Accelerated nitrogen cycling on Mediterranean seagrass leaves at volcanic CO2 vents.

Seagrass meadows form highly productive and diverse ecosystems in coastal areas worldwide, where they are increasingly exposed to ocean acidification (OA). Efficient nitrogen (N) cycling and uptake are essential to maintain plant productivity, but the effects of OA on N transformations in these systems are poorly understood. Here we show that complete N cycling occurs on leaves of the Mediterranean seagrass Posidonia oceanica at a volcanic CO2 vent near Ischia Island (Italy), with OA affecting both N gain and loss while the epiphytic microbial community structure remains largely unaffected. Daily leaf-associated N2 fixation contributes to 35% of the plant's N demand under ambient pH, while it contributes to 45% under OA. Nitrification potential is only detected under OA, and N-loss via N2 production increases, although the balance remains decisively in favor of enhanced N gain. Our work highlights the role of the N-cycling microbiome in seagrass adaptation to OA, with key N transformations accelerating towards increased N gain.

Examining the resistance and resilience of anode-respiring Shewanella oneidensis biohybrid using microsensors.

Immobilizing electro-active microbes within polymer matrices (thereby forming biohybrids) is a promising approach to accelerate microbial attachment to electrodes and increase the biofilm robustness. However, little is known on the fine scale chemical environment that develops within the electro-active biohybrids. Herein, we develop a biohybrid by immobilizing a culture of Shewanella oneidensis MR1 in agar matrix on the surface of a graphite electrode poised at +0.25 V. The resulting bioanode (3-6 mm thick) was grown under anoxic conditions and produced a steady current of 40 µA. Oxygen and pH distribution within the biohybrid were characterized in-situ using microsensors. As Shewanella is a facultative aerobe, it will halt the current production in the presence of oxygen. Thus, in addition, we investigated the alteration of the microenvironment during and after aeration of the medium to evaluate the oxygen tolerance of the system. During aeration, oxygen was effectively consumed in the top layers of the biofilm, leaving a 400-900 µm thick anoxic zone on the anode surface, that sustained >60% of the initial current. Current production recovered to pre-oxic condition within 5 h after the aeration was stopped, showing that immobilization can promote both high resistance and resilience of the system. Despite the absence of strong buffering conditions, pH profiles indicated a maximum drop of 0.2 units across the biohybrid. Characterizing the chemical microenvironment helps to elucidate the mechanistic functioning of artificial biofilms and hold a great potential for the designing of future, more effective biohybrid electrodes.

Microsensor for total dissolved sulfide (TDS).

Total Dissolved Sulfide (TDS) concentrations can either be derived from simultaneous measurement of pH and one of the sulfide species or determined indirectly in samples following an acidification step. Here we report a microsensor that allows for direct measurement of TDS in aquatic media without the need for pH monitoring. An acidic chamber placed in front of a commercial, amperometric H2S microsensor allows for the in-situ conversion of dissolved ionic sulfide species to H2S, which in turn is oxidized at the transducer anode. A typical sensor had a tip opening of 30 µm, a response time of <50 s and linear range between 0.5 and 650 µM. The sensor performance can be largely tuned by altering the geometry of the chamber. Sensors of different sensitivity (0.04-2.93 pA/µM) showed no noticeable change in zero current and sensitivity during continuous polarization over 7 weeks. The sensor was successfully applied to resolve microscale TDS gradients in freshwater and marine sediments. Other avenues of application include the online monitoring of industrial and urban sewers.

Nested interactions between chemosynthetic lucinid bivalves and seagrass promote ecosystem functioning in contaminated sediments.

