Compound specific isotope analysis of amino acids in freshwater ecosystems: insights and applications

Investigation of the δ^13 C and δ^15 N of individual compounds, commonly referred to as compound specific isotope analysis (CSIA), along with traditional techniques such as stomach content, dietary tracers, and bulk stable isotopic analysis (BSIA), provide a detailed description of trophic dynamics of organisms. CSIA analysis of amino acids (CSIA-AA) in particular, has been used successfully in marine, estuarine, and more recently, terrestrial systems to estimate trophic position (TP) among organisms. Despite the analytical potential of this technique, its application in freshwater systems has remained limited. The goal of this dissertation is to bridge this knowledge gap by assessing the validity and efficacy of the CSIA-AA technique, through studies ranging from laboratory microcosm experiments, to natural freshwater ecosystems in Alabama. In Chapter 2, patterns of trophic enrichment were investigated in a freshwater phytoplankton-grazer model through a controlled feeding experiment in microcosms. Trophic fractionation among amino acids was consistent with previously published results from marine studies. TP determined by CSIA-AA provided a better estimate of trophic status than BSIA, extending the potential of this technique in freshwater systems. Chapter 3 examined the isotopic underpinnings of the phenotypic plasticity in bluegills from the Sipsey River main channel and its backwater habitats, thereby extending CSIA-AA to fluvial and lacustrine environments. BSIA of δ^13 C indicated that the main channel bluegills were subsidized by allochthonous detrital carbon, compared to bluegills from the two floodplain lakes that relied on submerged macrophytic vegetation. Estimates of trophic length using CSIA placed bluegills from all three sites at comparable TP, between trophic levels 3 and 4, suggesting some degree of omnivory. In Chapter 4, the biotic factors affecting the significant difference in mercury concentrations between the largemouth bass from the Sipsey and Black Warrior Rivers were identified, using dual CSIA of triglyceride fatty acids (TGFA; δ^13 C) and amino acids (δ^15 N). Results indicate that increased presence of sulfate-reducing bacteria, coupled with detritus based food web, was the primary driver of the elevated mercury levels in the Sipsey bass. Data presented in this dissertation clearly demonstrate the potential of CSIA-AA and CSIA-TGFA to provide high-resolution insights into complex food web interactions. (Published By University of Alabama Libraries

Spatial and temporal variability in methane and carbon dioxide exchange at three coastal marshes along a salinity gradient in a northern Gulf of Mexico estuary

Carbon fluxes in tidal marshes vary spatially and temporally because of vegetation cover, subsurface biogeochemical processes, and environmental forcing. The objective of this study was to examine how ecosystem carbon exchange changes along an estuarine salinity gradient. I measured greenhouse gas fluxes, methane (CH_4) and carbon dioxide (CO_2), from three marshes along a salinity gradient (0-32 ppt) in the Mobile Bay estuary, Alabama, USA. CH_4 flux ranged from 1.2 to 2.4 mmol CH_4 m^-2 d^-1 with no significant differences across sites. Soil temperature, dissolved inorganic nitrate and nitrite, and ecosystem respiration of CO_2, not salinity, were correlated to CH_4 emissions. Midday net ecosystem exchange was greatest at the most fresh site (-4.8 ± 0.3 μmol CO_2 m^-2 s^-1), followed by the saline (-2.8 ± 1.0 μmol CO_2 m^-2 s^-1) and brackish (-1.4 ± 0.6 μmol CO_2 m^-2 s^-1) sites. However, net ecosystem exchange integrated over a diurnal time period using a shade cloth technique revealed each marsh to be a net CO_2 source to the atmosphere as a result of high ecosystem respiration with no difference across the fresh (105.5 ± 28.9 mmol CO_2 m^-2 d^-1), brackish (100.1 ± 36.5 mmol CO_2 m^-2 d^-1), and salt marsh (78.3 ± 28.6 mmol CO_2 m^-2 d^-1) sites. These findings lead to the conclusion that either the marshes are losing carbon or that they receive a subsidy of respirable carbon, possibly via tidal deposition. The extent to which sedimentation from tidal deposition contributes carbon to these ecosystems, however, remains unknown. Without such a subsidy, marshes in the study area will not be able to keep up with sea level rise. (Published By University of Alabama Libraries

The interactive effects of hurricanes and fire on plant productivity, accretion and elevation of a saltwater marsh at Grand Bay Nerr, Mississippi

