Use of carbon monoxide and hydrogen by a bacteria-animal symbiosis from seagrass sediments

Abstract

The gutless marine worm Olavius algarvensis lives in symbiosis with chemosynthetic bacteria that provide nutrition by fixing carbon dioxide (CO2 ) into biomass using reduced sulfur compounds as energy sources. A recent metaproteomic analysis of the O. algarvensis symbiosis indicated that carbon monoxide (CO) and hydrogen (H2 ) might also be used as energy sources. We provide direct evidence that the O. algarvensis symbiosis consumes CO and H2 . Single cell imaging using nanoscale secondary ion mass spectrometry revealed that one of the symbionts, the γ3-symbiont, uses the energy from CO oxidation to fix CO2 . Pore water analysis revealed considerable in-situ concentrations of CO and H2 in the O. algarvensis environment, Mediterranean seagrass sediments. Pore water H2 concentrations (89-2147 nM) were up to two orders of magnitude higher than in seawater, and up to 36-fold higher than previously known from shallow-water marine sediments. Pore water CO concentrations (17-51 nM) were twice as high as in the overlying seawater (no literature data from other shallow-water sediments are available for comparison). Ex-situ incubation experiments showed that dead seagrass rhizomes produced large amounts of CO. CO production from decaying plant material could thus be a significant energy source for microbial primary production in seagrass sediments.

Figure 1

O lavius algarvensis and the Mediterranean seagrass sediments it inhabits. A. The O . algarvensis environment is characterized by medium‐ to coarse‐grained silicate sediments and patches of the seagrass P osidonia oceanica. Subsurface roots and rhizomes (horizontal stems) stabilize the plants in the sediment. The roots and rhizomes form dense mats that are very stable, even after the seagrass has died, and can remain in the sediment for millennia (Mateo et al., 1997; Alcoverro et al., 2001; Duarte, 2002; Boudouresque et al., 2009; Gutiérrez et al., 2011). At the collection site for this study (Sant' Andrea in the north of the Island of Elba), reef‐like mats of dead rhizomes are buried underneath the sediment in the entire bay. The sediment overlying the rhizome mats is very poor in nutrients and energy sources (Kleiner et al., 2012b). B. Image of the O . algarvensis collection site showing sandy sediments surrounded by seagrass beds in 5–6 m water depth. C. Dead seagrass rhizomes from the O . algarvensis collection site. D. Olavius algarvensis, scale bar = 0.4 mm.

Figure 2

CO consumption by O . algarvensis. CO was consumed in incubations with live O . algarvensis worms, but not in controls. Consumption rates of live worms were calculated based on linear rates between 65 and 87 h (solid line). Mean values and standard deviations of three independent incubation bottles are plotted for each control and treatment.

Figure 3

Oxidation of ¹³ CO to ¹³ CO ₂ by O . algarvensis. ¹³ CO oxidation to ¹³ CO ₂ in live and dead O . algarvensis worms was measured over 70 h after the addition of ¹³ CO (7 μM at start of incubations) and ¹³ CO ₂ was produced. Mean values and standard deviations of four independent incubation bottles are plotted for each treatment and control. Standard deviations were very small in the first four time points and are therefore not visible.

Figure 4

H₂ consumption by the O . algarvensis symbiosis. A. H ₂ consumption by live worms began only after 40 h and was then completely consumed within 86 h (circled in red) in all three replicates (standard deviations at this time point were so small that they are not visible in this figure). A second injection of 80 μl of H ₂ to the incubations with live worms was monitored in shorter intervals and revealed a linear consumption of H ₂ by the O . algarvensis symbiosis. B. Close‐up of A after second H₂ injection (at 95.5 h after begin of incubations). Linear consumption is emphasized by solid line (R ² = 0.99). Mean values and standard deviations of three independent incubation bottles are plotted for each control and treatment.

Figure 5

¹³C‐content of single symbiont cells based on nanoSIMS analysis. For all symbionts and treatments cells from three worms were analysed except for the γ3‐symbiont in the H ₂ treatment, for which only cells from two worms were analysed. Horizontal bars with P‐values indicate significant differences based on a Kruskal–Wallis test. Due to the different ¹³ C‐contents of the four symbionts we used different scales for the y‐axis for optimal visualization of the data. AT%: atom percent [¹³ C / (¹² C + ¹³ C) × 100]; n: total number of symbiont cells analysed. ¹³ C isotope content values for all individual cells can be found in Table S1.

Figure 6

Comparison of ¹³ CO ₂ fixation by single O . algarvensis symbiont cells in the presence and absence of CO. Increased carbon fixation in the presence of CO was only visible in the γ3‐symbionts (bottom right image). Images in the left and right columns show in the top row epifluorescence micrographs of O . algarvensis symbionts on a filter, followed by the corresponding nanoSIMS images for sulfur (³² S ^‐, counts per pixel) in the second row, phosphorus (³¹ P ^‐, counts per pixel) in the third row and ¹³ C‐content as ¹³ C / (¹³ C + ¹² C) in the bottom row. In the epifluorescence images symbiont cells hybridized with the general eubacterial probe (EUB338I‐III) are green, the sulfur‐oxidizing symbionts targeted by the gammaproteobacterial probe (Gam42a) are red and the general DNA stain 4,6‐diamidino‐2‐phenylindole (DAPI) is shown in blue. The strong green fluorescence signal of the eubacterial probe (EUBI‐III) masks the red fluorescence signal of the Gam42a probe in the γ1‐symbiont (for images showing the single channels separately see Fig. S1).

Figure 7

(A) CO and (B) H ₂ concentrations at the O . algarvensis collection site. Concentrations were measured at 15 and 25 cm sediment depth, in the dead rhizome mats underlying the sediment, and in the seawater about 5 cm above the sediment. For each sediment depth and control at least eight independent samples were measured. Values are blank corrected.