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Vol. 519: 47–59, 2015 MARINE ECOLOGY PROGRESS SERIES Published January 20 doi: 10.3354/meps11062 Mar Ecol Prog Ser

Respiration quotient variability: bacterial evidence

V. Romero-Kutzner1,*, T. T. Packard1, E. Berdalet2, S. O. Roy3, J.-P. Gagné4, M. Gómez1

1EOMAR, Grupo de Ecofisiología de Organismos Marinos, Universidad de Las Palmas de Gran Canaria 35017, Spain 2Institut de Ciències del Mar (CSIC), Passeig Marítim de la Barceloneta, 37-49, 08003 Barcelona, Spain 310 Ryan Court, Embrun, Ontario K0A 1W0, Canada 4Université du Québec à Rimouski, Rimouski, Québec G5L 3A1, Canada

ABSTRACT: Respiratory was compared between 2 different physiological states of acetate- and pyruvate-grown cultures of Pseudomonas nautica and Vibrio natriegens. Here, we analyze 35 h and 520 h experiments in which time-courses of , pyruvate, acetate, respira-

tory CO2 production (RCO2), respiratory O2 consumption (RO2), isocitrate dehydrogenase (IDH) activity, and potential (Φ) were measured. Respiratory quotients (RQs) were calculated

as the ratio of the respiration rates (RCO2/RO2). Such RQs are widely used in ocean ecosystem mod- els, in calculations of flux, and in evaluations of the ocean’s metabolic balance. In all the cultures, the RQ tended to increase. In the case of P. nautica on acetate, the RQ rose nearly an order of magnitude from values below 1 during carbon-substrate sufficiency to values close to 10 during carbon-substrate deficiency. In all the cultures, the respiration rates during the growth period paralleled the biomass increase, but after the substrates were exhausted, the respiration rates fell. In contrast, through this same transition period, the IDH activity and the Φ remained rel- atively high for the first 10 h of carbon-substrate deprivation, and then, these enzyme activities fell slowly, along with the biomass, as the carbon-substrate deprivation continued. The nutritional state of the bacteria affected the RQ, rendering the RQ variable for physiological and ecological purposes. These results argue that ecosystem models, oceanographic calculations of carbon flux, and evaluations of the ocean’s metabolic balance that are influenced by bacterial metabolism need to be reconsidered in light of RQ variability.

KEY WORDS: O2 consumption · CO2 production · Isocitrate dehydrogenase · IDH · Electron trans- port system · ETS · Potential respiration · Growth

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INTRODUCTION investigator can differentiate -based biological oxidation (metabolism) from -based me-

The respiration quotient (RQ), the ratio of the CO2 tabolism. The majority of studies of respiration in produced to the O2 consumed in respiration, is an old aquatic ecosystems are based on RQ ranging from 0.7 concept, dating back to the 1860s. Armsby & Moul ton to 1.2 (Berggren et al. 2012), but in many ecological (1925) and Lusk (1928) discuss its use in animal hus- calculations of microbial respiration, an RQ of 1 is as- bandry and human physiology, presenting re search sumed (González et al. 2003, Bühring et al. 2006). of Pettenkofer & Voit (1866), Warburg (1926), and This practice occurs even though it is known that the others who were measuring RQ much earlier. RQ is a RQ varies from 0.65 to 1.4 in individual ecological dimensionless ratio, calculated per mole, and is microbial communities (del Giorgio et al. 2006). an index of the type of organic matter being oxidized Amado et al. (2013) argue that such an assumption is in respiration. From the shift in RQ from 0.7 to 1.0, an the only feasible option given the dearth of physio-

*Corresponding author: [email protected] © Inter-Research 2015 · www.int-res.com 48 Mar Ecol Prog Ser 519: 47–59, 2015

logical studies that document the RQ variability in non-pathogenic marine bacterium discovered by bacterial respiration and they conclude that more in- Payne et al. (1961) in a Georgia salt-marsh (Lee & vestigations of RQ variability are needed. A recent Levy 1987, Lee 1995). Carbon-substrate limitation study of respiration in yeast found an RQ range of 0.4 conditions are common in the ocean as marine micro- to 1.4 (Slavov et al. 2014). When an organism is oxi- bial communities pass from bloom to post-bloom con- dizing carbohydrate, its CO2 production rate and its ditions (Liu et al. 2013), and are likely dominant in O2 consumption rate are equal, i.e. the RQ = 1. When oligotrophic ecosystems where bacterial respiration an organism is oxidizing protein, the RQ is around accounts for up to 59% of plankton respiration 0.8, and when oxidizing , it is close to 0.7 (Can- (Robinson & Williams 2005). Here, we show that in tarow & Schepartz 1967, Guyton 1971, Hoar 1975, our cultures, the RQ can range higher than the upper Gnaiger 1983, Stanier & Forsling 1990). When burn- values reported above. To investigate this wide range, ing organic compounds richer in O2 than carbohy- we examine time-courses of the physiological respi- drates, such as carboxylic acids, and when converting ration rates (RO2 and RCO2) and the enzymatic activi- carbohydrate to fat, RQ values can be >1. When ties from the respiratory electron transport system metabolism consumes -rich (ETS) and isocitrate dehydrogenase (IDH). ETS and

(C2H2O4), RQ can rise to 4 (Dilly 2001). At the other IDH activities are the biochemical enzyme activities end of the scale, gluconeogenesis (glucose synthesis) that largely control the RO2 consumption and RCO2 occurs when RQ falls below 0.7 (Cantarow & Schep- production (Packard et al. 1996a, Nelson & Cox 2005, artz 1967). In this way, RQ variations are generally Gnaiger 2009). Via aerobic respiration, marine or- explained by the fact that the amounts of CO2 and O2 ganisms obtain the energy to live from a wide range produced are dependent on the oxidation state of the of compounds that are reduced in different, but well substrate and the pathways by which the substrate is coordinated biochemical pathways. Two of these key metabolized (Burton 1982, Kader 1987). Berggren et pathways are the Krebs or tricarboxylic acid (TCA) al. (2012) argue from their field studies of Quebec cycle and ETS. IDH is proposed to be a future ana- lakes that variability in RQ indicates shifts in bacterial lytical method for CO2 calculations in the ocean physiology and carbon consumption that cannot be (Packard et al. 1996a, Roy & Packard 2001), but it deduced from other measurements. However, in needs further investigation, improvement (Robinson , only a handful of RQ measurements & Williams 2005), and calibration. In our study, we have been made (Oviatt et al. 1986, Robinson et al. work with 2 substrates: acetate and pyruvate. Ace- 2002). Nevertheless, when the effort is made to deter- tate, a 2 carbon molecule, is transformed into acetyl- mine the RQ for each situation, as did Obernosterer et CoA at the entry of the TCA and, via IDH and alpha- al. (2008) in their Southern Ocean iron-fertilization keto glutarate dehydrogenase, loses both as − experiment, an RCO2 production or a carbon oxidation CO2 and produces 8 reducing equivalents (e ) that in calculation is more accurate. turn, lead to the consumption of 2 molecules of O2 + − Whether in the laboratory or in the field, an RQ is (O2 + 4H + 4e → 2H2O) at cytochrome oxidase in essential in calculating either organic carbon con- the ETS. Accordingly, the potential RQ for acetate sumption or CO2 production from RO2 consumption would be 1.0. When pyruvate (a 3-carbon molecule) measurements (Boucher et al. 1994, Bergström 2011, is cycled through the TCA, it produces 10 reducing