In seagrass sediments, lucinid bivalves and their chemoautotrophic bacterial symbionts consume H2S, relying indirectly on the plant productivity for the presence of the reduced chemical. Additionally, the role of lucinid bivalves in N provisioning to the plant (through N2 fixation by the symbionts) was hypothesized. Thus, lucinids may contribute to sediment detoxification and plant fitness. Seagrasses are subject to ever-increasing human pressure in coastal environments. Here, disentangling nested interactions between chemosynthetic lucinid bivalves and seagrass exposed to pollution may help to understand seagrass ecosystem dynamics and to develop successful seagrass restoration programs that consider the roles of animal-microbe symbioses. We evaluated the capacity of lucinid bivalves (Loripes orbiculatus) to promote nutrient cycling and seagrass (Cymodocea nodosa) growth during a 6-week mesocosm experiment. A fully crossed design was used to test for the effect of sediment contamination (metals, nutrients, and hydrocarbons) on plant and bivalve (alone or interacting) fitness, assessed by mortality, growth, and photosynthetic efficiency, and for the effect of their nested interaction on sediment biogeochemistry. Plants performed better in the contaminated sediment, where a larger pool of dissolved nitrogen combined with the presence of other trace elements allowed for an improved photosynthetic efficiency. In fact, pore water nitrogen accumulated during the experiment in the controls, while it was consumed in the contaminated sediment. This trend was accentuated when lucinids were present. Concurrently, the interaction between clams and plants benefitted both organisms and promoted plant growth irrespective of the sediment type. In particular, the interaction with lucinid clams resulted in higher aboveground biomass of C. nodosa in terms of leaf growth, leaf surface, and leaf biomass. Our results consolidate the notion that nested interactions involving animal-microbe associations promote ecosystem functioning, and potentially help designing unconventional seagrass restoration strategies that exploit chemosynthetic symbioses.

Snorkels enhance alkanes respiration at ambient and increased hydrostatic pressure (10 MPa) by either supporting the TCA cycle or limiting alternative routes for acetyl-CoA metabolism.

The impact of piezosensitive microorganisms is generally underestimated in the ecology of underwater environments exposed to increasing hydrostatic pressure (HP), including the biodegradation of crude oil components. Yet, no isolated pressure-loving (piezophile) microorganism grows optimally on hydrocarbons, and no isolated piezophile at all has a HP optimum <10 MPa (e.g. 1000 m below sea water level). Piezosensitive heterotrophs are thus largely accountable for oil clean up < 10 MPa, however, they are affected by such a mild HP increase in ways which are not completely clear. In a first study, the application of a bioelectrochemical system (called "oil-spill snorkel") enhanced the alkane oxidation capacity in sediments collected at surface water but tested up to 10 MPa. Here, the fingerprint left on transcript abundance was studied to explore which metabolic routes are 1) supported by snorkels application and 2) negatively impacted by HP increase. Transcript abundance was comparable for beta-oxidation across all treatments (also at a taxonomical level), while the metabolism of acetyl-CoA was highly impacted: at either 0.1 or 10 MPa, snorkels supported acetyl-CoA oxidation within the TCA cycle, while in negative controls using non-conductive rods several alternative routes for acetyl-CoA were stimulated (including those leading to internal carbon reserves e.g. 2,3 butanediol and dihydroxyacetone). In general, increased HP had opposite effects as compared to snorkels, thus indicating that snorkels could enhance hydrocarbons oxidation by alleviating in part the stressing effects imposed by increased HP on the anaerobic, respiratory electron transport chain. 16S rRNA gene analysis of sediments and biofilms on snorkels suggest a crosstalk between oil-degrading, sulfate-reducing microorganisms and sulfur oxidizers. In fact, no sulfur was deposited on snorkels, however, iron, aluminum and phosphorous were found to preferentially deposit on snorkels at 10 MPa. This data indicates that a passive BES such as the oil-spill snorkel can mitigate the stress imposed by increased HP on piezosensitive microorganisms (up to 10 MPa) without being subjected to passivation. An improved setup applying these principles can further support this deep-sea bioremediation strategy.

The role of epiphytes in seagrass productivity under ocean acidification.

Ocean Acidification (OA), due to rising atmospheric CO2, can affect the seagrass holobiont by changing the plant's ecophysiology and the composition and functioning of its epiphytic community. However, our knowledge of the role of epiphytes in the productivity of the seagrass holobiont in response to environmental changes is still very limited. CO2 vents off Ischia Island (Italy) naturally reduce seawater pH, allowing to investigate the adaptation of the seagrass Posidonia oceanica L. (Delile) to OA. Here, we analyzed the percent cover of different epiphytic groups and the epiphytic biomass of P. oceanica leaves, collected inside (pH 6.9-7.9) and outside (pH 8.1-8.2) the CO2 vents. We estimated the contribution of epiphytes to net primary production (NPP) and respiration (R) of leaf sections collected from the vent and ambient pH sites in laboratory incubations. Additionally, we quantified net community production (NCP) and community respiration (CR) of seagrass communities in situ at vent and ambient pH sites using benthic chambers. Leaves at ambient pH sites had a 25% higher total epiphytic cover with encrusting red algae (32%) dominating the community, while leaves at vent pH sites were dominated by hydrozoans (21%). Leaf sections with and without epiphytes from the vent pH site produced and respired significantly more oxygen than leaf sections from the ambient pH site, showing an average increase of 47 ± 21% (mean ± SE) in NPP and 50 ± 4% in R, respectively. Epiphytes contributed little to the increase in R; however, their contribution to NPP was important (56 ± 6% of the total flux). The increase in productivity of seagrass leaves adapted to OA was only marginally reflected by the results from the in situ benthic chambers, underlining the complexity of the seagrass community response to naturally occurring OA conditions.

