Marsh sediments chronically exposed to nitrogen enrichment contain degraded organic matter that is less vulnerable to decomposition via nitrate reduction.

Blue carbon habitats, including salt marshes, can sequester carbon at rates that are an order of magnitude greater than terrestrial forests. This ecosystem service may be under threat from nitrate (NO3-) enrichment, which can shift the microbial community and stimulate decomposition of organic matter. Despite efforts to mitigate nitrogen loading, salt marshes continue to experience chronic NO3- enrichment, however, the long-term consequence of this enrichment on carbon storage remains unclear. To investigate the effect of chronic NO3- exposure on salt marsh organic matter decomposition, we collected sediments from three sites across a range of prior NO3- exposure: a relatively pristine marsh, a marsh enriched to ~70 µmol L-1 NO3- in the flooding seawater for 13 years, and a marsh enriched between 100 and 1000 µmol L-1 for 40 years from wastewater treatment effluent. We collected sediments from 20 to 25 cm depth and determined that sediments from the most chronically enriched site had less bioavailable organic matter and a distinct assemblage of active microbial taxa compared to the other two sites. We also performed a controlled anaerobic decomposition experiment to test whether the legacy of NO3- exposure influenced the functional response to additional NO3-. We found significant changes to microbial community composition resulting from experimental NO3- addition. Experimental NO3- addition also increased microbial respiration in sediments collected from all sites. However, sediments from the most chronically enriched site exhibited the smallest increase, the lowest rates of total NO3- reduction by dissimilatory nitrate reduction to ammonium (DNRA), and the highest DNF:DNRA ratios. Our results suggest that chronic exposure to elevated NO3- may lead to residual pools of organic matter that are less biologically available for decomposition. Thus, it is important to consider the legacy of nutrient exposure when examining the carbon cycle of salt marsh sediments.

Microbial chemolithoautotrophs are abundant in salt marsh sediment following long-term experimental nitrate enrichment.

Long-term anthropogenic nitrate (NO3-) enrichment is a serious threat to many coastal systems. Nitrate reduction coupled with the oxidation of reduced forms of sulfur is conducted by chemolithoautotrophic microbial populations in a process that decreases nitrogen (N) pollution. However, little is known about the diversity and distribution of microbes capable of carbon fixation within salt marsh sediment and how they respond to long-term NO3- loading. We used genome-resolved metagenomics to characterize the distribution, phylogenetic relationships, and adaptations important to microbial communities within NO3--enriched sediment. We found NO3- reducing sulfur oxidizers became dominant members of the microbial community throughout the top 25 cm of the sediment following long-term NO3- enrichment. We also found that most of the chemolithoautotrophic genomes recovered contained striking metabolic versatility, including the potential for complete denitrification and evidence of mixotrophy. Phylogenetic reconstruction indicated that similar carbon fixation strategies and metabolic versatility can be found in several phylogenetic groups, but the genomes recovered here represent novel organisms. Our results suggest that the role of chemolithoautotrophy within NO3--enriched salt marsh sediments may be quantitatively more important for retaining carbon and filtering NO3- than previously indicated and further inquiry is needed to explicitly measure their contribution to carbon turnover and removal of N pollution.

Microbial Community Succession Along a Chronosequence in Constructed Salt Marsh Soils.