Giering et al. 2014). In this way, RQ becomes an equivalents and 3 molecules of CO2 and it consumes influential factor in ocean carbon-cycle studies, for 2.5 O2 molecules. This would result in an RQ of 1.2. calculating carbon flux from plankton metabolism Thus, the use of these different carbon substrates (Packard & Christensen 2004, Steinberg et al. 2008, becomes a tool to generate different values of RQ. Packard & Gómez 2013, Osma et al. 2014), and in In P. nautica, we found that acetate-grown cul - investigating whether the ocean is autotrophic or tures showed an RQ increase to 10 during carbon- heterotrophic (Ducklow & Doney 2013). Here, we limitation, whereas RQ in pyruvate-grown cultures measure the RQ in cultures of the marine bacteria remained near 1.0 (Roy et al. 1999). However, Pseudomonas nautica and Vibrio natriegens as they because of the continued lack of published physio- pass from carbon substrate sufficiency to carbon sub- logical information about RQ, we wanted to in - strate limitation. vestigate this topic with another species and over P. nautica is an oil-degrading bacterium from the longer periods of carbon-limitation. We expected that Gulf of Fos, France (Bonin et al. 1987a,b), and was different levels of carbon limitation and different used to advance studies of oil-spill bio-remediation durations of starvation would generate a wider range (Swannell et al. 1996). V. natriegens is a well-studied of RQs. Romero-Kutzner et al.: Respiration quotient variability in bacteria 49

MATERIALS AND METHODS triegens were experimentally established. They were

grown on 400 mM NaCl, 10 mM MgSO4·7H2O, Experimental design 10 mM CaCl2·2H2O, 10 mM KCl, 25 mM NH4Cl, 0.33 mM phosphate buffer, 0.01 mM FeSO4·7H2O. To investigate the RQ in different bacterial growth Initial of the culture-medium carbon- stages, time-course experiments were run on batch source was 30 mM sodium acetate or 20 mM pyru - cultures at 22°C, maintained on pyruvate or acetate vate. (Note that these would provide as described by Berdalet et al. (1995) and Packard et the same amount of organic carbon at the start of an al. (1996b). Short-term experiments were run for a experiment.) The mean coefficient of variation was maximum of 35 h, and a (different) set of long-term 2.1% (n = 20) for acetate and pyruvate. Reagents for experiments were run for 2 to 3 wk. The bacteria cul- the culture media were obtained from Sigma-Aldrich. tures were grown in 25 cotton-plugged 500 ml Erlen- All components were dissolved in 0.22 µm filtered meyer flasks containing 100 ml of media kept slowly deionized water (except FeSO4·7H2O and phosphate rotating on an ‘orbital shaker table’. At intervals over buffer), and pH was adjusted to 7.5 with 1 N NaOH. the different time courses, 2 flasks were chosen ran- To remove particles, the medium was filtered through domly, 25 ml of culture were transferred to the air- a GF/F glass fiber filter. Later, the medium was auto- tight flasks, and the respiration was claved for 45 min at 121°C. Then, to avoid precipita- measured. Then, samples of the remaining culture in tion during the autoclaving, the phosphate buffer the chosen flasks were taken in duplicate for growth (0.67 M, pH 7.5) and the iron sulphate (FeSO4· (optical density at 550 nm [OD550], meas- 7H2O, 0.1 mM) were prepared separately. The PO4 ured as absorbance at 550 nm), protein, pyruvate, buffer was sterilized by autoclaving, and the FeSO4 acetate, RCO2, RO2, IDH activity, and potential respira- solution was filtered through 0.22 µm acrodiscs. Final- tion (Φ) as the cultures grew exponentially, reached ly, both were kept frozen and were thawed steady state, and passed into senescence. and added to the culture medium just before use.

Bacterial cultures Protein measurements

Vibrio natriegens (ATCC 33788) and Pseudomonas For every flask, protein samples were taken in nautica (Strain 617 from Dr. P. Bonin, Université de la duplicate. Five to 10 ml of culture, depending on the Méditerranée, Marseille, France) were used for these level of biomass, were centrifuged at 10000 × g at 4°C cultures. Before any experiment, the bacteria had for 15 min and then frozen in liquid N2 (Ahmed et al. been adapted for a minimum of 15 generations to the 1976). Later, the bacterial pellets were defrosted, experimental media (pyruvate or acetate). To inocu- mixed well with 2 to 4 ml 1 N NaOH (at 22°C), and late the cultures, a sample from a mother culture in analyzed for protein in aliquots of 0.5 ml by the exponential or early stationary phase was used. Cul- Lowry method (Lowry et al. 1951) in according with tures were continuously agitated on an orbital shaker Berdalet et al. (1995). If the absorbance at 750 nm of at 100 rpm at 22°C, and growth was followed spec- the samples exceeded 0.4, the homogenates were trophotometrically at 550 nm (OD550). The OD550 diluted and analyzed again. For standardization, had an initial value after inoculation of 0.1 duplicate measurements of bovine serum albumin absorbance units. (BSA) from Sigma Chemical Company were used. Their range around the average of these duplicates increased from 2% during exponential growth to Culture media 14% during stationary phase (after 15 h). The mean of these ranges averaged 4.1%. The general culture procedures have been de- scribed by Berdalet et al. (1995) and Roy et al. (1999). More specifically, P. nautica was cultured according Biochemical parameters to the method of Packard et al. (1996a), and the medium for V. natriegens was developed from the For acetate and/or pyruvate and enzyme activity media of Niven et al. (1977), Baumann & Baumann (IDH and ETS), samples were also taken in duplicate (1981), King & Berman (1984), and Nissen et al. from every experimental flask and prepared as for (1987). The optimal conditions for growth of V. na- the protein samples (Berdalet et al. 1995). The super- 50 Mar Ecol Prog Ser 519: 47–59, 2015

−1 natant fluid was collected in an acid-rinsed Corex CO2. This is equivalent to 0.44 µmol l . The maxi- −1 tube and then stored in liquid N2 for acetate or pyru- mum sensitivity for the 2 rates was 8.9 nmol h . The vate analysis after centrifugation. Samples were instrument had a multiple sample chamber (for up to thawed and adjusted to pH 2 by adding concentrated 20 channels), a reference chamber, and a computer- phosphoric acid and analyzed by high performance ized data acquisition and analysis system. The Micro- liquid chromatography. Oxymax maintained aerobic conditions because it Pellets for IDH extraction were resuspended in periodically refreshed (replenished) the air in the

