Nitrogen is an essential nutrient limiting forest productivity. Plants cannot access atmospheric N2 directly and rely on diazotrophic bacteria to fix nitrogen into bioavailable forms such as ammonium. Within forests, biological nitrogen fixation (BNF) occurs primarily in soils and root nodules. However it is unclear whether the extensive microbial communities recently discovered in tree bark can also fix nitrogen. Here we combine field measurements, metagenomic profiling, and biogeochemical assays to show that bark-dwelling diazotroph communities are abundant and active across diverse tree species. Bark from eight Australian tree species showed exceptionally high C:N ratios and depleted nitrogen stable isotope signatures (δ15N) consistent with locally fixed nitrogen, suggesting strong selection for diazotrophs. Consistently, bark microbial communities harbour phylogenetically and physiologically diverse diazotrophs, averaging ∼1012 cells m-2 and in higher relative abundance than underlying soils. Canonical and alternative nitrogenases were detected across eight bacterial phyla, primarily bark-adapted Alphaproteobacteria, Acidobacteriota, and Verrucomicrobiota, with strong signatures of purifying selection. Stable isotope labelling experiments demonstrated that bark-dwelling diazotrophs fix nitrogen at rates varying with tree species and habitat. In line with the presence of methane-oxidising diazotrophs, BNF was strongly stimulated by methane addition and suppressed by methanotroph inhibitors. Initial upscaling suggests bark-associated nitrogen fixation contributes up to 3.8 Tg N yr-1 globally (∼6% of natural terrestrial BNF), with further studies required to constrain this budget and its contribution to tree nitrogen demands. Altogether, this discovery of substantial above-ground nitrogen inputs revises our understanding of forest nutrient cycles and redefines the functional scope of the caulosphere. ### Competing Interest Statement The authors have declared no competing interest. Australian Research Council, https://ror.org/05mmh0f86, DP210100096, DP210101595, DE240100338, DE250101210, FT240100502 Hermon Slade Foundation, https://ror.org/02ng9tz41 Holsworth Wildlife Foundation

Mangroves are valuable ecosystems that facilitate primary production, carbon sequestration, and regulation of greenhouse gas (GHG) cycles in coastal sediments, with microorganisms playing key roles. Specialized bacteria and archaea compete for energy and resources in mangrove sediments to inhabit optimal ecological niches and can produce or consume methane (CH4)-a potent GHG-in the process. CH4 cycling in mangroves has gained growing attention, yet uncertainties regarding functional and spatial distributions of microorganisms remain. Here, we demonstrate that in a pristine mangrove forest, CH4 concentrations and methanogen communities are concentrated within lower or below rhizosphere depths. We also reveal atypical niches for methanogens in the upper tidal salt marsh zone where vegetation is sparse and highest methanogens abundances were detected at deepest depths (4715 reads g(-1)) despite relatively high redox potentials (> 250 mV). Pore water CH4 concentrations were highest at the deepest depth within the mangrove forest (max. 3.40 & PLUSMN; 0.21 & mu;M) and coincided with the highest sediment CH4 fluxes (276.4 & PLUSMN; 54.2 & mu;mol m(-2)d(-1)) and methanotroph abundances at the surface (1309 reads g(-1)). Sediment CH4 oxidation fractions between the deepest (60 cm) and shallowest (5 cm) depths were estimated between 18.8% and 64.9%. Positive correlation between crab burrows and CH4 fluxes suggests that CH4 from deeper sediment and salt marsh niches can be transported via conduits to the atmosphere. The spatial data from this study highlights the importance of investigating CH4 dynamics across estuarine ecosystem gradients to better understand the complex roles of vital coastal vegetation zones in the face of a changing climate.