In this study, we examined the succession of soil microbial communities across a chronosequence of newly constructed salt marshes constructed primarily of fine-grained dredge material, using 16S rRNA amplicon sequences. Alpha diversity in the subsurface horizons was initially low and increased to reference levels within 3 years of marsh construction, while alpha diversity in the newly accumulating organic matter-rich surface soils was initially high and remained unchanged. Microbial community succession was fastest in the surface horizon (~ 24 years to reference equivalency) and became progressively slower with depth in the subsurface horizons (~ 30-67 years). Random forest linear regression analysis was used to identify important taxa driving the trajectories toward reference conditions. In the parent material, putative sulfate-reducers (Desulfobacterota), methanogens (Crenarchaeota, especially Methanosaeta), and fermenters (Chloroflexi and Clostridia) increased over time, suggesting an enrichment of these metabolisms over time, similar to natural marshes. Concurrently in the surface soils, the relative abundances of putative methane-, methyl-, and sulfide oxidizers, especially among Gammaproteobacteria, increased over time, suggesting the co-development of sulfide and methane removal metabolisms in marsh soils. Finally, we observed that the surface soil communities at one of the marshes did not follow the trajectory of the others, exhibiting a greater relative abundance of anaerobic taxa. Uniquely in this dataset, this marsh was developing signs of excessive inundation stress in terms of vegetation coverage and soil geochemistry. Therefore, we suggest that soil microbial community structure may be effective bioindicators of salt marsh inundation and are worthy of further targeted investigation.

Not All Nitrogen Is Created Equal: Differential Effects of Nitrate and Ammonium Enrichment in Coastal Wetlands.

Excess reactive nitrogen (N) flows from agricultural, suburban, and urban systems to coasts, where it causes eutrophication. Coastal wetlands take up some of this N, thereby ameliorating the impacts on nearshore waters. Although the consequences of N on coastal wetlands have been extensively studied, the effect of the specific form of N is not often considered. Both oxidized N forms (nitrate, NO3-) and reduced forms (ammonium, NH4+) can relieve nutrient limitation and increase primary production. However, unlike NH4+, NO3- can also be used as an electron acceptor for microbial respiration. We present results demonstrating that, in salt marshes, microbes use NO3- to support organic matter decomposition and primary production is less stimulated than when enriched with reduced N. Understanding how different forms of N mediate the balance between primary production and decomposition is essential for managing coastal wetlands as N enrichment and sea level rise continue to assail our coasts.

Introducing the Mangrove Microbiome Initiative: Identifying Microbial Research Priorities and Approaches To Better Understand, Protect, and Rehabilitate Mangrove Ecosystems.

Mangrove ecosystems provide important ecological benefits and ecosystem services, including carbon storage and coastline stabilization, but they also suffer great anthropogenic pressures. Microorganisms associated with mangrove sediments and the rhizosphere play key roles in this ecosystem and make essential contributions to its productivity and carbon budget. Understanding this nexus and moving from descriptive studies of microbial taxonomy to hypothesis-driven field and lab studies will facilitate a mechanistic understanding of mangrove ecosystem interaction webs and open opportunities for microorganism-mediated approaches to mangrove protection and rehabilitation. Such an effort calls for a multidisciplinary and collaborative approach, involving chemists, ecologists, evolutionary biologists, microbiologists, oceanographers, plant scientists, conservation biologists, and stakeholders, and it requires standardized methods to support reproducible experiments. Here, we outline the Mangrove Microbiome Initiative, which is focused around three urgent priorities and three approaches for advancing mangrove microbiome research.

Sulphide addition favours respiratory ammonification (DNRA) over complete denitrification and alters the active microbial community in salt marsh sediments.

The balance between nitrate respiration pathways, denitrification and dissimilatory nitrate (NO3 - ) reduction to ammonium (DNRA), determines whether bioavailable nitrogen is removed as N2 gas or recycled as ammonium. Saltwater intrusion and organic matter enrichment may increase sulphate reduction leading to sulphide accumulation. We investigated the effects of sulphide on the partitioning of NO3 - between complete denitrification and DNRA and the microbial communities in salt marsh sediments. Complete denitrification significantly decreased with increasing sulphide, resulting in an increase in the contribution of DNRA to NO3 - respiration. Alternative fates of NO3 - became increasingly important at higher sulphide treatments, which could include N2 O production and/or transport into intracellular vacuoles. Higher 16S transcript diversity was observed in the high sulphide treatment, with clear shifts in composition. Generally, low and no sulphide, coupled with high NO3 - , favoured the activity of Campylobacterales, Oceanospirillales and Altermonadales, all of which include opportunistic denitrifiers. High ∑sulphide conditions promoted the activity of potential sulphide oxidizing nitrate reducers (Desulfobulbaceae, Acidiferrobacteraceae and Xanthomonadales) and sulphate reducers (Desulfomonadaceae, Desulfobacteraceae). Our study highlights the tight coupling between N and S cycling, and the implications of these dynamics on the fate of bioavailable N in coastal environments susceptible to intermittent saltwater inundation and organic matter enrichment.

