Journal of Marine Research, 65, 545–560, 2007
Mismatch between community respiration and the
contribution of heterotrophic bacteria in the NE Atlantic
open ocean: What causes high respiration in
oligotrophic waters?
by Xosé Anxelu G. Morán1, Valesca Pérez2 and Emilio Fernández2
ABSTRACT
Heterotrophic bacteria have long been considered as the major respirers of the open ocean. During
a survey in the eastern N Atlantic Subtropical Gyre we measured 3H-leucine heterotrophic bacterial
production (BP), 14C particulate primary production (PPP) and the O2 community metabolism rates:
gross primary production (GPP), net community production (NCP) and community respiration (CR).
We estimated heterotrophic bacterial respiration (BR) from BP and literature models of bacterial
growth efficiency [BGE ⫽ BP/(BP ⫹ BR)]. BP was an order of magnitude lower than PPP
(integrated means of 17 ⫾ 2 and 207 ⫾ 29 mg C m-2 d-1, respectively). Although volumetric PPP and
GPP were significantly correlated (r ⫽ 0.51; p ⫽ 0.009; n ⫽ 25), CR (1120 ⫾ 122 mg C m-2 d-1) bore
no significant relationship with either primary or heterotrophic bacterial production and exceeded
GPP (578 ⫾ 117 mg C m-2 d-1) at 5 out of 6 sampling stations. Integrated values of CR were only
significantly correlated, but negatively, with chlorophyll a values (r ⫽ ⫺0.95; p ⬍0.001; n ⫽ 6).
Integrated BR tended to decrease with increasing CR and, with a mean estimated BGE of 6%, it
accounted on average for a much lower fraction of CR (33 ⫾ 7%) than currently assumed. By
estimating the contributions of other trophic groups a large amount (48 ⫾ 10%) of the measured CR
remained unaccounted for. This gap in respiration estimates casts some doubt on the magnitude of the
net heterotrophic balance frequently observed in oligotrophic waters from changes of O2 over 24 h
dark in vitro incubations.
1. Introduction
Determination of plankton-mediated biogeochemical fluxes remains a priority task for
biological oceanographers in the context of global change, especially in oceanic areas
poorly represented in sampling strategies. The balance between biological production and
consumption of organic matter in the upper layers of the ocean is an essential component of
models aiming at resolving the global cycling of elements. This metabolic balance in the
open ocean – summarized by the variable net community production (NCP) as the result of
gross primary production (GPP) minus community respiration (CR) – has generated one of
1. Instituto Español de Oceanografı́a, Centro Oceanográfı́co de Xixón, Camı́n de L’Arbeyal, s/n E-33212
Xixón, Spain. email: [email protected]
2. Facultad de Ciencias, Universidad de Vigo, Campus Lagoas-Marcosende, E-36200 Vigo, Spain.
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the most active debates in biological oceanography of the last years (Geider, 1997; del
Giorgio et al., 1997; Williams, 1998; Duarte and Agustı́, 1998). A focal geographical
location for this controversy has been the N Atlantic Subtropical Gyre, characterized by
very low values of phytoplankton biomass and production (Marañón et al., 2000; Teira et
al., 2005). The paucity of measurements extended over long periods and hence, our
inability to determine the scales of coupling between GPP and CR, has apparently resulted
in an overestimation of net heterotrophic periods relative to net autotrophic or balanced
ones (Karl et al., 2003). However, research in the eastern N. Atlantic has systematically
found that CR exceeds GPP for most of the year (Duarte et al., 2001; González et al., 2002;
Serret et al., 2001; Robinson et al., 2002a) apparently rendering this region a subsidized
system in terms of organic carbon loading.
In spite of the slowly growing dataset of oceanic CR data, considerable uncertainty on its
magnitude remains (Hansell et al., 2004), which becomes even larger when evaluating the
possible responses to global change (del Giorgio and Duarte, 2002). In autumn 2001 we
performed a cruise that crossed the subtropical and tropical provinces of the N. Atlantic
Subtropical Gyre (Longhurst, 1998) and the results add to the above-mentioned empirical
basis for a negative NCP in this region (Morán et al., 2004). Yet, for NCP to be a good
descriptor of the role of biota in biogeochemical carbon cycling we must be confident of its
two components. In spite of the well-known uncertainties associated to the radiocarbon
uptake technique, GPP can be compared with simultaneous measurements of 14C-based
photosynthesis (Bender et al., 1999; Laws et al., 2000), but CR is more difficult to
constrain with other variables. However, an attempt can be made from measured rates of
activity of heterotrophic and autotrophic plankton and published growth efficiencies of
different trophic groups (Robinson et al., 2002a, b; Robinson and Williams, 2005). This
comparative exercise has yielded estimated CR values within the ⫾50% range of measured
rates, hence supporting the validity of the approach despite the uncertainties associated to
each respiration estimate.
