Salt marsh nitrogen cycling: where land meets sea.

Salt marshes sit at the terrestrial-aquatic interface of oceans around the world. Unique features of salt marshes that differentiate them from their upland or offshore counterparts include high rates of primary production from vascular plants and saturated saline soils that lead to sharp redox gradients and a diversity of electron acceptors and donors. Moreover, the dynamic nature of root oxygen loss and tidal forcing leads to unique biogeochemical conditions that promote nitrogen cycling. Here, we highlight recent advances in our understanding of key nitrogen cycling processes in salt marshes and discuss areas where additional research is needed to better predict how salt marsh N cycling will respond to future environmental change.

Vegetation-Dependent Response to Drought in Salt Marsh Ammonia-Oxidizer Communities.

We investigated the impacts of drought on ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a salt marsh and compared the response to the total bacterial community. We analyzed abundance and community composition of amoA genes by QPCR and TRFLP, respectively, in three vegetation zones in 2014 (pre-drought), 2016 (drought), and 2017 (post-drought), and analyzed bacterial 16S rRNA genes by QPCR, TRFLP, and MiSeq analyses. AOA and AOB abundance in the Spartina patens zone increased significantly in 2016, while abundance decreased in the tall S. alterniflora zone, and showed little change in the short S. alterniflora zone. Total bacterial abundance declined annually in all vegetation zones. Significant shifts in community composition were detected in 2016 in two of the three vegetation zones for AOA and AOB, and in all three vegetation zones for total bacteria. Abundance and community composition of AOA and AOB returned to pre-drought conditions by 2017, while bacterial abundance continued to decline, suggesting that nitrifiers may be more resilient to drought than other bacterial communities. Finding vegetation-specific drought responses among N-cycling microbes may have broad implications for changes in N availability and marsh productivity, particularly if vegetation patterns continue to shift as predicted due to sea level rise.

Influence of local and regional drivers on spatial and temporal variation of ammonia-oxidizing communities in Gulf of Mexico salt marshes.

We characterized ammonia-oxidizing archaea (AOA) and bacteria (AOB) from salt marsh sediments in the Gulf of Mexico over 5 years to identify environmental drivers of nitrifying community patterns following the Deepwater Horizon oil spill. Samples were collected from oiled and unoiled sites in July of 2012-2016 from 12 marshes spanning three regions on the Louisiana coast. No consistent oil effect was detected for either AOA or AOB abundance or community composition. At the local scale, abundance was correlated with changes in marsh elevation, suggesting that oxygen may be an important driver. Regional differences in abundance were best explained by salinity and soil moisture, while interannual variation may be more linked to changes in climate and Mississippi River discharge. Variation of AOA communities was correlated with organic sediment nutrients, while AOB communities were correlated with soil extractable nutrients. AOA and AOB diversity and AOB abundance decreased in 2014 in all regions, suggesting that broad-scale drivers, such as climate, may explain synchronous shifts throughout the coastal area. Our results provide insights about large-scale disturbances on nitrifying microbes in the Gulf of Mexico, and suggest that nitrogen cycling may be controlled primarily by local factors, but large-scale drivers might override these localized differences at times.

Population Dynamics and Community Composition of Ammonia Oxidizers in Salt Marshes after the Deepwater Horizon Oil Spill.

The recent oil spill in the Gulf of Mexico had significant effects on microbial communities in the Gulf, but impacts on nitrifying communities in adjacent salt marshes have not been investigated. We studied persistent effects of oil on ammonia-oxidizing archaeal (AOA) and bacterial (AOB) communities and their relationship to nitrification rates and soil properties in Louisiana marshes impacted by the Deepwater Horizon oil spill. Soils were collected at oiled and unoiled sites from Louisiana coastal marshes in July 2012, 2 years after the spill, and analyzed for community differences based on ammonia monooxygenase genes (amoA). Terminal Restriction Fragment Polymorphism and DNA sequence analyses revealed significantly different AOA and AOB communities between the three regions, but few differences were found between oiled and unoiled sites. Community composition of nitrifiers was best explained by differences in soil moisture and nitrogen content. Despite the lack of significant oil effects on overall community composition, we identified differences in correlations of individual populations with potential nitrification rates between oiled and unoiled sites that help explain previously published correlation patterns. Our results suggest that exposure to oil, even 2 years post-spill, led to subtle changes in population dynamics. How, or if, these changes may impact ecosystem function in the marshes, however, remains uncertain.

Long-term impacts of disturbance on nitrogen-cycling bacteria in a New England salt marsh.

