Resumen de las obras

Coastal sediments are typically inhabited by rich macrofaunal communities, which can exert an important control on sedimentary biogeochemical cycling (Aller, 1988; Meysman et al., 2006). Our results revealed that apart from the effect of global environmental variables (water and sediment chl-a concentration, water temperature, organic C:N ratio in the sediment and grain size in space and time) on the link of microbial biodiversity and N-cycle processes in intertidal and subtidal sediments, this link was modulated by macrofauna through their effects on local environmental variables (DIN fluxes, O2 concentration and oscillations). Macrofauna affect the

environment mainly through their allogenic effects by bioturbation (particle mixing) and bio-irrigation (solute transfer) (Solan et al., 2004): (i) transferring water and solutes from the overlying water (Kristensen, 1988; Kristensen and Kostka, 2004) into the deeper sediment layers; (ii) extending the surface area available for diffusive solute exchange across the sediment-water interface (Howe et al., 2004, Nizzoli et

al., 2007); (iii) creating microniches within sediments and the burrow wall (Bertics and

Ziebis, 2009).

Our results further revealed the effect of macrofauna at the community and single species level (e.g. bio-irrigating polychaete, L. conchilega) on total microbial (bacteria

131 and archaea) communities and functional gene expression (amoA-carrying bacterial and archaeal nitrifiers and nosZ-carrying denitrifiers).

In the single species level such as Lanice conchilega, Lanice introduces oxygen-rich water in layers where oxygen is absent by bio-irrigating its tube (Forster and Graf, 1995) and also affects O2 concentration over short-time intervals through periodical

piston-pumping activity and intermittent ventilation (Forster and Graf, 1995; our study). We also found a density-dependent effect of Lanice bio-irrigation on the oxygen penetration depth within the Lanice reef. Increasing oxygen content and bringing oxygen deeper in the sediment by macrofauna enhance nitrification rates in the sediment which fuels denitrification (Howe et al., 2004). In addition, macrofaunal activity increases the surface for coupled nitrification-denitrification in the burrow wall (Howe et al., 2004; Birchenough et al., 2012) and probably in the sediment along the tube in tube building polychaetes (Kristensen and Kostka, 2005) such as L.

conchilega. Under conditions of O2 oscillation (such as the result of Lanice

intermittent ventilation activity), nitrifying and denitrifying bacteria react to oxygen and nitrate in the environment by coordinating their respective activities (Gao et al., 2010) and such conditions support higher abundance of denitrifying genes (nirS and nosZ) compared to oxic or anoxic conditions alone (Wittorf et al., 2016). The bio-irrigating behaviour of macrofauna (e.g. L. conchilega) can also affect directly other solute exchanges such as NO3− (Gilbert et al., 1997), which is used as a substrate for

denitrification.

Lanice conchilega manifests both autogenic and allogenic ecosystem engineering

characteristics (Godet et al., 2008). However, the effect of macrofauna on nitrogen cycling processes exclusively via autogenic effects (i.e. through their own physical structures) has not been reported yet (Braeckman et al., 2014a). Although our results showed measures of nosZ diversity indices were related to chl-a concentration and % mud in the sediment (Chapter 4), this was not affected by granulometric differences and differences in chl-a concentration resulted from autogenic activities of Lanice in our study site.

Previous studies of the effects of macrofauna on biogeochemical processes involved mainly manipulative experiments (e.g. introducing of organisms to homogenised and/or defaunated sediments) in laboratory contexts (Braeckman et al., 2010;

Laverock et al., 2010; Gilbertson et al., 2012; Stauffert et al., 2014) and the effect of single species of large burrowing macrofauna on the biogeochemical processes

132 (Bertics and Ziebis, 2009; Bertics et al., 2012; Laverock et al., 2014). In addition, our knowledge on interactions between macrofaunal and microbial communities is still very limited (Laverock et al., 2014). The impact of macrofaunal reworking activities on microbial communities was mainly assessed at the level of total bacterial community (Papaspyrou et al., 2005 and 2006; Bertics and Ziebis, 2009; Laverock et al., 2010;

Sapp et al., 2010) while less attention was paid to the archaeal community (Sapp et

