E ff ect of CO 2 enrichment on bacterial production and respiration and on bacterial carbon metabolism in Arctic waters

Effect of CO2 enrichment on bacterial production and respiration and on bacterial carbon metabolism in Arctic waters C. Motegi, T. Tanaka, J. Piontek, C. P. D. Brussaard, J. P. Gattuso, and M. G. Weinbauer Université Pierre et Marie Curie-Paris 6, Laboratoire d’Océanographie de Villefranche, 06230, Villefranche-sur-Mer Cedex, France Laboratoire d’Océanographie de Villefranche, CNRS, UMR7093, 06230, Villefranche-sur-Mer Cedex, France Helmholtz Centre for Ocean Research Kiel (GEOMAR), Germany Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany Royal Netherlands Institute for Sea Research (NIOZ), Dept. of Biological Oceanography, BP 59, 1790 AB Den Burg, The Netherlands


Introduction
Bacteria are the main organisms that incorporate and mineralize dissolved organic carbon in the ocean, recycling about 50 % of daily primary production.Since bacteria take up carbon into anabolic and catabolic processes, measuring both bacterial production (BP) and respiration (BR) is crucial to estimate carbon metabolism components (e.g.bacterial carbon demand and bacterial growth efficiency) for understanding impact of bacteria on global marine carbon flux (del Giorgio and Cole, 2000;Robinson, 2008).A previous study suggests that bacterial carbon metabolism components are mostly con- limitation) (del Giorgio et al., 2011).However, only a few studies have focused on effect of environmental condition on bacterial carbon metabolism.
Recent studies have reported that the world ocean is absorbing about 25 % of atmospheric partial pressure of CO 2 (pCO 2 ) and that pCO 2 will increase from 280 to nearly 384 µatm over the next 250 yr (IPCC 2007).The increase in pCO 2 reduces ocean pH (ocean acidification), which may threaten calcifying organisms (e.g.Riebesell et al., 2000) and primary production (reviewed by Liu et al., 2010); however, few studies have focused on pCO 2 effects on bacterial metabolism.Previous studies have examined effect of pCO 2 on microbial communities and found that pCO 2 potentially alters bacterial production (Coffin et al., 2004;Grossart et al., 2006;Yamada et al., 2010), growth rate (Grossart et al., 2006), enzymatic activity (Grossart et al., 2006;Piontek et al., 2010;Yamada and Suzumura, 2010) and community structure (Allgaier et al., 2008;Yamada et al., 2010); other studies have found little or no effect of pCO 2 on bacterial production (Grossart et al., 2006;Allgaier et al., 2008;Arnosti et al., 2011), abundance (Rochelle-Newall et al., 2004;Grossart et al., 2006;Allgaier et al., 2008;Arnosti et al., 2011) or chromophoric dissolved organic matter (Rochelle-Newall et al., 2004).A recent review paper suggests that unlike calcifying organisms, the effect of pCO 2 on biogeochemical processes driven by microbes or microbial function in the oceans might be minor (Joint et al., 2011), however, there is also evidence that some functions such as nitrification and bacterial production can be changed, which would influence biogeochemical processes (Liu et al., 2011).However, there is no study on the pCO 2 influence on anabolic and catabolic processes of carbon by bacteria and on bacterial carbon metabolism components.
In the present study, a mesocosm experiment (Svalbard 2010 mesocosm experiment of the European Project on Ocean Acidification (EPOCA) project) was performed that was designed to determine the potential impact of changes in pCO 2 on BP, BR and bacterial carbon metabolism in Kongsfjorden, Svalbard.Particularly, we focused on how changes in pCO 2 may influence (1) bacterial cell production, ( 2 assimilated (i.e.bacterial growth efficiency, BGE), (4) amount of organic C assimilated by bacteria (i.e.bacterial carbon demand, BCD), and (5) the ratio of biomass produced to substrate assimilated (i.e.Leucine : Thymidine ratio) in the Arctic Ocean.