Recent studies on the impacts of disturbance on microbial communities indicate communities show differential responses to disturbance, yet our understanding of how different microbial communities may respond to and recover from disturbance is still rudimentary. We investigated impacts of tidal restriction followed by tidal restoration on abundance and diversity of denitrifying bacteria, ammonia-oxidizing bacteria (AOB), and ammonia-oxidizing archaea (AOA) in New England salt marshes by analyzing nirS and bacterial and archaeal amoA genes, respectively. TRFLP analysis of nirS and betaproteobacterial amoA genes revealed significant differences between restored and undisturbed marshes, with the greatest differences detected in deeper sediments. Additionally, community patterns indicated a potential recovery trajectory for denitrifiers. Analysis of archaeal amoA genes, however, revealed no differences in community composition between restored and undisturbed marshes, but we detected significantly higher gene abundance in deeper sediment at restored sites. Abundances of nirS and betaproteobacterial amoA genes were also significantly greater in deeper sediments at restored sites. Porewater ammonium was significantly higher at depth in restored sediments compared to undisturbed sediments, suggesting a possible mechanism driving some of the community differences. Our results suggest that impacts of disturbance on denitrifying and ammonia-oxidizing communities remain nearly 30 years after restoration, potentially impacting nitrogen-cycling processes in the marsh. We also present data suggesting that sampling deeper in sediments may be critical for detecting disturbance effects in coastal sediments.

Differential responses of ammonia-oxidizing archaea and bacteria to long-term fertilization in a New England salt marsh.

Since the discovery of ammonia-oxidizing archaea (AOA), new questions have arisen about population and community dynamics and potential interactions between AOA and ammonia-oxidizing bacteria (AOB). We investigated the effects of long-term fertilization on AOA and AOB in the Great Sippewissett Marsh, Falmouth, MA, USA to address some of these questions. Sediment samples were collected from low and high marsh habitats in July 2009 from replicate plots that received low (LF), high (HF), and extra high (XF) levels of a mixed NPK fertilizer biweekly during the growing season since 1974. Additional untreated plots were included as controls (C). Terminal restriction fragment length polymorphism analysis of the amoA genes revealed distinct shifts in AOB communities related to fertilization treatment, but the response patterns of AOA were less consistent. Four AOB operational taxonomic units (OTUs) predictably and significantly responded to fertilization, but only one AOA OTU showed a significant pattern. Betaproteobacterial amoA gene sequences within the Nitrosospira-like cluster dominated at C and LF sites, while sequences related to Nitrosomonas spp. dominated at HF and XF sites. We identified some clusters of AOA sequences recovered primarily from high fertilization regimes, but other clusters consisted of sequences recovered from all fertilization treatments, suggesting greater physiological diversity. Surprisingly, fertilization appeared to have little impact on abundance of AOA or AOB. In summary, our data reveal striking patterns for AOA and AOB in response to long-term fertilization, and also suggest a missing link between community composition and abundance and nitrogen processing in the marsh.

Abundance of ammonia-oxidizing archaea and bacteria along an estuarine salinity gradient in relation to potential nitrification rates.

Abundance of ammonia-oxidizing Archaea (AOA) was found to be always greater than that of ammonia-oxidizing Bacteria along an estuarine salinity gradient, and AOA abundance was highest at intermediate salinity. However, AOA abundance did not correlate with potential nitrification rates. This lack of correlation may be due to methodological limitations or alternative energy sources.

Distribution and diversity of archaeal and bacterial ammonia oxidizers in salt marsh sediments.

Diversity and abundance of ammonia-oxidizing Betaproteobacteria (beta-AOB) and archaea (AOA) were investigated in a New England salt marsh at sites dominated by short or tall Spartina alterniflora (SAS and SAT sites, respectively) or Spartina patens (SP site). AOA amoA gene richness was higher than beta-AOB amoA richness at SAT and SP, but AOA and beta-AOB richness were similar at SAS. beta-AOB amoA clone libraries were composed exclusively of Nitrosospira-like amoA genes. AOA amoA genes at SAT and SP were equally distributed between the water column/sediment and soil/sediment clades, while AOA amoA sequences at SAS were primarily affiliated with the water column/sediment clade. At all three site types, AOA were always more abundant than beta-AOB based on quantitative PCR of amoA genes. At some sites, we detected 10(9) AOA amoA gene copies g of sediment(-1). Ratios of AOA to beta-AOB varied over 2 orders of magnitude among sites and sampling dates. Nevertheless, abundances of AOA and beta-AOB amoA genes were highly correlated. Abundance of 16S rRNA genes affiliated with Nitrosopumilus maritimus, Crenarchaeota group I.1b, and pSL12 were positively correlated with AOA amoA abundance, but ratios of amoA to 16S rRNA genes varied among sites. We also observed a significant effect of pH on AOA abundance and a significant salinity effect on both AOA and beta-AlphaOmicronBeta abundance. Our results expand the distribution of AOA to salt marshes, and the high numbers of AOA at some sites suggest that salt marsh sediments serve as an important habitat for AOA.

