Chain elongation may occur in protein mixed-culture fermentation without supplementing electron donor compounds

Jou nal o En i onmen al Chemical Enginee ing 10 (2022) 106943 A ailable online 3 Decembe 2021 2213-3437/© 2021 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). 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 Jou nal o En i onmen al Chemical Enginee ing jou nal homepage: www.else ie .com/loca e/jece h ps://doi.o g/10.1016/j.jece.2021.106943 Recei ed 7 Oc obe 2021; Recei ed in e ised o m 22 No embe 2021; Accep ed 1 Decembe 2021 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. 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