Our experiments allowed us to confirm that a fraction of DOM released by heterotrophic prokaryotes is labile, promoting significant prokaryotic growth in all incubations, in line with previous studies [10, 31]. In contrast to our hypothesis, clear differences in DOM lability released under P-repletion and P-limitation (B-DOM and U-DOM, respectively) could not be evidenced based on the cell growth, and P-limitation was not demonstrated to decrease the lability of the released DOM.
Fdom Consumption
Although clear differences in cell growth were not observed between U-DOM and B-DOM, different patterns in FDOM consumption were detected. The protein-like component C340, usually considered as a proxy of labile DOM [33, 37], was only partially used on the time scale of our incubations. Lønborg et al. [31], using long term incubations, suggested that a fraction of bacterial derived protein-like FDOM could be refractory. However, in contrast to our initial hypothesis, the microbial humic-like component C398 was consumed by the communities from the Mediterranean Sea in some of the incubations. This component has been considered refractory that resists bacterial degradation [28, 29], however we evidence that the microbial humic-like FDOM fluorophore released by HP is not always refractory and can be partially used when this DOM component dominates. Previous studies in marine and freshwater environments reported bacterial consumption of similar humic-like fluorescence (summarised in Table 3).
Table 3
Comparaison to previous studies reporting the consumption of microbial humic like fluorescence originating from different sources.
Study
|
FDOM peak
Ex Em
|
DOM Producer / source
|
Consumer
|
% of consumed FDOM
|
[38]
|
310 392
|
Single Phytoplankton
|
Natural HP community
|
|
Skeletonema cotatum
|
30.3%
|
Chaetoceros sp.
|
5%
|
Micromonas pusilla
|
10%
|
[39]
|
310 420
|
Stream water
(white Clark Creek)
|
Stream water Bacteria
|
16%
|
[10]
|
320 410
|
Single bacteria strains
|
HP communities collected in:
|
|
Photobacterium angustum
|
500 m, March
|
20%
|
5 m, March
|
30%
|
5 m, April
|
59%
|
5 m, December
|
0%
|
S. alaskensis
|
5 m, April
|
57%
|
5m, December
|
11%
|
[40]
|
320 410
|
Mesopelagic North Atlantic water fractionated to low (L) and high (H) molecular weight DOM
|
Natural HP community
|
|
LDOM
|
0%
|
L + H DOM
|
2%
|
[41] *
|
330 386
|
Coastal HP
|
Coastal HP
|
52%
|
Actual study
|
315 398
|
Single bacteria strains
|
HP community collected in
|
|
S. alaskensis under P replete condition
|
5 m, February
|
32.5%
|
S. alaskensis under P limited condition
|
60.9%
|
Fall HP communities under P-replete
|
5 m, November
|
0%
|
Fall HP communities under P-limited
|
78.3%
|
Spring HP communities under P-replete
|
5 m, May
|
0%
|
Spring HP communities under P-limited
|
0%
|
* in the present studies fluorescence consumption was estimated as the decrease from the maximum of fluorescence along the same incubation.
Based on these studies, the humic-like fluorescence was used to different extent (Table 3) depending on its molecular weight [40], source [38] and or the degrading community [10]. In our incubations, only 32.5–78.3% of the humic-like DOM was consumed. The percentages of prokaryotic degradation of microbial humic-like FDOM derived either from bacteria or phytoplankton (Table 3) are higher than the degradation of DOM directly isolated from natural waters [39, 40]. This is most likely because humic-like DOM isolated from natural systems is more processed than DOM produced over short time incubations. Together with our results, this also suggests that similar humic-like FDOM peaks could be of different bioavailability and likely different chemical composition since fluorescence spectroscopy captures only a bulk signal from DOM. Further DOM analyses (e.g. high-resolution mass spectrometry) would be needed to elucidate this point.