Experimental set-up and sample collection
The mesocosm experiment was conducted over a period of 30 days, between June 7 (day t0) and July 7 (day t30) 2010, at Kongsfjord, Svalbard (78 • 56,2 N, 11 • 53,6 E).Details of the mesocosm set-up are described by Riebesell et al. (2012).Briefly, nine Kiel off-shore mesocosms (KOSMOS) were deployed at t-10, and seven days after closing of the mesocosms, a stepwise addition of CO 2 saturated water was applied between t-1 to t4 to obtain 8 different level of CO 2 : 185 µatm (M3 and M7, 2 controls of no CO 2 saturated water addition mesocosms), 270 µatm (M2), 375 µatm (M4), 480 µatm (M8), 685 µatm (M1), 820 µatm (M6), 1050 µatm (M5) and 1420 µatm (M9).No further CO 2 manipulation was done after reached initial pCO 2 levels (for details see Riebesell et al., 2012).Due to gas exchange and photoautotrophic uptake, pCO 2 levels declined in the mesocosms during the experiment, and final levels of CO 2 were range from 160 to 855 µatm.At day 13 of the experiment (t13), inorganic nutrients (nitrate, silicate and phosphate: 5, 2.5, and 0.32 µmol l −1 , respectively) were added.Subsamples for BP and BR were obtained every 2 and 4 days.Water samples were collected using clean depth integrated sampler (12-l volume) at depths between the surface and 12 m for all mesocosms, transferred to 2-l polycarbonate bottle (Nalgen) and brought back to the laboratory.Containers and plastic wares used for the sampling were rinsed before use with 1.

Bacterial production (BP)
Bacterial production rates were determined from the incorporation rate of 3 H-thymidine (BP TdR , Kirchman 2001) and 14 C-leucine (BP Leu , for a detailed method description see Piontek et al., 2012) using a centrifuge method.Triplicate subsamples (1.5 ml) and 1 trichloroacetic acid (TCA)-killed control were spiked with [methyl-3 H] TdR (1.77 TBq mmol −1 , PerkinElmer, NET027W, final conc. 10 nM) and incubated for 1 h at 2 • C in the dark.Extraction by precipitations with 5 % cold TCA was followed by cold ethanol rinsing using a temperature controlled desktop centrifuge (18 000 × g at 4 • C for 10 min for each run; Sigma, 1-15 K).The extracts were then completely dried and mixed with scintillation cocktail (1 ml, Ultima Gold, PerkinElmer) for the radioassay using a Packard Tri-Carb 1600CA liquid scintillation counter with corrections for quenching.The coefficient of variations (CVs) of the triplicate measurement were 0 to 41 %.The 3 H-TdR incorporation rates were converted to cell production by the conversion factor 2 × 10 18 cells per mole of TdR (Fuhrman and Azam, 1982).Net bacterial growth rate (sBP, d −1 ) was estimated as BP TdR (cells l −1 d −1 ) divided by bacterial abundance (cells l −1 ).Bacterial production rates of free-living (BP Free ) were determined 3 H-TdR incorporation rates of 0.8-µm (Nucleopore, Millipore) filtered water, and of attached fraction (BP Att ) were estimated by BP Free subtracted from BP TdR .To estimate the Leucine (Leu; pmol Leu l −1 d −1 ) and TdR incorporation (pmol TdR l −1 d −1 ) ratio (the Leu : TdR ratio), BP Leu data was obtained from Piontek et al. (2012).

Bacterial respiration (BR)
BR was determined from the decrease of dissolved oxygen concentration during 48-h incubations of water samples (< 0.8 µm pore-size filter).Sample water was filtered through 0.8-µm filter (Nucleopore, Millipore) by applying a weak positive pressure (< 67 cm Hg) with an air pressure pump and dispensed into biochemical oxygen demand bottles (60-ml capacity).Dissolved oxygen concentration was determined by Winkler titration using an automated titrator with a potentiometric end-point detector 15217 Introduction

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Bacterial growth efficiency (BGE) and bacterial carbon demand (BCD)
BGE and BCD were estimated with the following equations: where BR (O 2 consumption rate was converted to C flux by assuming that the respiratory quotient = 1; del Giorgio and Cole 1998) and BP (TdR incorporation was converted to C flux by assuming a conversion factor of 20 fg C per cell, Cho and Azam, 1990) were estimated as described above.