We focus here on the relationship between the respiration of the whole planktonic
community and heterotrophic bacterial respiration (BR), as estimated from measured
heterotrophic bacterial production (BP) and from computed bacterial growth efficiency
[BGE ⫽ BP/(BP⫹BR)] values based on empirical models. The rationale of this comparison is that heterotrophic bacteria have been frequently considered as the main respirers of
open-ocean microbial communities (e.g., Williams, 1981; Pomeroy et al., 1995; Rivkin
and Legendre, 2001). The demand for a better knowledge of oceanic CR and the need to
enlarge the as yet scarce dataset available (Williams and del Giorgio, 2005) must be
accompanied by periodic assessments of the reliability of the techniques used. If, as we
show here, BR turns out to be much lower than CR, it can be due to an underestimation of
BR, an overestimation of CR or both. Each type of measurement has its own assumptions
and possible sources of error, but most importantly, BR and CR were obtained with
substantially different incubation periods. We relate our findings with the known effect of
long incubations on enclosed microbial plankton communities. Our results show that the
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Morán et al.: Mismatch between community & bacterial respiration
547
absolute values of CR measured in this area may be difficult to reconcile with independent
estimates of the contribution of the various microbial plankton groups.
2. Material and methods
Data of community metabolism, particulate primary production and bacterial activity
were collected in successive days at 5 to 7 depths of 6 stations extending from 29 to 10N in
autumn 2001 during the cruise CIRCANA-I on board the R/V Hespérides. More information on hydrography, size-fractionated chlorophyll a and the abundance of microbial
plankton can be found in Morán et al. (2004). A short description of the most relevant
findings is included in Section 3.
Gross primary production (GPP), net community production (NCP) and dark community respiration (CR) were determined from in vitro changes in dissolved oxygen after light
and dark bottle incubations over 24 h. At each depth 4 replicate 120 mL borosilicate bottles
were fixed immediately for initial O2 concentrations, 4 bottles were kept in the dark and 4
bottles were incubated under in situ-simulated irradiance at surface temperature in an on
deck incubator. After the incubation, dissolved O2 concentration was measured with an
automated Winkler titration system using a potentiometric end point (Pomeroy et al.,
1994). Aliquots of fixed samples were delivered with a 50-ml overflow pipette. Production
and respiration rates were calculated from the difference between the averaged light and
dark replicates and time zero analyses: NCP ⫽ measured ⌬O2 in light bottles (mean 24 h
[O2] – mean initial [O2]); CR ⫽ measured ⌬O2 in dark bottles (mean initial [O2] – mean
24 h [O2]); GPP ⫽ NCP ⫹ CR. Since all bottles were flushed with surface seawater, a Q10
of 2 was applied for correcting rates of deep samples, which were incubated at higher
values than their in situ temperature. Although the choice of photosynthetic (PQ) and
respiratory (RQ) quotients for converting oxygen to carbon units may result in different
estimates, this is a minor source of error (Williams and del Giorgio, 2005). For comparative reasons we used a PQ of 1.25 and a RQ of 0.89 (Robinson et al., 2002a; Robinson and
Williams, 2005). The comparison of CR measurements with estimates of respiration of
bacteria and other planktonic trophic groups were made in C units.
The rate of particulate primary production (PPP) was estimated with the 14C method as
the sum of the production in three size-fractions (⬎20, 2 - 20, ⬍2 ␮m). Three clear plus
one black 70 ml polypropylene bottles were filled with seawater collected from 5 - 6 depths
from the surface down to the 1% irradiance level. After addition of 740 KBq of NaH14CO3
to each bottle, they were incubated for 5-7 h starting at ⬃1100 local time in the same
incubator used for oxygen measurements. After incubation, samples were vacuum-filtered
at ⬍50 mm Hg and collected onto polycarbonate filters. Inorganic 14C was eliminated after
exposure to concentrated HCl fumes for 12 h. Filters were placed into vials and 4 ml of
scintillation cocktail was added before measurement of radioactivity in a LKB Winspectral
1414 liquid scintillation counter. Disintegrations per minute (dpm) were converted into carbon
units by using a constant concentration of dissolved inorganic carbon of 25700 mg C m-3 and
an isotope discrimination factor of 1.06. The same temperature correction used for oxygen was
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Table 1. Details on the calculations of the contribution of each trophic group to total community
respiration discussed in the text. *Calculations shown in Table 2. BP, bacterial production. BGE,
bacterial growth efficiency. chl a, chlorophyll a. Temp, temperature. PPP, particulate primary
production. GPP, gross primary production.