Dissimilatory nitrate reduction by a freshwater cable bacterium.

Cable bacteria (CB) are filamentous Desulfobulbaceae that split the energy-conserving reaction of sulfide oxidation into two half reactions occurring in distinct cells. CB can use nitrate, but the reduction pathway is unknown, making it difficult to assess their direct impact on the N-cycle. Here we show that the freshwater cable bacterium Ca. Electronema sp. GS performs dissimilatory nitrate reduction to ammonium (DNRA). 15NO3--amended sediment with Ca. Electronema sp. GS showed higher rates of DNRA and nitrite production than sediment without Ca. Electronema sp. GS. Electron flux from sulfide oxidation, inferred from electric potential (EP) measurements, matched the electron flux needed to drive CB-mediated nitrate reduction to nitrite and ammonium. Ca. Electronema sp. GS expressed a complete nap operon for periplasmic nitrate reduction to nitrite, and a putative octaheme cytochrome c (pOCC), whose involvement in nitrite reduction to ammonium remains to be verified. Phylogenetic analysis suggests that the capacity for DNRA was acquired in multiple events through horizontal gene transfer from different organisms, before CB split into different salinity niches. The architecture of the nitrate reduction system suggests absence of energy conservation through oxidative phosphorylation, indicating that CB primarily conserve energy through the half reaction of sulfide oxidation.

Novel method to immobilize phosphate in lakes using sediment microbial fuel cells.

Phosphate pollution in lakes poses an intractable remediation challenge. Accumulated stocks of phosphorus in sediments cause high concentrations in the overlying water despite elimination of external sources. We propose to use sediment microbial fuel cells (SMFCs) for lake remediation by sediment phosphorus immobilization. The hypothesis is that SMFCs can increase sediment redox potential at the top layer, and that such changes will allow the sediment to retain phosphorus as immobile species. This study placed an emphasis on scalability, practicality, and use of low-cost materials. Stainless steel net was selected as electrode material, and modifications were tested: (i) chronoamperometric operation with anode poised at +399 mV (versus standard hydrogen potential); (ii) injection of graphite slurry; and (iii) coating with nickel-carbon matrix. Stainless steel electrodes were implemented in laboratory microcosms (1.3 L) and at field scale in a eutrophic freshwater lake. All tests were carried out in untreated sediment and water from Lake Søllerød, Denmark. Phosphate immobilization was shown at lab scale, with 85% decrease in overlying water using steel electrodes. At field scale maximum phosphate decrease of 94% was achieved in the water body above a 16 m2 stainless steel SMFC electrode. Results are promising and warrant further study, including remediation trials at full scale. Added benefits include degradation of sediment organic matter and pollutants, inhibition of methane and sulfide emission and production of electricity.

Effect of salinity on cable bacteria species composition and diversity.

Cable bacteria (CB) are Desulfobulbaceae that couple sulphide oxidation to oxygen reduction over centimetre distances by mediating electric currents. Recently, it was suggested that the CB clade is composed of two genera, Ca. Electronema and Ca. Electrothrix, with distinct freshwater and marine habitats respectively. However, only a few studies have reported CB from freshwater sediment, making this distinction uncertain. Here, we report novel data to show that salinity is a controlling factor for the diversity and the species composition within CB populations. CB sampled from a freshwater site (salinity 0.3) grouped into Ca. Electronema and could not grow under brackish conditions (salinity 21), whereas CB from a brackish site (salinity 21) grouped into Ca. Electrothrix and decreased by 93% in activity under freshwater conditions. On a regional scale (Baltic Sea), salinity significantly influenced species richness and composition. However, other environmental factors, such as temperature and quantity and quality of organic matter were also important to explain the observed variation. A global survey of 16S rRNA gene amplicon sequencing revealed that the two genera did not co-occur likely because of competitive exclusion and identified a possible third genus.

