6. Summarizing Discussion and Concluding Remarks
6.2. Carbon Uptake by the Sediment Microbial Community
The findings complement earlier studies that investigated the role of bacterial chemoautotrophs in coastal sediments (Kepkay and Novitsky, 1980) by providing quantitative data on their in situ abundance and phylogenetic affiliation. The identified chemoautotrophs feed a microbial foodweb apart from infiltrated organic matter and benthic microalgal communities (Billerbeck et al., 2007) and thus promote primary production in marine sediments.
Figure 8 Epifluorescence microscopy images of substrate assimilating cells.
Microautoradiography combined with FISH visualizes Gammaproteobacteria (green) that assimilated (A, B) 14CO2 or (C, D) 14 C-acetate. The precipitation of silver grains (dark granules) associated with single cells and cell aggregates indicated substrate uptake. Due to efficient ultrasonic treatment cell aggregates were only occasional detected. In future studies high-resolution nanoSIMS analysis may visualize different activity patterns of individual cells clustered in aggregates.
Figure 9 Incorporation of inorganic and organic carbon by the sediment microbial community.
The relative abundances of (A) 14CO2 or (B) 14C-acetate incorporating microbial cells (▪) and Gammaproteobacteria ( ) were ▪ assessed by MAR-FISH. The proportion of cells that did not incorporate the substrate ( ) and the proportion of substrate ▪ incorporating cells other than Gammaproteobacteria (▫) are indicated. Surface sediment sampled in June 2009 was incubated in slurries under oxic conditions.
In addition, the assimilation of organic carbon (Fig. 8 and 9) was quantified. Acetate serves as a carbon source for several giant sulfur oxidizers (Nelson and Castenholz, 1981; Schulz and Schulz, 2005; Hogslund et al., 2009) and Thiobacilli and Thiomicrospira species (Kuenen and Veldkamp, 1973; Wood and Kelly, 1989) and can be linked to sulfide and thiosulfate oxidation (Hagen and Nelson, 1996; Schulz and de Beer, 2002; Otte et al., 1999; Nielsen et al., 2000). Here, we can not directly link in situ acetate uptake to sulfur-oxidizing activity of the labeled Gammaproteobacteria. However, some of the acetate utilizing cells might represent lithoheterotrophic or mixotrophic populations. As organic compounds are readily available in the surface sediment of Janssand, it is likely that prevailing SOP use both sulfide and acetate.
Similarly, RCB contribute to turnover of acetate. The observed assimilation under oxic conditions is consistent with their high in situ abundance in the surface layers of Janssand site and an aerobic lifestyle.
In deeper permanently anoxic sediment layers RCB accounted for lower proportions of the microbial community. We hypothesize prevailing cells to be of low activity or inactive. Here, our cultivation experiments yielded evidences for a facultative anaerobic metabolism of sedimentary RCB. Both,
metagenomic information and cultivation experiments indicated the use of dimethylsulfoxide and nitrate as alternative electron acceptors. In addition, Koepke (2007) and Sass and colleagues (2009) isolated facultative anaerobic RCB from nearby tidal flats that grew anaerobic with acetate as electron donor and trimethylamine-N-oxide (TMAO) and dimethyl sulfoxide (DMSO) as terminal electron acceptor. These findings add evidence for an adaptation of sedimentary RCB to anoxic conditions. Alonso and Pernthaler (2005) who observed glucose uptake of pelagic RCB under anaerobic conditions proposed that respective physiology represents a special adaptation to temporally anoxia which aggregate-associated RCB face upon burial in sediments. Similarly, the facultative anaerobic lifestyle of sedimentary RCB likely facilitates a response to temporal oxygen limitation in the tidal surface sediment. Whether it enables life under permanently anoxic conditions in deeper sediment layers needs be explored by further experiments.
