Importance and implication of respiration Quotient (RQ): a bacterial experimental assay with pseudomonia náutica and Vibrio natrigiensis

Impo ance and implica ions o espi a ion quo ien (RQ): Bac e ial expe imen s wi h Pseudomonas nau ica and Vib io na iegens Vanesa Rome o Ku zne Más e en Oceanog a ía Uni e sidad de Las Palmas de G an Cana ia Di ec o es: Ted Packa d y May Gómez Diciemb e 2013 1 Impo ance and implica ions o espi a ion quo ien (RQ): Bac e ial expe imen s wi h Pseudomonas nau ica and Vib io na iegens Vanesa Rome o-Ku zne *, Ted T. Packa d, May Gómez Plank on Ecophysiology G oup, Ins i u o de Oceanog a ía y Cambio Global. Uni e sidad de Las Palmas de G an Cana ia, Spain *Co esponding au ho . E-mail add ess: anesa. o[email p o ec ed] 2 Abs ac Respi a o y me abolism in wo di e en physiological s a es o ace a e and py u a e g own cul u es o Pseudomonas nau ica and Vib io na iegens we e compa ed. He e we analyze 35 hou s expe imen s and 520 hou s expe imen s in which ime-cou ses o p o ein, py u a e, ace a e, espi a o y CO2 p oduc ion (RCO2), espi a o y O2 consump ion (RO2), isoci a e dehyd ogenase (IDH) ac i i y, and po en ial espi a ion we e measu ed. The cul u es we e moni o ed h ough hei exponen ial g ow h, hei s eady s a e, and hei senescence phases. Respi a o y quo ien s (RQs) we e calcula ed om he a io o he espi a ion a es (RCO2/RO2). Such RQs a e widely used in ocean ecosys ems models, calcula ions o ca bon lux, and in e alua ions o he au o ophic-he e o ophic na u e o he ocean, i s me abolic balance. In all cul u es, ega dless o bac e ial species and ca bon sou ce, he RQ ended o ise nea ly an o de o magni ude om alues below 1 du ing nu ien su iciency o alues close o 10 du ing nu ien de iciency. The espi a ion a es du ing he g ow h pe iod pa alleled he biomass inc ease bu a e he nu ien s we e exhaus ed he espi a ion a es ell. Th ough his same ansi ion pe iod he IDH ac i i y and he po en ial espi a ion (φ) e mained ela i ely high o i s 10 hou s o nu ien dep i a ion and hen ell slowly, along wi h he biomass, as he nu ien dep i a ion con inued. Du ing his s a a ion pe iod he biomass speci ic IDH and φ dec eased. This inding challenges he idea ha IDH and he espi a o y elec on anspo sys ems (ETS) a e cons i u i e and can be used o biomass p oxies. We conclude ha he physiological s a e o he bac e ia a ec s he RQ. These esul s a gue ha many ecosys ems models, oceanog aphic calcula ions o ca bon lux, and e alua ions o he ocean’s me abolic balance need o be econside ed in ligh o his newly disco e ed RQ a iabili y. Keywo ds: O2 consump ion, CO2 p oduc ion, IDH, po en ial espi a ion (Φ), g ow h 3 1. In oduc ion Respi a ion (R) in he ocean has many ace s. In he deep wa e s o he Black Sea i p oduces hyd ogen sul ide, in he anoxic in e media e wa e s o he Pe u coas i p oduced ni i e, ni ous oxide and ni ogen gas, in mos o he wo ld’s ocean i simply consumes oxygen and p oduces wa e . Howe e , in all cases, h oughou he ocean espi a ion is ubiqui ous and p oduces CO2. He e, o he oxic pa s o he wo ld ocean, we ocus on he ela ionship be ween CO2 p oduc ion and he concu en consump ion o O2 (R) du ing ae obic espi a ion. R oughly balances oxygenic pho osyn hesis, p oduc ion (P), in he ma ine ca bon cycle, bu un il he Winkle O2 echnique was modi ied (B yan e al., 1976) R was no well esea ched because i is so di icul o measu e. Wi h his imp o ed echnology, oceanog aphe s began o no e an excess o R o e P in some pa s o he ocean gi ing ise o he cu en con o e sy abou he me abolic s a e o he ocean (Ducklow and Doney, 2013). The quanda y is whe he he oligo ophic ocean is au o ophic o he e o ophic. Williams e al. (2012) epo ed in si u obse a ions whe e he esul s using na u al seawa e iso ope composi ion clea ly showed an au o ophic s a e whe eby esul s om modi ied Winkle analysis on incuba ed seawa e samples showed a he e o ophic one. One