Trophic group
Bacteria
Phytoplankton
Bacterivores
Herbivores
Method
Resp ⫽ (BP/BGE) – BP
BGE ⫽ 1 – 1/[0.73 ⫻ chl a/
(chl a ⫹ 4.08) ⫹ 1.02)] *
BGE ⫽ 0.037 ⫹ (0.65 ⫻ BP)/
(1.8⫹BP)
BGE ⫽ 0.374 – 0.0104 ⫻ Temp
Resp ⫽ 0.40 ⫻ PPP *
Resp ⫽ 0.35 ⫻ GPP
Resp ⫽ BP ⫻ 0.80 ⫻ 0.75 *
Resp ⫽ 0.80 ⫻ PPP ⫻ 0.80 ⫻ 0.75
*
Reference
López-Urrutia and Morán, 2007
del Giorgio and Cole, 1998
Rivkin and Legendre, 2001
Marra and Barber, 2004
Duarte and Cebrián, 1996
Zubkov et al., 2000; Nagata, 2000;
Robinson et al., 2002a; Straile, 1997
Quevedo and Anadón, 2001; Robinson
et al., 2002a; Straile, 1997
applied to deep, lower temperature samples. Daily values were obtained by multiplying hourly
rates by total hours of sunlight.
The net production of heterotrophic prokaryotes, commonly termed bacterial production
(BP) although the method does not distinguish between Bacteria and Archaea, was
estimated from 3H-leucine incorporation rates as described in Smith and Azam (1992).
Four replicates ⫹ 2 immediately killed controls of 1 mL samples were inoculated with
50 nmol 3H-leucine and incubated for 1 - 2 h in the dark at surface temperature. We
performed three extra experiments at station 6 comparing light and dark incubations and
found that incubation in the light resulted in slightly higher leucine incorporation rates in
two out of three experiments, with an overall mean value of 15% ⫾ 13%. Leucine
incorporation rates were corrected for differences of temperature following Zubkov et al.
(2000). To convert Leu to C units, we used a value of 0.73 kg C mol Leu-1, which was the
average of three empirical determinations carried out in surface waters on two different
cruises crossing the region (see Morán et al. (2004) for details). The use of a single factor
for all data is justified since the three original conversion factors were rather similar (0.80,
0.95 and 0.46 kg C mol Leu-1) and clearly well below the theoretical ones (1.55 or
3.1 kg C mol Leu-1). Two additional determinations during those cruises carried out at 150
and 250 m, 0.50 and 0.73 kg C mol Leu-1, respectively, give further support to the use of a
common factor for all depths. Since bacterial heterotrophic respiration (BR) was not
measured in the cruise, we calculated it as BP/BGE – BP. There are several models
available in the literature that predict bacterial growth efficiency (BGE) from other
physical or biological variables (Table 1). When applied to our data, individual BGE
values obtained with these models ranged from 2.1 to 23.8%. The specific choice for
carbon budget calculations is discussed below.
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Morán et al.: Mismatch between community & bacterial respiration
549
Table 1 also includes the calculations used to estimate the contribution of other
planktonic trophic groups to total community respiration. Although incubations with the
14
C method for estimating PPP yields values somewhere between gross and net primary
production, there is experimental evidence relating PPP and GPP measurements with
corresponding phytoplankton respiration. Phytoplankton (including Prochlorococcus and
Synechococcus cyanobacteria) was thus assumed to respire 40% (2 x 20%) of daylight PPP
when exposed to a 12:12 h light:dark period (Marra and Barber, 2004), or 35% of GPP
(Duarte and Cebrián, 1996). For bacterian grazers we assumed that they consume all the
daily BP (Zubkov et al., 2000), that 20% of the assimilated carbon is egested (Nagata,
2000; Robinson et al., 2002a) and that they use the remaining fraction with a gross growth
efficiency of 25% (Straile, 1997). The contribution of herbivores was made on the
observation that they take up on average 80% of daily PPP in the suptropical Atlantic
(Quevedo and Anadón, 2001) assuming the same egested fraction and growth efficiency as
for the bacterivores.