82%
47%
53%
18%
B
78%
54%
46%
22%
A
Uptake of additional organic substrates
The consumption of particular dissolved organic matter fractions in the sea has been the subject of intense MAR-FISH investigations on marine bacterioplakton communities. However, nearly nothing is known about assimilation patterns of different substrates by sediment microbial communities, nor has the relative contribution of various phylogenetic groups to substrate uptake been investigated so far.
Accordingly, surface sediment sampled in 2009 was incubated with various organic substrates, including the sugars glucose and n-acetylglucosamine, the amino acid leucine and the organosulfonate taurine.
Between 10 and 20% of the total microbial community incorporated the different substrates (Fig. 10).
Consistent with previous incubations (Zerjatke, 2009) numerous cells incorporated glucose under oxic and anoxic conditions. Preliminary analysis revealed that members of the Gammaproteobacteria accounted for substantial proportions of substrate-assimilating cells for all carbon compounds (Fig. 11).
Initial results indicate that RCB and Bacteroidetes assimilate glucose and N-acetylglucosamin. Leucine, a commonly used tracer of bacterial protein synthesis was chosen as a substrate for dissolved free amino acid incorporation. Similar to acetate incorporation patterns, Gammaproteobacteria and RCB accounted for the highest fractions of leucine-active cells. In contrast, only very few Deltaproteobacteria and cells of Desulfobacteraceae assimilated the substrate (<2% of total probe targets). Bacteroidetes and
Planctomycetales did not incorporate leucine. The findings are consistent with investigation on pelagic RCB that have been found to account for substantial proportions of glucose and leucine incorporating cells in coastal North Sea waters {Alonso, 2006 #3747; Alonso, 2006 #4566}. Similarly, pelagic
Gammaproteobacteria and Bacteroidetes contributed to glucose turnover {Alonso, 2006 #4566}. Further analysis has to assess community bulk incorporation rates for applied substrates and the fraction and identity of substrate-incorporating cells in the different phylogenetic groups.
Figure 10 Assimilation of additional organic substrates by the microbial community.
The relative proportion of cells that incorporated the substrates is given in % of total microbial cells.
Generally sediment (0-1 cm) was incubated oxically. For glucose, sediment (10-11 cm depth) was additionally incubated anoxically.
Bars illustrate mean values of duplicate incubations. Exposure times to microautoradio-graphic emulsion: glucose, leucine, taurine 10 days; N-acetylglucosamine 4 days Figure 11 Assimilation of additional organic substrates by Gamma-proteobacteria.
The relative proportion of substrate assimilating Gammaproteobacteria is given in % of total substrate assimilating cells (preliminary data from one parallel of two replicates).
0 5 10 15 20 25 30 35 40 45
Glucose oxic Glucose anoxic Leucine N-Acetylglucosamine Taurine MAR+ Gammaproteobacteria in [%] of total MAR+ cells
0 5 10 15 20
Glucose oxic Glucose anoxic Leucine N-Acetylglucosamine Taurine MAR+ cells in [%] of total microbial cells
Gammaproteobacteria as abundant and active sediment group
In summary, Gammaproteobacteria accounted for the highest proportion of substrate assimilating cells among prevailing sediment bacteria (Fig. 9 and 11). In addition, they constituted the largest clone fractions within all 16S libraries and represented the numerically dominant group in situ throughout the study period.
Figure 12 Taxonomic composition of the sediment microbial community of geographically distant tidal sites.
The charts illustrate the (A) affiliation of sequences obtained from different 16S rRNA gene clone libraries of Janssand sediment and (B) those obtained from pyrosequencing of Dongmak tidal flat sediment, Korea (data taken from Kim et al., 2008).