eason o his disc epancy could be he assumed alue o he espi a ion quo ien (RQ) used o con e R om he Winkle analysis in o espi a o y CO2 p oduc ion (RCO2). Wi hou his con e sion espi a ion canno be compa ed o pho osyn hesis o plank on p oduc i i y. Consequen ly, a deep unde s anding and ca e ul measu emen o RQ is essen ial o p og ess in esol ing, no only his oceanog aphic quanda y, bu also o making many o he ma ine ecosys ems calcula ions, including ca bon lux (Packa d and Ch is ensen, 2004; S einbe g e al., 2008; Packa d and Gómez, 2013). He e, we epo measu emen s o RQ in wo species o ma ine bac e ia g owing sepa a ely on wo di e en ca bon sou ces, ace a e and py u a e. Fu he mo e, we show ha RQ changes wi h he s a a ion le el, he ca bon sou ce, and he bac e ial species. Via ae obic espi a ion ma ine o ganisms ob ain he ene gy o li e om a wide ange o compounds ha a e educed in di e en , bu well coo dina ed biochemical pa hways. Two o hese key pa hways a e he K ebs o ica boxylic acid (TCA) cycle and he espi a o y elec on anspo sys em (ETS). The TCA cycle accep s py u a e om glycolysis and ace a e om β- oxida ion o a y acids, condenses hem in sepa a e eac ions wi h oxaloace a e, and oxidizes 4 he esul ing ci ic acids o di e en ica boxylic acids such as glu amic, succinic, uma ic, and malic acid. This oxida ion p ocess yields h ee CO2 om py u a e, bu only wo om ace a e. Concu en ly py u a e oxida ion by he TCA yields 10 educing equi alen s (10H+) o he ETS while ace a e oxida ion esul s in only 8H+. These educing equi alen s en e he ETS ia complex I and complex II and a e apidly ans e ed o ubiquinone (Q), educing i o ubiquinol (QH2). In he case o ace a e, he combined s oichiome y o his sequence o eac ions, glycolysis, TCA cycle and he ETS esul s in he p oduc ion o wo molecules o CO2 and he consump ion o wo O2 molecules. The a io be ween he p oduced CO2 and he consumed O2, he RQ , would be 1 (RQ= Δ CO 2/- Δ O2= 1). In he case o py u a e, he combined s oichiome y o his sequence o eac ions, glycolysis, TCA cycle and he ETS esul s in he p oduc ion o h ee molecules o CO2 and he consump ion o 2.5 O2 molecules. The a io be ween he p oduced CO2 and he consumed O2, he RQ, would be 1.2. The majo i y o s udies o espi a ion in he aqua ic ecosys ems a e based on assumed RQ alues anging om 0.7 o 1.2 (Be gg en e al., 2012), howe e , depending on he nu i ion, he RQ can a y o e a b oade ange. Fo example, nu i ion based on oxalic acid can esul in an RQ o 4. To calcula e a ue RQ alue i is impo an o accu a ely calcula e CO2 p oduc ion. In he pas his was di icul and expensi e and so was a ely done. Now, wi h he de elopmen o he CO2 op ode, RCO2 (Mills e al., 1992) measu emen s a e mo e easible (Be gg en e al., 2012). In Oceanog aphy he e a e ela i ely ew measu emen s o RQ (O ia e al., 1986; Robinson e al., 2002). As a esul , RQs a e calcula ed heo e ically by analyzing he s oichiome y o he comple e oxida ion o subs a es (Takahashi e al., 1985; Ande son, 1995; and Hedges e al., 2002). In his app oach, issues o whole o ganisms oxidizing pu e ca bohyd a e ha e an RQ o 1.0, hose oxidizing a , an RQ o 0.7, and hose oxidizing p o ein, an RQ o abou 0.8 (Can a ow and Schepa z, 1967; Guy on, 1971; Hoa , 1975; S anie and Fo sling, 1990). Values ou side his ange (i.e. 0.7–1.0) a e ela i ely a ely encoun e ed, al hough RQs below 0.7 can be associa ed wi h gluconeogenesis, and ansien inc eases abo e 1.0 can esul om he con e sion o ca bohyd a e in o a (Can a ow and Schepa z, 1967). This a iabili y in RQ can indica e undamen al shi s in bac e ial physiology and ca bon 5 consump ion ha may occu along he en i onmen al g adien s and ha canno be deduced om o he measu emen s (Be gg en e al., 2012). He e we show ha RQ can ange highe he ange epo ed abo e. To in es iga e his wide ange, we examine ime-cou ses o he physiological espi a ion a es (RO2 and RCO2) and ac i i ies o