Bacterial abundance
Bacterial abundance was determined by flow cytometer.Details are described in Brussaard et al. (2012).

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Full a substantial increase (2.4 to 5.5-fold) at t10 in all treatments (Fig. 1a).Although there was no pronounced enhancement by nutrient addition, BP TdR gradually increased towards the end of the experiment (Fig. 1a).In contrast to BP TdR , BR did not vary greatly and no clear pattern was observed during the experiment (Fig. 1b).sBP and sBR at the beginning of experiment ranged between 0.16 ± 0.02 to 0.22 ± 0.01 d −1 and 5.43 ± 6.47 to 23.55 ± 6.76 fg C cell −1 d −1 , respectively (Fig. 2a, b).sBP gradually increased after closing the mesocosms and showed a substantial increase at t10; however, after nutrient addition sBP gradually decreased towards the end of the experiment (Fig. 2a).In contrast to BR, sBR did not show any clear pattern during the experiment (Fig. 2b).

Temporal variations of BP Free and BP Att
The percentage of BP Free dominated in all mesocosms except at t3 and t20 (Fig. 4).Averaged % of BP Free at t-1, t7, t16, t24 and t28 was 89 ± 22 % (± SD), while the average % of BP Free at t3 and t20 was 58 ± 11 %.At t3 (the end of pCO 2 manipulation) and t20 (the second Chlorophyll a peak, Schulz et al., 2012), percentage of BP Att ranged from 24-56 and 23-56 % (Fig. 4b, e).The percentage of BP Att of CO 2 manipulated mesocosms tended to be lower than control mesocosm at t3. BP Free was significantly influenced by changes in pCO 2 at t24 (Linear regression: r 2 = 0.67, p < 0.05, n = 9).

Temporal variations of Leu : TdR ratio, BGE and BCD
The Leu : TdR ratio averaged 10.4 ± 4.05 (range 3.74 to 14.7) at the beginning of experiment, and it increased to 25.8 ± 8.10 (range 15.1 to 35.2) on t5.However, the ra-Introduction

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BGE and BCD were estimated based on TdR and Leu incorporation rate.BGE Leu and BGE TdR at the beginning of experiment ranged between 2 ± 1 to 18 ± 2 % and 13 ± 4 to 31 ± 5 %, respectively, and varied greatly from 3 to 61 % and 4 to 75 %, respectively, during the experiment (Fig. 6).BCD Leu and BCD TdR at the beginning of experiment ranged between 15.0 ± 2.5 to 52.4 ± 14.8 µg C l −1 d −1 and 18.4 ± 2.8 to 58.7 ± 15 µg C l −1 d −1 , and varied from 3.5 to 46.6 µg C l −1 d −1 and 2.6 to 47.2 µg C l −1 d −1 (Fig. 7).There were significant relations between BGE Leu and BGE TdR and between BCD Leu and BCD TdR during the experiment (Liner regression, BGE; r 2 = 0.825, p < 0.001, n = 37, BCD; r 2 = 0.983, p < 0.001, n = 37), but no obvious trends were observed by the effect of pCO 2 or time of experiment.(HDNA) declined, thus, suggesting that this decline was most likely due to viral lysis (Brussaard et al., 2012).Middelboe et al. (1996) demonstrated that viral lysates support growth of non-infected bacteria and this indicates that viral lysates are potentially labile compounds.Our results suggest that decline of BP TdR and bacterial abundance potentially attributed by viruses until t7 and viral lysates might enhance BP TdR at t10.Furthermore, peaks of chlorophyll a (Schulz et al., 2012), picophytoplankton II and nanophytoplankton I (Brussaard et al., 2012) were found at t6 to t8 and t4 to t6.In addition to the effect of viral lysates of bacteria, these results suggest that phytoplankton exudation of carbon and viral lysates of nanophytoplankton could have also stimulated BP TdR at t10.In support of this notion, average Leu : TdR ratio decreased from 25.8 ± 8.10 at t5 to 7.81 ± 3.09 at t10 potentially as a consequence of labile dissolved organic matter release by phytoplankton (i.e.balanced growth, see discussion below).Moreover, viral lysis of nanoplankton blooms accounts for a portion of our BCD (e.g.6-28 %, Brussaard et al., 2012).Interestingly, the bacterial diversity index determined by T-RFLP showed that species richness and diversity index increased during pCO 2 manipulation and decreased at t10 when we observed the peak of BP TdR (Zhang et al., 2012).This suggests that active bacteria might have dominated at t10 of incubation.After nutrient addition at t13, chlorophyll a (Schulz et al., 2012) showed 2 peaks, while BP TdR exhibited a gradual increase.However, this pattern was inconsistent and could be due to changes in bacterial community composition after nutrient addition (Zhang et al., 2012).
Furthermore, after nutrient addition, bacterial abundance increased and reached its maximum at the end of experiment (Brussaard et al., 2012), while net bacterial growth rate gradually decreased.This discrepancy might be attributed to lower bacterial losses rather than increased gross bacterial growth.Although high viral abundance was observed at end of experiment (Brussaard et al., 2012) the percentage of BP Att increased and reached a percentage similar to BP Free at t20).Allgaier et al. (2008) reported similar rates of free-living and attached bacterial production, both of which were tightly coupled to a phytoplankton bloom.Although BP Leu was positively correlated with primary production in the present study (Piontek et al., 2012), no coupling between phytoplankton and rates of free-living and attached bacterial production was observed.Hence, temporal variation of BP Free and BP Att was potentially uncoupled from phytoplankton, e.g.due to grazing or viral lysis (Brussaard et al., 2012).
Our data on BR are within the range reported for the western Arctic Ocean (Kirchman et al., 2009).In contrast to BP TdR , BR and sBR did not show any trend during the experiment.L ópez-Urrutia and Mor án (2007) found that bacterial respiration and production responded similarly to changes in temperature, while an extensive dataset of concurrent measurement of bacterial production and respiration revealed that bacterial respiration is much less variable than production across marine systems (del Giorgio and Cole, 2000).Our results are consistent with the general pattern of a small variability of bacterial respiration.

