Biogeochemical Contributions of Tree Islands to Everglades Wetland Landscape Nitrogen Cycling During Seasonal Inundation

In the Florida Everglades, tree islands are conspicuous heterogeneous elements in the herbaceous wetland landscape. We characterized the biogeochemical role of a seasonally flooded tree island during wet season inundation, specifically examining hydrologically mediated flows of nitrogen (N) and N retention by the tree island. We estimated ecosystem N standing stocks and fluxes, soil and litter N transformation rates, and hydrologic fluxes of N to quantify the net ecosystem N mass flux. Results showed that hydrologic sources of N were dominated by surface water loads of nitrate (NO3) and ammonium (NH4). Nitrate immobilization by soils and surficial leaf litter was an important sink for surface water dissolved inorganic N (DIN). We estimated that the net annual DIN retention by a seasonally flooded tree island was 20.5 ± 5.0 g m−2 during wet season inundation. Based on the estimated tree island surface water DIN loading rate, a seasonally flooded tree island retained 76% of imported DIN. As such, seasonally flooded tree islands have the potential to retain 55% of DIN entering the marsh landscape via upstream canal overland flow in the wet season. By increasing reactive surface area and DOC availability, we suggest that tree islands promote convergence of elements that enhance DIN retention. Tree islands of this region are thus important components of landscape-scale restoration efforts that seek to reduce sources of anthropogenic DIN to downstream estuaries.

N2 fixation in eddies of the eastern tropical South Pacific Ocean

Mesoscale eddies play a major role in controlling ocean biogeochemistry. By impacting nutrient availability and water column ventilation, they are of critical importance for oceanic primary production. In the eastern tropical South Pacific Ocean off Peru, where a large and persistent oxygen-deficient zone is present, mesoscale processes have been reported to occur frequently. However, investigations into their biological activity are mostly based on model simulations, and direct measurements of carbon and dinitrogen (N2) fixation are scarce. We examined an open-ocean cyclonic eddy and two anticyclonic mode water eddies: a coastal one and an open-ocean one in the waters off Peru along a section at 16°S in austral summer 2012. Molecular data and bioassay incubations point towards a difference between the active diazotrophic communities present in the cyclonic eddy and the anticyclonic mode water eddies. In the cyclonic eddy, highest rates of N2 fixation were measured in surface waters but no N2 fixation signal was detected at intermediate water depths. In contrast, both anticyclonic mode water eddies showed pronounced maxima in N2 fixation below the euphotic zone as evidenced by rate measurements and geochemical data. N2 fixation and carbon (C) fixation were higher in the young coastal mode water eddy compared to the older offshore mode water eddy. A co-occurrence between N2 fixation and biogenic N2, an indicator for N loss, indicated a link between N loss and N2 fixation in the mode water eddies, which was not observed for the cyclonic eddy. The comparison of two consecutive surveys of the coastal mode water eddy in November 2012 and December 2012 also revealed a reduction in N2 and C fixation at intermediate depths along with a reduction in chlorophyll by half, mirroring an aging effect in this eddy. Our data indicate an important role for anticyclonic mode water eddies in stimulating N2 fixation and thus supplying N offshore.

Carbon and Nitrogen Stable Isotopic Patterns in South Florida Coastal Ecosystems: Modern and Paleoceanographic Perspectives

Long term management plans for restoration of natural flow conditions through the Everglades increase the importance of understanding potential nutrient impacts of increased freshwater delivery on coastal biogeochemistry. The present study sought to increase understanding of the coastal marine system of South Florida under modern conditions and through the anthropogenic changes in the last century, on scales ranging from individual nutrient cycle processes to seasonal patterns in organic material (OM) under varying hydrodynamic regime, to century scale analysis of sedimentary records. In all applications, carbon and nitrogen stable isotopic compositions of OM were examined as natural recorders of change and nutrient cycling in the coastal system. High spatial and temporal variability in stable isotopic compositions were observed on all time scales. During a transient phytoplankton bloom, ä15N values suggested nitrogen fixation as a nutrient source supporting enhanced productivity. Seasonally, particulate organic material (POM) from ten sites along the Florida Reef Tract and in Florida Bay demonstrated variable fluctuations dependent on hydrodynamic setting. Three separate intra-annual patterns were observed, yet statistical differences were observed between groupings of Florida Bay and Atlantic Ocean sites. The POM ä15N values ranged on a quarterly basis by 7‰, while ä13C varied by 22‰. From a sediment history perspective, four cores collected from Florida Bay further demonstrated the spatial and temporal variability of the system in isotopic composition of bulk OM over time. Source inputs of OM varied with location, with terrestrial inputs dominating proximal to Everglades freshwater discharge, seagrasses dominating in open estuary cores, and a marine mixture of phytoplankton and seagrass in a core from the boundary zone between Florida Bay and the Gulf of Mexico. Significant shifts in OM geochemistry were observed coincident with anthropogenic events of the 20th century, including railroad and road construction in the Florida Keys and Everglades, and also the extensive drainage changes in Everglades hydrology. The sediment record also preserved evidence of the major hurricanes of the last century, with excursions in geochemical composition coincident with Category 4-5 storms.