he enzyme, isoci a e dehyd ogenase (IDH) and he espi a o y ETS (po en ial espi a ion, φ) in ace a e and py u a e-g own ma ine bac e ia, Pseudomona nau ica and Vib io na iegens cul u es. 2. Ma e ial and Me hods 2.1 Expe imen al design. To in es iga e he RQ in di e en bac e ial g ow h s ages, ime-cou se expe imen s we e un on ba ch cul u es, main ained on py u a e o ace a e as desc ibed in Be dale e al. (1995) and Packa d e al. (1996b). Sho e m expe imen s we e un o maximum 35 hou s, long e m ones we e un o 2-3 weeks. The bac e ia cul u es we e g own in 25 co on-plugged 500 ml E lenmeye lasks con aining 100 ml o media. The op ical densi y a 550 nm (OD550) had an ini ial alue a e inocula ion o 0,1 abso bance uni s. A 2 h in e als, 2 lasks we e chosen andomly, 25 ml o cul u e we e ans e ed o he Oxymax ( espi ome e ) lasks, and he espi a ion was measu ed. Then cul u e samples we e aken in duplica e o OD550 ( u bidi y measu ed as abso bance a 550 nm), p o ein, py u a e, ace a e, RCO2, RO2, IDH ac i i y, and po en ial espi a ion (φ) as he cul u es g ew exponen ially, eached s eady s a e, and passed in o senescence. 2.2 Bac e ial cul u es. Vib io na iegens (ATCC 33788) and Pseudomona nau ica (S ain 617 om D . P. Bonin, Uni e si é de la Médi e anée, Ma seille, F ance) we e used o hese expe imen s. The cul u ing has been desc ibed by Be dale e al. (1995) and Roy e al. (1999). Be o e an expe imen , he bac e ia had been adap ed o a minimum o 15 gene a ions o he expe imen al media (py u a e o ace a e). To inocula e he expe imen s exponen ial o 6 ea ly s a iona y phase we e used. Cul u es we e con inuously agi a ed on an o bi al shake a 100 pm a 22ᴼC and g ow h was ollowed spec opho ome ically a 550 nm (OD550). 2.3 Cul u e media. Pseudomona nau ica was cul u ed acco ding o Packa d e al. (1996a) and he medium o Vib io na iegens was de eloped om he media o Ni en e al. (1977), Baumann and Baumann (1981), King and Be man (1984) and Nissen e al. (1987). La e , he op imal condi ions o g ow h o Vib io na iegens we e es ablished a e he expe imen s wi h: 400 mM NaCl, 10 mM MgSO4 ◦7H20, 10 mM CaCl2 ◦2H20, 10 mM KCl, 25 mM NH4Cl, 0.33 mM phospha e bu e , 0.01 mM FeSO4◦7H20, and 30 mM sodium ace a e o 20 mM py u a e. Reagen s o he cul u e media we e ob ained om Sigma-Ald ich Co. All componen s we e dissol ed in 0.22 µm il e ed deionized wa e , excep ing FeSO4◦7 H20 and phospha e bu e and pH was adjus ed o 7.5 wi h 1 N NaOH. To emo e pa icles he solu ion was il e ed h ough a GF/F glass ibe il e . La e he solu ion was au ocla ed o 45 min a 121°C. Then, o a oid p ecipi a ion du ing he au ocla ing, he phospha e bu e (0.67 M, pH 7.5) and he i on sulpha e solu ion (FeSO4◦7H2O, 0.1 mM) we e p epa ed sepa a ely. The PO4 bu e was s e ilized by au ocla ing and FeSO4 solu ion was il e ed h ough 0.22 µm ac odiscs. Finally, bo h solu ions we e kep ozen and we e added o he cul u e medium be o e use. 2.4 P o ein measu emen s. The bac e ial pelle s we e de os ed, well mixed, and analyzed o p o ein in aliquo s o 0.5 ml by he Low y Me hod (Low y e al., 1951) acco ding o Be dale e al. (1995). I he abso bance a 750 nm o he samples exceeds 0.4 he homogena es we e dilu ed and analyzed again. Fo s anda diza ion, duplica e measu emen s o Bo ine Se um Albumin (BSA) om Sigma Chemical Company we e used. 2.5 Respi a ion measu emen s. Mic o-Oxymax espi ome e (Columbus Ins umen s In e na ional Co po a ion, Columbus, OH, USA) measu ed changes in he concen a ion o CO2 and O2, in he head space o he expe imen al lasks wi h an oxygen de ec o based on he p inciple o a PbO2 7 uel cell. This ins umen has an in a ed de ec o (sensi i e o he 2000 µm abso p ion peak o CO2), a mul iple sample chambe ( o up o 20 channels), a e e ence chambe and a compu e ized da a acquisi ion and analysis sys em. Mic o-Oxymax wo ks in ae obic condi ions because he appa a us can change he ai in he head space i he O2 le el ell below 19,3 %. Respi a ion is gi en as µmol O2 /min /l. A no mal measu emen akes abou 30 min. The calib a ion o he oxygen de ec o was done wi h high p ecision gas s anda ds. The espi ome y o all he o iginal measu emen s was desc ibed by Be dale e al. (1995). 