Dilution limits dissolved organic carbon utilization in the deep ocean

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Science  17 Apr 2015:
Vol. 348, Issue 6232, pp. 331-333
DOI: 10.1126/science.1258955

Dilution solves the recalcitrance question

The deep ocean is full of dissolved organic carbon, some of which has remained unchanged for thousands of years. What makes these compounds so resistant to microbial degradation? Perhaps their chemical structures make them intrinsically difficult to metabolize? In contrast, Arrieta et al. show that they are simply too dilute to be viable sources of energy for microorganisms (see the Perspective by Middleburg). Further experiments show that if these seemingly recalcitrant organic molecules are concentrated, the ambient microbes can consume them.

Science, this issue p. 331; see also p. 290


Oceanic dissolved organic carbon (DOC) is the second largest reservoir of organic carbon in the biosphere. About 72% of the global DOC inventory is stored in deep oceanic layers for years to centuries, supporting the current view that it consists of materials resistant to microbial degradation. An alternative hypothesis is that deep-water DOC consists of many different, intrinsically labile compounds at concentrations too low to compensate for the metabolic costs associated to their utilization. Here, we present experimental evidence showing that low concentrations rather than recalcitrance preclude consumption of a substantial fraction of DOC, leading to slow microbial growth in the deep ocean. These findings demonstrate an alternative mechanism for the long-term storage of labile DOC in the deep ocean, which has been hitherto largely ignored.

The accepted paradigm is that recalcitrant dissolved organic carbon (DOC) is ubiquitous in the ocean and makes up the bulk of the DOC pool at depths of >1000 m and at DOC concentrations below 42 μmol C liter−1 (1). However, most of the components of the recalcitrant DOC pool remain unidentified (1, 2), and there is little evidence of structural properties that could make these compounds unavailable to microbial degradation. Conversely, the dilution hypothesis (3, 4) postulates that most organic substrates in the deep ocean are labile but cannot be used by prokaryotes at concentrations below the levels matching the energetic investment required for their uptake and degradation. An early study (5) tested the dilution hypothesis by looking for microbial consumption in concentrates of natural DOC from deep waters, but found no substantial changes in DOC concentrations after a 2-month incubation. Those results led to the conclusion that deep-water DOC is composed of recalcitrant molecules and therefore to the dismissal of the dilution hypothesis. Here, we revisit the dilution hypothesis using a simple experimental approach, similar to that used by Barber in 1968 (5) but using methodologies not available at that time. Specifically, we tested the hypothesis that no significant increase in prokaryotic growth should be detectable when increasing DOC concentrations, which is as expected if deep oceanic DOC were structurally refractory.

Natural prokaryotic communities collected at 14 stations between 1000 to 4200 m in the Pacific and Atlantic Ocean (fig. S1) were exposed to ambient, 2-, 5- and 10-fold concentrations of natural DOC collected from their original location by means of solid phase extraction (6, 7) and incubated at in situ temperature. A consistent increase in prokaryotic abundance over time was observed in response to increasing concentrations of DOC in all 14 experiments (Fig. 1 and fig. S2). Maximum prokaryotic abundances obtained at ~10-fold DOC concentrations were 3.6 to 11.7 times higher than those observed in the corresponding controls (Fig. 1 and fig. S2). Unamended controls showed much lower, sometimes undetectable, prokaryotic growth, comparable with the values observed in deep layers of the ocean in other studies (8, 9), whereas specific growth rates in the higher DOC enrichments showed values up to 0.4 days−1, which is typical of productive surface waters (Fig. 2 and fig. S3). No significant differences (t test, P > 0.05) in prokaryotic growth were observed between unamended controls and extraction controls, confirming that the observed growth was due to the materials being extracted from seawater and not the result of a contamination with labile organics during the extraction procedure (fig. S4). The solid phase extraction method used to concentrate natural dissolved organic matter (DOM) may have introduced some compositional bias toward small and polar compounds while losing a substantial part of the DOM pool (extraction efficiency ~40%), but this does not change the fact that increasing the concentration of the extractable components of natural DOC resulted in enhanced prokaryotic growth. Chemical alterations of DOC, such as the disruption of supramolecular arrangements (10) or mild hydrolysis produced during the concentration procedure, are an unlikely explanation for the observed response because the treatments in which DOC concentration was doubled by adding concentrated DOC showed little or no enhancement of prokaryotic growth as compared with that of controls. Hence, we validated the dilution hypothesis tested, showing that dilution limits C utilization in the deep ocean.

Fig. 1 Prokaryotic abundance in experimental treatments containing approximately 2, 5, and 10 times the in situ DOC concentration versus controls containing unnamended seawater.

