al esidues: ca bon sou ce composi ion egula es communi y unc ions,
389
Bio echnol. Bio uels, 2015, 8, 192.
390
5 H. Ma, H. Liu, L. Zhang, M. Yang, B. Fu and H. Liu, No el insigh in o he ela ionship
391
be ween o ganic subs a e composi ion and ola ile a y acids dis ibu ion in acidogenic co-
392
e men a ion, Bio echnol. Bio uels, 2017, 10, 137.
393
6 W. Fang, X. Zhang, P. Zhang, J. Wan, H. Guo, D. S. M. Ghasimi, X. C. Mo e a and T. Zhang,
394
O e iew o key ope a ion ac o s and s a egies o imp o ing e men a i e ola ile a y
395
acid p oduc ion and p oduc egula ion om sewage sludge, J. En i on. Sci., 2020, 87, 93–
396
111.
397
7 P. Keh ein, M. an Loosd ech , P. Osseweije , M. Ga í, J. Dewul and J. Posada, A c i ical
398
e iew o esou ce eco e y om municipal was ewa e ea men plan s – ma ke supply
399
po en ials, echnologies and bo lenecks, En i on. Sci. Wa e Res. Technol., 2020, 6, 877–
400
910.
401
8 U. Jayak ishnan, D. Deka and G. Das, Enhancing he ola ile a y acid p oduc ion om ag o-
402
indus ial was e s eams h ough sludge p e ea men , En i on. Sci. Wa e Res. Technol.,
403
2019, 5, 334–345.
404
9 A. M. B eu e, K. A. Mooijman and J. G. an Andel, P o ein deg ada ion in anae obic
405
diges ion: in luence o ola ile a y acids and ca bohyd a es on hyd olysis and acidogenic
406
e men a ion o gela in, Appl. Mic obiol. Bio echnol., 1986, 24, 426–431.
407
10 G. Tommaso, R. Ribei o, M. B. A. Va esche, M. Zaia and E. Fo es i, In luence o mul iple
408
subs a es on anae obic p o ein deg ada ion in a packed-bed bio eac o , Wa e Sci.
409
Technol., 2003, 48, 23–31.
410
11 R. González-Cabalei o, J. M. Lema and J. Rod íguez, Me abolic Ene gy-Based Modelling
411
Explains P oduc Yielding in Anae obic Mixed Cul u e Fe men a ions, PLoS One, 2015, 10, 1–
412
17.
413
12 M. F. Temudo, R. Klee ebezem and M. C. M. an Loosd ech , In luence o he pH on (open)
414
mixed cul u e e men a ion o glucose: A chemos a s udy, Bio echnol. Bioeng., 2007, 98,
415
69.
416
13 A. R. Gou eia, E. B. F ei as, C. F. Galinha, G. Ca alho, A. F. Duque and M. A. M. Reis,
417
Dynamic change o pH in acidogenic e men a ion o cheese whey owa ds
418
polyhyd oxyalkanoa es p oduc ion: Impac on pe o mance and mic obial popula ion, N.
419
Bio echnol., 2017, 37, 108–116.
420
14 R. Be ilacqua, A. Reguei a, M. Mau icio-Iglesias, J. M. Lema and M. Ca balla, P o ein
421
composi ion de e mines he p e e en ial consump ion o amino acids du ing anae obic
422
mixed-cul u e e men a ion, Wa e Res., 2020, 183, 115958.
423
15 R. Be ilacqua, A. Reguei a, M. Mau icio-Iglesias, J. M. Lema and M. Ca balla, S ee ing he
424
con e sion o p o ein esidues o ola ile a y acids by adjus ing pH, Bio esou . Technol.,
425
2021, 320, 124315.
426
16 A. Reguei a, J. M. Lema, M. Ca balla and M. Mau icio-Iglesias, Me abolic modeling o
427
p edic ing VFA p oduc ion om p o ein- ich subs a es by mixed-cul u e e men a ion,
428
Bio echnol. Bioeng., 2020, 117, 73–84.
429
17 A. Zhou, Z. Guo, C. Yang, F. Kong, W. Liu and A. Wang, Vola ile a y acids p oduc i i y by
430
anae obic co-diges ing was e ac i a ed sludge and co n s aw: E ec o eeds ock
431
p opo ion, J. Bio echnol., 2013, 168, 234–239.
432
18 R. Be ilacqua, A. Reguei a, M. Mau icio-Iglesias, J. M. Lema and M. Ca balla, Unde s anding
433
he e ec o ace elemen s supplemen a ion on ola ile a y acids p oduc ion om
434
p o eins, J. En i on. Chem. Eng., , DOI:10.1016/j.jece.2021.105934.
435
19 D. P. R. He lemann, M. Lab enz, K. Jü gens, S. Be ilsson, J. J. Waniek and A. F. Ande sson,
436
T ansi ions in bac e ial communi ies along he 2000 km salini y g adien o he Bal ic Sea,
437
ISME J., 2011, 5, 1571–1579.
438
20 C. Quas , E. P uesse, P. Yilmaz, J. Ge ken, T. Schwee , P. Ya za, J. Peplies and F. O. Glöckne ,
439
The SILVA ibosomal RNA gene da abase p ojec : imp o ed da a p ocessing and web-based
440
ools, Nucleic Acids Res., 2013, 41, D590–D596.
441
21 G. Tchobanoglous, Lau ie M. F anklin, E. Bu on, H. D. S ensel. Me cal & Eddy, Was ewa e
442
enginee ing : ea men and euse, Fou h edi ion / e ised by Geo ge Tchobanoglous,
443
F anklin L. Bu on, H. Da id S ensel. Bos on : McG aw-Hill, [2003] ©2003, 2014.
444
22 A. Aguila , C. Casas, J. La uen e and J. M. Lema, Kine ic modelling o isome iza ion and
445
anae obic deg ada ion o n- and i-bu y a e, J. Fe men . Bioeng., 1990, 69, 261–264.
446
23 A. Reguei a, R. Be ilacqua, J. M. Lema, M. Ca balla and M. Mau icio-Iglesias, A me abolic
447
model o a ge ed ola ile a y acids p oduc ion by co e men a ion o ca bohyd a es and
448
p o eins, Bio esou . Technol., 2020, 298, 122535.
449
24 A. Reguei a, J. L. Rombou s, S. A. Wahl, M. Mau icio-Iglesias, J. M. Lema and R.
450
Klee ebezem, Resou ce alloca ion explains lac ic acid p oduc ion in mixed-cul u e anae obic
451
e men a ions, Bio echnol. Bioeng., 2021, 118, 745–758.
452
25 L. T. Angenen , H. Rich e , W. Buckel, C. M. Spi i o, K. J. J. S einbusch, C. M. Plugge, D. P. B.
453
T. B. S ik, T. I. M. G oo schol en, C. J. N. Buisman and H. V. M. Hamele s, Chain Elonga ion
454
wi h Reac o Mic obiomes: Open-Cul u e Bio echnology o P oduce Biochemicals, En i on.
455
Sci. Technol., 2016, 50, 2796–2810.
456
26 S. Liang and C. Wan, Ca boxylic acid p oduc ion om B ewe ’s spen g ain ia mixed cul u e
457
e men a ion, Bio esou . Technol., 2015, 182, 179–183.
458
27 L. Le én, A. R. B. E iksson and A. Schnü e , E ec o p ocess empe a u e on bac e ial and
459