2.6 Biochemical pa ame e s. Duplica e samples we e aken o ace a e and/o py u a e om e e y lask. La e 5 o 10 ml o cul u e, depending o he le el o biomass, was cen i uged a 10000 x g o a 4°C o 15 min. The supe na an luid was collec ed in an acid- insed Co ex ube and hen s o ed in liquid ni ogen o ace a e o py u a e analysis (Ahmed e al., 1976). High Pe o mance Liquid Ch oma og aphy (HPLC) was used by J-P Gagné as desc ibed by Be dale e al. (1995) and Packa d e al. (1996a), o sepa a e and quan i y ace a e and py u a e in hei acid. 2.7 In i o IDH ac i i y (AIDH). IDH assay was de e mina e spec opho ome ically a 340 nm ollowing he NADPH p oduc ion (Ree es e al., 1971, 1972; Holms & Benne , 1971; Be dale e al., 1995). This is he eac ion e ealing he CO2 p oduced om he isoci a e oxida ion. Isoci a e + NADP+ ↔ α-ke oglu a a e + NADPH + CO2; Resul s a e gi en as µm CO2 (min)-1 (li e o cul u e)-1. The eac ion was s a ed by addi ion o NADP+. The IDH ac i i y is calcula ed om he eg ession line o OD340 e sus ime. NADPH is used as he s anda d o con e ing OD340 o CO2 (µmol) because, om he equa ion abo e, NADPH p oduc ion is s ochiome ically equal o CO2 p oduc ion. 8 2.8 Po encial espi a ion (φ). Depending on he biomass, samples om 5 o 10 ml o cul u e we e cen i uged a 10,000 ×g o 15min a 4 °C and he pelle s we e s o ed in liquid ni ogen. La e hey we e esuspended a 0 o 4 °C in 2 ml o he homogenizing bu e , and measu ed kine ically o ETS ac i i y wi h a modi ica ion o he Packa d and Williams (1981) as desc ibed in Packa d and Ch is ensen (2004). Final esul s a e con e ed om ETS uni s o μmol e−min−1 l−1 o po en ial espi a ion uni s in μmol O2 min−1 l−1 o cul u e di iding by 4 (4e−+4H++O2→2H2O). 2.9 S a is ical analysis Da a we e analyzed using he p og am R om he R De elopmen Co e Team 2010 (R Founda ion o S a is ical Compu ing, Vienna, Aus ia). Rela ionships be ween RCO2/RO2 and IDH/Φ in di e en ime scales (sho and long- e m expe imen s) we e ob ained om he eg ession equa ions, using con idence limi s o 95% and he Pea son co ela ion coe icien s. ANCOVA we e applied o de e mine s a is ical di e ences be ween slopes and o dina es in he eg ession lines. No mali y o esiduals was con i med by Shapi o-Wilk es . 3. Resul s 3.1 Time cou se o espi a ion, enzyme ac i i y, biomass, and ca bon sou ce. Two ypes o expe imen s we e analyzed: Sho e m (Day) expe imen s ha could ex end o 35 hou s and long e m (2-3 week) expe imen s ha anged be ween 330 and 520 hou s. In hose expe imen s we wo k wi h wo bac e ia species, Ps. nau ica and V. na iegens g owing on wo di e en subs a es (ace a e and py u a e). In Figu e 1 measu emen s o subs a e (ca bon sou ce), p o ein, enzyme ac i i y (IDH and φ), physiological espi a ion (RCO2 and RO2) a e shown. On he o he hand we ha e he same measu emen s o long e m expe imen s in Figu e 2. Measu emen s a e ep esen ed on he same scale, so ha con e sion ac o s ha e o be applied. As wi h many o he esul s, he g aphics a e plo ed wi h di e en scales o ocus on he beha io o he a iables o e ime. 15 pnpy da a; he equa ion is: RCO2= 0,7295 RO2 -0,5969, (R2=0,9882, n=10). The slope o he equa ion (0,7295) is he mean RQ alue. Fo he ace a e-based cul u es, he da a below he line a e nu ien su icien ; he da a abo e he line a e nu ien deple ed, equa ions o s a a ion condi ions a e shown in Table 1. F om he equa ions in Table 1, i is clea ha he s a ed ace a e-based cul u es ha e di e en RQs (slopes) han he s a ed py u a e- based cul u es. The di e ence be ween he wo s a ed ace a e-based cul u es is mo e sub le, bu ANCOVA analysis e ealed ha a p<0.05, he slopes, and he RQs we e