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The Azolla-Anabaena symbiosis is a mutualistic association between the eukaryotic water fern Azolla and the prokaryotic, nitrogen-fixing cyanobacterium Anabaena. This association has gained attention, because it may represent a viable alternative to chemical nitrogen fertilizers. The growth and development of endosymbiotic Anabaena azollae are greatly influenced by the host fern. Although numerous physiological and biochemical studies on Azolla-Anabaena have been described, there is a paucity of information on ultrastructural characteristics of the cyanobacterial endosymbiont. Azolla can reproduce either sexually via sporocarp formation or asexually via vegetative fragmentation. Each megasporocarp contains one megasporangium (consisting of a single megaspore and the megaspore apparatus) and a distally located colony of Anabaena. Azolla microsporocarps are globular structures that are significantly larger than megasporocarps. The symbiotic association between Azolla and Anabaena is maintained throughout both sexual and asexual modes of reproduction. The cyanobacterial endophytes of all Azolla species are members of the Nostocaceae family and are collectively referred to as Anabaena azollae Strasburger.

On-site wastewater treatment systems (OWTSs or septic systems) are designed to treat and dispose effluents on the same property that produces the wastewater. Approximately 25% of the U.S. population is served by such facilities. Nevertheless, studies on the treatment efficiency and discharge of organic contaminants through septic effluents are lacking. This pilot study showed the occurrence of organic contaminants including pharmaceuticals and personal care products (PPCPs), perfluoroalkyl surfactants (PFASs), polybrominated diphenyl ethers (PBDEs), and polychlorinated biphenyls (PCBs) in septic effluents, adjacent lake water samples, household drinking water in homes that use lake water or a well adjacent to the lake as a source of drinking water, and offshore lake water samples. Septic effluent as well as lake and tap water samples were collected from several households with OWTSs around Skaneateles Lake located in central New York. The advanced on-site systems were installed in some households for the purpose of limiting nutrient levels in the effluent to protect the local surface water. Additionally, because many of these systems serve homes with limited land, advanced treatment systems were needed. The median concentrations of ten PPCPs (ranged from 0.45 to 388 ng/L) and eleven PFASs (ranged from 0.20 to 14.6 ng/L) in septic water were significantly higher ( p ≤ 0.01) than in lake water samples. The median concentrations of PPCPs and PFASs in lake and tap water samples were not significantly different ( p ≥ 0.65). The median concentrations of ∑PBDEs in septic, lake, and tap water samples were 7.47, 3.49, and 2.22 ng/L, respectively, and those for ∑PCBs were 33.1, 29.2, and 28.6 ng/L, respectively. The mass flux of PPCPs (i.e. the mass flow of PPCPs per unit area per unit time) through the disposal of treated septic effluent from textile biofilter and aerobic treatments to the dispersal unit ranged from 12 (carbamazepine) to 66900 μg/m 2 /day (caffeine) whereas that for PFASs ranged from 7.0 (perfluorobutanesulfonate) to 833 μg/m 2 /day (perfluorooctanoic acid). Based on the ratio of measured concentrations and method detection limit, triclocarban, perfluorooctanoic acid, and perfluorooctanesulfonate have the potential to be used as chemical tracers of septic water contamination in Skaneateles Lake. The median concentrations of atenolol, a beta-blocker drug, in septic water were significantly ( ρ = 0.86, p = 0.01) correlated with enterococci counts.

Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers. Six flow chambers were also inoculated with an Escherichia coli isolate obtained from potable water. The effect of disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of hypochlorous acid (ClOH) for 67 min or 4 ppm of monochloramine (NH 2 Cl) for 155 min. To test the ability of bacterial populations to initiate biofilm formation in the presence of disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of ClOH or 4 ppm of NH 2 Cl. Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of ClOH after 5 days of growth in the absence of disinfectant. Whole-cell in situ hybridizations using fluorescently tagged, 16S rRNA-targeted oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species. The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of disinfectants. Specifically, Legionella pneumophila , E. coli , and β and δ proteobacteria were identified within biofilms both before and after treatment. Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing E. coli within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems. The persistence of diverse bacterial species within disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting waterborne disease.

Transmission electron microscopy and immunocytological labeling were used to localize the 32-kilodalton (kDa) protein (DI polypeptide) of photosystem II in different cell types of the cyanobionts within leaf cavities ofAzolla caroliniana Willd. The 32-kDa protein binds the secondary electron acceptor QB, and is highly conserved between plants and cyanobacteria. Three antisera, specific for different epitopes of the 32-kDa protein, were used as primary antibodies. Immunologically recognizable 32-kDa protein was localized on membranes ofAzolla chloroplasts, vegetative cyanobacterial cells, akinetes, and heterocysts that were at all stages of the differentiation process. The 32-kDa protein was not detected in nonphotosynthetic endosymbiotic bacteria found within leaf cavities. The amount of the 32-kDa protein observed in different cyanobacterial cell types was dependent upon the primary antiserum used and membrane orientation within a cell with respect to the plane of sectioning. Therefore, although 32-kDa protein was present in all three cyanobacterial cell types and clear trends in labeling patterns could be elucidated, it was not possible to quantitate the amounts of protein with respect to either cell type or leaf-cavity age.

A simple technique in which rRNA-targeted oligodeoxynucleotide probes are used to identify bacteria immobilized on membranes is described. By using colony lifts, bacteria are directly transferred from plates to untreated nitrocellulose membranes. Alternatively, cells resuspended from colonies can be applied to membranes by using a vacuum manifold under high-salt conditions. Blotted cells are baked and hybridized under stringent conditions by using standard protocols. Treatment of blotted cells with sodium dodecyl sulfate, urea, formaldehyde, or protease had no apparent effect on hybridization signals. Hybridization to rRNA from cells that had been stored refrigerated for 6 days was readily detected; however, fivefold more cells (approximately 10(7) cells) were required to obtain hybridization signals comparable to those generated by cells not subjected to storage. The sequences of oligonucleotide probes specific for Pseudomonas cepacia, Comamonas testosteroni, and Acinetobacter calcoaceticus and a group probe identifying Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas fluorescens, Comamonas acidovorans, and "Flavobacterium" lutescens are presented. In conjunction with the colony lift hybridization procedure, bacteria isolated from river water were identified by using these probes.

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.