Bacterioplankton in the Chesapeake Bay: Genetic diversity, population dynamics and community proteomics

Although the ecosystem of the Chesapeake Bay has been studied extensively, little is known about the genetic diversity, population dynamics and metabolic activity of bacterioplankton living in the Bay. In this study, clone libraries containing the rRNA operon (16S rRNA-ITS-23S rRNA) were constructed from samples collected from the Chesapeake Bay to study spatial and temporal dynamics of estuarine bacterioplankton. Major bacterial groups changed dramatically between cold and warm seasons. In the summer, Alpha- and Gammaproteobacteria, Bacteroidetes (Flavobacterium-Bacteroidetes-Cytophaga), Cyanobacteria and Actinobacteria were the dominant groups while in the winter, Alpha- and Betaproteobacteria, and Actinobacteria were commonly found. Clone library analysis also revealed dramatic shifts in bacterial species composition between seasons. Unique SAR11, SAR86, and Roseobacter clades were discovered in the Chesapeake Bay, suggesting the ecological adaptation of organisms endemic to the Bay or perhaps, large temperate estuaries. The bacterioplankton populations were monitored from 2002 to 2004 by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Remarkable seasonal shifts and repeatable annual patterns were identified. Temporal variation of bacterial communities was best explained by the change of chlorophyll a (Chl a) and water temperature, while other factors such as dissolved oxygen, ammonia, nitrite and nitrate, and viral abundance also contributed to the seasonal succession of bacterial populations. In order to understand ecological functions of microbes living in the natural environment, a community-based proteomic approach was developed. Typically, a few hundred-protein spots were visualized based on two-dimensional gel electrophoresis (2-DGE) from Chesapeake Bay microbial communities (0.2 to 3.0 µm filtered fractions). Distinct seasonal patterns and noticeable spatial variations of Chesapeake Bay metaproteomes were observed and the metaproteomic patterns correlated with genetic fingerprints based on 16S rRNA-DGGE. Six protein spots were characterized by LC-MS/MS and three of them were most closely related to the genes in the Sargasso Sea metagenomic database. We proved for the first time that metaproteomics could be applied to a complex marine microbial community. Our results indicate that community proteomics has great potential to unveil novel microgeochemical functions and to link microbial functions to their population structures

Spatial Variability in Streambed Microbial Community Structure across Two Watersheds

Both spatial and temporal variability are key attributes of sedimentary microbial communities, and while spatial effects on beta-diversity appear to dominate at larger distances, the character of spatial variability at finer scales remains poorly understood, especially for headwater stream communities. We investigated patterns of microbial community structure (MCS) in biofilms attached to streambed sediments from two watersheds across spatial scales spanning ,1 m within a single stream to several hundred kilometers between watersheds. Analyses of phospholipid fatty acid (PLFA) profiles indicated that the variations in MCS were driven by increases in the relative abundance of microeukaryotic photoautotrophs and their contribution to total microbial biomass. Furthermore, streams within watersheds had similar MCS, underscoring watershed-level controls of mi- crobial communities. Moreover, bacterial community structure assayed as either PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints or PLFA profiles edited to remove microeukaryotes indicated a distinct watershed-level biogeography. No distinct stream order-level distributions were identified, although DGGE analyses clearly indicated that there was greater variability in community structure among 1st-order streams than among 2nd- and 3rd-order streams. Longitudinal gradients in microbial biomass and structure showed that the greatest variations were associated with 1st-order streams within a watershed, and 68% of the variation in total microbial biomass was explained by sediment atomic carbon-to-nitrogen ratio (C:N ratio), percent carbon, sediment surface area, and per- cent water content. This study confirms a distinct microbial biogeography for headwater stream communities driven by environmental heterogeneity across distant watersheds and suggests that eukaryotic photoautotrophs play a key role in structuring bacterial communities on streambed sediments

Distinct Distribution of Archaea From Soil to Freshwater to Estuary: Implications of Archaeal Composition and Function in Different Environments

In addition to inhabiting extreme territories, Archaea are widely distributed in common environments spanning from terrestrial to aquatic environments. This study investigated and compared archaeal community structures from three different habitats (representing distinct environments): agriculture soils (from farming system trials FST, PA, United States), freshwater biofilms (from White Clay Creek, PA, United States), and estuary water (Chesapeake Bay, United States). High-throughput sequencing of 16S rRNA genes indicated that Thaumarchaeota, Euryarchaeota, Nanoarchaeota, Crenarchaeota, and Diapherotrites were the commonly found dominant phyla across these three environments. Similar to Bacteria, distinct community structure and distribution patterns for Archaea were observed in soils vs. freshwater vs. estuary. However, the abundance, richness, evenness, and diversity of archaeal communities were significantly greater in soils than it was in freshwater and estuarine environments. Indicator species (or amplicon sequence variants, ASVs) were identified from different nitrogen and carbon cycling archaeal groups in soils (Nitrososphaerales, Nitrosotaleales, Nitrosopumilales, Methanomassiliicoccales, Lainarchaeales), freshwater biofilms (Methanobacteria, Nitrososphaerales) and Chesapeake Bay (Marine Group II, Nitrosopumilales), suggesting the habitat-specificity of their biogeochemical contributions to different environments. Distinct functional aspects of Archaea were also confirmed by functional predictions (PICRUSt2 analysis). Further, co-occurrence network analysis indicated that only soil Archaea formed stable modules. Keystone species (ASVs) were identified mainly from Methanomassiliicoccales, Nitrososphaerales, Nitrosopumilales. Overall, these results indicate a strong habitat-dependent distribution of Archaea and their functional partitions within the local environments