di e en excep o bac e ia g own on py u a e in s a ed condi ions. Table 1. Reg ession lines and 2 o sho e m s a a ion condi ions(*) shown in Fig.6. pnac= Ps. nau ica on ace a e, nac= V. na iegens on ace a e, pnpy= Pseudomnas nau ica on py u a e, npy= V. na iegens on py u a e. S ain Equa ion 2 pnac* y= 5,2692x + 3,4891 0,92 nac* y= 2,0564x - 0,4225 0,95 pnpy* y= 0,5675x + 1,95 0,98 npy* y= 0,5737x + 1,7177 0,98 Long e m expe imen In Figu e 2 we see ha all physiological espi a o y ac i i y (RCO2 and RO2) ends wi hin he i s 100 hou s o he expe imen . We can see di e en shi s in he da a as nu ien limi a ion se s in. Ps. nau ica in ace a e (Figu e 2a) has highe alues o RO2 un il s a a ion begins. Then, a he momen o s a a ion, RO2 doubles, om 25 o 50µmol O2/min/l, bu a e his ise, he RCO2 alues a e la ge han RO2. In he case o V. na iegens in ace a e all RCO2 and RO2 alues a e almos he same and only he RCO2 ises a he momen ha nu ien limi a ion begins. Ps. nau ica in py u a e is he only long e m expe imen whe e a jump in he RCO2 and RO2 co-occu s a s a a ion poin (Figu e 2c). V. na iegens on py u a e jumps be o e s a a ion begins (Figu e 2 d). Expe imen s on py u a e show highe RCO2 alues han RO2 du ing he expe imen s (Figu e 2 c and d). 16 All RCO2/RO2 o he long e m expe imen s a e shown in Figu e 7, we see a good co ela ion in all cases excep in V. na iegens on ace a e (Table 2). The slope o hese unc ions a e he gene al RQ, his is a quan i a i e way o es ima e RQ. Howe e , i mus be emembe ed ha each poin ep esen s a physiological measu emen made a di e en ime in he li e o he cul u e. ANCOVA analysis (Table 2) be ween cul u es which we e ed wi h he same nu ien (i.e. Ps. nau ica o V. na iegens on ace a e) shows ha he slopes a e no signi ican di e en (p>0.05). On he o he hand, o he same s ain, bu o di e en subs a es (i.e. Ps. nau ica on ace a e o py u a e) he slopes appea o be signi ican ly di e en (p<0.05). Compa ing slopes (RQs) in Fig. 7 du ing s a a ion condi ions inds all o hem o be signi ican ly di e en (p<0.05) (Table 2). Figu e 7. Long e m expe imen s. pnac= Ps. nau ica in ace a e, pnpy= Ps. nau ica in py u a e, nac= V. na iegens in ace a e, npy= V. na iegens in py u a e. No e ha he ca bon sou ces dic a e he pa allelism o he cu es, no he bac e ial species. In addi ion, no e ha he RQ (slope) is highe o he h ee-ca bon subs a e (py u a e) han o he wo ca bon one (ace a e). 17 Table 2. Reg ession lines and 2 o long e m expe imen s shown in Fig. 7. No e ha pnac = Ps. nau ica on ace a e, nac = V. na iegens on ace a e, pnpy = Pseudomnas nau ica on py u a e, npy = V. na iegens on py u a e. *S a a ion condi ions in long e m expe imen s. S ain Equa ion 2 pnac y= 0,3681x + 1,5454 0,93 pnac* y= 4,2758x – 0.1633 0,76 nac y= 0,3152x + 0,5364 0,68 nac* y= 0,8582x + 0,1265 0,97 pnpy y= 1,204x + 0,3718 0,99 pnpy* y= 1,204x + 0,3665 0,99 npy y= 1,0624x + 0,0701 0,99 npy* y= 1,0541x + 0,1333 0,99 3.4 IDH and Φ no malized by biomass (p o ein) Sho e m expe imen s Time cou ses o p o ein-speci ic enzyma ic ac i i y a e shown in Figu e 8. Enzyma ic ac i i y is p esen e en du ing s a a ion. Φ in Ps. nau ica on ace a e (Figu e 8a) is highe han he co esponding IDH alues. IDH dec eases be o e s a a ion begins, bu Φ ho lds almos cons an . Again, IDH ises in ace a e–based cul u es o V. na iegens be o e nu ien limi a ion begins (Figu e 8b) as we saw in Ps. nau ica on ace a e (Figu e 8a). In his case, alues o IDH and Φ a e mo e o less simila . Ps. nau ica expe imen s display a be e co ela ion in ace a e and in py u a e o IDH and Φ. In Ps. nau ica on py u a e, we see again highe alues o Φ. In he case o Ps. nau ica in py u a e (Fig. 8c) ha e he mos simila alues o IDH and Φ and b e o e s a a ion begins IDH ises and Φ ac i i y alls. V. na iegens on py u a e displays an IDH ise un il s a a ion begins and hen a slow d op (Figu e 8 d), Φ ac i i y ises oo and has a mo e