Effect of pCO 2 on bacterial carbon metabolism
We focused on day-to-day results rather than dividing the experimental period into 4 phases (other papers, this issue) because averaged results did not show trends for microbial parameters and their relationship to pCO 2 concentrations.
BP TdR was significantly correlated with pCO 2 at t5.This indicates that pCO 2 enhanced BP TdR rapidly after the pCO 2 manipulation, which is consistent with Grossart et al. (2006) that bacterial production was enhanced in the highest pCO 2 mesocosm (700 ppmV).However, although no pronounced enhancement of BP TdR by addition of nutrient was found in the present study, BP TdR decreased with increasing pCO 2 at t14.Suppression of bacterial production by the effect of an increase in pCO 2 or decrease in pH was previously reported in experiments using deep sea water (Coffin et al., 2004;Yamada et al., 2010).Yamada et al. (2010) stimulated acidification through enrichments with high CO 2 air or artificial chemical buffers, and found suppression of 15222 Introduction

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Full prokaryotic activities at lower pH (pH ≤ 7.0).This suppression was more profound under the enrichment treatment with artificial chemical buffers, perhaps because artificial chemical buffers contain organic matter.Consistent with this, our results indicate that the effects of changes in pCO 2 on BP TdR were potentially linked to nutrient status.
In addition to BP TdR , the Leu:TdR ratio was significantly influenced by changes in pCO 2 .The Leu : TdR ratio is the indicator of the relative importance of protein and nucleic acid synthesis and it may reflect the balance of bacterial growth (Chin-Leo and Kirchman, 1988;Kirchman, 1992;Gasol et al., 1998;Ducklow, 2000;del Giorgio et al., 2011).In our experiment, the initial Leu : TdR ratio was relatively low (i.e.10.3 ± 4.3, average ± SD) compared to the whole water column (0 to 80 m) average from May to September in the subarctic Pacific (i.e.16.8, n = 481; Kirchman, 1992); however, the average ratio in the low (M3, 7, 2), medium (M4, 8, 1) and high (M6, 5, 9) pCO 2 mesocosms were slightly enhanced (i.e.low : 13.1, medium : 12.3, high : 15.1) during the experiment.In particular, the Leu:TdR ratio decreased with increasing pCO 2 concentration at t5 and t7 but this trend changed at end of the experiment.Previous studies have suggested that under favorable environmental conditions (e.g.rich in organic matter or temperature increase), bacteria might optimize DNA duplication over protein synthesis to maximize reproduction (i.e.balanced growth), resulting in a decline in the Leu : TdR ratio.But, under unfavorable environmental conditions (i.e.unbalanced growth), the Leu : TdR ratio would increase because bacteria might concentrate on biomass accumulation rather than reproduction to maximize the chance of survival (Shiah and Ducklow, 1997;Gasol et al., 1998).In this regard, our result imply that after the pCO 2 manipulation, bacterial growth became more unbalanced with increasing pCO 2 ; however this trend changed and bacterial growth became more balanced towards the end of experiment.Previous mesocosm studies showed that the Leu : TdR ratio was more likely associated with algal bloom rather than pCO 2 (Grossart et al., 2006;Arnosti et al., 2011), and a connection between primary production and BP Leu was found in this experiment (Piontek et al., 2012;Engel et al., 2012); however, we did not observe a significant connection between the Leu : TdR ratio and phytoplankton Figures