2 ml of buffer at 0 to 4°C, and activity was deter- headspace if the O2 level fell below 19.3% to avoid mined spectrophotometrically at 340 nm following oxygen limitation. Cultures were maintained at 22°C the NADPH production (Reeves et al. 1971, 1972, and continuously shaken (to optimize ). −1 −1 Holms & Bennett 1971, Berdalet et al. 1995) after Respiration is given as µmol O2 min l and µmol + −1 −1 NADP addition. IDH catalyzes the reaction produc- CO2 min l . Note that here, RCO2 refers to CO2 gen- ing CO2 during isocitrate oxidation. erated by intact bacteria suspended in their growth medium, and R refers to O consumed under the Isocitrate + NADP+ ↔α-ketoglutarate + NADPH + CO O2 2 2 same conditions. A normal measurement took −1 Results are given as µm CO2 min per liter of cul- ~30 min. Measurements over 30 min intervals were ture. The IDH activity was calculated from the referenced against a baseline and a control and were regression line of OD340 versus time. NADPH was made in duplicate. The calibration of the oxygen used as the standard converting OD340 to CO2 detector was done with high-precision gas standards. (µmol) because, from the equation above, NADPH The respirometry for all the original measurements production is stoichiometrically equal to CO2 produc- was described by Berdalet et al. (1995) and Packard tion (1:1). Each data point represented 4 analyses. et al. (1996b). The mean coefficient of variation was

The mean coefficient of variation was 5.0% (n = 20). 5.1% (n = 20) for RO2 and 5.2% (n = 20) for RCO2. Samples for ETS were resuspended at 0 to 4°C in 2 ml of homogenizing buffer and measured kinetically for ETS activity with a modification of the Packard & Statistical analysis Williams (1981) method as described by Packard & Christensen (2004). To calculate Φ, ETS in µmol e− Data were analyzed using the program R (R Devel- −1 −1 − + min l was divided by 4 (4e + 4H + O2 → 2H2O) to opment Core Team 2010). Relationships between −1 −1 Φ give µmol O2 min l of culture. Again, each data RCO2/RO2 and IDH/ over different time scales (short- point represents 4 analyses. In the case of ETS, the and long-term experiments) were obtained from the mean coefficient of variation was 7.2% (n = 20). regression equations, using confidence limits of 95% and Pearson correlation coefficients. ANCOVA was applied to determine statistical differences between Respiration measurements slopes and ordinates in the regression lines. Normal- ity of residuals was confirmed using the Shapiro-

RCO2 and RO2 were simultaneously measured by a Wilks test. Micro-Oxymax system (Columbus Instruments Inter- national). The Micro-Oxymax is a computer-con- trolled closed-circuit respirometer. The system moni- RESULTS tors gas concentrations in the headspace above the culture (www.colinst.com) via 2 well-sealed tubes. Data summary The culture itself was sampled by syringe through a septum in each experimental flask. Calculations of Two types of experiments were conducted. Short- incremental and accumulated values for consump- term experiments that ran up to 35 h and long-term tion of O2 and production of CO2 were possible (2 to 3 wk) experiments that ran for 330 and 520 h. In because of periodic sensing of the gas concentration. all these experiments, we used the 2 bacterial species Oxygen measurements were carried out with an oxy- Pseudomonas nautica and Vibrio natriegens growing gen detector based on the principle of a PbO2 fuel on either acetate or pyruvate. In Fig. 1, measure- cell. CO2 measurements were made with an infrared ments of substrate (carbon source), protein, enzyme Φ CO2 detector (sensitive to the 2000 µm absorption activity (IDH and ), RCO2, and RO2 are shown. Fig. 2 peak of CO2). Both were part of the Micro-Oxymax presents comparable long-term experiments in which system. The detection limit was 10 ppm of O2 and we made the same measurements. Data for both fig- Romero-Kutzner et al.: Respiration quotient variability in bacteria 51

Substrate Protein IDH P. nautica V. natriegens 50 300 50 300 RO2 AB ) ) RCO2 IDH, RCO IDH, RCO –1 –1 φ 250 250 φ , RO 40 40 , RO 2 2

200 200 (µmol O (µmol O 2 2

(µmol CO 30 30 (µmol CO ), Protein (mg l ), Protein (mg l 150 150 –1 –1 2 2

20 min 20 min 2 2

min min –1 100 100 –1

l l –1 –1 –1 –1 ) )

l l –1 10 10 –1 50 50 ) ) Acetate (mmol l Acetate (mmol l 0 0 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Time (h) Time (h) 50 C 300 50 500

) D ) –1 IDH, RCO IDH, RCO –1 l

250 φ φ , RO 40 40 400 , RO 2 2

200 (µmol O 2 (µmol O 2

(µmol CO 30 30 300 (µmol CO ), Protein (mg l ), Protein (mg

150 –1 –1 2 2

min 20 20 200 min 2 2

min min –1

100 –1

l l –1 –1 –1 –1 ) )

l l

10 –1 10 100 –1 50 ) ) Pyruvate (mmol l Pyruvate (mmol l 0 0 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Time (h) Time (h)

Fig. 1. Short-term experiments. Time courses of respiration, enzyme activity, biomass, and carbon source: (A) Pseudomonas nautica in acetate, (B) Vibrio natriegens in acetate, (C) P. nautica in pyruvate, (D) V. natriegens in pyruvate. Note that, depend- ing on the experiment, the cultures are substrate-sufficient in the first 5 to 15 h and substrate-limited (starved) afterwards. For display clarity, the protein data have been divided by 10, and R and R have been multiplied by 5. IDH: isocitrate dehy- CO2 O2 drogenase; φ: potential respiration; R : O consumed; R : CO generated O2 2 CO2 2 ures are represented on the same scale so that all the biomass because both the enzyme activities and the time-courses could be shown in the same graph. biomass trend in parallel with culture age (Fig. 1). However, this required the use of multipliers, as During the same conditions of substrate limitation, explained in the legends for Figs. 1 & 2. IDH in the V. natriegens cultures retains similarly high activity levels. Long-term experiments. Enzymatic processes in Enzyme activity: IDH and Φ the long-term experiments (Fig. 2) after 24 h varied little. In all experiments, enzymatic activity was Short-term experiments. Enzymatic activity is observable even during carbon substrate limitation. present even during starvation (Fig. 1). In general, Almost all IDH and Φ values were similar; IDH −1 −1 the IDH activity and Φ rise during the hours of sub- ranged from 0.5 to 66 µmol CO2 min l of culture, −1 −1 strate sufficiency. After substrate limitation sets in, while Φ ranged from 1.3 to 284 µmol O2 min l of both parameters in the P. nautica cultures stay rela- culture. In P. nautica on acetate (Fig. 2A), IDH rose tively constant with Φ higher than IDH. This can be with carbon substrate limitation and then decreased seen when either enzyme activity is normalized by with Φ until ~350 h. In V. natriegens growing on 52 Mar Ecol Prog Ser 519: 47–59, 2015

