Stable Isotope Probing Identifies Bacterioplankton Lineages Capable of Utilizing Dissolved Organic Matter Across a Range of Bioavailability

Abstract

Bacterioplankton consume about half of the dissolved organic matter (DOM) produced by phytoplankton. DOM released from phytoplankton consists of a myriad of compounds that span a range of biological reactivity from labile to recalcitrant. Linking specific bacterioplankton lineages to the incorporation of DOM compounds into biomass is important to understand microbial niche partitioning. We conducted a series of DNA-stable isotope probing (SIP) experiments using ¹³C-labeled substrates of varying lability including amino acids, cyanobacteria lysate, and DOM from diatom and cyanobacteria isolates concentrated on solid phase extraction PPL columns (SPE-DOM). Amendments of substrates into Sargasso Sea bacterioplankton communities were conducted to explore microbial response and DNA-SIP was used to determine which lineages of Bacteria and Archaea were responsible for uptake and incorporation. Greater increases in bacterioplankton abundance and DOC removal were observed in incubations amended with cyanobacteria-derived lysate and amino acids compared to the SPE-DOM, suggesting that the latter retained proportionally more recalcitrant DOM compounds. DOM across a range of bioavailability was utilized by diverse prokaryotic taxa with copiotrophs becoming the most abundant ¹³C-incorporating taxa in the amino acid treatment and oligotrophs becoming the most abundant ¹³C-incorporating taxa in SPE-DOM treatments. The lineages that responded to SPE-DOM amendments were also prevalent in the mesopelagic of the Sargasso Sea, suggesting that PPL extraction of phytoplankton-derived DOM isolates compounds of ecological relevance to oligotrophic heterotrophic bacterioplankton. Our study indicates that DOM quality is an important factor controlling the diversity of the microbial community response, providing insights into the roles of different bacterioplankton in resource exploitation and efficiency of marine carbon cycling.

Keywords: DOM; Sargasso Sea; bioavailability; copiotrophs; labile; oligotrophs; recalcitrant; stable isotope probing.

FIGURE 1

Flow chart of SIP sample processing and data analysis steps.

FIGURE 2

NMR 2D ¹³C-¹H HSQC plots (¹H on x axis and ¹³C on y axis) of superimposed ¹³C Syn lysate (red positive contours) and ¹³C Syn lysate_PPL (green positive contours) samples in deuterated DMSO. Noise negative contours are in blue. Circles are functional groups enriched in Syn lysate or Syn lysate_PPL samples.

FIGURE 3

(A–C) Bacterioplankton abundance (BA) change with incubation time (* indicates significant difference compared to control). (D) BA fold change from T0 to the end (letters indicate significant difference).

FIGURE 4

(A–C) DOC change with incubation time. DOC concentrations at T0 in the M TW lysate_PPL treatment (open symbol, connected with next data point in dashed line) were calculated based on measured TDAA C concentration and assuming a TDAA C: DOC ratio of 0.64 as measured for the amended substrate in the surface treatment (see text for details). Actual measured DOC at T0 in the M TW lysate_PPL treatment was in bracket as it was possibly underestimated due to sample preservation or storage problems. (D) Amended DOC removal percentage from T0 to the end (letters indicate significant difference).

FIGURE 5

(A,B) TDAA C change in TW and Syn DOM treatments and their corresponding control treatments with incubation time. (C) Amended TDAA C removal percentage from T0 to the end (letters indicate significant difference).

FIGURE 6

Left panel shows ranges of raw relative abundance data of each corresponding family taxon in the right panel. Right panel is a heatmap of z-score of major bacterioplankton family taxa (>0.5%) in every treatment at time point T0 and TF (2–4 days, see Table 1) of unfractionated samples. Z-score (standardized relative abundance by family taxa) was calculated as relative abundance of one taxon in the sample minus mean of relative abundance of the same taxon across all treatment samples and then divided by the standard deviation of relative abundance of that taxon across all treatment samples. This sets relative abundance of each taxon to a normalized scale to avoid skewing by only a few most abundant taxa like Alteromonadaceae. Higher z-score for each family taxon means relatively higher proportion of that taxon in the sample among all treatments.

FIGURE 7

Non-metric Multidimensional Scaling plot of bacterioplankton community structure of unfractionated samples at T0 and TF (final DNA harvesting time point) based on Bray-Curtis distance (Alteromonadaceae were excluded to avoid bias by its dominance at TF samples). Five clusters were grouped based on hierarchical clustering analysis and simprof analysis (α = 0.05) at threshold of 70% dissimilarity (identified as in Supplementary Figure 4).

FIGURE 8

Method validation of identifying ¹³C-incorporating family taxa. Relative abundance vs. CsCl density and unamended control normalized and log2 transformed relative abundance vs. CsCl density plots for examples of well-labeled, partially labeled and unlabeled taxa among ¹³C-TW lysate_PPL, ¹²C-TW lysate_PPL, and unamended control treatments. Red lines and arrows indicated shift to heavier density in the ¹³C treatment compared to the ¹²C and unamended control treatments. Since lightest and heaviest fractions are mixed with water or CsCl, DNA quantities were low and densities deviated from normal range (as shown in left panel x break axis), thus these two fractions were excluded in following analyses.

FIGURE 9

Mean CsCl density where each bacterioplankton taxon in Table 4 was resolved at TF among treatments. The shading on the taxa represents the following: orange-shaded, copiotroph; blue-shaded, oligotroph; purple-shaded, archaea. Warm colors represent the mean CsCl density associated with taxon that likely incorporated more ¹³C in that treatment.

FIGURE 10

Relative abundance at family level of members in panels (A) SAR11 clade, (B) SAR202 clade, (C) SAR86 clade, (D) Acidimicrobiales OM1 clade, (E) Salinisphaeraceae, and (F) Acidimicrobiales Sva0996 clade in top 300 m at BATS station in the Sargasso Sea before (January), during (April), and shortly after (June) convective mixing event in 2017.