Subterranean estuaries (STEs) are important coastal biogeochemical reactors facilitating unique niches for microbial communities. A common approach in determining STE greenhouse gas and nutrient fluxes is to use terrestrial endmembers, not accounting for microbially mediated transformations throughout the STE. As such, the microbial ecology and spatial distribution of specialists that cycle compounds in STEs remain largely underexplored. In this study, we applied 16S rRNA amplicon sequencing with paired biogeochemical characterisations to spatially evaluate microbial communities transforming greenhouse gases and nutrients in an STE. We show that methanogens are most prevalent at the terrestrial end (up to 2.81% relative abundance) concomitant to the highest porewater methane, carbon dioxide and dissolved organic carbon concentrations (0.41 ± 0.02 μM, 273.31 ± 6.05 μM and 0.51 ± 0.02 mM, respectively). Lower ammonium concentrations corresponded with abundant nitrifying and ammonia-oxidising prokaryotes in the mixing zone (up to 11.65% relative abundance). Methane, ammonium and dissolved organic carbon concentrations all decreased by >50% from the terrestrial to the oceanic end of the 15 m transect. This study highlights the STE's hidden microbiome zonation, as well as the importance of accounting for microbial transformations mitigating nutrient and greenhouse gas fluxes to the coastal ecosystems.

Estuaries provide valuable ecosystem services, such as carbon storage and nutrient retention, which may be affected by episodic rainfall events. This study aimed to investigate the short-term effect of episodic rainfall on alkalinity, dissolved carbon and nutrient biogeochemistry in a small, urbanised and mangrove-fringed estuary. High temporal resolution sampling (1.5-h interval) at upper and lower estuary sites, as well as groundwater sampling, were conducted over two weeks to assess estuarine source/sink dynamics of total alkalinity (TAlk), organic alkalinity (OAlk), dissolved inorganic carbon (DIC), dissolved organic carbon (DOC), nitrate, nitrite, ammonium and phosphate. Rapid, short-term changes in estuarine biogeochemistry and mixing were triggered by two episodic rainfall events, which delivered 26 mm over 2 h and 39 mm over 21 h. The estuary was a source for TAlk and DIC exporting 2.2 +/- 1.9 and 2.2 +/- 1.5 mmol/m(2) catchment/d, respectively, to the coastal ocean during the observation period. On average, OAlk accounted for 8% of TAlk at the upstream and 3% at the downstream site. Unlike pristine mangrove systems, the estuary was a net sink for DOC, equivalent to similar to 23% of the DIC source. Rainfall increased catchment nutrient inputs into the estuary, which was a source for ammonium, but a sink for nitrate and nitrite (NOx) throughout the study period. In contrast, phosphate dynamics were less clear. Estuarine biogeochemical transformations affected the exchange with the coastal ocean, driving net TAlk export and by acting as a sink for catchment-derived nutrients. Our high-temporal resolution results suggest that rainfall events rapidly modify estuarine biogeochemistry and mixing, altering the net fluxes of TAlk, dissolved carbon and nutrients to the coastal ocean.

Tree stem mediated methane emissions represent a potentially important yet poorly constrained source of atmospheric methane. Here we present the first ever quantification of tree stem methane emissions from Melaleuca quinquenervia, a widespread iconic Australian lowland tree and globally invasive species. Under two distinct hydrological conditions (wet and dry) we captured 431 tree stem flux measurements encompassing six different vertical stem heights along a 50 m topo-gradient transect, separated into three distinct hydrological zones (upper, transitional and lower). The tree stem methane fluxes closely reflected local topography/hydrology and ranged from − 30.0 to 123,227 µmol m−2 day−1, with the maximum values amongst the highest values reported to date. The highest methane emissions were observed during wet conditions, within the inundated lower zone and from near the tree stem bases and water table. The average methane flux per tree (scaled to 1 m of stem) for the transitional and lower zones was 52-fold and 46-fold higher during wet conditions compared to dry, whereas the upper zone emissions changed little between seasons. Adjacent soil fluxes followed similar trends along the hydrology gradient with the upper zone tree stem emissions offsetting the adjacent soil methane sink capacity. A clear trend of sharply decreasing methane emissions with stem-height suggests a soil methane origin. A 45-h time-series of two trees within the lower zone revealed three to fourfold diel variability, with elevated morning-time fluxes. Overall, the study revealed that seasonal hydrological conditions and topo-gradient substantially regulated the methane emissions from M. quinquenervia and that this previously overlooked pathway should be accounted for within wetland methane budgets, especially during inundated conditions.