50 P. nautica 500 50 V. natriegens 200 A Substrate B ) Protein ) IDH, RCO IDH, RCO –1 IDH –1 φ φ , RO 40 φ 400 40 , RO RO2 150 2 2

(µmol O RCO2 (µmol O 2 2

(µmol CO 30 300 (µmol CO 30 ), Protein (mg l ), Protein (mg l 100 –1 2 –1 2

min

20 200 20 min 2 2

–1

min min –1 l

l –1 –1 –1

–1 50 ) ) l 10 100 10 l –1 –1 ) ) Acetate (mmol l Acetate (mmol l 0 0 0 0 0 100 200 300 400 500 0 100 200 300 400 500 Time (h) Time (h)

50 500 50 200 ) C ) D IDH, RCO IDH, RCO –1 –1 φ φ , RO 40 400 40 , RO 150 2 2

2 (µmol O 2 (µmol O

(µmol CO 30 300 30 (µmol CO ), Protein (mg l ) ,Protein (mg l

–1 –1 100 2 2

min 20 200 20 min 2 2

min min –1 –1 l

l –1 –1 –1 50 –1 ) ) l 10 100

10 l –1 –1 ) ) Pyruvate (mmol l Pyruvate (mmol l 0 0 0 0 0 100 200 300 400 500 0 100 200 300 400 500 Time (h) Time (h)

Fig. 2. Long-term experiments. Time courses of respiration, enzyme activity, biomass, and carbon source: (A) Pseudomonas nautica in acetate, (B) Vibrio natriegens in acetate, (C) P. nautica in pyruvate, (D) V. natriegens in pyruvate. Note that, except for the first 30 to 35 h, the cultures are continually starved for carbon; they are substrate-limited. During this period, the bio- mass and the enzyme activities decline, while the respiration (R and R ) is barely measureable. For display clarity, protein O2 CO2 data have been divided by 10, have R and R been multiplied by 10. Dotted lines symbolize absent data. IDH: isocitrate O2 CO2 dehydrogenase; φ: potential respiration; R : O consumed; R : CO generated O2 2 CO2 2

Φ acetate, both IDH and dropped after carbon sub- Physiological measurements: RCO2 and RO2 strate limitation to levels lower than 4.7 µmol CO2 −1 −1 −1 −1 min l and 6.2 µmol O2 min l (Fig. 2B), respec- Short-term experiments. In all short-term experi-

tively. In experiments with P. nautica growing on ments, RO2 was higher than RCO2 before starvation pyruvate, Φ and IDH remained almost constant for was reached (Fig. 1). Experimental results with P. 350 h after carbon substrate limitation (Fig. 2C). In nautica and V. natriegens on acetate were similar, experiments with V. natriegens growing on pyruvate, but the 2 curves were out of phase. The peaks were Φ −1 −1 maintained activity around 72 µmol O2 min l of displaced in time. In these experiments, RO2 rose culture for the first 100 h and then decreased to rapidly, but as starvation began, around 15 h, it fell.

barely detectable levels of activity after 500 h. IDH During this RO2 fall, RCO2 peaked and then rapidly activities decreased more rapidly in the first 100 h to declined (Fig. 1A,B). In the 2 pyruvate-based experi- −1 −1 13 µmol CO2 min l of culture and then more ments, pyruvate de creased from a initial level of −1 −1 −1 slowly in the next 400 h to 4.5 µmol CO2 min l of 20 mmol l to nearly zero after ~10 h (Fig. 1C,D). In

culture (Fig. 2D). Substrate deficiency characterized the P. nautica culture, both RCO2 and RO2 rose and fell most of the culture’s life. Note that both IDH and Φ in parallel (Fig. 1C). The decrease in respiration tended to decrease with time as the bacterial cultures occurred before carbon substrate depletion. In the starved. comparable V. natriegens pyruvate culture (Fig. 1D), Romero-Kutzner et al.: Respiration quotient variability in bacteria 53

40 pnac s vnac s vnpy w pnpy pnac pnpy pnac w vnac w vnpy s vnac vnpy 40 ) -1 l

30 -1 35 min ) 2 -1 l 30 20 -1

25 (µmol CO

2 10

20 RCO

15 0 10 0 102030405060

RCO2 (µmol CO2 min -1 -1 RO2 (µmol O2 min l ) 5 Fig. 4. Long-term experiments. R production ver- CO2 sus R consumption with regression equations that O2 include all data points. pnac: R = 0.3681 R + 0 CO2 O2 1.5454; r2 = 0.93; n = 9; p < 0.05; pnpy: R = 1.204 R 0 102030405060 CO2 O2 + 0.3718; r2 = 0.99; n = 7; p < 0.05; vnac: R = 0.3103 –1 –1 CO2 RO2 (µmol O2 min l ) R + 0.5962; r2 = 0.65; n = 6; p = 0.05; vnpy: R = O2 CO2 1.0624 R + 0.0701; r2 = 0.99; n = 7; p < 0.05. Note that Fig. 3. Short-term experiments. R production versus R con- O2 CO2 O2 the carbon sources dictate the parallelism of the sumption; w is for well-fed (substrate-sufficient), and s is for curves, not the bacterial species. In addition, note that starvation conditions (substrate-starved). pnac: Pseudomonas the respiration quotient (slope) is higher for the 3-C nautica in acetate, pnpy: P. nautica in pyruvate, vnac: Vibrio substrate (pyruvate) than for the 2-C substrate natriegens in acetate, vnpy: V. natriegens in pyruvate. For (acetate). This difference cannot be seen in the short- pnpy, the well-fed and starved conditions had the same slope term experiments (Fig. 3) the respiration signals were out of Table 1. Minimum, maximum, and mean (RQ) from phase; however, in both of the ace tate- exponential phase (w: well fed) and stationary phase (s: starvation) for short- term and long-term experiments. (Note that in all experiments, the transition based cultures, they were even more points between the well-fed and starved conditions were excluded). Regression out of phase. equations correspond to R = mR + b CO2 O2 In comparing the RCO2 to RO2 in the short-term experiments (Figs. 1 & 3), Bact- Cond- ———— RQ ————— Regression a r2 n the relationship between RCO2 and RO2 eria ition MIN. MAX. Mean SD y = differs with each experiment. High variability is clear (Fig. 3). However, Short-term experiments pnac w 0.3 0.5 0.4 0.06 0.4054x − 0.4045 0.92 6 note that in the P. nautica in pyruvate s 5.7 9.59 7.2 1.66 5.2962x + 3.4891 0.92 4 (pnpy) culture (Figs. 1C & 3), the data vnac w 0.3 0.3 0.3 0.02 0.2769x + 0.3332 0.98 9 from both the carbon-sufficient and the s 1.6 2.4 2.0 0.32 2.0564x − 0.4225 0.95 4 carbon-limited phases follows the same pnpy w 0.6 0.8 0.6 0.07 0.7598x − 1.2327 0.99 5 correlation (RCO2 = 0.7295 RO2 − 0.5969, s 0.6 1.1 0.9 0.3 2 2 n = 10, r = 0.9882, p < 0.05). For all the vnpy w 0.7 1.0 0.9 0.16 1.126x − 5.0411 0.97 4 experiments, if the substrate-starved s 0.7 1.3 1.0 0.34 0.5737x + 1.7177 0.99 3 and the substrate-sufficient data are Long-term experiments considered separately, 2 different rela- pnac w 0.3 0.8 0.5 0.25 0.383x + 0.1797 0.99 3 tionships appear (Fig. 4). From the s 2.4 7.8 4.7 2.23 4.2758x − 0.1633 0.76 5 equations in Table 1, it is clear that the vnac w 0.2 0.7 0.4 0.33 2 s 0.6 1.2 0.9 0.29 0.928x − 0.0242 0.99 3 starved acetate-based cul tures have pnpy w 1.2 1.9 1.6 0.34 1.1533x + 1.5905 0.99 3 different RQs (slopes) than the starved s 0.6 1.2 0.9 0.30 0.3266x + 0.3467 0.98 3 pyruvate-based cultures. vnpy w 0.8 1.1 1.0 0.18 2 Long-term experiments. RCO2 and s 1.0 4.5 2.4 1.64 0.86x + 0.3836 0.99 4 R decreased to low values within the O2 aRegression lines: p-values < 0.05 in all cases first 150 h of the experiment (Fig. 2). 54 Mar Ecol Prog Ser 519: 47–59, 2015