Comparison of nitrogen isotopic values from different preparation methods

We are writing to comment on the work of Tamburini et al. (2003, doi:10.1029/2000PA000616). During the course of subsequent discussions between the authors and ourselves, it has become clear that the published sedimentary nitrogen isotopic values for Ocean Drilling Program (ODP) Site 724 are in error. Our reanalysis of sediment samples from the same intervals has revealed a significant offset from the original d15N data, requiring a revised assessment of their initial interpretation. The purposes of this comment are to (1) address the origin of these errors; (2) outline a protocol for future validation of nitrogen isotopic analyses; and (3) provide revised interpretations of the sedimentary d15N data in terms of the regional relative contributions of denitrification and nitrogen fixation and mean state of the southwest monsoon. (2) Nitrogen isotopic values measured on late Quaternary sediments at Arabian Sea ODP Site 724 by Tamburini et al. (2003, doi:10.1029/2000PA000616) are inexplicably different from a number of published records of d15N from very nearby on the Oman margin (Altabet et al., 1995, doi:10.1038/373506a0; 1999, doi:10.1029/1999PA900035; 2002, doi:10.1038/415159a; Higginson et al., 2004, doi:10.1016/j.gca.2004.03.015) and elsewhere in the Arabian Sea (Reichart et al., 1998, doi:10.1029/98PA02203). These data were generated using similar instrumentation (elemental analyzer coupled with an isotope ratio mass spectrometer) and analytical methodology to those already published. Concerned by this clear discrepancy, we analyzed aliquots of sediment from the same depth intervals for nitrogen abundance and bulk sedimentary nitrogen isotopes. We have been unable to duplicate the values published by Tamburini et al. (2003, doi:10.1029/2000PA000616 ), even after analysis of multiple replicates and due consideration of natural sediment heterogeneities and postrecovery sample storage.

Spatially explicit structural equation modeling

Structural equation modeling (SEM) is a powerful statistical approach for the testing of networks of direct and indirect theoretical causal relationships in complex data sets with intercorrelated dependent and independent variables. SEM is commonly applied in ecology, but the spatial information commonly found in ecological data remains difficult to model in a SEM framework. Here we propose a simple method for spatially explicit SEM (SE-SEM) based on the analysis of variance/covariance matrices calculated across a range of lag distances. This method provides readily interpretable plots of the change in path coefficients across scale and can be implemented using any standard SEM software package. We demonstrate the application of this method using three studies examining the relationships between environmental factors, plant community structure, nitrogen fixation, and plant competition. By design, these data sets had a spatial component, but were previously analyzed using standard SEM models. Using these data sets, we demonstrate the application of SE-SEM to regularly spaced, irregularly spaced, and ad hoc spatial sampling designs and discuss the increased inferential capability of this approach compared with standard SEM. We provide an R package, sesem, to easily implement spatial structural equation modeling.

Spatial and temporal distribution of tropical phytoplankton species and biomass in the Gulf of Carpenteria, Australia

The biomass and species composition of tropical phytoplankton in Albatross Bay, Gulf of Carpentaria, northern Australia, were examined monthly for 6 yr (1986 to 1992). Chlorophyll a (chl a) concentrations were highest (2 to 5.7 mu g l(-1)) in the wet season at inshore sites, usually coinciding with low salinities (30 to 33 ppt) and high temperatures (29 to 32 degrees C). At the offshore sites chi a concentrations were lower (0.2 to 2 mu g l(-1)) and did not vary seasonally. Nitrate and phosphate concentrations were generally low (0 to 3.68 mu M and 0.09 to 3 mu M for nitrate and phosphate respectively), whereas silicate was present in concentrations in the range 0.19 to 13 mu M. The phytoplankton community was dominated by diatoms, particularly at the inshore sites, as determined by a combination of microscopic and high-performance liquid chromatography (HPLC) pigment analyses. At the offshore sites the proportion of green flagellates increased. The cyanobacterium genus Trichodesmium and the diatom genera Chaetoceros, Rhizosolenia, Bacteriastrum and Thalassionema dominated the phytoplankton caught in 37 mu m mesh nets; however, in contrast to many other coastal areas studied worldwide there was no distinct species succession of the diatoms and only Trichodesmium showed seasonal changes in abundance. This reflects a stable phytoplankton community in waters without pulses of physical and chemical disturbances. These results are discussed in the context of the commercial prawn fishery in the Gulf of Carpentaria and the possible effect of phytoplankton on prawn larval growth and survival.