Stratification is a distinct property of the eastern N. Atlantic upper ocean, so this
ecosystem might be better split into two self-contained layers, the mixed layer and that
below the thermocline. However, our analysis would be seriously compromised by
restricting it to the upper mixed layer, with only two to three depths sampled within it.
Integrated values are, therefore, given for the photic layer (1% of surface irradiance),
which ranged from 60 to 120 m during this survey. This also allows a direct comparison
with most previous reports for the region (Duarte et al., 2001; Serret et al., 2001; Agustı́ et
al., 2001; González et al., 2002).
3. Results and discussion
In a strongly stratified water-column, with a 4.7 to 15.1°C difference between the surface and
100 m depth, both integrated chlorophyll a and particulate primary production (PPP) were
consistently low, with respective ranges of 16-28 mg m-2 and 135-321 mg C m-2 d-1. PPP
measurements for the region usually do not exceed 300 mg C m-2 d-1 except in winter
(Marañón et al., 2000; Teira et al., 2005). Picoplanktonic organisms were the dominant
contributors to total phytoplankton biomass (70%) but not production (38%, cf. 43%
nanoplankton, Morán et al., 2004). Differential picophytoplanktonic contributions to
biomass and production are a persistent feature of these waters (Fernández et al., 2003).
The abundance of heterotrophic bacteria in the photic layer averaged 9.4 ⫾ 1.3 105 cells
mL-1 with most of the cells belonging to the low nucleic acid (LNA) group (55 ⫾ 2%),
suggesting a low overall activity of the community (Gasol et al., 1999). Indeed, integrated
heterotrophic bacterial production (BP) was on average one order of magnitude lower than
PPP (15 ⫾ 2 mg C m-2 d-1). BP values were somewhat lower than those found in previous
surveys conducted farther north (González et al., 2001; Teira et al., 2003) or in the Atlantic
Meridional Transects (AMT) 3 and 4 (Zubkov et al., 2000), but similar to the AMT-11
(Pérez et al., 2005). The large difference between BP and PPP, in agreement with previous
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work (Hoppe et al., 2002; Teira et al., 2003), was also reflected in the estimated biomass of
both planktonic compartments with a phytoplankton:heterotrophic bacteria biomass ratio
of 1.32 ⫾ 0.16, using the C to chl a empirical model of Marañón et al. (2000). Our CR
values (104.9 ⫾ 11.4 mmol O2 m-2 d-1) were in good agreement with previous measurements (González et al. 2001, 2002; Robinson et al., 2002a; Serret et al., 2001, 2002),
confirming a relative constancy at ⬃ 100-150 mmol O2 m-2 d-1. CR greatly exceeded GPP
at all but one station, with a mean negative NCP of -43.6 ⫾ 14.9 mmol O2 m-2 d-1. CR was
also less variable (CV⫽27%) than GPP (CV-50%) or NCP (CV⫽83%), as found by other
studies (Duarte and Agustı́, 1998; Serret et al., 2001; Arı́stegui and Harrison, 2002;
Robinson and Williams, 2005). Therefore, our community metabolism data support the
overall net heterotrophic nature of open-ocean, oligotrophic waters of the N. Atlantic noted
by these and other researchers (Agustı́ et al., 2001).
A comparison of pooled volumetric data of community metabolism and particulate
primary production expressed in carbon units is shown in Figure 1. GPP was higher than
CR at only 3 out of 26 determinations (Fig. 1A). Although in situ primary production rate
has been claimed as one of the main drivers of CR (del Giorgio et al., 1997; del Giorgio and
Duarte, 2000) no significant correlation was found between PPP or GPP and CR, either for
volumetric (Fig. 1A) or integrated units (data not shown) in our dataset. The uncoupling
between primary production and respiration has also been reported by Arı́stegui and
Harrison (2002) for a region farther north. Being a related variable, NCP was as expected
significantly correlated with GPP (Fig. 1B, r ⫽ 0.60; p ⫽ 0.001; n ⫽ 25). Likewise,
volumetric GPP was also significantly and positively correlated with independent measurements of PPP (Fig. 1C, r ⫽ 0.51; p ⫽ 0.009; n ⫽ 25), although it exceeded it 3-fold on
average, similar to the findings of Pérez et al. (2005) for the equatorial Atlantic. Both
methods of estimating primary productivity often yield similar differences (Bender et al.,
1999; Laws et al., 2000; Robinson et al., 2002a). With a PQ of 1.25, the mean molar ratio
of PPP to GPP was 0.57 ⫾ 0.10, slightly higher to that obtained in other open ocean studies
using a PQ of 1.2 (Bender et al., 1999; Laws et al., 2000; Pérez et al., 2005). Among the
causes explaining this discrepancy are the high rates of DOC production in oligotrophic
waters (Karl et al., 1998), which may make up to 60% of total photosynthetic carbon
fixation. Dissolved primary production would be measured as GPP but obviously not in
PPP. In addition, phytoplankton respiration is included in GPP but not in PPP determinations. Although the fraction of primary production directly usable by bacteria was not
measured, PPP was still significantly correlated with BP (see Fig. 8A in Morán et al.,
2004), as expected from a direct dependence of heterotrophic bacterial growth on recently
fixed photosynthate.