P. nautica V. natriegens 40 10 50 3 substrate A RQ B RQ (µmol CO

8 RQ (µmol CO 40 30 2 6 30 20 2 /µmol O 2 4 /µmol O 20 1

10 2 ) 2 Acetate (mM of culture) ) 2 Acetate (mM of culture) 10

0 0 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Time (h) Time (h) 20 2.0 20 2.0 C D RQ (µmol CO RQ (µmol CO 15 1.5 15 1.5

10 2 1.0 2 10 1.0 /µmol O /µmol O 2 2 5 5 0.5 )

0.5 ) Pyruvate (mM of culture) Pyruvate (mM of culture)

0 0.0 0 0.0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Time (h) Time (h) Fig. 5. Short-term experiments: RQ and substrate (carbon source) versus time. Note the carbon substrate sufficiency on the left and carbon substrate depletion (starvation) on the right of each panel. Top panels are acetate-based cultures; bottom panels are pyruvate-based cultures. Left panels are Pseudomonas nautica, right panels are Vibrio natriegens. In all experiments, RQ rises during starvation

After that time, RCO2, although low, steadily slopes appeared to be significantly different (p < increased in relation to RO2. This trend contrasted 0.05). All the slopes (RQs) during starvation condi- greatly with both the biomass and the enzyme activ- tions were significantly different (p < 0.05). ity data, all of which maintained relatively high val- ues throughout substrate limitation. All the slopes between the 2 respiration measure- Respiration quotient ments (RCO2 and RO2) for the long-term experiments were compared (Fig. 4) and displayed a good corre- Short-term experiments. RQ measurements in the lation in all cases except in V. natriegens on acetate. short-term experiments are shown in Fig. 5. When The slope of these functions was the overall RQ. This the cultures were substrate-sufficient, RQs were low, is another way to estimate RQ, but requires sufficient but when carbon substrate limitation started in the samples collected during the same culture condition. acetate cultures of both P. nautica and V. natriegens, ANCOVA analysis between cultures that were grow- the RQ values rose dramatically (Table 1, Fig. 5A,B). ing on the same carbon substrate (i.e. P. nautica or V. In P. nautica on pyruvate, the RQ rose only at the end natriegens on acetate) shows that the slopes were not of the experiment (Fig. 5C). In contrast to the con- significantly different (Fig. 4; p > 0.05). In contrast, stant pre-starvation RQ values in the first 3 experi- for the same bacterial species but for different sub- ments, in the V. natriegens culture growing on pyru- strates (i.e. P. nautica on acetate or pyruvate), the vate (Fig. 5D), the signal displayed noticeable noise Romero-Kutzner et al.: Respiration quotient variability in bacteria 55

50 3 50 A P. nautica10 V. natriegens substrate B

40 RQ (µmol CO 40 RQ 8 RQ (µmol CO 2 30 6 30 2 2 /µmol O 20 4 /µmol O 20 1 2 Acetate (mM of culture) 2 RQ (µmol CO ) Acetate (mM of culture)

10 RQ (µmol CO 2 ) 10

0 0 0 0 0 100 200 300 400 500 0 100 200 300 400 500 Time (h) Time (h)

50 2.0 50 5 C D

40 40 4 1.5

30 30 3 2

1.0 /µmol O 2 /µmol O 20 20 2 2 0.5 ) 2 Pyruvate (mM of culture) )

Pyruvate (mM of culture) 10 10 1

0 0.0 0 0 0 100 200 300 400 500 0 100 200 300 400 500 Time (h) Time (h)

Fig. 6. Long-term experiments: RQ and substrate (carbon source) versus time. Top panels are acetate-based cultures; bottom panels are pyruvate-based cultures. Left panels are Pseudomonas nautica, right panels are Vibrio natriegens. In all panels, RQ increases during the late starvation period between Hours 7 and 12 of the experiment. After- and carbon substrate limitation set in after 65 h, the wards, at 25 h, it rose to an RQ of 1.4. RQ rose. However, it rose at a lower rate than in the In summary, these short-term experiments show a second experiment (V. natriegens) on pyruvate in distinct shift in the RQ when either bacteria species which RQ rose to 4.5 at the experiment end (Fig. 6D). was growing on acetate (Figs. 5A,B). In contrast, In V. natriegens growing on acetate (Fig. 6B), RQ rose when growing on pyruvate, both species displayed to 1.2 after acetate exhaustion; however, this RQ was an RQ shift, but it was less pronounced than in lower than the high value (8.0) found in the P. nautica Figs. 5C,D. culture growing on acetate (Fig. 6A, Table 1). Long-term experiments. The time-course signature of the long-term experiments (Fig. 6) is comparable to that of the short-term experiments (Fig. 5). Of the 4 DISCUSSION experiments, P. nautica on acetate (Fig. 6A) behaved most similarly to the short-term experiments. In all RQ and enzyme activity long-term experiments, carbon substrate limitation began within the first 50 h. We see an RQ jump when RQ shifts can result from the variable activities of carbon substrate limitation was reached, with a sub- different biochemical pathways. In the Krebs cycle, sequent rise to the end of the experiment (Fig. 6A). RCO2 is the collective activity of 3 enzymes: isocitrate When P. nautica was growing on pyruvate (Fig. 6C) dehydrogenase (IDH, EC 1.1.1.42), α-ketoglutarate 56 Mar Ecol Prog Ser 519: 47–59, 2015