Leaf chlorophyll concentration relates to transpiration efficiency in peanut

Two pot experiments were conducted in two different seasons at the University of Agricultural Science, Bangalore, India, to study (a) the relationship between chlorophyll concentration (by measuring the leaf light-transmittance characteristics using a SPAD metre) and transpiration efficiency (TE) and (b) the effect of leaf N on chlorophyll and TE relationship in peanut. In Experiment (Expt) I, six peanut genotypes with wide genetic variation for the specific leaf area (SLA) were used. In Expt II, three non-nodulating isogenic lines were used to study the effect of N levels on leaf chlorophyll concentration–TE relationship without potential confounding effects in biological nitrogen fixation. Leaf N was manipulated by applying N fertiliser in Expt II. Chlorophyll concentration, TE (g dry matter kg−1 of H2O transpired, measured using gravimetric method), specific leaf nitrogen (g N m−2, SLN), SLA (cm2 g−1), carbon isotope composition (Δ13C) were determined in the leaves sampled during the treatment period (35–55 days after sowing) in the two experiments. Results showed that the leaf chlorophyll concentration expressed as soil plant analytical development (SPAD) chlorophyll metre reading (SCMR) varied significantly among genotypes in Expt I and as a result of N application in Expt II. Changes in leaf N levels were strongly associated with changes in SCMR, TE and Δ13C. In both the experiments, a significant positive relationship between SCMR and TE with similar slopes but differing intercepts was noticed. However, correction of TE for seasonal differences in vapour pressure deficit (VPD) between the two experiments resulted in a single and stronger relationship between SCMR and TE. There was a significant inverse relationship between SCMR and Δ13C, suggesting a close linkage between chlorophyll concentration and Δ13C in peanut. This study provides the first evidence for a significant positive relationship between TE and leaf chlorophyll concentration in peanut. The study also describes the effect of growing environment on the relationships among SLA, SLN and SCMR.

Declining soil phosphorus reserves on sustainable agricultural production systems

PhD scholarship investigating the relative sensitivity of nitrogen fixation in adapted grain and ley legume species to low soil phosphorus.

Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy

An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy (Oryza sativa L.)

An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.

The role of phosphorus as a regulator of bloom-forming diazotrophic cyanobacteria in the Baltic Sea