Large fractions of Gammaproteobacteria-related clones (Fig. 12) have been repeatedly observed for coastal (Kim et al., 2004; Polymenakou et al., 2005; Hong et al., 2006; Bi-Wei et al., 2009), aquaculture (Asami et al., 2005) and polar sediment (Bowman et al., 2005; Li et al., 2009; Teske et al., 2011) as well as permeable shelf (Hunter et al., 2006), subseafloor (Santelli et al., 2008) and hydrothermal sites (López-García et al., 2003). Similarly, high FISH counts were reported for the investigated (Ishii et al., 2004) and related North Sea sites (Llobet-Brossa et al., 1998; Musat et al., 2006) or different sediments worldwide (Ravenschlag et al., 2001; Perner et al., 2007; Schauer et al., 2010). However, few studies have defined specific gammaproteobacterial populations (Ravenschlag et al., 2001) and the metabolic potential of its versatile members has been rarely studied by molecular tools. Edlund and colleagues (2008) found major portions of Gammaproteobacteria in 16S rRNA gene libraries constructed from bromodeoxyuridine (BrdU)-labeled DNA and from cDNA obtained by reverse transcription polymerase chain reaction (rt-PCR) of RNA and thus provided evidence for their in situ growth and activity in Baltic Sea sediments. Teske and colleagues (2011) studied the 16S rRNA gene diversity and extracellular enzyme activities of
heterotrophic sediment communities in surface and subsurface sediment. They suggested
Gammaproteobacteria to contribute to extracellular enzymatic hydrolysis of polysaccharides and algae extracts (Teske et al., 2011) but lacked a direct link to the identity of substrate hydrolyzing microbes.
Additional SIP based studies repeatedly recovered gammproteobacterial sequences from coastal 12%
6%
3%
4%
14%
40%
19%
2%
61%
A
15%
3%
3%
0%
21%
48%
37%
10%
3%
2%
58%
B
sediment demonstrating metabolic activity by in situ substrate incorporation of phytodetritus (Gihring et al., 2009) and low molecular weight compounds (Miyatake et al., 2009; Webster et al., 2010).
Overall, our MAR-FISH experiments expand previous findings as they provide first evidence for a numerical relevance of the active Gammaproteobacteria under oxic and anoxic conditions in different sediment layers of a coastal site. Given their global distribution our findings support a central role in sulfur and carbon cycling in marine sediments worldwide.
MAR-FISH concluding remarks
Application of the MAR-FISH method provided first insights into the in situ abundance of autotrophic and heterotrophic Gammaproteobacteria. However, we could hardly resolve CO2-fixation and acetate
incorporation to the level of distinct gammaproteobacterial populations. Bacteria of the heterotrophic NOR5/OM60 clade, which did not take up acetate, might prefer other carbon compounds. However, preliminary attempts also failed to detect incorporation of alternative substrates such as glucose (Zerjatke, 2009). In case of the WS-Gam446 population the lack of substrate uptake might be addressed to their low in situ abundance (<1% of all cells). Assuming that on average only 20-30% of hybridized target cells are labeled, as found for the CO2-incoporating Gam209 group, the proportion of WS-Gam446 cells with associated silver grain precipitates detected by manual analysis would not allow a reliable quantification. Here, automated high-throughput microscopy holds a promising improvement for elaborative manual analysis of sediment bacteria (Zeder et al., 2009; Zeder and Pernthaler, 2009). The current preparation of Janssand samples will allow a fully automated enumeration of MAR and FISH signals in the near future (M. Zeder, personal communication). In addition, the application of density gradient centrifugation prior to MAR-FISH can significantly increase the cell density on membrane filters and facilitate a more efficient automated microscopic analysis.
The specific probes applied here cover approximately one third of all Gammaproteobacteria. Accordingly the proportion of cells not targeted by the probes might account for the observed uptake. During the course of the study the entire gammaproteobacterial community composition could not be resolved. The high overall diversity and the insufficient sequence coverage along with the limited set of applied probes hampered further resolution. Similar findings for sulfate reducing Deltaproteobacteria have been
reported (Mussmann et al., 2005). Thus, an array of low abundant species likely contributes to the observed substrate uptake and accounts for the overall gammaproteobacterial diversity in situ. Adequate resolution of the gammaproteobacterial community structure by FISH might be achieved by application of extended probe sets and modified counting protocols (Gomez-Pereira et al., 2010), which allow the reliable detection and quantification of scarce species.