Sediment-Nitrogen (N) connectivity: suspended sediments in streams as N exporters and reactors for denitrification and assimilatory N uptake during storms

Nitrogen (N) pollution in riverine ecosystems has substantial environmental, economic, and policy consequences. Various riverine N removal processes include permanent dissimilatory sinks such as denitrification (Uden) and temporary assimilatory sink such as microbial N uptake (Uassim). Both processes have been extensively evaluated in benthic sediments but only sparsely in the water column, particularly for storm flows producing high suspended sediment (SS) concentrations. Stormflows also increase the sediment bound N (Sed-N) export, and in turn, the overall N exports from watersheds. The balance between N removal by Uden and Uassim vs. Sed-N export has not been studied and is a key knowledge gap. We assessed the magnitude of Uden and Uassim against stormflow Sed-N exports for multiple storm events of varying magnitude and across two drainage areas (750 ha and 15,330 ha) in a mixed landuse mid-Atlantic US watershed. We asked: How do the Uden and Uassim sinks compare with Sed-N exports and how do these N fluxes vary across the drainage areas for sampled storms on the rising and falling limbs of the discharge hydrograph? Mean Uden and Uassim as % of the Sed-N exports ranged between 0.1–40% and 0.6–22%, respectively. Storm event Uassim fluxes were generally slightly lower than the corresponding Uden fluxes. Similarly, comparable but slightly higher Uden fluxes were observed for the second order vs. the fourth order stream, while Uassim fluxes were slightly higher in the fourth-order stream. Both of these N sinks were higher on the falling vs. rising limbs of the hydrograph. This suggests that while the N sinks are not trivial, sediment bound N exports during large stormflows will likely overshadow any gains in N removal by SS associated denitrification. Understanding these N source-sink dynamics for storm events is critical for accurate watershed nutrient modeling and for better pollution mitigation strategies for downstream aquatic ecosystems. These results are especially important within the context of climate change as extreme hydrological events including storms are becoming more and more frequent

Ghosts of Landuse Past: Legacy Effects of Milldams for Riparian Nitrogen (N) Processing and Water Quality Functions

Milldams and their legacies have significantly influenced fluvial processes and geomorphology. However, less is known about their effects on riparian zone hydrology, biogeochemistry, and water quality. Here, we discuss the potential effects of existing and breached milldams on riparian nitrogen (N) processing through multiple competing hypotheses and observations from complementary studies. Competing hypotheses characterize riparian zone processes that remove (sink) or release (source) N. Elevated groundwater levels and reducing soil conditions upstream of milldams suggest that riparian zones above dams could be hotspots for N removal via denitrification and plant N uptake. On the other hand, dam removals and subsequent drops in stream and riparian groundwater levels result in drained, oxic soils which could increase soil nitrification and decrease riparian plant uptake due to groundwater bypassing the root zone. Whether dam removals would result in a net increase or decrease of N in riparian groundwaters is unknown and needs to be investigated. While nitrification, denitrification, and plant N uptake have typically received the most attention in riparian studies, other N cycle processes such as dissimilatory nitrate reduction to ammonium (DNRA) need to be considered. We also propose a novel concept of riparian discontinuum, which highlights the hydrologic and biogeochemical discontinuities introduced in riparian zones by anthropogenic structures such as milldams. Understanding and quantifying how milldams and similar structures influence the net source or sink behavior of riparian zones is urgently needed for guiding watershed management practices and for informed decision making with regard to dam removals

Streambank Legacy Sediments in Surface Waters: Phosphorus Sources or Sinks?

Streambank legacy sediments can contribute substantial amounts of sediments to Mid-Atlantic waterways. However, there is uncertainty about the sediment-bound P inputs and the fate of legacy sediment P in surface waters. We compared legacy sediment P concentrations against other streambank sediments and upland soils and evaluated a variety of P indices to determine if legacy sediments are a source or sink of P to surface waters. Legacy sediments were collected from 15 streambanks in the mid-Atlantic USA. Total P and M3P concentrations and % degree of phosphorus saturation (DPS) values for legacy sediments were lower than those for upland soils. % DPS values for legacy sediments were below the water quality threshold for P leaching. Phosphorus sorption index (PSI) values for legacy sediments indicated a large capacity for P sorption. On the other hand, equilibrium phosphorus concentration (EPC0) for legacy sediments suggested that they could be a source or a sink depending on stream water P concentrations. Anoxic conditions resulted in a greater release of P from legacy sediments compared to oxic conditions. These results suggest that legacy sediment P behavior could be highly variable and watershed models will need to account for this variability to reliably quantify the source-sink behavior of legacy sediments in surface waters