Coastal wetlands are subject to large-scale disturbance by fire and hurricanes, which are predicted to increase in frequency and intensity with climate change. A field study and mesocosm experiment examined effects of fire and hurricanes on a Juncus roemerianus dominated marsh at Grand Bay NERR, MS. Field plots were established along transects, which spanned low, mid and high marsh (July 2008). Bimonthly measurements of accretion, plant biomass and porewater physicochemistry were collected from plots. Study plots were affected by Hurricanes Gustav and Ike (September 2008), which deposited 15.4 ± 2.4 mm of sediment in low marsh. Half of the transects were subjected to a low-intensity burn (January 2009). Accretion was influenced by initial input of hurricane sediment, particularly in the low marsh. Following fire, above-ground biomass was significantly reduced, and recovered to pre-burn levels over the subsequent year, with low marsh recovering more readily than mid or high marsh. Distance from shoreline and elevation determined flooding and sulfide stress, which likely influenced plant recovery. Mesocosms were established with sods of soil and vegetation from the study marsh. In a controlled experiment, hurricane and fire disturbances were simulated through removal of biomass, burning, and deposition of sediment and ash on soil surfaces. Data was collected on elevation change, above- and below-ground biomass, and bioavaliable nutrients for a year. Initial die-off of mesocosms masked many impacts of disturbance, as treatment effects were not significant for biomass variables. Control mesocosms had the greatest biomass, suggesting vegetation's intolerance to disturbance. Although many mesocosms lost all vegetation, sod elevations did not collapse completely, suggesting that these sediment driven ecosystems may be more resistant to subsidence from root zone collapse. As climate change contributes to sea-level rise, understanding impacts of multiple disturbances on ecosystem processes will be imperative for the long-term persistence and management of coastal ecosystems. (Published By University of Alabama Libraries

Presence of vegetation and tidal heights influence rates of denitrification in a natural and restored marsh in the northern Gulf of Mexico

Salt marshes act as coastal filters, permanently removing nitrate through denitrification. This study determined denitrification rates in a typical northern Gulf of Mexico (GOM) salt marsh and the factors that influenced these rates. Salt marsh denitrification rates were compared to rates within an adjacent tidal creek to determine the impact of vegetation on denitrification and to rates at a nearby restored salt marsh to evaluate the extent to which restored habitats contribute to N removal 21 years post construction. Rates of denitrification were measured with the isotope pairing technique on a membrane inlet mass spectrometer. The highest overall denitrification rates were measured in the restored marsh followed by the natural marsh site, with the lowest rates at the creek site (p=0.0005). Rates displayed a temporal pattern with peaks in the summer (Restored: 63±11 μmol m-2 hr-1; Marsh: 46±4 μmol m-2 hr-1; Creek: 35±3 μmol m-2 hr-1). The presence of vegetation likely enhanced denitrification by supplying oxygen supply to the rhizosphere, thereby allowing for increased coupled nitrification-denitrification. Porewater hydrogen sulfide concentrations remained low at the Marsh (0-184 μM) and Restored Marsh (0-2 μM) sites, but were greater at the Creek site (50-860 μM). Lower H2S concentrations in vegetated sediments likely resulted from its greater oxidation through root oxygen inputs in vegetated zones, a process, which also promotes coupled denitrification. Overall, the restored marsh efficiently removed N at rates comparable to, or higher than, a natural marsh. However, the height of the restored marsh platform limited its inundation period to half as much as the natural marsh, reducing its effectiveness in removing anthropogenically-derived nitrate from infiltrating waters and mitigating nitrogen loading to offshore waters. (Published By University of Alabama Libraries

Factors influencing the distribution of soil Streptomyces spp. and determination of species capable of producing a neurodegenerative metabolite

The distribution of the common soil bacteria, Streptomyces, is believed to be influenced by soil characteristics. Studies have indicated that soil properties can be influenced by land use, suggesting different communities of Streptomyces may be found in soils from varying land use patterns. Additionally, previous research has demonstrated the potential of some species of Streptomyces to produce a neurodegenerative compound. With higher incidence of Parkinson's disease, a neurodegenerative disorder, being reported from rural areas, identifying the diversity and prevalence of compound-producing Streptomyces spp. could lead to a greater understanding of the etiology of Parkinson's. This study characterized the cultivable Streptomyces communities within soil from different land uses and examined the distribution and prevalence of neurodegenerative compound-producing species. Various Streptomyces-specific and soil-specific media were used for cultivation and isolates were confirmed as Streptomyces by the generation of appropriately sized amplification products using Streptomyces-specific PCR primers. Box PCR was employed to differentiate strains, allowing evaluation of the diversity of Streptomyces spp. present within a sample. The results indicated that distribution of Streptomyces was affected by differing land uses and edaphic properties. Greater recovery of Streptomyces was found for soils used for cultivation, with less recovery from urban and undeveloped soils. The only significant difference in recovery existed between agricultural and undeveloped soils. Production of the neurodegenerative compound did not follow the observed distribution of Streptomyces spp. and was instead broadly distributed across physiography and land use patterns. While soil pH correlated with streptomycete recovery, the amount of soil organic matter and soil texture did not exhibit a strong influence on the distribution of Streptomyces spp. or compound production. Approximately 32% of isolates were capable of producing the neurodegenerative compound and, based on the genomic fingerprints, this capability is widespread throughout the genus. This study provides needed information on the diversity of Streptomyces communities and yield insights into potential environmental links to human neurodegenerative diseases. (Published By University of Alabama Libraries