al., 2010; Stauffert et al., 2014) and specific functional gene groups, such as nitrifiers and denitrifiers (Gilbertson et al., 2012; Laverock et al., 2014). The investigation of metabolically active microorganisms at RNA or protein level has also been rarely performed (technically challenging). In addition, an integrated study investigating simultaneously macrofauna, microbes and N-cycling processes (i.a. as done in this thesis, Chapter 3) was missing so far. We used the bioturbation potential of a community (BPc) as well as density and biomass to investigate the impact of macrofaunal communities. BPc is an index based on the classification of invertebrate taxa into discrete functional groups (Solan et al., 2004; Birchenough et al., 2012; Queirós et al., 2013). It is rather the functional biodiversity than taxonomic biodiversity that matters for benthic ecosystem functioning (Emmerson and Raffaelli 2000; Leno et al., 2006; Cardinale et al., 2012). The functional biodiversity can be estimated by calculating the contribution of each species using e.g. bioturbation potential of each species (BPi), which is at the basis of the bioturbation potential of a community (BPc) (Solan et al., 2004; Queirós et al., 2013). Therefore, this index reflects the capacity of macrofaunal communities to mix sediments (Solan et al., 2004; Queirós et al., 2013) and is related to the apparent redox potential discontinuity depth (aRPD) (Birchenough et al., 2011), which is known as biogenic mixing depth as well (Solan et al., 2004; Birchenough et al., 2011). Our results (Chapters 2 and 3) showed that macrofaunal activities (BPc) and/or abundance indeed were related to diversity indices of metabolically active β-AOB and AOA and also to the rates of nitrification in space and time. The models also indicated the significant importance of macrofaunal activities (BPc) to modulate the link between N-mineralization and total bacterial community affecting both bacteria and their mineralization activities. In addition, BPc was retained in our models for denitrification reflecting an increase in denitrification rates by maybe providing directly the nitrate from the overlying water (Nizzoli et al., 2007) and/or an increase in the coupled nitrification-denitrification processes especially in fine sands characterized by a rich BPc (Chapters 2 and 3).

133 However, BPc also has some drawbacks. BPc cannot predict all bioturbation attributes: while this index successfully predicts bioturbation distance (average distance travelled by sediment particles), it is not related to the other attributes such as bioturbation depth, activity and biodiffusive transport (Queirós et al., 2015). Ventilation of the burrow (bio-irrigation) is a mechanism which is also not accounted for in BPc. Bio-irrigating animals such as L. conchilega have a low score for M (mobility) and R (sediment reworking mode). However, bio-irrigation activity was shown to have indeed an important effect on benthic biogeochemical processes (Braeckman et al., 2010; Woodin et al., 2016). In Chapter 4, we also found density- dependent effects of the bio-irrigation activity of L. conchilega on the sediment heterogeneity and the expression of the nosZ gene, involved in the final step of the denitrification process.

Interaction between species is also not accounted for in BPc as BPc is a summation of single species effects, biomass, and abundance (Solan et al., 2004). However, despite the mentioned drawbacks of BPc, this index appears to explain the variability in measured fluxes and microbial processes much better than macrobenthic density or biomass alone (see Chapter 2; Van Colen et al., 2012).For future investigations, however, we suggest to develop better descriptions of bioturbation potential or extend the BPc index with a measure for bio-irrigation in the European Marine Strategy Framework Directive (MSFD).

5.4. Conclusions

This PhD increases the knowledge of factors controlling the microbial-mediated N- cycle processes in shallow coastal sediments and provides an important step in filling the existing gap how the functional traits of macroorganisms, through the interactions with microorganisms, affect microbial-mediated ecosystem functioning in coastal benthic ecosystems. We showed that the links between microorganisms and benthic biogeochemical N-cycle processes are affected by the density and/or activity of the macrofaunal communities causing heterogeneity in sedimentary environmental variables over local spatial and temporal scales as well as by changes in global environmental variables. We investigated this link in both intertidal and subtidal sediments (muddy, fine sandy and permeable sands) of shallow coastal areas and were able to relate the integrated effect of total macrofaunal activity and also bio- irrigation by a single macrofaunal species on sediment heterogeneity with functional

134 aspects of microbial communities. We showed that functional gene transcriptions (amoA-carrying bacterial and archaeal nitrifiers and nosZ-carrying denitrifiers) reveals better insights in the macrofauna – microbe – particular process interactions compared to the total microbial community (16S rDNA gene) investigation. Our results also indicated the effects of macrofauna on this link at both horizontal large scales (km and m) and small (cm) vertical scales.

Lanice conchilega in high densities affects denitrifying organisms causing an

increase in the relative abundance of nosZ transcripts at anoxic depth layers in the sediment, which may stimulate the rates of denitrification at depth. They do this via their allogenic effects by increasing the oxygen content at the top sediment layer, increasing oxygen penetration depth and stimulating oscillation in oxygen concentrations, which all lead to increasing coupled nitrification-denitrification at depth. Bio-irrigation behaviour can further affect the exchanges of other solutes across the sediment-water interface, including NO3− which is a substrate for

denitrification.

In our study area, the Belgian Part of the North Sea, the links between microbial biodiversity and ecosystem functioning in sediments in space and time are influenced by the annual spring phytoplankton bloom (water and sediment chl-a concentration), water temperature, sediment organic C:N ratio and grain size as well as larger fauna density and/or functional diversity. The importance of macrofauna depends on the sediment type. Fine sandy sediments are characterized by highest macrofauna densities, biomass and bioturbation potential (BPc). While nitrification and denitrification rates in permeable and muddy sediments were low, in the fine sandy sediments denitrification rates were high throughout the year. During summer, nitrification rates in fine sandy sediments were also higher than in muddy and permeable sediments. This was concomitant with the highest richness of metabolically active β-AOB and AOA in September. In fine sediments, AOA and β- AOB seem both to play roles in sedimentary nitrification in September but differentiation of their individual contributions was not investigated. Generally, our results revealed that bacteria (total and β-AOB) showed more spatio-temporal variation than archaea (total and AOA) as sedimentation of organic matter and the subsequent changes in the environment had a stronger impact on their community composition and diversity indices.

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