Meiofauna improve oxygenation and accelerate sulfide removal in the seasonally hypoxic seabed.

Oxygen depleted areas are widespread in the marine realm. Unlike macrofauna, meiofauna are abundant in hypoxic sediments. We studied to what extent meiofauna affect oxygen availability, sulfide removal and microbial communities. Meiofauna were extracted alive and added to intact sediments simulating abundance gradients previously reported in the area. A total of 324 porewater microprofiles were recorded over a 3-week incubation period and microbial community structure and cable bacteria densities were determined at the end of the experiment. At high abundances meiofauna activity deepened oxygen penetration by 85%, 59%, and 62% after 5, 14, and 22 days, respectively, compared to control sediment with scarce meiofauna. After 6 days, meiofauna increased the volume of oxidized, sulfide-free sediment by 68% and reduced sulfide fluxes from 8.8 to 0.4 mmol m-2 d-1. After 15 days, the difference with the control attenuated due to the presence of a cable bacteria population, which facilitated sulfides oxidation in all treatments. 16S rRNA gene analysis revealed that meiofauna affected microbial community structure (beta diversity). Thus, meiofauna bioturbation plays an important role in deepening oxygen penetration, counteracting euxinia and in structuring microbial diversity of hypoxic sediments. Co-existence with cable bacteria demonstrates neutralism interaction between these two ecosystem engineers.

Uncovering diversity and metabolic spectrum of animals in dead zone sediments.

Ocean deoxygenation driven by global warming and eutrophication is a primary concern for marine life. Resistant animals may be present in dead zone sediments, however there is lack of information on their diversity and metabolism. Here we combined geochemistry, microscopy, and RNA-seq for estimating taxonomy and functionality of micrometazoans along an oxygen gradient in the largest dead zone in the world. Nematodes are metabolically active at oxygen concentrations below 1.8 µmol L-1, and their diversity and community structure are different between low oxygen areas. This is likely due to toxic hydrogen sulfide and its potential to be oxidized by oxygen or nitrate. Zooplankton resting stages dominate the metazoan community, and these populations possibly use cytochrome c oxidase as an oxygen sensor to exit dormancy. Our study sheds light on mechanisms of animal adaptation to extreme environments. These biological resources can be essential for recolonization of dead zones when oxygen conditions improve.

Electrogenic sulfide oxidation mediated by cable bacteria stimulates sulfate reduction in freshwater sediments.

Cable bacteria are filamentous members of the Desulfobulbaceae family that oxidize sulfide with oxygen or nitrate by transferring electrons over centimeter distances in sediments. Recent studies show that freshwater sediments can support populations of cable bacteria at densities comparable to those found in marine environments. This is surprising since sulfide availability is presumably low in freshwater sediments due to sulfate limitation of sulfate reduction. Here we show that cable bacteria stimulate sulfate reduction in freshwater sediment through promotion of sulfate availability. Comparing experimental freshwater sediments with and without active cable bacteria, we observed a three- to tenfold increase in sulfate concentrations and a 4.5-fold increase in sulfate reduction rates when cable bacteria were present, while abundance and community composition of sulfate-reducing microorganisms (SRM) were unaffected. Correlation and ANCOVA analysis supported the hypothesis that the stimulation of sulfate reduction activity was due to relieve of the kinetic limitations of the SRM community through the elevated sulfate concentrations in sediments with cable bacteria activity. The elevated sulfate concentration was caused by cable bacteria-driven sulfide oxidation, by sulfate production from an indigenous sulfide pool, likely through cable bacteria-mediated dissolution and oxidation of iron sulfides, and by enhanced retention of sulfate, triggered by an electric field generated by the cable bacteria. Cable bacteria in freshwater sediments may thus be an integral component of a cryptic sulfur cycle and provide a mechanism for recycling of the scarce resource sulfate, stimulating sulfate reduction. It is possible that this stimulation has implication for methanogenesis and greenhouse gas emissions.

Parallel artificial and biological electric circuits power petroleum decontamination: The case of snorkel and cable bacteria.