Individual heterotrophic bacterial production rates ranged from 0.002 to 0.69 mg C m-3 d-1,
with less variable integrated values (10.4-22.5 mg C m-2 d-1). Using López-Urrutia and
Morán (2007) model of BGE (Table 1), mean integrated carbon demand (BP⫹BR) was
303 ⫾ 28 mg C m-2 d-1, more than the estimated supply of recent photosynthate (Morán et
al., 2004). Most of the carbon taken up by heterotrophic bacteria was respired, with the
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Morán et al.: Mismatch between community & bacterial respiration
551
Figure 1. Relationship between 14C- and O2-based volumetric measurements of primary production
and respiration at the 6 stations, using a PQ of 1.25 and a RQ of 0.89. Notice the different scale of
the Y-axis in the 3 plots. Continuous lines represent 1:1 values and dashed lines represent
significant correlations.
corresponding BR ranging more than two-fold from 189 to 453 mg C m-2 d-1 (Table 2). The
relationship between BR and CR was just the opposite of that expected (Fig. 2A), i.e., BR
decreased with CR, although the correlation was not significant due to the small number of
samples (r ⫽ ⫺0.64; p ⫽ 0.17; n ⫽ 6). Mean ⫾ SE contribution of heterotrophic
bacterioplankton to total respiration was 33 ⫾ 7% (Fig. 2B, Table 2). One could argue that
the choice of BGE seriously influenced our analysis. In fact, there is appreciable
disagreement between different BGE models (Table 1). Using the model of del Giorgio and
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Table 2. Estimated respiration rates and relative contribution to total community respiration (among
parentheses, %) of different plankton trophic groups at the 6 stations. Details and assumptions of
the calculations are given in Table 1. Mean ⫾ SE are also given. ?: unaccounted for.
Station
1
2
3
4
5
6
Mean ⫾ SE
Community
respiration
Bacteria
1407
762
762
1415
1251
1126
1120 ⫾ 122
(100)
268 (19.0)
453 (59.4)
370 (48.5)
357 (25.2)
189 (15.1)
346 (30.7)
330 ⫾ 37
(33.0 ⫾ 7.1)
Phytoplankton Bacterivores
(mg C m-2 d-1)
54 (3.8)
57 (7.5)
128 (16.8)
87 (6.1)
99 (7.9)
73 (6.5)
83 ⫾ 11
(8.1 ⫾ 1.8)
6 (0.4)
9 (1.1)
13 (1.7)
10 (0.7)
8 (0.6)
10 (0.9)
9⫾1
(0.9 ⫾ 0.2)
Herbivores
?
65 (4.6)
68 (9.0)
154 (20.2)
104 (7.4)
118 (9.5)
87 (7.7)
99 ⫾ 47
(9.7 ⫾ 2.2)
1014 (72.1)
175 (23.0)
97 (12.7)
858 (60.6)
838 (66.9)
610 (54.1)
599 ⫾ 156
(48.2 ⫾ 10.0)
Cole (1998), a weighted BGE value of 2.4% was obtained. A higher value (12.3%) was
obtained with the model of Rivkin and Legendre (2001). The model we used (LópezUrrutia and Morán, 2007) lay in between these two widespread models (Fig. 2B), with a
weighted BGE value of 5.8%. Broad empirical relationships may not be representative of
small-scale studies such as this one, but even with the range of possible BGE values shown
in Figure 2B the differences between BR and CR persist. BGE values rarely exceed 20% in
open ocean oligotrophic waters (del Giorgio and Cole, 1998). In a recent survey in the NE
Atlantic, Alonso-Sáez et al. (2007) found an average BGE for offshore stations of 9%, very
similar to the mean they obtained in a review for oligotrophic open ocean waters (12%,
range 1-43%). Therefore, we feel that the range of BGE values used (4.4-7.2%) was
reasonable for constraining the upper limit of BR in the study region. Any higher BGE
would obviously further decrease the share of heterotrophic bacteria to CR shown in Figure 2B.