α dehydrogenase ( -KGDH, EC 1.2.7.3), and pyruvate the RQ to rise. As starvation sets in, RO2 decreases dehydrogenase (PDH, EC 1.2.2.2) (Walsh & Koshland before RCO2, causing the RQ rise (Fig. 1A,B). Why this 1984, Holms 1986a,b). RO2, although largely con- uncoupling occurs is not obvious. Recent research trolled by the ETS, is also influenced by all the oxi- with nematodes has shown that starvation decreases dases, oxygenases, and hydroxylases in a cell. In RO2 without decreasing the activity of ETS (Chin et Figs. 1 & 2, we see that in starvation, IDH and Φ al. 2014). This impact was traced to increases in the activities are still present at relatively high levels, concentration of alpha-keto glutarate (α-KG), the and an IDH dominance is not detectable. Conse- product of IDH activity. Since CO2 is also a product of quently, the RQ elevation (Figs. 5 & 6) cannot be IDH activity, perhaps RCO2 is also stimulated, result- attributed to the relative activity trends of IDH and ing in a rise in the RQ. Here, we saw the decrease in

ETS. In nature, microorganisms are able to react RO2 and stability in the ETS but not an increase in the quickly to environmental changes, adapting their IDH activity and RCO2. Nevertheless, could starvation metabolic profiles to organic inputs (Martinez et al. have caused an increase in the α-KG by another 1996, Mudryk & Donderski 1997, Sala & Güde 2004, mechanism in our cultures? Additional experimenta- Mudryk & Skórczewski 2006). Still, such a mecha- tion is required to address this question. nism requires time-consuming enzyme induction or Another observed trend in our results was that the activation. Here, the maintenance of IDH and Φ lev- temporal signatures of the RQs differed between els after starvation suggests that Vibrio natriegens the acetate- and the pyruvate-grown cultures, inde- and Pseudomonas nautica are prepared to bypass pendent of the species. This difference occurred in this time-consuming step to be ready to react imme- short-term experiments but not in the long-term ones diately via Michaelis-Menten kinetics to any fortu- (Figs. 5 & 6). A comparison of the slopes in the itous availability of substrates. Thus, the differential RCO2-RO2 regressions (Fig. 4) showed that the bio- impact of declining substrates (NADP+, isocitrate, chemistry, not the bacterial species, determined the NADH, and succinate) induced by substrate-limita- RQ. An ANCOVA test confirmed it by showing that tion could provide the explanation for the declining there was no significant difference in the slopes of P. physiological rates. nautica and V. natriegens growing on the same sub- strate (Fig. 4). Because acetate and pyruvate are products of lipid and carbohydrate metabolism, RQ variability and carbon limitation respectively, the short-term experimental results (Table 1) confirmed that carbohydrate consumption In this study, we observed a rising RQ after nutrient leads to higher RQs (0.7 to 1) than does lipid con- depletion in cultures of 2 marine bacteria. The RQ sumption (0.4 to 0.3). This is consistent with the his- ranged from a low of 0.3 during exponential growth torical use of the RQ. to a high of 9.6 during senescence and nutrient depletion (Fig. 5A). In comparison, the RQs in fresh waters of Quebec ranged from 0.25 to 2.26 (Berggren Future research et al. 2012). Those authors argue that the common assumptions of 0.8 to 1.0 RQ values in the literature In the present study, we document the accompany- are not justified. Furthermore, they argue that the ing RQ changes that occurred in bacterial culture use of a constant RQ depresses estimations of the experiments designed to detect the shift in the metabolic balance between respiration and photo- relationship between respiratory enzyme activity synthesis, i.e. the P/R ratio. They maintain that high and respiration as exponentially growing bacteria RQs have a physiological explanation based on the encounter carbon-substrate limitation. The RQ rose different biochemical pathways of DOC degradation. after the carbon limitation set in. We had seen this However, it is not obvious how RQ could rise to 10 before with short-term experiments of P. nautica, but (Fig. 5A) by a biochemical mechanism. This would here, this rise is confirmed in long-term experiments mean that O2 consumption is severely repressed or with P. nautica and with both short-term and long- that CO2 production is highly stimulated. It is difficult term experiments with V. natriegens. In future exper- to see how starvation could cause those shifts. Possi- iments, it would be interesting to focus on the shift in bly, vegetative cells are being transformed into RQ during the opposite transition, from nutrient- spores, but what reactions are involved are depletion (senescence) to carbon-sufficiency, but unknown. Here, the 2 physiological signals of the adding more detailed measurements of the organic respiration do appear to shift in the right direction for components of the culture media. Learning the Romero-Kutzner et al.: Respiration quotient variability in bacteria 57