In coastal aquatic ecosystems, prokaryotic communities play an important role in regulating the cycling of nutrients and greenhouse gases. In the coastal zone, estuaries are complex and delicately balanced systems containing a multitude of specific ecological niches for resident microbes. Anthropogenic influences (i.e. urban, industrial and agricultural land uses) along the estuarine continuum can invoke physical and biochemical changes that impact these niches. In this study, we investigate the relative abundance of methanogenic archaea and other prokaryotic communities, distributed along a land use gradient in the subtropical Burnett River Estuary, situated within the Great Barrier Reef catchment, Australia. Microbiological assemblages were compared to physicochemical, nutrient and greenhouse gas distributions in both pore and surface water. Pore water samples from within the most urbanised site showed a high relative abundance of methanogenic Euryarchaeota (7.8% of all detected prokaryotes), which coincided with elevated methane concentrations in the water column, ranging from 0.51 to 0.68 μM at the urban and sewage treatment plant (STP) sites, respectively. These sites also featured elevated dissolved organic carbon (DOC) concentrations (0.66 to 1.16 mM), potentially fuelling methanogenesis. At the upstream freshwater site, both methane and DOC concentrations were considerably higher (2.68 μM and 1.8 mM respectively) than at the estuarine sites (0.02 to 0.66 μM and 0.39 to 1.16 mM respectively) and corresponded to the highest relative abundance of methanotrophic bacteria. The proportion of sulfate reducing bacteria in the prokaryotic community was elevated within the urban and STP sites (relative abundances of 8.0%– 10.5%), consistent with electron acceptors with higher redox potentials (e.g. O2, NO3-) being scarce. Overall, this study showed that ecological niches in anthropogenically altered environments appear to give an advantage to specialized prokaryotes invoking a potential change in the thermodynamic landscape of the ecosystem and in turn facilitating the generation of methane–a potent greenhouse gas.

For Pseudomonas aeruginosa, levels of cyclic di-GMP (c-di-GMP) govern the transition from the planktonic state to biofilm formation. Type IV pili (T4P) are crucial determinants of biofilm structure and dynamics, but it is unknown how levels of c-di-GMP affect pilus dynamics. Here, we scrutinized how c-di-GMP affects molecular motor properties and adhesive behavior of T4P. By means of retraction, T4P generated forces of ∼30 pN. Deletion mutants in the proteins with known roles in biofilm formation, swarming motility, and exopolysaccharide (EPS) production (specifically, the diguanylate cyclases sadC and roeA or the c-di-GMP phosphodiesterase bifA) showed only modest effects on velocity or force of T4P retraction. At high levels of c-di-GMP, the production of exopolysaccharides, particularly of Pel, is upregulated. We found that Pel production strongly enhances T4P-mediated surface adhesion of P. aeruginosa, suggesting that T4P-matrix interactions may be involved in biofilm formation by P. aeruginosa Finally, our data support the previously proposed model of slingshot-like "twitching" motility of P. aeruginosaIMPORTANCE Type IV pili (T4P) play various important roles in the transition of bacteria from the planktonic state to the biofilm state, including surface attachment and surface sensing. Here, we investigate adhesion, dynamics, and force generation of T4P after bacteria engage a surface. Our studies showed that two critical components of biofilm formation by Pseudomonas aeruginosa, T4P and exopolysaccharides, contribute to enhanced T4P-mediated force generation by attached bacteria. These data indicate a crucial role for the coordinated impact of multiple biofilm-promoting factors during the early stages of attachment to a surface. Our data are also consistent with a previous model explaining why pilus-mediated motility in P. aeruginosa results in characteristic "twitching" behavior.