Our results indicate that humic-like FDOM use is also related to the lack of other DOM compounds. In our experiment, the humic-like FDOM component was consumed only when the presumably labile protein-like component was initially not prevailing, implying that heterotrophic prokaryotes are able to use less labile carbon sources when the presumably labile ones are scarce. This is in line with the results by [43], that followed the bioavailability of Synechococcus-derived DOM to estuarine and coastal bacteria for 180 days and showed that a similar humic-like fluorophore can be produced by some bacterial groups when labile Synechococcus-derived DOM is available, then re-used by other bacterial groups after the labile DOM is depleted. Microbial humic-like consumption was also accompanied by a consistently high alkaline phosphatase activity. Although high alkaline phosphatase activity could be interpreted as a response to P-limitation, this was not the case in our incubations since inorganic P was added (Table S2). Studies demonstrated that prokaryotes can use alkaline phosphatases to unbind bioavailable organic carbon [44]. We hypothesize that alkaline phosphatase in our experiments was used to break P-containing DOM polymers to use them as a substrate.
Selection Of Community Composition According To Hp-dom Quality
HP-DOM quality driven by P availability appeared to change the taxonomic composition of the Mediterranean Sea communities growing on it. Both HP-DOM released under P-repletion and P-limitation (B- and U-DOM) lead to the growth of diverse communities, suggesting that HP-DOM is composed of a multitude of substrates. Previous studies using ultra-high resolution mass spectroscopy revealed a large number of molecules in bacterially derived DOM [24, 45]. Landa et al., [46] discussed different effects of DOM generated by diatoms and cyanobacteria on the composition and diversity of bacterial communities. According to their results, the more chemically diverse the DOM is, the higher the diversity of the community growing on it.
Even though prokaryotic diversity did not differ between B- and U-DOM, community composition analyses revealed different taxonomic groups growing on the different HP-DOM sources, indicating the different composition of the HP-DOM released under P-repletion vs. P-limitation. Few families, belonging mainly to Alphaproteobacteria and Gammaproteobacteria and few Bacteroidia, dominated the HP-DOM incubations. However, the ASVs differed among the experiments and the treatments, suggesting that community composition plays an important role in determining DOM lability. Members of Rhodobacteraceae, Alteromonadaceae (Pseudophaeobacter, Alteromonas) were the main indicators of the protein-like rich treatment in SOLA-fall B-DOM. Some members of the Alteromonadales class have already been shown to be related to the protein-like peak T, corresponding to our component C340, in bacterial incubations of high and low molecular weight DOM suggesting their preference for labile DOM [40]. DOM rich in humic-like components, namely S. alaskensis DOM and SOLA-fall U-DOM, selected different indicator taxa, including mainly Methylophilaceae, Marinomonadaceae, Rhodobacteraceae and Alteromonadaceae. A coupling between FDOM Peak M (corresponding to our C398) and several members of Rhodobacterales, was observed in communities growing on low molecular weight DOM [40] or on viral lysates of cyanobacteria (Xiao et al., 2021). Members of Marinomonadaceae, particularly Marinomonas, have been shown to degrade complex organic matter such as terrestrial DOM inputs in high latitudes [48]. But they are also known to be associated with Cyanobacteria lysates [49]. This suggests that they can respond positively to various carbon sources including complex DOM or humic-like DOM as shown here. In addition, Alteromonadales, Burkholderiales (Methylophilaceae), and Rhodobacterales are among most abundant heterotrophic marine bacteria that might hydrolyse dissolved organic phosphorus outside the cytoplasmic membrane using alkaline phosphatase [50], in line with our results showing high alkaline phosphatase activities in these treatments.
Overall, our results illustrate how P availability shapes HP-DOM degradation patterns, suggesting that P would play a key role shaping prokaryote-mediated DOM fluxes in the ocean. Although the growth of Mediterranean Sea communities did not differ between P-replete and P-limited derived DOM, different metabolisms and taxonomic groups were selected for the different DOM conditions. We could show that a considerable portion of humic-like DOM might be bioavailable when this DOM pool prevails as a substrate for HP. This bioavailability is also controlled by the releasing conditions, and the consumers community composition. Our results emphasize that DOM lability is context dependent, and implies that the impact of P-limitation on DOM fluxes via the MCP should be placed on its local environmental context rather than generalized to a global context.