(A to E) Error bars represent the standard error of the mean of triplicate cultures. Only the five experiments carried out in the North Pacific (experiments A to E) are represented. The results of the 14 experiments are shown in fig. S2.

Fig. 2 Specific growth rates at different concentrations of DOC.

Horizontal error bars represent the standard deviation of the mean initial DOC concentration measured in the triplicate cultures, and vertical error bars represent the standard deviation of the mean of the specific growth rates estimated for each of the triplicate cultures. Only the five experiments carried out in the North Pacific (experiments A to E) are shown. The results of the 14 experiments are available in figure S3.

Specific growth rates increased with DOC concentrations following a classical Monod model [coefficient of determination (R2) of 0.71 to 0.98] at all the locations studied (Fig. 2, fig. S3, and table S2), further confirming the hypothesis that prokaryotic growth in deep waters is limited by the concentration of DOC. In 9 out of 14 experiments, the initial in situ DOC concentration was low enough to capture the lower part of the curve and thus to give an estimate of the minimum DOC concentration necessary to support prokaryotic maintenance metabolism (Fig. 2, fig. S3, and table S2). According to these estimates, concentrations of natural DOC below 30.7 ± 5.4 μmol C L−1 (average ± SE, n = 9 measurements) would not be sufficient to support prokaryotic metabolism, a value not significantly different (t test, P > 0.05) from the lowest concentrations of DOC around 34 μmol C liter−1 reported for the deep ocean (11).

Prokaryotic growth efficiency (PGE) in the unamended controls was always lower than 3%, which is similar to the values reported for deep-water masses (9). No statistically significant differences among treatments were detected in our PGE estimates because of the accumulation of errors propagating from the original measurements (one-way analysis of variance, P > 0.05). However, growth efficiency estimates show a consistent tendency to increase with increasing DOC concentration in all the experiments (fig. S5), suggesting a positive effect of concentration that could be related to a relief from substrate limitation (12). An increasing growth efficiency with increasing DOC concentrations cannot be explained if the bulk of the DOC components were structurally recalcitrant. The differences between our results and those reported by Barber in 1968 are probably due to a combination of methodological improvements. We used only dissolved materials, whereas Barber used a concentrate of everything with nominal size >500 daltons, including particulate matter. Thus, true DOC concentrations in Barber’s experiments were probably not as high as intended. Also, a large fraction of marine DOC consists of molecules <500 daltons (fig. S7) (13, 14); thus, the composition was biased toward higher-molecular-weight compounds in Barber’s experiments, meaning that the molar enrichment in his fivefold C concentration treatment was probably much lower than in our experiments.

In the four experiments carried out in the North Atlantic, incubations were kept on the ship for an additional month after the cruise until the ship returned to the harbor (fig. S2, K to N). Prokaryotic abundance remained essentially constant during the additional month in all but one of the experiments (M), in which a second phase of intense growth was observed in the most concentrated treatments (fig. S2). Although it is unclear why this happened, we can rule out contamination because growth occurred in all replicates, and a pronounced decrease in DOC was found in these samples (fig. S6). The appearance of large cells in the flow cytograms, indicating substantial growth of heterotrophic protists (15) in the late stages of the more concentrated cultures, and the fact that the prokaryotic carbon demand inferred from the increase in abundance and growth efficiency was much lower than the measured decrease in DOC concentration indicate that labile DOC was still being consumed at the end of the experiments, even when no increase in prokaryotic abundance was detectable owing to enhanced prokaryotic mortality.

A corollary to the dilution hypothesis is that bulk DOC is composed of a large diversity of individual molecules. Indeed, molecular characterization by means of Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) (fig. S7) (13, 14) shows that marine DOC is composed of a very large variety of small molecules (200 to 700 daltons) present in very low concentrations and adding up to a large pool of apparently recalcitrant DOC. We characterized the changes caused by microbial activity on the molecular composition of deep oceanic DOM in two additional experiments (O and P) in unamended seawater and in samples in which the concentration of in situ DOC was raised to approximately five times the original concentration. Molecular characterization by means of FT-ICR-MS showed a highly reproducible pattern of microbial utilization among replicate-independent incubations (fig. S8), affecting a large number of different molecules (fig. S9). Significant utilization (relative signal in replicate incubations significantly lower than in the corresponding initial samples; t test P < 0.05) was found for 2095 and 1753 different compounds in the controls and for 2846 and 936 different compounds in the 5×-concentrated samples for experiments O and P, respectively. Consistent differences could be observed between the set of molecules used in concentrated versus unamended samples, resulting in a total of 3950 different molecules consumed in either the concentrated samples or the controls for experiment O and 2140 in experiment P. Moreover, the sum of the normalized signal of all the peaks in the FT-ICR-MS fingerprint in which significant consumption was detected in either concentrates or controls was >70% in experiment O and >40% in experiment P, indicating that a major fraction of the original DOC consisted of labile compounds.