organic composition of the media during the ‘nutri- after nutrients are exhausted, both respiration rates ent-depletion’ phase would be especially interesting. fall rapidly. This drop in the respiration rates occurs In addition, investigation should be extended to much sooner and more rapidly than the slow decline bacteria cultures growing on different substrates; to in the enzyme activities. cultures of phytoplankton, macroalgae, zooplankton, (5) IDH activity and potential respiration (ETS and benthic invertebrates; as well as to natural as- activity expressed in terms of oxygen) can maintain semblages of these organisms in field samples. Ex - high levels after nutrients are depleted (Figs. 1 & 2), ploiting mesocosms, as is done in the KOSMOS pro- but after 10 h of starvation, these values and the cell grams (Riebesell et al. 2013), is an excellent way of protein begin to slowly decline as substrate-defi- achieving the later experiments. ciency persists. The decline in the enzyme activities Finally, RQ variability could be used to study bio- is much slower after the point of nutrient exhaustion logical energy production. At present, RQ is used to than is the rapid decline in the physiological respira- differentiate different types of biological oxidation. tion rates. However, in the future, RQ could also be used as an index of the cost, in terms of organic carbon, of gen- Acknowledgements. This work was supported by BIOMBA erating a unit of energy (energy production cost). project CTM 2012-32729/MAR granted to M.G., the Univer- Oxygen consumption is directly related, in aerobic sity Foundation of Las Palmas by the program ‘Innova metabolism, to ATP production, and the molar ratio Canarias 2020’ financed by Endesa granted to V.R.-K., and a of the ATP production to the reduction of an oxygen CIE Canarias tri-Continental Atlantic Campus (#CEI10/ 00018) contract to T.T.P. atom (ATP/2e− ratio) (Ferguson 2010) ranges from 2.5 (NADH oxidation) to 1.5 (succinate oxidation). Accordingly, high RQs would indicate that more LITERATURE CITED organic matter is oxidized to produce a unit of ATP than in cases involving low RQs. An inverted RQ Ahmed SI, Kenner RA, King FD (1976) Preservation of enzy- Δ Δ matic activity in marine plankton by low- [1/RQ = ( O2 consumed)/( CO2 produced)] is an freezing. Mar Chem 4: 133−139 index of energy production efficiency. Future re - Amado AM, Meirelles-Pereira F, Vidal LO, Sarmento H and search should explore using the RQ to assess both others (2013) Tropical freshwater ecosystems have lower energy production costs and energy production bacterial growth efficiency than temperate ones. Front Microbiol 4: 167 efficiencies. Armsby HP, Moulton CR (1925) The animal as a converter of matter and energy: a study of the role of livestock in food production. Am Chem Soc Monogr Ser, Chemical Cata- CONCLUSIONS log Co., New York, NY Baumann P, Baumann L (1981) The marine Gram-negative Eubacteria: Genus Photobacterium, Beneckea, Altero - (1) RQ changes with nutritional impact on the monas, Pseudomonas and Alcaligenes. In: Starr S, Trüper physiological state. Bacteria under substrate-limita- HG, Balows A, Schelegel HG (eds) The prokaryotes: a tion have RQs higher than when they are growing handbook on the biology of bacteria: ecophysiology, iso- under substrate-sufficient conditions. lation, identification, applications. Springer-Verlag, New York, NY, p 1352−1394 (2) RQs rise by nearly a factor of 10 (Fig. 5A) during Berdalet E, Packard T, Legacé B, Roy S, St-Amand L, Gagné substrate-deficiency (senescence). This suggests that JP (1995) CO2 production, O2 consumption and isocitrate the nutritional conditions of bacteria should be con- dehydrogenase in the marine bacterium Vibrio sidered, in the future, when selecting an RQ for cal- natriegens. Aquat Microb Ecol 9: 211−217 culation purposes. Berggren M, Lapierre J, Del Giorgio PA (2012) Magnitude and regulation of bacterioplankton respiratory quotient (3) Should this level of RQ variability (Fig. 3) be across freshwater environmental gradients. ISME J 6: found in phytoplankton, macroalgae, zooplankton, 984−993 and benthic invertebrates, then ecosystem models, Bergström I (2011) Carbon gas fluxes from boreal aquatic ocean carbon flux calculations, and predictions of the sediments. Monogr Boreal Environ Res 38 Bonin P, Gilewicz M, Bertrand JC (1987a) Denitrification by balance between ocean autotrophy and heterotrophy a marine bacterium Pseudomonas nautica strain 617. could be impacted. Ann Inst Pasteur Microbiol 138:371−383 (4) Respiration under starved and well-fed condi- Bonin P, Barbotin JN, Dhulster P, Bertrand J (1987b) Nitrate tions of acetate- and pyruvate-based cultures of P. reduction in simulated microniches by a denitrifying marine bacterium. Can J Microbiol 33: 276−279 and show that rates of O con- nautica V. natriegens 2 Boucher G, Clavier J, Garrigue C (1994) Oxygen and carbon sumption and CO2 production during substrate-suffi- dioxide fluxes at the water-sediment interface of a tropi- ciency parallels the biomass increase (Fig. 1), but that cal lagoon. Mar Ecol Prog Ser 107: 185−193 58 Mar Ecol Prog Ser 519: 47–59, 2015

Bühring SI, Lampadariou N, Moodley L, Tselepides A, Witte crude oil in sandy beaches. Proc 1987 Oil Spill Conf. U (2006) Benthic microbial and whole-community re- American Petroleum Institute, Washington, DC, p sponses to different amounts of 13C-enriched algae: in 411−416 situ experiments in the deep Cretan sea (eastern Medi- Liu M, Xiao T, Sun J, Wei H, Wu Y, Zhao Y, Zhang W (2013) terranean). Limnol Oceanogr 51: 157−165 Bacterial community structures associated with a natural Burton WG (1982) Post-harvest physiology of food crops. spring phytoplankton bloom in the Yellow Sea, China. Longman, New York, NY Deep-Sea Res II 97:85−92 Cantarow A, Schepartz B (1967) Biochemistry, 4th edn. W. B. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Pro- Saunders, Philadelphia, PA tein measurement with the Folin phenol reagent. J Biol Chin RM, Fu X, Pai MY, Vergnes L and others (2014) The Chem 193: 265−275 metabolite α-ketoglutarate extends lifespan by inhibit- Lusk G (1928) The elements of the science of nutrition, 4th ing ATP synthase and TOR. Nature 510: 397−401 edn. WB Saunders, Philadelphia, PA del Giorgio PA, Pace ML, Fischer D (2006) Relationship of Martinez J, Smith DC, Steward GF, Azam F (1996) Variabil- bacterial growth efficiency to spatial variation in bacter- ity in ectohydrolytic enzyme activities of pelagic marine ial activity in the Hudson River. Aquat Microb Ecol 45: bacteria and its significance for substrate processing in 55−67 the sea. Aquat Microb Ecol 10: 223−230 Dilly O (2001) Microbial respiratory quotient during basal Mudryk Z, Donderski W (1997) The occurrence of heterotro- metabolism and after glucose amendment in soils and lit- phic bacteria decomposing some macromolecular com- ter. Soil Biol Biochem 33: 117−127 pounds in shallow estuarine lakes. Hydrobiologia Ducklow HW, Doney SC (2013) What is the metabolic state 342−343: 71−78 of the oligotrophic ocean? A debate. Annu Rev Mar Sci 5: Mudryk ZJ, Skórczewski P (2006) Enzymatic activity and 525−533 degradation of organic macromolecules by neustonic Ferguson SJ (2010) ATP synthase: from sequence to ring and planktonic bacteria in an estuarine lake. Pol J Ecol size to the P/O ratio. Proc Natl Acad Sci USA 107: 54: 3−14 16755−16756 Nelson DL, Cox MM (2005) Lehninger Principles of bio- Giering SLC, Sanders R, Lampitt RS, Anderson TR and oth- chemistry. WH Freeman and Company, New York, NY ers (2014) Reconciliation of the carbon budget in the Nissen H, Heldal M, Norland S (1987) Growth, elemental ocean’s twilight zone. Nature 507: 480−483 composition, and formation of polyphosphate bodies in Gnaiger E (1983) Calculation of energetic and biochemical Vibrio natriegens cultures shifted from phosphate-lim- equivalents of respiratory oxygen consumption. In: ited to phosphate-pulsed media. Can J Microbiol 33: Gnaiger E, Forstner H (eds) Polarographic oxygen sen- 583−588 sors. Springer-Verlag, Berlin, p 337−345 Niven DF, Collins PA, Knowles CJ (1977) Adenylate energy Gnaiger E (ed) (2009) Mitochondrial pathways and respira- charge during batch culture of Beneckea natriegens.J tory control, 2nd edn. OROBOROS MiPNet Publications, Gen Microbiol 98: 95−108 Innsbruck Obernosterer I, Christaki U, Lefèvre D, Catala P, Van González N, Anadón R, Viesca L (2003) Carbon flux through Wambeke F, Lebaron P (2008) Rapid bacterial mineral- the microbial community in a temperate sea during sum- ization of organic carbon produced during a phytoplank- mer: role of bacterial metabolism. Aquat Microb Ecol 33: ton bloom induced by natural iron fertilization in the 117−126 southern ocean. Deep-Sea Res II 55:777−789 Guyton AC (1971) Textbook of medical physiology, 4th edn. Osma N, Fernández-Urruzola I, Packard TT, Postel L, W. B. Saunders, Philadelphia, PA Gómez M, Pollehne F (2014) Short-term patterns of ver- Hoar WS (1975) General and comparative physiology, 2nd tical particle flux in northern Benguela: a comparison edn. Prentice Hall, Upper Saddle River, NJ between sinking POC and respiratory carbon consump- Holms WH (1986a) The central metabolic pathways of tion. J Mar Syst 140(B):150–162 Escherichia coli: relationship between flux and control at Oviatt CA, Rudnick DT, Keller AA, Sampou PA, Almquist a branch point, efficiency of conversion to biomass, and GT (1986) A comparison of system (O2 and CO2) and C- excretion of acetate. Curr Top Cell Regul 28: 69−105 14 measurements of metabolism in estuarine mesocosms. Holms WH (1986b) Evolution of the glyoxylate bypass in Mar Ecol Prog Ser 28:57−67 Escherichia coli —An hypothesis which suggests an Packard TT, Christensen JP (2004) Respiration and vertical alternative to the Krebs cycle. FEMS Microbiol Lett 34: carbon flux in the Gulf of Maine water column. J Mar Res 123−127 62: 93−115 Holms WH, Bennett PM (1971) Regulation of isocitrate Packard TT, Gómez M (2013) Modeling vertical carbon flux dehydrogenase activity in Escherichia coli on adaptation from zooplankton respiration. Prog Oceanogr 110: 59−68 to acetate. J Gen Microbiol 65:57−68 Packard TT, Williams PJ LeB (1981) Rates of respiratory oxy- Kader AA (1987) Respiration and gas exchange of vegeta- gen consumption and electron transport in surface sea- bles. In: Weichmann J (ed) Postharvest physiology of water from the northwest Atlantic. Oceanol Acta 4: vegetables. Marcel Dekker, New York, NY, p 25−43 351−358 King GM, Berman T (1984) Potential effects of isotopic dilu- Packard TT, Berdalet E, Blasco D, Roy SO and others (1996a) 14 tion on apparent respiration in C heterotrophy experi- CO2 production predicted from isocitrate dehydrogenase ments. Mar Ecol Prog Ser 19: 175−180 activity and bisubstrate enzyme kinetics in the marine Lee K (1995) Bioremediation studies in low-energy shoreline bacterium Pseudomonas nautica. Aquat Microb Ecol 11: environments. Proc 2nd Int Oil Spill Res Development 11−19 Forum. International Maritime Organization, 23−26 May Packard TT, Berdalet E, Blasco D, Roy SO and others 1995, London (1996b) Oxygen consumption in the marine bacterium Lee K, Levy EM (1987) Enhanced biodegradation of a light Pseudomonas nautica predicted from ETS activity and Romero-Kutzner et al.: Respiration quotient variability in bacteria 59