Nitrate addition stimulates microbial decomposition of organic matter in salt marsh sediments.

Salt marshes sequester carbon at rates more than an order of magnitude greater than their terrestrial counterparts, helping to mitigate climate change. As nitrogen loading to coastal waters continues, primarily in the form of nitrate, it is unclear what effect it will have on carbon storage capacity of these highly productive systems. This uncertainty is largely driven by the dual role nitrate can play in biological processes, where it can serve as a nutrient-stimulating primary production or a thermodynamically favorable electron acceptor fueling heterotrophic metabolism. Here, we used a controlled flow-through reactor experiment to test the role of nitrate as an electron acceptor, and its effect on organic matter decomposition and the associated microbial community in salt marsh sediments. Organic matter decomposition significantly increased in response to nitrate, even at sediment depths typically considered resistant to decomposition. The use of isotope tracers suggests that this pattern was largely driven by stimulated denitrification. Nitrate addition also significantly altered the microbial community and decreased alpha diversity, selecting for taxa belonging to groups known to reduce nitrate and oxidize more complex forms of organic matter. Fourier Transform-Infrared Spectroscopy further supported these results, suggesting that nitrate facilitated decomposition of complex organic matter compounds into more bioavailable forms. Taken together, these results suggest the existence of organic matter pools that only become accessible with nitrate and would otherwise remain stabilized in the sediment. The existence of such pools could have important implications for carbon storage, since greater decomposition rates as N loading increases may result in less overall burial of organic-rich sediment. Given the extent of nitrogen loading along our coastlines, it is imperative that we better understand the resilience of salt marsh systems to nutrient enrichment, especially if we hope to rely on salt marshes, and other blue carbon systems, for long-term carbon storage.

Method to differentiate asn deamidation that occurred prior to and during sample preparation of a monoclonal antibody.

Asparagine (Asn) deamidation is a major source of antibody instability and micro heterogeneity. For this reason, it is critical to accurately characterize both the levels and the sites of Asn deamidation in therapeutic antibodies. Asn deamidation is normally quantified by analyzing antibodies at the peptide level by liquid chromatography-mass spectrometry. This requires denaturation, reduction, alkylation, and enzyme digestion of the antibody prior to analysis. These steps in sample preparation may directly contribute to the total levels of Asn deamidation detected. Therefore, to obtain accurate levels and sites of Asn deamidation, it is important to determine if any deamidation occurred during the sample preparation steps. However, this could be challenging because deamidation that occurred prior to and during sample preparation resulted in peptides with the same retention times and the same molecular weight increase of 1 Da. Sample preparation was carried out in (18)O-water in the current study to differentiate between the two events of Asn deamidation. Using this method, deamidation that occurred during sample preparation resulted in a molecular weight increase of 3 Da instead of 1 Da. This molecular weight difference was readily detected by inspection of the isotopic peak cluster of the peptides containing the deamidation products, isoAsp and Asp residues. It enabled discrimination of deamidation that was due to analytical artifacts and thus determination of the level of deamidation that was present in the samples.

Characterization of the glycosylation state of a recombinant monoclonal antibody using weak cation exchange chromatography and mass spectrometry.

Recombinant monoclonal antibody heterogeneity is inherent due to various enzymatic and non-enzymatic modifications. In this study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH(2) domain was analyzed by weak cation exchange chromatography. Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states of antibody with zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with either zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes in large proteins such as monoclonal antibodies, caused by different types of neutral oligosaccharides as well as the absence of oligosaccharides, can be differentiated by cation exchange column chromatography.