Determining drivers of plant community composition in a restored marsh: a complementary field and greenhouse study

As climate change continues, tidal marshes are increasingly vulnerable to degradation or loss from sea-level rise (SLR). Restoration projects can recover ecosystem services provided by tidal marshes, but initial planting effort and continued SLR may influence the success of such projects. To test these effects on ecosystem structure, three fringing marshes were restored in Weeks Bay, Alabama by transplanting intact sods of Juncus roemerianus from a nearby donor marsh into restored marshes at varying percent efforts (0, 25, 50, 75, 100%). SLR effects were tested by installing weirs in half of the plots (+SLR) and leaving the other half as controls (-SLR). Subsequent changes in plant community structure were monitored over two growing seasons. In a companion study, intact sods of J. roemerianus were transplanted to the greenhouse facility at the University of Alabama in Tuscaloosa, AL. These sods were exposed to three inundation (-5, 0, +5 cm) and salinity (0, 4, 8 psu) levels in a full factorial experiment to gain further insight into the plant community changes observed in the field. Plant species richness increased in the field and greenhouse studies (from 1 to 25 and 8 total, respectively). In the greenhouse experiment, the increase in species richness was not affected by inundation or salinity. In the field experiment, the increase in species richness was dependent on initial cover, but was independent of inundation. This suggests that species richness and shifts in community composition were not driven by flooding and salinity associated with SLR, but by the larger propagule bank and colonization space for species other than J. roemerianus. However, the relative abundances of the species present in the field study did differ with SLR and initial planting cover. J. roemerianus and Sagittaria lancifolia increased with increasing initial effort treatments, but the relative abundance of J. roemerianus was greater within control plots while S. lancifolia was greater within weirs. Furthermore, after two growing seasons, plant cover was similar among the 50, 75, and 100% initial effort treatments, suggesting that moderate levels of initial effort can regain marsh structure and function within a few years. (Published By University of Alabama Libraries

Cyclic occurrence of fire and its role in carbon dynamics along an edaphic moisture gradient in longleaf pine ecosystems

Fire regulates the structure and function of savanna ecosystems, yet we lack understanding of how cyclic fire affects savanna productivity and carbon dynamics. Furthermore, it is largely unknown how predicted changes in climate may impact the interaction between fire and carbon cycling in these systems. This study utilizes a novel combination of prescribed fire, eddy covariance (EC) and statistical techniques to investigate carbon dynamics in frequently burned longleaf pine savannas that lie along a gradient of soil moisture availability (mesic, intermediate and xeric). Results over three years of EC measurement of net ecosystem exchange (NEE) show that the mesic site was a net carbon sink (NEE = -248.3 g C m^-2 yr^-1), while intermediate and xeric sites were net carbon sources (NEE = 157.5 and 146.2 g C m^-2 yr^-1, respectively), but when carbon losses due to fuel consumption were taken into account, all three sites were carbon sources (1077.9, 795.0 and 969.0 g C m^-2 yr^-1 at the mesic, intermediate and xeric sites, respectively). Nonetheless, rates of NEE returned to pre-fire levels 1-2 months following fire. Loss of leaf area drove the reduction in NEE following fire, but evolutionary adaptations to frequent fire allowed the ecosystem to quickly recover carbon uptake capacity. While losses due to fire affected carbon balances, drought conditions over the final two years of the study were a more important factor driving net carbon loss during the study. In this work, we found that cyclic fire in pine savanna ecosystems maintains structure and carbon dynamics, but also that complex interactions between water availability, ecosystem structure and fire influence carbon dynamics on multi-year timescales. Longer-term observations over greater environmental variability and multiple fire cycles and/or the development of process models would help to more precisely examine the complex interactions between fire and climate and make future prediction about carbon dynamics in these systems. (Published By University of Alabama Libraries