Bacterial Community Composition and Chromophoric Dissolved Organic Matter Differs with Culture Time of Skeletonema dohrnii

Skeletonema dohrnii is a common red tide microalgae occurring in the coastal waters and throughout the world. The associated heterotrophic or autotrophic bacteria play vital roles in regulating algal growth, production, and physiology. In this study, we investigated the detailed bacterial community structure associated with the growth of S. dohrnii’s using high-throughput sequencing-based on 16S rDNA. Our results demonstrated that Bacteroidetes (48.04%) and Proteobacteria (40.66%) in all samples accounted for the majority of bacterial populations. There was a significant linear regression relationship between the abundance of bacterial phyla and culture time. Notable shifts in bacterial community composition were observed during algal growth: Flavobacteriales accounted for the vast majority of sequences at the order level. Furthermore, the relative abundance of Rhodobacterales was gradually reduced during the whole growth process of S. dohrnii (0–12 days). However, beyond that, the relative abundance of Marinobacter was slowly increasing. It is noteworthy that five fluorophores (Peaks T1, T2, I, M, and A) were detected during the growth stage of S. dohrnii. The characteristic indexes (fluorescence index, humification index, and biological index) of chromophoric dissolved organic matter (CDOM) also varied with the culture time. In addition, the taxa of bacteria had certain effects on CDOM and they were inextricably linked to each other

Increasing Occurrence of High Fecal Indicator Bacteria (FIB) in Headwater Streams Within the Lower Delaware River Watershed

According to United States’ Environmental Protection Agency (EPA), bacteria and pathogen contamination has been ranked as the leading cause for impaired and threatened waters nationwide including streams in the Delaware River Watershed. Historic bacteria samples from the lower Delaware River Basin have primarily been collected from larger streams at the base of contributing sub-watersheds. As a result, there is little information on the status of bacterial contamination in headwater streams in this region. Headwaters constitute more than three quarters of the stream length in these watersheds and significantly influence water quantity and quality (physical, chemical and biological). In this study, we monitored fecal indicator bacteria (FIB) including total coliform, E. coli and Enterococci at 46 upstream sites across Delaware River Watershed, including the White Clay Creek, Red Clay Creek, Brandywine Creek, and the Schuylkill River. These data indicated an increasing occurrence of high FIB in the watershed. The concentrations of total coliform, E. coli and Enterococci were significantly higher than the EPA standards, suggesting a rising public health threat, a potential risk for surface-fed drinking water suppliers, and a challenge for watershed managers. Relationships between concentrations of FIB and landuses and other stream and watershed physical factors (e.g., watershed size, population density, location of known point sources) are also explored and discussed. Finally, molecular source tracking methods were used to identify the possible sources for FIB contamination, and our results indicated that headwaters are more susceptible to local landuses, and the bacterial contaminations are likely related to agriculture, urbanization, mushroom operations, and wildlife

Seasonal Dynamics of Soil Microbial Community Structure in the Proximal Area of Tree Boles: Possible Influence of Stemflow

Diversity and structure of the soil microbial community (SMC) significantly affects soil ecosystem services including, nutrient cycling, C-mineralization, and stabilization/formation of soil structure. SMC also responds to changes in soil moisture and chemistry. Stemflow, the concentration of precipitation to soils at the trunk base, is well known to alter these edaphic conditions near the stem. Thus, the aim of this study is to investigate the potential link between stemflow and SMC structure. Soil samples collected near stems of two tree species of contrasting stemflow character (Fagus grandifolia [AB] and Liriodendron tulipifera [YP]) were evaluated for changes in chemical properties and SMC structure via PCR-DGGE (denaturant gradient gel electrophoresis) in summer, winter, and spring. Variations within the SMC DGGE banding profiles were determined by GelCompare II software and Nonmetric Multidimensional Scaling (NMDS) analysis. Sequencing and phylogenetic analyses of dominant and unique DGGE bands was conducted to identify shifts in significant SMC organisms. Our results demonstrate that stemflow can alter edaphic conditions near individual tree trunks, and that temporal soil moisture trends, soil pH, PO43-, SO42-, and Ca2 can further vary in response to species-specific stemflow supply characteristics. This, in turn, may explain SMC structural and diversity differences in the proximal area (\u3c 0.5 m) of different SMC species’ near tree trunks. Specifically, SMC diversity (particularly bacterial) increases where stemflow resources are unevenly input for YP but decrease for AB which has more consistent stemflow inputs. Soil chemistry and microbial community differences between species’ proximal areas also indicate the potential for stemflow processes to influence soil pH, and therefore nutrient status, including SOM. Seasonal patterns in DGGE banding patterns and intensity further show that as stemflow characteristics change with canopy phenology and meteorological conditions, so do specific clades or species (e.g., ectomycchorizal and saprophytic fungi, or proteo- and actinobacteria)