Archaeal diversity and the prevalence of Crenarchaeota in salt marsh sediments.

Crenarchaeal 16S rRNA sequences constituted over 70% of the archaeal clones recovered from three salt marsh sites dominated by different grasses. Group I.1a Crenarchaeota dominated at two sites, while group I.3b Crenarchaeota sequences were most abundant at a third site. Abundances of 16S rRNA genes related to "Candidatus Nitrosopumilus maritimus" differed by site and sampling date.

Functionally distinct communities of ammonia-oxidizing bacteria along an estuarine salinity gradient.

The relationship between ammonia-oxidizing bacteria (AOB) and potential nitrification rates was examined along a salinity gradient in a New England estuary in spring and late summer over 3 years. Ammonia-oxidizing bacteria abundance was estimated by measuring gene copies of the ammonia monooxygenase catalytic subunit (amoA) using real-time polymerase chain reaction. Ammonia-oxidizing bacteria abundance ranged from below detection to 6.0 x 10(7)amoA copies (gdw sediment)(-1). Mean potential nitrification rates ranged from 0.5 to 186.5 nmol N (gdw sediment)(-1) day(-1). Both AOB abundance and potential rates were significantly higher in spring than late summer. Correlations between rates and abundance varied significantly among sites, but showed site-specific ammonia oxidation kinetics related to AOB community structure. The effect of salinity on potential nitrification rates was evaluated by incubating sediment from each site under four salinity conditions (0, 5, 10 and 30 psu). At all sites, rates were generally highest in the intermediate salinity treatments, but rates at the upstream site were inhibited at high salinity, while rates at the two downstream sites were inhibited at the lowest salinity. Although salinity appears to be an important factor in determining AOB distribution, it may not be the primary factor as AOB exhibited a broad range of salinity tolerance in our experiments. Our results indicate that there are significant differences in abundance and community composition of AOB along the salinity gradient, and the differences are reflected in community function.

Microbial community dynamics based on 16S rRNA gene profiles in a Pacific Northwest estuary and its tributaries.

We analyzed bacterioplankton community structure in Tillamook Bay, Oregon and its tributaries to evaluate phylogenetic variability and its relation to changes in environmental conditions along an estuarine gradient. Using eubacterial primers, we amplified 16S rRNA genes from environmental DNA and analyzed the PCR products by length heterogeneity polymerase chain reaction (LH-PCR), which discriminates products based on naturally occurring length differences. Analysis of LH-PCR profiles by multivariate ordination methods revealed differences in community composition along the estuarine gradient that were correlated with changes in environmental variables. Microbial community differences were also detected among different rivers. Using partial 16S rRNA sequences, we identified members of dominant or unique gene fragment size classes distributed along the estuarine gradient. Gammaproteobacteria and Betaproteobacteria and members of the Bacteroidetes dominated in freshwater samples, while Alphaproteobacteria, Cyanobacteria and chloroplast genes dominated in marine samples. Changes in the microbial communities correlated most strongly with salinity and dissolved silicon, but were also strongly correlated with precipitation. We also identified specific gene fragments that were correlated with inorganic nutrients. Our data suggest that there is a significant and predictable change in microbial species composition along an estuarine gradient, shifting from a more complex community structure in freshwater habitats to a community more typical of open ocean samples in the marine-influenced sites. We also demonstrate the resolution and power of LH-PCR and multivariate analyses to provide a rapid assessment of major community shifts, and show how these shifts correlate with environmental variables.

Isolation of an autotrophic ammonia-oxidizing marine archaeon.