Hence, contrary to the analysis of Robinson et al. (2002a), who estimated a four-fold
greater BP from bacterial abundances and subsequent application of the del Giorgio and
Cole (1998) model, our results strongly indicate that the bulk CR in the region was not
readily attributable to BR. In order to arrive at an average BR:CR ratio of ⬃0.90, a rough
average of the estimation given by Robinson et al. (2002a) for these waters, the mean BGE
should have been as low as 1.3%. Yet, it seems difficult to accept that heterotrophic
bacteria respire 99% of the assimilated organic carbon for long periods of time.
The mismatch between BR and CR lead us to look for other possible contributions to the
measured respiration rates as shown in Table 2. We decided to estimate phytoplankton
respiration with the independently measured PPP rather than a variable that results from
the same variable of interest (GPP⫽NCP⫹CR). Respiration by autotrophs thus amounted
to a mean 8% of CR (Table 2), and a maximum of 17%. Based on GPP measurements,
mean ⫾ SE phytoplankton contribution to CR (data not shown) would have been 20 ⫾ 5%.
Besides the lack of relationship with GPP or PPP, CR was highly negatively correlated
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553
Figure 2. (A) Relationship between integrated values of heterotrophic bacterial respiration and
community respiration using the bacterial growth efficiency (BGE ⬃ 6%) model of López-Urrutia
and Morán (2007). Continuous line represents the 1:1 relationship. Error bars represent standard
errors. (B) Relationship between the percent contribution of heterotrophic bacteria to community
respiration (BR:CR) for the data pairs shown in (A) (filled squares) and for del Giorgio and Cole
(1998, BGE ⬃ 2%, open circles) and Rivkin and Legendre (2001, BGE ⬃12%, open squares)
models of bacterial growth efficiency, and community respiration. Only Y-standard errors of filled
squares are plotted. Exponential fits to data are only shown for visual reference.
with chlorophyll a (Fig. 3, r ⫽ ⫺0.95; p ⬍0.001; n ⫽ 6), contrary to the findings of
Robinson et al. (2002a, b), strongly suggesting a negligible contribution of phytoplankton
to total CR in our samples. Autotrophic contribution is only a major portion of CR during
phytoplankton blooms (Robinson et al., 2002a, b; Robinson and Williams, 2005) or net
autotrophic periods, where CR has been shown to covary with chl a (Navarro et al., 2004).
Following Robinson et al. (2002a), the remaining contribution of heterotrophs other than
bacteria was split into two groups, bacterivores and herbivores, with micro- and mesozooplankton included in the latter group. We used a common conservative growth efficiency
value of 25% (Straile, 1997) with the aim of estimating the maximum possible contribution
of grazers to CR. The contribution of bacterivores would range from 0.4 to 1.7% of CR,
with as low a mean as 0.9% (Table 2). Although higher that that of bacterivores, mean
contribution of herbivores would not exceed 10% (range 5-20%, Table 2). The sum of the
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Figure 3. Relationship between integrated chlorophyll a concentration and community respiration.
Dashed line represents model I linear regression. Details are given in the text.
different contributions would not reach the measured CR at any station, leaving a mean
0.6 g C m-2 d-1 of respired carbon unaccounted for. These values imply that on average
48% (range 13–72%) of total community respiration could not be explained by the
constituent microbial plankton trophic groups (Table 2).