Degradation of petroleum hydrocarbons (HC) in sediments is often limited by the availability of electron acceptors. By allowing long-distance electron transport (LDET) between anoxic sediments and oxic overlying water, bioelectrochemical snorkels may stimulate the regeneration of sulphate in the anoxic sediment thereby accelerating petroleum HC degradation. Cable bacteria can also mediate LDET between anoxic and oxic sediment layers and thus theoretically stimulate petroleum HC degradation. Here, we quantitatively assessed the impact of cable bacteria and snorkels on the degradation of alkanes in marine sediment from Aarhus Bay (Denmark). After seven weeks, cable bacteria and snorkels accelerated alkanes degradation by +24 and +25%, respectively, compared to control sediment with no cable bacteria nor snorkel. The combination of snorkels and cable bacteria further enhanced alkanes degradation (+46%). Higher degradation rates were sustained by LDET-induced sulphide removal rather than, as initially hypothesized, sulphate regeneration. Cable bacteria are thus overlooked players in the self-healing capacity of crude-oil contaminated sediments, and may inspire novel remediation treatments upon hydrocarbon spillage.

Zebra Mussel Holobionts Fix and Recycle Nitrogen in Lagoon Sediments.

Bivalves are ubiquitous filter-feeders able to alter ecosystems functions. Their impact on nitrogen (N) cycling is commonly related to their filter-feeding activity, biodeposition, and excretion. A so far understudied impact is linked to the metabolism of the associated microbiome that together with the host constitute the mussel's holobiont. Here we investigated how colonies of the invasive zebra mussel (Dreissena polymorpha) alter benthic N cycling in the shallow water sediment of the largest European lagoon (the Curonian Lagoon). A set of incubations was conducted to quantify the holobiont's impact and to quantitatively compare it with the indirect influence of the mussel on sedimentary N transformations. Zebra mussels primarily enhanced the recycling of N to the water column by releasing mineralized algal biomass in the form of ammonium and by stimulating dissimilatory nitrate reduction to ammonium (DNRA). Notably, however, not only denitrification and DNRA, but also dinitrogen (N2) fixation was measured in association with the holobiont. The diazotrophic community of the holobiont diverged substantially from that of the water column, suggesting a unique niche for N2 fixation associated with the mussels. At the densities reported in the lagoon, mussel-associated N2 fixation may account for a substantial (and so far, overlooked) source of bioavailable N. Our findings contribute to improve our understanding on the ecosystem-level impact of zebra mussel, and potentially, of its ability to adapt to and colonize oligotrophic environments.

Capping with activated carbon reduces nutrient fluxes, denitrification and meiofauna in contaminated sediments.

Sediment capping with activated carbon (AC) is an effective technique used in remediation of contaminated sediments, but the ecological effects on benthic microbial activity and meiofauna communities have been largely neglected. This study presents results from a 4-week experiment investigating the influence of two powdered AC materials (bituminous coal-based and coconut shell-derived) and one control material (clay) on biogeochemical processes and meiofauna in contaminated sediments. Capping with AC induced a 62-63% decrease in denitrification and a 66-87% decrease in dissimilatory nitrate reduction to ammonium (DNRA). Sediment porewater pH increased from 7.1 to 9.0 and 9.7 after addition of bituminous AC and biomass-derived AC, respectively. High pH (>8) persisted for at least two weeks in the bituminous AC and for at least 24 days in the coconut based AC, while capping with clay had no effect on pH. We observed a strong impact (nitrate fluxes being halved in presence of AC) on nitrification activity as nitrifiers are sensitive to high pH. This partly explains the significant decrease in nitrate reduction rates since denitrification was almost entirely coupled to nitrification. Total benthic metabolism estimated by sediment oxygen uptake was reduced by 30 and 43% in presence of bituminous coal-based AC and coconut shell-derived AC, respectively. Meiofauna abundances decreased by 60-62% in the AC treatments. Taken together, these observations suggest that AC amendments deplete natural organic carbon, intended as food, to heterotrophic benthic communities. Phosphate efflux was 91% lower in presence of bituminous AC compared to untreated sediment probably due to its content of aluminum (Al) oxides, which have high affinity for phosphate. This study demonstrates that capping with powdered AC produces significant effects on benthic biogeochemical fluxes, microbial processes and meiofauna abundances, which are likely due to an increase in porewater pH and to the sequestration of natural, sedimentary organic matter by AC particles.