For years, microbiologists characterized the Archaea as obligate extremophiles that thrive in environments too harsh for other organisms. The limited physiological diversity among cultivated Archaea suggested that these organisms were metabolically constrained to a few environmental niches. For instance, all Crenarchaeota that are currently cultivated are sulphur-metabolizing thermophiles. However, landmark studies using cultivation-independent methods uncovered vast numbers of Crenarchaeota in cold oxic ocean waters. Subsequent molecular surveys demonstrated the ubiquity of these low-temperature Crenarchaeota in aquatic and terrestrial environments. The numerical dominance of marine Crenarchaeota--estimated at 10(28) cells in the world's oceans--suggests that they have a major role in global biogeochemical cycles. Indeed, isotopic analyses of marine crenarchaeal lipids suggest that these planktonic Archaea fix inorganic carbon. Here we report the isolation of a marine crenarchaeote that grows chemolithoautotrophically by aerobically oxidizing ammonia to nitrite--the first observation of nitrification in the Archaea. The autotrophic metabolism of this isolate, and its close phylogenetic relationship to environmental marine crenarchaeal sequences, suggests that nitrifying marine Crenarchaeota may be important to global carbon and nitrogen cycles.

Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system.

Ammonia-oxidizing bacteria (AOB) play an important role in nitrogen cycling in estuaries, but little is known about AOB diversity, distribution and activity in relation to the chemical and physical changes encountered in estuary systems. Although estuarine salinity gradients are well recognized to influence microbial community structure, few studies have examined the influence of varying salinity on the diversity and stability of AOB populations. To investigate these relationships, we collected sediment samples from low-, mid- and high-salinity sites in Plum Island Sound estuary, MA, during spring and late summer over 3 years. Ammonia-oxidizing bacteria distribution and diversity were assessed by terminal restriction fragment length polymorphism (TRFLP) analysis of the ammonia monooxygenase (amoA) gene, and fragments were identified by screening amoA clone libraries constructed from each site. Most striking was the stability and low diversity of the AOB community at the high-salinity site, showing little variability over 3 years. Ammonia-oxidizing bacteria at the high-salinity site were not closely related to any cultured AOB, but were most similar to Nitrosospira spp. Ammonia-oxidizing bacteria at the mid- and low-salinity sites were distributed among Nitrosospira-like sequences and sequences related to Nitrosomonas ureae/oligotropha and Nitrosomonas sp. Nm143. Our study suggests that salinity is a strong environmental control on AOB diversity and distribution in this estuary.

Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification.

The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.

Flow cytometry-assisted cloning of specific sequence motifs from complex 16S rRNA gene libraries.

A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.

Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays.

Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.

Molecular approaches to microbiological monitoring: fecal source detection.

Molecular methods are useful both to monitor natural communities of bacteria, and to track specific bacterial markers in complex environments. Length-heterogeneity polymerase chain reaction (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) of 16S rDNAs discriminate among 16S rRNA genes based on length polymorphisms of their PCR products. With these methods, we developed an alternative indicator that distinguishes the source of fecal pollution in water. We amplify 16S rRNA gene fragments from the fecal anaerobic genus Bacteroides with specific primers. Because Bacteroides normally resides in gut habitats, its presence in water indicates fecal pollution. Molecular detection circumvents the complexities of growing anaerobic bacteria. We identified Bacteroides LH-PCR and T-RFLP ribosomal DNA markers unique to either ruminant or human feces. The same unique fecal markers were recovered from polluted natural waters. We cloned and sequenced the unique markers; marker sequences were used to design specific PCR primers that reliably distinguish human from ruminant sources of fecal contamination. Primers for more species are under development. This approach is more sensitive than fecal coliform assays, is comparable in complexity to standard food safety and public health diagnostic tests, and lends itself to automation and high-throughput. Thus molecular genetic markers for fecal anaerobic bacteria hold promise for monitoring bacterial pollution and water quality.

Application of a rapid method for identifying fecal pollution sources in a multi-use estuary.

We demonstrate the application of a new PCR assay to detect and differentiate human and ruminant sources of fecal pollution in natural water samples. We tested samples collected from Tillamook Bay, Oregon, which has a long history of fecal pollution levels that exceed acceptable standards. The most likely sources are from dairy operations and ineffective sewage treatment. Using a suite of three PCR primer pairs specific for human or ruminant bacterial 16S ribosomal DNA markers, we detected at least one marker in 17 of 22 samples. In general, host-specific fecal markers were detected in areas that are heavily impacted by anthropogenic activities. Nine out of 11 sites classified as either urban or near a sewage point source were positive for the human marker while only five of these same sites were positive for ruminant markers. Conversely, 12 out of 21 sites classified as rural or agricultural use were positive for ruminant markers, while only six of these sites were positive for human pollution. This suite of host-specific genetic markers holds promise for identifying non-point source fecal pollution in coastal waters.