It is obvious that all these calculations rely on the accuracy of the original measurements
of PPP and BP. As far as PPP estimates are concerned, our results are well within the range
of published values (Jochem and Zeitzschel, 1993; Marañón et al., 2000, 2001; Robinson
et al., 2002a; Teira et al., 2005). In contrast, BP lay at the lower end within the range for
open ocean systems (Ducklow, 2000). There is generally more uncertainty when comparing BP estimates because of the need for converting leucine incorporation into carbon
production. Low leucine uptake rates (⬍100 pmol Leu L-1 h-1), however, are typically
found in oligotrophic, subtropical waters of the Atlantic (Carlson et al., 1996; Zubkov et
al., 2000; H. G. Hoppe, pers. comm.) and the Pacific (Sherr et al., 2001) oceans. Values
similar to ours have also been found in other open ocean environments (JGOFS Arabian
Sea and Equatorial Pacific cruises data bases had mean ⫾ SD values of 25.1 ⫾ 32.3 and
20.4 ⫾ 13.7 pmol Leu L-1 h-1, respectively). Since leucine incorporation rates were
determined during the cruise (mean 11.2 ⫾ 8.0 pmol Leu L-1 h-1), the other possible major
source of error was circumvented by the use of an empirically-determined conversion
factor rather than a published one. Although many studies have used the theoretical
conversion factor (3.1 kg C mol Leu-1, Simon and Azam (1989) or 1.55 kg C mol Leu-1 if
we assume no isotope dilution), there is growing evidence that this value is far from
realistic in oligotrophic environments. The CF we used (0.73 kg C mol Leu-1, see also
Section 2) was, as expected, markedly lower than the theoretical one and similar to other
values obtained in the NE Atlantic: 0.58 by Agustı́ et al. (2001) and 0.02-1.26 by
Alonso-Sáez et al. (2007).
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Heterotrophic bacteria have long been regarded as the major contributors to community
respiration in the open-ocean (Williams, 1981; Biddanda et al., 1994; Pomeroy et al.,
1995; Rivkin and Legendre, 2001). They were indeed more important respirers than
phytoplankton, heterotrophic protists and metazoa according to the calculations shown in
Table 2. Interestingly, heterotrophic bacteria would account for a mean 63 ⫾ 4% of the
summed contributions of the four microbial groups considered here, a value much closer to
those reported by the above mentioned authors. Heterotrophic bacterial contribution to
measured CR would only be greater than 50% if the effective BGE values were lower than
2%. Although some authors have revised upwards the contribution of microzooplankton to
total respiration (Calbet and Landry, 2004), even their highest estimate of ⬃60% of PPP
being respired by micrograzers would leave a mean 50% of CR unaccounted for with the
examined trophic groups. It is, therefore, hard to envision how the estimated contributions
can be revised so as to meet 100% of measured CR. It is necessary to note that the errors
associated to the sum of estimated respirations of individual plankton groups might reach
the 48% difference between measured and estimated respiration (Table 2), but the apparent
uncoupling between BR and CR (Fig. 2A) was not reported in any previous studies using
this approach (Robinson et al. 2002a, b; Robinson and Williams, 2005). A large discrepancy between both estimates, such that bacteria fail to account for more than 30% of total
O2 consumption, was recently confirmed in an extensive review of the planktonic carbon
budget in the subtropical NE Atlantic (Marañón et al., 2007). The point can be made that it
is the respiration of heterotrophic bacteria and other components that was severely
underestimated. However, as argued before, neither higher Leu to C conversion factors nor
lower BGEs are justified in our region in order to raise the estimate of total respiration
attributable to heterotrophic bacteria. Dark incubations might have a role in the low BP
values measured in open ocean waters if the results of Church et al. (2004) in the NE
Pacific are general. However, significantly lower leucine incorporation rates in the light in
oligotrophic environments have also been reported (Morán et al., 2002). Furthermore, our
results of three experiments comparing light and dark uptake were not supportive of
significant enhancement of aminoacid incorporation in the light. With a mean underestimation of 13% ⫾ 12% at station 6, the possible underestimation of BP in the rest of the
stations should have been much lower than the 32%-48% obtained by Church et al.(2004).
In an attempt to reconcile this discrepancy of our budget calculation it might be useful to
discuss the old problem of long in vitro incubations. Enclosure in bottles, especially at
oligotrophic sites, may artificially increase heterotrophic bacterial abundance (Pomeroy et
al., 1994; Gattuso et al., 2002) and activity (Sherr et al., 1999). Important losses of primary
producers (⬎50%) have been frequently observed either as chlorophyll a or individual
group abundance (Fernández et al., 2003) with in situ planktonic populations remaining
stable over long periods (Marañón et al., 2000; Zubkov et al., 2000). Furthermore,
nonlinear increases of CR or BR in dark bottles (Pomeroy et al., 1994; Briand et al., 2004)
have been documented, although linearity responses are also frequent (Robinson et al.,
2002a). We hypothesize that extending the incubation time from 1 - 2 h (as in most BP
556
Journal of Marine Research
[65, 4
measurements) to 24 h (as in most CR experiments) might exacerbate this opposite
behavior of autotrophs and heterotrophs confined in bottles. It stems from the available
evidence that we could not discard an underestimation of PPP in experiments lasting ⬃6 h,
usually the minimum period for 14C incubations in oligotrophic waters.