Transient bottom water oxygenation creates a niche for cable bacteria in long-term anoxic sediments of the Eastern Gotland Basin.

Cable bacteria have been reported in sediments from marine and freshwater locations, but the environmental factors that regulate their growth in natural settings are not well understood. Most prominently, the physiological limit of cable bacteria in terms of oxygen availability remains poorly constrained. In this study, we investigated the presence, activity and diversity of cable bacteria in relation to a natural gradient in bottom water oxygenation in a depth transect of the Eastern Gotland Basin (Baltic Sea). Cable bacteria were identified by FISH at the oxic and transiently oxic sites, but not at the permanently anoxic site. Three species of the candidate genus Electrothrix, i.e. marina, aarhusiensis and communis were found coexisting within one site. The highest filament density (33 m cm-2 ) was associated with a 6.3 mm wide zone depleted in both oxygen and free sulphide, and the presence of an electric field resulting from the electrogenic sulphur oxidizing metabolism of cable bacteria. However, the measured filament densities and metabolic activities remained low overall, suggesting a limited impact of cable bacteria at the basin level. The observed bottom water oxygen levels (< 5 µM) are the lowest so far reported for cable bacteria, thus expanding their known environmental distribution.

Effect of settled diatom-aggregates on benthic nitrogen cycling.

The marine sediment hosts a mosaic of microhabitats. Recently it has been demonstrated that the settlement of phycodetrital aggregates can induce local changes in the benthic O2 distribution due to confined enrichment of organic material and alteration of the diffusional transport. Here, we show how this microscale O2 shift substantially affects benthic nitrogen cycling. In sediment incubations, the settlement of diatom-aggregates markedly enhanced benthic O2 and NO3- consumption and stimulated NO2- and NH4+ production. Oxygen microprofiles revealed the rapid development of anoxic niches within and underneath the aggregates. During 120 h following the settling of the aggregates, denitrification of NO3- from the overlying water increased from 13.5 µmol m-2 h-1 to 24.3 µmol m-2 h-1, as quantified by 15N enrichment experiment. Simultaneously, N2 production from coupled nitrification-denitrification decreased from 33.4 µmol m-2 h-1 to 25.9 µmol m-2 h-1, probably due to temporary inhibition of the benthic nitrifying community. The two effects were of similar magnitude and left the total N2 production almost unaltered. At the aggregate surface, nitrification was, conversely, very efficient in oxidizing NH4+ liberated by mineralization of the aggregates. The produced NO3- was preferentially released into the overlying water and only a minor fraction contributed to denitrification activity. Overall, our data indicate that the abrupt change in O2 microdistribution caused by aggregates stimulates denitrification of NO3- from the overlying water, and loosens the coupling between benthic nitrification and denitrification both in time and space. The study contributes to expanding the conceptual and quantitative understanding of how nitrogen cycling is regulated in dynamic benthic environments.

Electric coupling between distant nitrate reduction and sulfide oxidation in marine sediment.

Filamentous bacteria of the Desulfobulbaceae family can conduct electrons over centimeter-long distances thereby coupling oxygen reduction at the surface of marine sediment to sulfide oxidation in deeper anoxic layers. The ability of these cable bacteria to use alternative electron acceptors is currently unknown. Here we show that these organisms can use also nitrate or nitrite as an electron acceptor thereby coupling the reduction of nitrate to distant oxidation of sulfide. Sulfidic marine sediment was incubated with overlying nitrate-amended anoxic seawater. Within 2 months, electric coupling of spatially segregated nitrate reduction and sulfide oxidation was evident from: (1) the formation of a 4-6-mm-deep zone separating sulfide oxidation from the associated nitrate reduction, and (2) the presence of pH signatures consistent with proton consumption by cathodic nitrate reduction, and proton production by anodic sulfide oxidation. Filamentous Desulfobulbaceae with the longitudinal structures characteristic of cable bacteria were detected in anoxic, nitrate-amended incubations but not in anoxic, nitrate-free controls. Nitrate reduction by cable bacteria using long-distance electron transport to get privileged access to distant electron donors is a hitherto unknown mechanism in nitrogen and sulfur transformations, and the quantitative importance for elements cycling remains to be addressed.