Eutrophication favours harmful algal blooms worldwide. The blooms cause toxic outbreaks and deteriorated recreational and aesthetic values, causing both economic loss and illness or death of humans and animals. The Baltic Sea is the world s only large brackish water habitat with recurrent blooms of toxic cyanobacteria capable of biological fixation of atmospheric nitrogen gas. Phosphorus is assumed to be the main limiting factor, along with temperature and light, for the growth of these cyanobacteria. This thesis evaluated the role of phosphorus nutrition as a regulating factor for the occurrence of nitrogen-fixing cyanobacteria blooms in the Baltic Sea, utilising experimental laboratory and field studies and surveys on varying spatial scales. Cellular phosphorus sources were found to be able to support substantial growth of the two main bloom forming species Aphanizomenon sp. and Nodularia spumigena. However, N. spumigena growth seemed independent of phosphorus source, whereas, Aphanizomenon sp. grew best in a phosphate enriched environment. Apparent discrepancies with field observations and experiments are explained by the typical seasonal temperature dependent development of Aphanizomenon sp. and N. spumigena biomass allowing the two species to store ambient pre-bloom excess phosphorus in different ways. Field experiments revealed natural cyanobacteria bloom communities to be predominantly phosphorus deficient during blooms. Phosphate additions were found to increase the accumulation of phosphorus relatively most in the planktonic size fraction dominated by the nitrogen-fixing cyanobacteria. Aphanizomenon sp. responded to phosphate additions whereas the phosphorus nutritive status of N. spumigena seemed independent of phosphate addition. The seasonal development of phosphorus deficiency is different for the two species with N. spumigena showing indications of phosphorus deficiency during a longer time period in the open sea. Coastal upwelling introduces phosphorus to the surface layer during nutrient deficient conditions in summer. The species-specific ability of Aphanizomenon sp. and N. spumigena to utilise phosphate enrichment of the surface layer caused by coastal upwelling was clarified. Typical bloom time vertical distributions of biomass maxima were found to render N. spumigena more susceptible to advection by surface currents caused by coastal upwellings. Aphanizomenon sp. populations residing in the seasonal thermocline were observed to be able to utilise the phosphate enrichment and a bloom was produced with a two to three week time lag subsequent to the relaxation of upwelling. Consistent high concentrations of dissolved inorganic phosphorus, caused by persistent internal loading of phosphorus, was found to be the main source of phosphorus for large-scale pelagic blooms. External loads were estimated to contribute with only a fraction of available phosphorus for open sea blooms. Remineralization of organic forms of phosphorus along with vertical mixing to the permanent halocline during winter set the level of available phosphorus for the next growth season. Events such as upwelling are important in replenishing phosphate concentrations during the nutrient deplete growth season. Autecological characteristics of the two main bloom forming species favour Aphanizomenon sp. populations in utilising the abundant excess phosphate concentrations and phosphate pulses mediated through upwelling. Whilst, N. spumigena displays predominant phosphorus limited growth mode and relies on more scarce cellular phosphorus stores and presumably dissolved organic phosphorus compounds for growth. The Baltic Sea is hypothesised to be in an inhibited state of recovery due to the extensive historical external nutrient loading, extensive internal phosphorus loading and the substantial nitrogen load caused by cyanobacteria nitrogen fixation. This state of the sea is characterised as a vicious circle .

Diversitet och tillväxtfrämjande egenskaper hos Stenotrophomonas-endofyter isolerade från Calliandra calothyrsus Meisn. rotknölar

The bacterial genus Stenotrophomonas comprises 12 species. They are widely found throughout the environment and particularly S. maltophilia, S. rhizophila and S. pavanii are closely associated with plants. Strains of the most common Stenotrophomonas species, S. maltophilia, promote plant growth and health, degrade natural and man-made pollutants and produce biomolecules of biotechnological and economical value. Many S. maltophilia –strains are also multidrug resistant and can act as opportunistic human pathogens. During an INCO-project (1998-2002) rhizobia were collected from root nodules of the tropical leguminous tree Calliandra calothyrsus Meisn. from several countries in Central America, Africa and New Caledonia. The strains were identified by the N2-group (Helsinki university) and some strains turned out to be members of the genus Stenotrophomonas. Several Stenotrophomonas strains induced white tumor- or nodule-like structures on Calliandra?s roots in plant experiments. The strains could, besides from root nodules, also be isolated from surface sterilized roots and stems. The purpose of my work was to investigate if the Stenotrophomonas strains i) belong to a new Stenotrophomonas species, ii) have the same origin, iii) if there are other differences than colony morphology between phase variations of the same strain, iv) have plant growth-promoting (PGP) activity or other advantageous effects on plants, and v) like rhizobia have ability to induce root nodule formation. The genetic diversity and clustering of the Stenotrophomonas strains were analyzed with AFLP fingerprinting to get indications about their geographical origin. Differences in enzymatic properties and ability to use different carbon and energy sources were tested between the two phases of each strain with commercial API tests for bacterial identification. The ability to infect root hairs and induce root nodule formation was investigated both using plant tests with the host plant Calliandra and PCR amplification of nodA and nodC genes for nodulation. The PGP activity of the strains was tested in vitro mainly with plate methods. The impact on growth, nitrogen content and nodulation in vivo was investigated through greenhouse experiments with the legumes Phaseolus vulgaris and Galega orientalis. Both the genetic and phenotypic diversity among the Stenotrophomonas strains was small, which proposes that they have the same origin. The strains brought about changes on the root hairs of Calliandra and they also increased the amount of root hairs. However, no root nodules were detected. The strains produced IAA, protease and lipase in vitro. They also showed plant a growth-promoting effect on G. orientalis, both alone and together with R. galegae HAMBI 540, and also activated nodulation among efficient rhizobia on P. vulgaris in greenhouse. It requires further research to get a better picture about the mechanisms behind the positive effects. The results in this thesis, however, confirm earlier studies concerning Stenotrophomonas positive impact on plants.