In an experiment aimed at evaluating changes in picoplankton populations in 125 mL O2
bottles over 24 h we found that autotrophic biomass decreased by 68% in the dark bottles
compared with a ⬃3500 L mesocosm in a temperate, mesotrophic ecosystem (Rı́a de Vigo,
NW Spain) while it remained virtually unchanged in the light ones (12% decrease;
Calvo-Dı́az, Marañón and Morán, unpublished data). In this case, the biomass of heterotrophic bacteria was comparable in the mesocosm and in both bottle types (⫾ 20%). These
results indicate that the combination of bottle confinement plus 24 h darkness can cause
serious changes in community composition at the end of long incubations with respect to
initial values. Evidence for dramatic changes in the picoplankton size-fraction after 2 h
bottle enclosure were obtained in the same cruise (Fernández et al., 2003), and serious
disruptions of Prochlorococcus cell-cycles due to continuous dark confinement have been
reported (Jacquet et al., 2001). As a consequence, metabolic rates from dark bottle
incubations may not be fully representative of natural oxygen and carbon fluxes We can
only speculate on this, but a decrease in algal biomass could be accompanied by an
artificial increase in POC and DOC consumption and hence greater heterotrophic bacterial
respiration, which was not taken into account in our estimates of BR based on ⬃2 h BP
incubations.
According to Takahashi et al. (2002) the subtropics are a weak sink of CO2, hence
contradicting the net heterotrophy indicated by the current method of measuring
changes in dissolved O2 in bottles along 24 h. In a more recent paper, by evaluating all
organic carbon inputs and outputs, Hansell et al. (2004) concluded that the net
heterotrophy in the NE Atlantic was much lower than previously found from incubation procedures, and that the system could indeed be considered as metabolically
balanced. Although part of the consistently high negative values obtained from O2
incubations in the NE Atlantic may be attributable to temporal (González et al., 2002;
Karl et al., 2003) or regional variations (Serret et al., 2002), the evidence presented
here of a serious mismatch between CR and the relative contribution of the different
trophic groups casts some doubt on the actual magnitude of the net heterotrophy of the
region. Interestingly, by comparing the estimated rates of community respiration
(Table 2) with those of gross primary production, the metabolic balance would still be
net heterotrophic on average (CR:GPP ratio of 1.48 ⫾ 0.53), but of a considerable
lower magnitude compared with measured CR and GPP rates (3.74 ⫾ 1.41). It must be
noted that not only CR, but also GPP and PPP (to a variable extent depending on the
incubation length) would be affected by the observed decreases in phytoplankton
biomass (Fernández et al., 2003), stressing the need for checking possible changes in
standing stocks and metabolic rates within incubation bottles.
2007]
Morán et al.: Mismatch between community & bacterial respiration
557
4. Concluding remarks
In conclusion, the expected positive linear relationship between integrated O2 consumption and the estimated contribution of bacteria was not found, with heterotrophic bacterial
respiration accounting on average for one third of community respiration given a mean,
low bacterial growth efficiency of 6%. In addition, community respiration was strongly and
negatively correlated with phytoplankton biomass. The heterotrophic metabolic balance in
the oligotrophic NE Atlantic could not be totally explained by the summed contributions of
individual trophic groups, leaving a mean 48% of community respiration unaccounted for.
The cause for the apparent uncoupling between autotrophic and heterotrophic activity and
respiration remains to be explained. From these considerations, we can safely conclude that
either measurements of heterotrophic bacterial production, community respiration, or both,
are in some error in oligotrophic environments. Unless there is a missing major respirer, a
possible hypothesis is that 24 h dark in vitro incubations, by means of opposite changes in
the biomass and activity of autotrophs and heterotrophs, may contribute to enhance the
apparent negative net metabolic balance of oligotrophic, open ocean waters. Long in vitro
confinements should be systematically controlled for community changes over the incubation period in order to determine the true coupling between autotrophic and heterotrophic
processes and ultimately better constrain carbon fluxes in the upper ocean.
Acknowledgments. We are grateful to P. Estévez for her help with sampling and chlorophyll a
measurements, and to the scientific support staff, captain and crew of the R/V Hespérides. Á.
López-Urrutia, R. Scharek and one anonymous referee provided useful comments on earlier drafts.
This work was supported by the Spanish Ministry of Science and Technology (MCYT) grant
CIRCANA (MAR1999-1072-C03-01) and the special action REN2001-3973-E.
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Received: 8 December, 2006; revised: 2 August, 2007.