d ama ic d op in nu ien limi a ion condi ions. In all cases excep V. na iegens in py u a e (Fig 8 d) we see IDH and Φ ac i i y 18 ises be o e nu ien limi a ion begins. Acco ding o he ANCOVA analysis he di e ence be ween IDH and φ is signi ican (p<0.05). Figu e 8. Time cou ses in he sho - e m expe imen s o enzyme ac i i ies no malized by biomass (IDH/p o ein an d φ/p o ein). A) Ps. nau ica in ace a e , b) V. na iegens in ace a e. c) Ps. nau ica in py u a e d) V. na iegens in py u a e. As wi h he o he ime- cou se Figu es, he nu ien -su icien condi ion is on he le while he nu ien -de icien condi ion is on he igh . No e ha , i hese enzymes we e good p oxies o p o ein biomass o i hey we e cons i uen pa s o he bac e ial cell, bo h IDH/p o ein and φ/p o ein would end o be s aigh lines, independen o he cul u e age. 19 Long e m expe imen s We see ha enzyma ic p ocesses in he long e m expe imen s (Figu e 9) a y less han in he sho e m expe imen s (Figu e 8). In all expe imen s we see enzyma ic ac i i y e en du ing nu ien limi a ion. Almos all IDH and Φ alues a e simila . In Ps. nau ica on ace a e (Figu e a) we see a li le IDH ump a e nu ien limi a on is eached and hen i dec eases un l 350 h whe e i s a s o inc ease wi h Φ. V. na iegens on ace a e jumps in IDH and Φ a e nu ien limi a ion and hen he wo enzyma ic ac i i ies dec ease oge he o le els lowe han 0,1 µmol CO2/min/l and µmol O2/min/l (Figu e 9 b). In expe imen s on py u a e we obse e ha Φ ac i i y ises a e nu ien limi a ion condi ions and ha IDH ac i i y d ops (Figu e 9 b and d). In Ps. nau ica on py u a e Φ and IDH ise a 250 h and la e hey d op oge he o lowe alues (Figu e 9 c). V. na iegens on py u a e is he only expe imen whe e Φ ac i i y ises om 0 µmol O2/min/l (be o e 100h o expe imen is eached) o 0,45 µmol O2/min/l ( 200h) (Figu e 9 d). He e again compa ison o he slopes o IDH and φ by ANCOVA analysis e eals a signi ican di e en (p<0.05). Figu e 9. Time cou ses in he long- e m expe imen s o IDH/p o ein and φ/p o ein. A) Ps. nau ica in ace a e, b) V. na iegens in ace a e, c) Ps. nau ica in py u a e, and d) V. 20 na iegens in py u a e . No e ha he nu ien -su icien condi ion e mina es on he le when a p ojec ion o he g een line in e sec s wi h he abscissa. Nu ien -de iciency cha ac e izes mos o he cul u e’s li e. No e ha , bo h IDH/p o ein and φ/p o ein end o dec ease wi h ime as he bac e ial cul u es s a e. 4. Discussion 4.1 RQ a iabili y and ca bon limi a ion. In his s udy we obse ed high a iabili y in RQ alues. In esh wa e lakes and ponds o Quebec, Be gg en e al. (2012) ound RQs a ying om 0.25-2.26 and a gued ha he common assump ions o 0.8-1.0 RQ alues a e no jus i ied by he li e a u e. Fu he mo e, hey a gue ha he use o a cons an RQ dep esses es ima ions o he me abolic balance be ween espi a ion and pho osyn hesis, i.e., P/R a io. They also a gued ha high RQs could be physiologically explained by biogeochemical pa hways o DOC deg ada ion. Howe e , i is no ob ious how he communi y-le el RQ could ise o alues as high as 10 in esponse o cell biochemis y as ou s udy shows (Figu e 1 a). S a a ion, in he o m o ca bon-sou ce dep i a ion, had a s ong impac and be o e ou s udy, his a iable and i s impac on RQ had no been well s udied. Fo example, Figu e 5 a and b shows how RQ ises du ing s a a ion hough a combina ion o a sligh RCO2 ise and a d ama ic d op in RO2. Again In Fig. 6, he RQ shi s up om nu ien su iciency o nu ien limi a ion. Lowe RQ le els occu in he i s phase o he expe imen s when he bac e ia we e well ed, la e he RQ ises as ca bon s a a ion se s in. 4.2. Ca bon sou ce and RQ In long e m expe imen s compa ing RCO2/RCO2 a ios we ound ou ha wo di e en bac e ia g own on he same subs a e a e ela ed, ANCOVA- es showed ha in hese cases he e is no signi ican di e ence be ween Ps. nau ica and V. na iegens ela ed o hei slopes (Table 2). I is clea om Fig. 3 ha Ps. nau ica and V. na