Sediments play a key role in the marine nitrogen cycle and can act either as a source or a sink of biologically available (fixed) nitrogen. This cycling is driven by a number of microbial remineralization reactions, many of which occur across the oxic/anoxic interface near the sediment surface. The presence and activity of large burrowing macrofauna (bioturbators) in the sediment can significantly affect these microbial processes by altering the physicochemical properties of the sediment. For example, the building and irrigation of burrows by bioturbators introduces fresh oxygenated water into deeper sediment layers and allows the exchange of solutes between the sediment and water column. Burrows can effectively extend the oxic/anoxic interface into deeper sediment layers, thus providing a unique environment for nitrogen-cycling microbial communities. Recent studies have shown that the abundance and diversity of micro-organisms can be far greater in burrow wall sediment than in the surrounding surface or subsurface sediment; meanwhile, bioturbated sediment supports higher rates of coupled nitrification–denitrification reactions and increased fluxes of ammonium to the water column. In the present paper we discuss the potential for bioturbation to significantly affect marine nitrogen cycling, as well as the molecular techniques used to study microbial nitrogen cycling communities and directions for future study.
- microbial community structure
- nitrogen cycle
- nutrient flux
- oxic/anoxic interface
Nitrogen cycling in marine sediments
Microbially driven cycling of nitrogen (Figure 1) in the world's oceans is a key process regulating biological productivity. Sediments play a fundamental role in recycling fixed nitrogen to the water column: it is estimated that up to 80% of the nitrogen needed by primary producers in shallow shelf seas is provided by benthic (sea floor) remineralization reactions . Conversely, marine sediments also represent an important sink for fixed nitrogen via the burial of organic matter; and by either the reduction of nitrate into N2 or N2O via denitrification; and/or the conversion of nitrite and ammonium into N2 via anaerobic ammonium oxidation (anammox) [2,3]. According to current nitrogen budgets, benthic processes account for two-thirds of the total marine conversion of fixed nitrogen into N2: 300 Tg·year−1 of a total 450 Tg·year−1 . Sediments are therefore a key site for marine nitrogen cycling, because they provide a largely anoxic environment in which anaerobic reduction reactions, such as denitrification, can take place, often within a few millimetres of the sediment/water interface . Sediments also provide a large repository for sinking organic matter from the water column, with the fate of this organic matter, once it reaches the sea floor, being dependent upon the compartmentalization of the different remineralization processes occurring within the sediment . Once nitrogen within organic matter is remineralized through ammonification, ammonium is then oxidized to nitrite and nitrate via nitrification. This occurs within the oxic zone comprising a few millimetres at the sediment/water interface (Figure 1). These oxidized compounds are then either released to the water column or used to fuel reductive processes such as denitrification. Thus nitrification performs several important functions, linking (i) the oxic and anoxic reactions within the nitrogen cycle, (ii) the production of new compounds to the loss of fixed nitrogen, and (iii) the decomposition of organic matter to the release of nutrients from the sediment [2,3,6]. For this reason, the oxic/anoxic interface occurring at the sediment surface is of particular importance in the marine nitrogen cycle .
Investigating the marine nitrogen cycle
Despite its obvious importance, our understanding of the marine nitrogen cycle is far from complete. In particular, our knowledge concerning the identity, distribution and function of the key microbial organisms involved is rapidly evolving. For example, the recent discoveries of both anaerobic ammonium oxidation (anammox) and of archaea as key drivers of ammonia oxidation have sparked considerable research effort into the distribution and importance of these processes in the marine realm. These discoveries have been reviewed extensively elsewhere [7–9]. However, controversy remains over the comparative importance of denitrification and anammox, especially within the oxygen minimum zones of the world's oceans . Much of this research has relied on the application of genetic markers to study the micro-organisms responsible for the different nitrogen cycling processes (Table 1) combined with isotope-pairing techniques  to study rates of nitrification, denitrification and anammox. These, combined with the advent of high-throughput sequencing technologies, are allowing us to explore more fully the marine nitrogen cycle in order to address many of the current knowledge gaps.
Bioturbation: an introduction
Macrofauna living in or on marine sediments can significantly alter both the physical structure and chemical composition of the sediment through their burrowing and irrigation activities. For this reason, they have long been considered ‘ecosystem engineers’ , and their presence can create unique microniches for sediment micro-organisms to inhabit [22–24]. To date, most bioturbation studies have concentrated on burrowers such as polychaete worms and crustaceans (e.g. [25–29]). However, bioturbation may be carried out by other benthic macrofauna, including molluscs and echinoderms, as well as pelagic bottom-feeders such as walrus, fish and stingrays, and meiofaunal communities such as larvae and nematode worms .