bisubstrate enzyme kinetics. J Plankton Res 18: Roy SO, Packard TT (2001) CO2 production rate predicted 1819−1835 from isocitrate dehydrogenase activity, intracellular sub- Payne WJ, Eagon RG, Williams AK (1961) Some observa- strate concentrations and kinetic constants in the marine tions on the physiology of Pseudomonas natriegens nov. bacterium Pseudomonas nautica. Mar Biol 138:1251−1258 spec. Antonie Leeuwenhoek 27: 121−128 Roy SO, Packard TT, Berdalet E, St-Amand L (1999) Impact Pettenkofer MJ, Voit K (1866) Untersuchungen über den of acetate, pyruvate, and physiological state on respira- Stoffverbrauch des normalen Menschen. Z Biol 2: tion and respiratory quotients in Pseudomonas nautica. 458−573 Aquat Microb Ecol 17: 105−110 R Development Core Team (2010) R: a language and envi- Sala MM, Güde H (2004) Ectoenzymatic activities and het- ronment for statistical computing. R Foundation for Sta- erotrophic bacteria decomposing detritus. Arch Hydro- tistical Computing, Vienna. www.r-project.org biol 160: 289−303 Reeves HC, Rabin R, Wegener WS, Ajl SJ (1971) Assays of Slavov N, Budnik BA, Schwab D, Airoldi EM, van Oude- enzymes of the tricarboxylic acid and glyoxylate cycles. naarden A (2014) Constant growth rate can be supported In: Norris JR, Ribbons DW (eds) Methods in microbiol- by decreasing energy flux and increasing aerobic glycol- ogy, Vol 6A. Academic Press, New York, NY, p 425−462 ysis. Cell Reports 7: 705−714 Reeves HC, Daumy GO, Lin CC, Houston M (1972) NADP+- Stanier M, Forsling M (1990) Physiological processes. specific isocitrate dehydrogenase of Escherichia coli. I. McGraw-Hill, London Purification and characterization. Biochim Biophys Acta Steinberg DK, Van Mooy BAS, Buesseler KO, Boyd PW, 258: 27−39 Kobari T, Karl DM (2008) Bacterial vs. zooplankton con- Riebesell U, Gattuso J, Thingstad TF, Middelburg JJ (2013) trol of sinking particle flux in the ocean’s twilight zone. Preface: Arctic ocean acidification: pelagic ecosystem Limnol Oceanogr 53: 1327−1338 and biogeochemical responses during a mesocosm study. Swannell RPJ, Lee K, McDonagh M (1996) Field evaluations Biogeosciences 10: 5619−5626 of marine oil spill bioremediation. Microbiol Rev 60: Robinson C, Williams PJ Le B (2005) Respiration and its 342−365 measurement in surface marine waters. In: del Giorgio Walsh K, Koshland DE Jr (1984) Determination of flux PA, Williams PJ le B (eds) Respiration in aquatic ecosys- through the branch point of two metabolic cycles. The tems. Oxford University Press, Oxford, p 147−180 tricarboxylic acid cycle and the glyoxylate shunt. J Biol Robinson C, Serret P, Tilstone G, Teira E, Zubkov MV, Rees Chem 259: 9646−9654 AP, Woodward EM (2002) Plankton respiration in the Warburg O (1926) Über die Wirkung des Kohlenoxyds auf Eastern Atlantic Ocean. Deep-Sea Res I 49:787−813 den Stoffwechsel der Hefe. Biochem Z 177: 471−486

Editorial responsibility: Antonio Bode, Submitted: April 4, 2014; Accepted: October 1, 2014 A Coruña, Spain Proofs received from author(s): December 11, 2014