iegens cul u es g own on ace a e o py u a e RQ´s a e comple ely di e en . Ace a e and py u a e a e p oduc s o lipid and 21 ca bohyd a e me abolism, espec i ely; he esul s sugges ha pai ed measu emen s o RCO2 and RO2 could se e as a diagnos ic o de ec ing di e en ypes o me abolism in si u s udies (Roy e al., 1999). High RQ would indica e ca bohyd a e me abolism which indica es a s ong p esence o ca bohyd a es, o he wise low RQ would indica e lipid me abolism. Ou esul s sugges ed ha , in Ps. nau ica cul u es, he RQ is always lowe han 1.0 be o e ace a e o py u a e is exhaus ed. Howe e , Ps. nau ica in he ace a e-g own cul u e, when ace a e was exhaus ed, he RQ ose abo e 1.0 and eached a alue highe han 10. Tha inc ease o RQ was caused by a as educ ion o RO2, a e ace a e exhaus ion when RCO2 was s ill high (Figu e 5 a). Again, he ime a ia ions o RQ while he cul u es passed om a well- nou ished s a e o a nu ien -s a ed s a e a gue o a econside a ion o he p ac ice o assuming a cons an RQ o 1.0. In igu e 7 we deduce om he slopes ha ace a e gi es a simila RQ o he wo bac e ia and like on py u a e g own cul u es, he same was obse ed om Be dale e al. 1995. 4.3 RQ and biochemical pa hways. The RQ is heo e ically egula ed by biochemical pa hways o me abolism, in his way K ebs-cycle CO2 p oduc ion is he esul o h ee enzymes: isoci a e dehyd ogenase (IDH, EC 1.1.1.42), α-ke oglu a a e dehyd ogenase (α -KGDH, EC 1.2.7.3), and py u a e dehyd ogenase (PDH, EC 1.2.2.2) (Walsch and Koshland 1984; Holms 1986 a,b; Packa d e al. 1996 a). The K ebs cycle is nea ly uni e sal in mic obes. In ou esul s we see ha enzyma ic ac i i y s ill wo ks in s a a ion condi ions (Figu e 8). In na u e mic o-o ganism a e able o eac quickly o en i onmen al changes, adap ing hei me abolic p o iles o o ganic inpu s (Ma inez e al., 1996; Mud yk and Donde ski, 1997; Sala and Gude, 2004; Mud yk and Sko czewski, 2006) in his s udy we sugges an adap a ion o enzyma ic ac i i y o he physiological s a es o bac e ia. Ca uso e al. (2013) poin ed ou ha he e o ophic me abolism and Bac e ioplanc on espi a ion shows di e en seasonal and in e annual cycles. Va ia ions in he mic obial ac i i y le els a e equen along ophic g adien s (Hoppe e al., 1998; Ca uso e al., 2005; Ch ós and Siuda, 2006; Williams and Jochem, 2006; Cunha e al., 2010). Pe haps conside ing hese cycles we can unde s and how enzyma ic ac i i y o mic obes is able o adap in o di e en condi ions 22 in an in- i o expe imen . Also (Be gg en e al., 2012) a gues ha na u al Bac e ioplanc on u ilize complex subs a e mix u es and he cells hemsel es ha e a wide ange o physiological s a es o i also can be explained ha high enzyma ic ac i i y a es can egene a e nu ien s om dissol ed subs a es, an making a ailable C, N, P which a e needed o ul ill mic obial me abolic demand (Be dale e al., 1995). 4.4. IDH/P and RQ a iabili y Ace a e, a 2 ca bon molecule, is ans o med in o ace yl-CoA and loses bo h ca bons when cycled h ough he TCA. Du ing g ow h on ace a e IDH is pa ially inac i a ed (El-Mansi e al., 1985) in o de o allow he ope a ion o he glyoxyla e cycle. While ace a e se es o ep ess IDH ac i i y, py u a e (a 3 ca bon molecule) se es o ac i a e i (Holms e al., 1971), so IDH ac i i y on ace a e-based cul u es should be lowe han on py u a e cul u es, and lowe IDH means lowe CO2 p oduc ion and lowe RQ bu in ou esul s we ha e no obse ed his beha io (Figu e 3a and 8a and igu e 1 b and d) ins ead o ha we see ha IDH eaches highe alues o expe imen s o Vib io na iegens (Figu e 8b and d). In long e m expe imen s we do no see his beha io , alues o IDH a e mo e o less cons an . IDH is he esponsible enzyme o he low o ca bon h ough he K ebs cycle o p oduce ene gy and CO2, and o he low o ca bon h ough he glyoxyla e cycle o p oduce cell cons i uen and i is known ha many bac e ia s ains a e able o main ain hei enzyma ic machine y when he ca bon sou