Infaunal tubes and burrows are semi-permanent or permanent structures that differ in size, appearance and composition according to the mode of life and habits of the inhabitant. A previous study has calculated the global volume of bioturbated sediment to be >20 700 km3 (based on an estimated ocean surface area of 360 million km2) ; clearly, the sheer abundance of these ‘ecosystem engineers’ means that they have the potential to have an impact on a substantial proportion of the world's shallow shelf marine habitats via their bioturbating activities. It has long been known that the macrofaunal burrow environment can support different physicochemical properties from those experienced in both the sediment surface and the surrounding ambient sediment [32,33]. In particular, the burrow may be frequently or intermittently ventilated by its inhabitant, thereby introducing fresh oxygenated water into the burrow; allowing greater solute exchange and an extension of the oxic/anoxic interface into deeper sediment [5,34]. For example, it is estimated that the presence of thalassinidean shrimp burrows can increase the sediment surface area by up to nine times . Additionally, it is thought that the physical mixing of sediment during burrow building and maintenance activities can contribute to increased biodiversity of both meiofaunal  and microbial communities [29,37].
Microbial community structure within sediment burrows
The layer of sediment, organic matter and biopolymers that make up the burrow wall has the potential to affect the benthic nitrogen cycle. First, bacterial abundance and activity can be 10-fold higher in the macrofaunal burrow wall compared with surrounding sediment [27,32]. Secondly, the composition of the microbial communities inhabiting burrow walls can also be significantly different from those in the surrounding sediment, in terms of both their structure and diversity [28,29,38]. More specifically, Satoh et al.  found that the ammonia-oxidizing and nitrite-oxidizing communities in the burrow walls of a polychaete worm were similar to those in surface sediment and the abundance of gene markers for these functional guilds was much higher in the burrow wall than in the bulk sediment. However, it should be noted that the exact effects of bioturbation on microbial community structure may be dependent upon the individual burrow builder. For example, the burrows of specific nereidid worms , thalassinidean shrimp  and fiddler crabs  were found to be more similar to the surrounding subsurface (anoxic) sediment than to surface sediment in terms of microbial community structure. Conversely, the burrows of some polychaete worms  and thalassinidean shrimp [28,29] were found to contain microbial communities more similar to those in the oxygenated surface sediment than those in subsurface ambient sediment. It is therefore probable that the individual burrowing and irrigation behaviours of different macrofaunal burrowers contribute to creating a physicochemical environment that may in itself be unique, but more similar to either the surface or subsurface sediment, depending on the burrow inhabitant [23,32].
Influence of bioturbation on nutrient fluxes and nitrogen cycling rates
Nutrient flux rates depend on many factors, including the type of sediment substrate and the mode and extent of bioturbation. Burrow walls effectively act as an extension of the oxic surface of the sediment, which has particular relevance to nitrification–denitrification reactions coupled across the oxic/anoxic interface . Fluxes of O2, total CO2 and dissolved inorganic nitrogen across the sediment/water interface have been observed to increase by 2.5–3.5 times in bioturbated sediment relative to control non-bioturbated sediment [5,26,42]. The concentration profiles for ammonium, nitrite and nitrate are linked with changes in oxygen concentration. Nedwell and Walker  showed that the presence of amphipods in Antarctic sediments doubled the production of NH4+ by direct release or stimulation of vertical transport processes, and Aller and Aller  demonstrated that the net remineralization rate for NH4+ increases as the efficiency of solute exchange with the overlying water increases. Meanwhile, several studies have shown that bioturbation activity increases denitrification by up to 400% [26,45,46]. However, this is not simply a direct consequence of macrofaunal bioturbation, rather the result of environmental alterations which affect microbially driven biogeochemical processes. For instance, Howe et al.  noted that most of the increase in denitrification stimulated by the presence of burrowing shrimp was fuelled by nitrification occurring within the burrow walls, suggesting that it is the elevated rates of nitrifying bacteria utilizing the increased fluxes of O2 and NH4+, rather than the direct supply of NO3−, that is the driving factor. It is generally accepted that the most important role of bioturbation in stimulating remineralization reactions, such as denitrification, is the introduction of oxygen into subsurface sediments which can stimulate the decay of organic matter by a factor of ten .