A two-stage bioreactor for the production of polyhydroxyalkanoates

The efficacy of ethyl 3-ethoxybutyrate (EEB) to serve as a biofuel additive has recently been investigated. This biofuel, which can be blended with gasoline or diesel, is made from polyhydroxyalkanoates (PHA). However, PHA are currently too expensive to produce for EEB to be economically viable. To reduce the cost of PHA production, we propose a two-stage bioreactor to convert waste hay into PHA using two separate mixed microbial cultures. We found that both communities were relatively stable over the year and a half experimental period. The maximum percent PHA achieved was 10.7% and the maximum amount of biomass accumulated was 1.545 g. While this showed promise, the maximum percent PHA occurred at the beginning of the experimental period and all attempted experimental manipulations failed to improve, and many times decreased, PHA yields. Using the knowledge gained from these failures, we propose a different method to accumulate PHA in future studies. (Published By University of Alabama Libraries

Blue crab residency and migration in the Mobile Bay estuary: a stable isotope study investigating connectivity

The blue crab (Callinectes sapidus) is an important commercial species throughout Gulf of Mexico. We used carbon and nitrogen stable isotopes from fast and slow turnover tissues to investigate residency and migration of blue crabs in Mobile Bay. A laboratory diet switch experiment was conducted to estimate tissue turnover. By day 83 of the experiment, hepatopancreas tissue turnover averaged 94%, while muscle turnover averaged 43%. Results confirmed that hepatopancreas and muscle tissues are indicators of recent and past diets, respectively. Therefore, these two tissue types were sampled from individual crabs from the Delta, mid-bay (Fowl River), and coastal sites to investigate residency. Average divergence in δ13C values between the two tissues from crabs in the delta (-0.41 /) and Fowl River (-0.31 /) was small, while for crabs in the coastal sites such as Fort Morgan featured a large average divergence (2.39 /). The convergence of hepatopancreas and muscle tissues to similar δ13C values are indicative of residency, while a large divergence between the tissues is characteristic of migratory crabs. Additionally, we found that the Fowl River site is a hot spot for female crabs that delay their spawning migration to coastal waters. Blue crabs and other migratory species link the food webs in the delta and Gulf of Mexico. A greater understanding the role of migratory species as agents of connectivity is critical for fisheries management in response to climate and human induced changes. (Published By University of Alabama Libraries

The contribution of benthic nutrient fluxes to water column primary production in Weeks Bay, Alabama

In shallow estuaries benthic and pelagic processes are tightly coupled such that the nutrients fueling primary production are regenerated from the sediments by the decomposition of organic matter. Weeks Bay, Alabama, a National Estuarine Research Reserve (NERR) site is characterized by high rates of gross primary production and is one of several shallow sub-estuaries, which has the potential to alter nutrients before exporting them to Mobile Bay. The overarching goal of the study was to determine the role of the sediments in providing nutrients for water column primary production in Weeks Bay. The study was conducted over a one year period (April 2009 - March 2010) at two sites, Magnolia River and Mid Bay. Monthly measurements of water column dissolved oxygen (DO), salinity, temperature, chlorophyll-a (chla), nutrients, and porewater profiles of dissolved inorganic nitrogen (DIN) and phosphate (PO_4 ^3- ) were made from 0-10 cm over 1 cm intervals. In addition, intact sediment cores were used to measure benthic fluxes of DIN (NO_3 ^- , NH_4 ^+ , and PO_4 ^3- ), sediment oxygen consumption (SOC), and di-nitrogen gas (N_2 ). Denitrification potential rates were measured using the acetylene inhibition method to determine if NO_3 ^- was limiting to denitrification. Net Ecosystem Metabolism (NEM), gross primary production, and respiration were calculated using the open water method from continuous dissolved oxygen concentrations measured at two sites. In general, there was an uptake of NO_3 ^- by the sediments. There was a seasonal pattern with NH_4 ^+ fluxes with the greatest effluxes occurring during the warmer months. N_2 fluxes were variable and in general there was an uptake into the sediments. There was no detectable site or seasonal pattern in SOC or PO_4 ^3- . Respiration exhibited a strong seasonal pattern in both sites and increased from spring to summer, peaked in the summer, and decreased in the fall and winter. There was not a strong seasonal pattern for gross primary production. Net ecosystem metabolism revealed net heterotrophy year-round. Phytoplankton nitrogen demand (PND) calculated from gross primary production suggest the sediments provide approximately 9% of PND. (Published By University of Alabama Libraries