Experimental design and nutrient concentrations.
An elaborated description of the experimental set-up is given in Materials and Methods. For better traceability throughout the manuscript, a graphical overview of the experimental design is given as
Fig. 1. Briefly, incubation bottles were filled with prefiltered coastal water from the eastern Mediterranean, amended with cyanobacterium and diatom DOM, and placed in flowthrough tanks in the dark (
Fig. 1). To monitor inorganic nutrient concentrations during the experiment, we measured PO
43–, NO
x−, and NH
4+. PO
43– concentrations were ca. 0.1 to 0.2 μM in the control and the diatom DOM treatment, whereas ca. 1 μM PO
43– were accidently added with the cyanobacterium DOM. However, neither cyanobacterium DOM treatments with high PO
43– concentration, nor control or diatom DOM treatments showed pronounced changes in PO
43– concentrations during the experiment (see Fig. S1 in the supplemental material). NO
x− concentrations at the start of the experiment were ~2.5 μM and remained constant throughout the experiment in the control and cyanobacterium DOM treatments, but decreased after 48 h in the diatom DOM treatment (Fig. S1). NH
4+ concentrations ranged between 1 and 15 μM but did not reveal obvious trends over time. Nevertheless, control samples showed slightly higher NH
4+ concentrations than the DOMp (diatom and cyanobacterium) treatments, which were, however, significantly higher only after 24 h of exposure (Fig. S1).
Bacterial community responses to DOMp additions.
In order to evaluate the consequences of different DOMp producers and the succession of DOMp on the richness of the active bacterial communities, we calculated Shannon diversities for the individual treatments and time points. In all samples, the diversity remained in a narrow margin, with values ranging from ca. 5.4 to 6.2 (
Fig. 3). Nevertheless, after 24 and 48 h, samples in the diatom DOM treatments revealed significantly higher diversities than the cyanobacterium DOM and control samples at this time point (
Fig. 3). The increased diversity in diatom samples was consistent also after 72 h of incubation, but not in a significant way (
P = 0.055, Table S1). Next, we examined shared and unique amplicon sequence variants (ASVs) between treatments at different time points using Venn analyses. When shared ASVs between all treatments (control, diatom DOM, cyanobacterium DOM) were compared with each other, no trend over time was obvious (13%, 22%, 20%, and 20% after 6, 24, 48, and 72 h of incubation, respectively), whereas ASVs that were only shared between both DOMp treatments (i.e., DOMp generalists) showed a slight increase over time, with shares of 2.3%, 3.5%, 4.3%, and 4.4% for 6 h, 24 h, 48 h, and 72 h of incubation, respectively (Fig. S2). On the other hand, at the different time points (6, 24, 48, 72 h) exclusive (i.e., specialist) ASVs in cyanobacterium- (22%, 20%, 20%, and 19%) and diatom-derived DOM (18%, 23%, 26%, and 25%, respectively) revealed slightly decreasing (cyanobacterium) and variable (diatom) trends over time (Fig. S2).
We then explored the impact of the different treatments and proceeding times on the active bacterial communities with nonmetric multidimensional scaling (NMDS) and Bray-Curtis dissimilarities. Different treatments (
t0, control, diatom DOM, cyanobacterium DOM;
Fig. 4A) showed significantly different bacterial communities (analysis of similarity [ANOSIM],
R = 0.3,
P = 0.001), and this difference remained if the comparison was performed only between the DOMp types (ANOSIM,
R = 0.22,
P = 0.001;
Fig. 4A). If treatments were pooled together and samples grouped into different incubation times (0 h, 6 h, 24 h, 48 h, and 72 h), significant differences in communities between the different time points could be detected (ANOSIM,
R = 0.4,
P = 0.001;
Fig. 4A).
In all treatments, time-dependent changes in the active bacterial community were significantly higher in the first 24 h of exposure (Bray-Curtis dissimilarities between
t0 and 6 h as well as between 6 h and 24 h) than dissimilarities between later time points (
Fig. 4B). Analogous to comparisons between different time points in the same treatments, community differences between different treatments at the same time point were most pronounced after 6 h, and the overall highest difference was obtained between diatom and cyanobacterium DOM treatments (
Fig. 4C). The differences between bacterial communities in diatom and cyanobacterium DOM treatments, however, diminished after 24 h but successively increased again after 48 and 72 h (
Fig. 4C).
General patterns revealing bacterial community differences between different treatments and incubation times were confirmed by abundance distributions of the overall 100 most abundant ASVs (defined as the sums of the subsampled reads for all samples;
Fig. 5). A dendrogram of the ASVs yielded three major clusters, which were largely driven by taxonomy, with
Saccharospirillaceae,
Spongiispira, SAR11, and KI89a ASVs in cluster I,
Ascidiaceihabitans,
Glaciecola, and
Aureimarina ASVs dominating cluster II, and a diverse consortium of ASVs (including
Synechococcus,
Rhodobacteraceae, and others) in cluster III (
Fig. 5). However, if the 100 most abundant ASVs were analyzed for the dominant ASVs in the different treatments (
t0, control, cyanobacterium, and diatom DOM, where all time points of the three latter were pooled),
t0 samples were dominated by ASVs from SAR11 and
Synechococcus, whereas 11 ASVs of
Saccharospirillaceae, 1
Spongiispira ASV and 1
Glaciecola ASV dominated the controls (
Fig. 5; Fig. S3). On the other hand, the cyanobacterium DOM treatments were exclusively dominated by ASVs of
Glaciecola, whereas two
Flavobacteriaceae ASVs, one
Ascidiaceihabitans ASV, one SAR11 ASV, and four
Glaciecola ASVs dominated the diatom DOM samples (
Fig. 5; Fig. S3A). Additional tests for DOMp generalists, i.e., ASVs that dominate DOMp samples independent of the producer species (assigned treatments: all samples containing DOMp,
t0, control), yielded 21
Glaciecola and 2
Flavobacteriaceae ASVs (Fig. S3B). If different incubation times were analyzed for their dominant ASVs (and the different treatments pooled),
t0 samples were defined by a broad array of bacterial genera and ASVs, with the phototrophic
Synechococcus and heterotrophic SAR11, the OM43 clade,
Ascidiaceihabitans, AEGEAN-169, and the NS2b, NS4, NS5, and NS9 marine group contributing several ASVs. The 6 h incubation samples were especially dominated by ASVs of the
Rhodobacteraceae, 24 h incubations solely by
Glaciecola ASVs, 48 h incubations by 7 ASVs of
Glaciecola and 1 ASV from
Pseudoalteromonas, and the 72 h incubations by 11 ASVs of
Saccharospirillaceae and 1
Aureimarina ASV (
Fig. 5; Fig. S3C).