The impact of bioturbation on microbial activity is complicated further by the existence of microniches within sediment systems and within the burrow wall in particular . The burrow can be viewed as a unique environment which experiences fluctuations in its biogeochemistry as a result of the irrigation, burrow building and burrow maintenance behaviours of its inhabitant; this may indeed lead to unique communities of micro-organisms inhabiting the burrow wall . Burrow walls are therefore not geochemically uniform structures, and often exhibit patterns of zonation and heterogeneity . For example, nitrogen fixation is normally assumed to be unimportant in burrows because of the high levels of oxygen and ammonium, both of which inhibit the activity of the nitrogenase enzyme. However, Bertics et al.  observed far greater levels of nitrogenase activity in sediments that were heavily populated by burrowing ghost shrimp. This activity was apparently linked to the presence of nifH genes (Table 1) in SRB (sulfate-reducing bacteria), suggesting that the SRB were able to exploit anoxic microniches within the shrimp burrows, and contribute to increased benthic nitrogen fixation and sulfate reduction. The authors suggest that in benthic nitrogen cycling models, bioturbated sediments could be a source of fixed nitrogen as well as a loss term due to stimulated denitrification .
A greater understanding of the occurrence and activity of these microbial microniches could significantly change our understanding of the effects of bioturbation on the marine nitrogen cycle. This may be particularly important when we consider that the changes in sediment biogeochemistry and microbial diversity brought about by bioturbation may have indirect effects on the nitrogen cycle via changes to the anaerobic respiration pathways that occur in sediments. For example, any increase in sulfate reduction is likely to have an inhibitive effect on both nitrification and denitrification in the burrow . Meanwhile, it appears that within fiddler crab burrows in salt marsh sediments, iron-reducing bacteria may out-compete SRB . This repartitioning of remineralization reactions could be important because the inhibitory effects of sulfide on nitrification may be ameliorated by increased amounts of iron(III) present in the burrow wall, which rapidly scavenges sulfide from the environment [23,49]. The spatial overlap of respiration processes, including denitrification, sulfate reduction and iron reduction, may therefore be encouraged by the presence of burrow microniches [23,24,50].
Challenges and future directions
We are still at the beginning of our understanding of how bioturbating macro-organisms interact with the sediment and its microbial communities, and the effects this interaction has on important biogeochemical cycles in the marine realm. It is clear from the numerous studies on nutrient flux measurements and biogeochemical rates that microbial activity is enhanced within the unique burrow environment. Several studies (as cited previously) have also shown that microbial diversity and abundance may be greater in burrow walls, which may also contain a different microbial community from that of the surrounding sediment. However, it is important now to investigate more deeply the taxonomy and functional diversity of burrow communities, specifically in relation to the abundance and activity of key nitrogen cycling genes.
Sampling marine sediments is challenging, especially when it is important to retain as much as possible the in situ biogeochemical conditions of the sediment. Many studies have sampled in situ infaunal burrows, mostly at periods when they are exposed by low tides (e.g. ). Some have also used mesocosms to investigate the burrow in the laboratory environment (e.g. ), whereas others have used ‘mimic’ burrow structures in the laboratory (e.g. ). All of these techniques have their attendant benefits and drawbacks, and often the choice will be down to logistical questions as well as knowledge of which technique will allow the most effective sampling for the analyses to be done. However, it is important to note that mesocosm studies will inevitably differ from those conducted in situ [23,52], and, where possible, it is essential to take into account the changes in macrofaunal and microbial activity that occur with season, temperature, salinity, sediment type and organic loading, and to compare any laboratory results with relevant in situ data.
Finally, given the clear relationship between bioturbating activity and microbial nutrient cycling, it would be beneficial to consider the factors that may affect this relationship. In particular, it is known that many bioturbators may be physiologically and/or behaviourally sensitive to environmental changes such as increasing seawater temperature and decreasing pH (e.g. [53–55]). In addition, it seems that there is increasing eutrophication within coastal waters from industrial and agricultural run-off , and an expansion of the world's oceans' oxygen minimum zones, which are themselves important sites for pelagic denitrification and anammox . Considering the relatively rapid changes that are occurring within our oceans, a key question should be what knock-on effects these changes will have on macrofaunal bioturbator behaviour, and hence on the essential functioning of nitrogen cycling micro-organisms in the sediment.
B.L. acknowledges funding from a Natural Environment Research Council (NERC) algorithm Ph.D. Studentship [NE/F008864/1] and from the NERC-funded programme Oceans 2025 (Theme 3: Coastal and Shelf Processes).
Enzymology and Ecology of the Nitrogen Cycle: A Biochemical Society Focused Meeting held at University of Birmingham, U.K., 15–17 September 2010. Organized and Edited by Jeff Cole (University of Birmingham, U.K.), Rosa María Martínez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick Watmough (University of East Anglia, Norwich, U.K.).
Abbreviations: SRB, sulfate-reducing bacteria
- © The Authors Journal compilation © 2011 Biochemical Society