The dilution hypothesis provides an alternative framework with which to explain observations of the apparent recalcitrance of DOC and lends a physiological meaning to the operationally defined “semi-labile” and “semi-refractory” fractions (16, 17). We hypothesize that under the dilution hypothesis, very heterogeneous mixtures of labile compounds appear semirefractory, whereas increasingly less diverse DOM assemblages containing larger concentrations of some substrates will present higher microbial growth and DOC turnover rates, resulting in increasing degrees of apparent lability. The microbial generation of apparently recalcitrant material (18) from labile substrates in a process recently dubbed the “microbial carbon pump” (19) can also be explained with the dilution hypothesis. Microbial utilization of abundant, labile compounds results in hundreds of different metabolites (20), which are subsequently consumed down to the lowest utilizable concentration. This mechanism explains observations of relatively concentrated, labile materials being transformed into apparently recalcitrant matter through microbial consumption (18) but does not necessarily imply the formation of structurally recalcitrant molecules. Indeed, “recalcitrant” DOC is not defined structurally, but operationally, as the DOC pool remaining after long experimental incubations or as the fraction transported in an apparently conservative manner with the ocean circulation (1). Thus, the dilution hypothesis severely limits the feasibility of geoengineering efforts to enhance carbon storage in the deep ocean (21) by using the microbial carbon pump.

FT-ICR-MS characterization of DOC from different oceans (13, 14, 22, 23) and also from this study (fig. S5) shows no indication of prevalent, intrinsically recalcitrant compounds accumulating in substantial amounts. Conversely, FT-ICR-MS data show that oceanic DOC is a complex mixture of minute quantities of thousands of organic molecules, which is in good agreement with the dilution hypothesis. Mean radiocarbon ages of deep oceanic DOC in the range of 4000 to 6000 years have been considered as evidence for its recalcitrant nature (24, 25). However, these are average ages of a pool containing a mixture of very old molecules >12,000 years old but also featuring a large proportion of contemporary materials (26). Moreover, elevated radiocarbon ages only demonstrate that these old molecules are not being newly produced at any appreciable rate—because that would lower their isotopic age—but does not necessarily imply that they are structurally recalcitrant. Furthermore, it is unlikely that natural organic molecules can accumulate in the ocean in substantial concentrations and remain recalcitrant or be preserved for millennia when degradation pathways for novel synthetic pollutants evolve soon after these compounds are released in nature (27).

Although there might be a truly recalcitrant component in deep oceanic DOC, our results clearly show that the concentration of individual labile molecules is a major factor limiting the utilization of a substantial fraction of deep oceanic DOC. These results provide, therefore, a robust and parsimonious explanation for the long-term preservation of labile DOC into one of the largest reservoirs of organic carbon on Earth, opening a new avenue in our understanding of the global carbon cycle.

Supplementary Materials

Materials and Methods

Figs. S1 to S9

Tables S1 and S2

References (2835)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: This is a contribution to the Malaspina 2010 Expedition project, funded by the CONSOLIDER-Ingenio 2010 program of the from the Spanish Ministry of Economy and Competitiveness (Ref. CSD2008-00077). J.M.A. was supported by a “Ramón y Cajal” research fellowship from the Spanish Ministry of Economy and Competitiveness. E.M. was supported by a fellowship from the Junta para la Ampliación de Estudios program of CSIC. G.J.H. and R.L.H. were supported by the Austrian Science Fund (FWF) projects I486-B09 and P23234-B11 and by the European Research Council (ERC) under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement 268595 (MEDEA project). We thank A. Dorsett for assistance with DOC analyses, participants in the Malaspina Expedition and the crews of the BIO Hespérides, and RV Pelagia and the personnel of the Marine Technology Unit of CSIC for their invaluable support. Original data sets are available online at J.M.A. designed the experimental setup, carried out part of the experiments, measured prokaryotic abundance, analyzed the data, and wrote the manuscript. E.M. carried out part of the experiments and data analysis. C.M.D. designed the Malaspina 2010 Expedition, was responsible for DOC analyses, and together with G.J.H. contributed to the design of the experiments and discussion of results. R.L.H. and T.D. analyzed the FT-ICR-MS samples. All authors discussed the results and contributed to the manuscript.
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