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Full in our experiment.Changes of pCO 2 can modify the quality and quantity of dissolved organic matter production by phytoplankton (Engel et al., 2004), therefore bacterial activity could be indirectly influenced by changes in pCO 2 (Robinson, 2008).These studies suggest that changes in pCO 2 potentially alter bacterial production and balance of bacterial growth in this study.
The effect of pCO 2 on bacterial respiration, BGE and BCD of bulk community was examined for the first time in the present study; however, no clear trend was observed.Teira et al. (2012) examined effect of CO 2 on 2 bacterial strains, Roseobacter and Cytophaga, and demonstrated that respiration of Cytophaga was significantly lower and BGE was higher in the elevated CO 2 treatment (1000 ppmV) than control (380 ppmV), while Roseobacter did not show any trend.Their study showed that different bacterial strains responded differently to CO 2 ; however, bacterial community structure varied during the experiment (i.e. one month, Zhang et al., 2012), so our measurement of bacterial carbon metabolism of whole community potentially hid the effect of pCO 2 on different strains.Although bacterial respiration does not vary greatly compared to production (del Giorgio and Cole, 2000), previous studies have shown that there is a significant relationship between temperature and bacterial respiration (del Giorgio and Cole, 2000;Robinson, 2008) or BGE (Rivkin and Legendre, 2001).Since ocean acidification is expected to occur concurrently with temperature increase, further studies are required to examine the combined effect of pCO 2 and temperature on bacterial respiration, BCD, BGE and total carbon flux through bacteria.

Summary
The goal of our study was to determine the potential impact of changes in pCO 2 on bacterial production, respiration and carbon metabolism in the Arctic water.In the present study, we found changes in pCO 2 influenced bacterial cell production, bacterial production of the free-living community, net growth rate and Leu : TdR ratio during mesocosm experiment.On the contrary, no clear trend of the effect of pCO 2 on bacterial BGD Full  Full nected and potentially influenced by environmental condition (i.e.temperature, energy Discussion Paper | Discussion Paper | Discussion Paper | ) bacterial respiration, (3) the amount of new bacterial biomass produced per unit of organic C substrate Discussion Paper | Discussion Paper | Discussion Paper | 2 N HCl followed by vigorous rinsing with Milli-Q water.During sample collection and handling, gloves were worn, and care was taken to minimize contamination.Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | variations of BP TdR and BR BP TdR decreased after closure of the mesocosms but strongly increased at t10 in all mesocosms.Concomitantly with BP TdR , abundance of high nucleic acid bacteria Discussion Paper | Discussion Paper | Discussion Paper | , viral mediated bacterial mortality declined towards the end of experiment (M.G. Weinbauer, personal communication, 2012), suggesting that bacteria might become resistant to viral infection.The free-living community usually dominated during the experiment.Temporal variations of free-living and attached bacterial production were found in pCO 2 manipulated experiments (i.e. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |
respiration, BGE and BCD was observed.Overall results suggest that although there was temporal variation, changes in pCO 2 potentially influence bacterial production and growth balance rather than the conversion of dissolved organic matter into CO 2 .Introduction