ce is exhaus ed (Roy e al. 2001). The IDH/P ac i i y and Φ/P ac i i y a ios did no ascend like he RQ did when he nu ien was limi ed (Figu e 8 and 9). El-Mansi e al. (1985) in his s udy showed ha , du ing g ow h on ace a e, IDH is pa ially inac i a ed o acili a e glyoxyla e cycle ac i i y. This beha io o IDH ac i i y/Φ ac i i y in ace a e and py u a e expe imen s sugges s ha espi a o y enzyme concen a ions a e no esponsible o RQ di e ences obse ed in nu ien limi a ion. 23 5. Conclusion 1. Respi a ion unde s a ed and well- ed condi ions o ace a e and py u a e-based cul u es o Pseudomonas nau ica and Vib io na iegens show ha espi a ion a es du ing nu ien -su iciency pa allels he biomass inc ease, bu ha a e nu ien s a e exhaus ed, he espi a ion a es all. 2. IDH ac i i y and po en ial espi a ion can main ain high le els as nu ien s a e deple ed, bu a e 10 h o s a a ion hey, and he cell-p o ein all slowly, as nu ien - de iciency pe sis s. 3. Du ing s a a ion biomass-speci ic IDH and φ d ec eases and his la e inding challenges he idea ha IDH and he espi a o y elec on anspo sys ems (ETS) a e cons i u i e and can be used o biomass p oxies. 4. RQs, calcula ed om he espi a o y da a, ise nea ly by a ac o o 10 du ing he shi om nu ien -su iciency o nu ien -de iciency. Because o his, i is clea ha he nu i ional condi ions o he bac e ia g ea ly changes he RQ. 5. The ac ha RQ a ies so much will g ea ly impac ecosys ems models, ocean ca bon lux calcula ions, and p edic ions o he balance be ween ocean-au o ophy and he e o ophy. Acknowledgemen s Thanks o my di ec o s: Ted Packa d and May Gómez and o Begoña González, Angelo San ana del Pino, o hei assis ance in he s a is ical analysis. Thanks o José Ma Zubeldia and San iago Ramí ez Jiménez o hei uncondi ional suppo . This wo k was suppo ed by BIOMBA p ojec (CTM 2012-32729/MAR) g an ed o M. Gómez 24 Re e ences Ahmed S I, Kenne R A, King F D (1976) P ese a ion o enzyma ic ac i i y in ma ine plank on by low- empe a u e eezing. Ma Che, 4(2):133-139 Ande son L A (1995) On he hyd ogen and oxygen con en o ma ine phy oplank on. Deep-Sea Res Pa I 42(9):1675-1680 Baumann P, Baumann L (1981) The Ma ine G am-nega i e Eubac e ia: Genus Pho obac e ium, Beneckea, Al e omonas, Pseudomonas and Alcaligenes, In S a S, T üpe H G, Balows A, Schelegel (eds). The p oka yo es: a handbook on he biology o bac e ia: ecophysiology, isola ion, iden i ica ion, applica ions. Sp inge -Ve lag, New Yo k, p 1352-1394 Be dale E, Packa d T, Lagace B, Roy S, S -Amand L,Gagne J P (1995) CO2 p oduc ion, O2 consump ion and isoci a e dehyd ogenase in he ma ine bac e ium Vib io na iegens. Aqua Mic o Ecol 9(3):211-217 Be gg en M, Lapie e J,Del Gio gio P A (2012) Magni ude and egula ion o bac e ioplank on espi a o y quo ien ac oss eshwa e en i onmen al g adien s. ISME Jou nal 6(5):984-993 B yan J R, Rlley J P, Williams P J L (1976) A winkle p ocedu e o making p ecise measu emen s o oxygen concen a ion o p oduc i i y and ela ed s udies. J Exp Ma Biol and Ecol 21(3):191-197 Can a ow A, Schepa z B (1967) Biochemis y, 4 h edn, W. B. Saunde s Co., Philadelphia Ca uso G, Mon icelli L, Azza o F, Azza o M, Decemb ini F, La Fe la R, Leona di M, Zaccone R (2005) Dynamics o ex acellula enzyma ic ac i i ies in a shallow Medi e anean ecosys em (Tinda i ponds, Sicily). Ma and F esh Res 56(2):173-188 Ca uso G, Azza o F, Azza o M, Decemb ini F, La Fe la R, Maimone G, De Pasquale F, Mon icelli L S. Zaccone R, Zappalà G, Leona di M (2013) En i onmen al a iabili y in a ansi ional Medi e anean sys em (Oli e i-Tinda i, I aly): Focusing on he esponse o mic obial ac i i ies and p oka yo ic abundance, Es ua ine, Coas al and Shel Science. 95-114 Ch ós R J, Siuda W (2006) Mic obial p oduc ion, u iliza ion, and enzyma ic deg ada ion o o ganic ma e in he uppe ophogenic laye in he pelagial zone o lakes along a eu ophica ion g adien . Limnol and Oceanog 51(1 II): 749-762