Heterotrophic bacteria and bacterivorous protozoa in oceanic macroaggregates.

Oceanic macroaggregates (marine snow and Rhizosolenia mats) sampled from the Sargasso Sea are associated with bacterial and protozoan populations up to four orders of magnitude greater than those present in samples from the surrounding water. Filamentous, curved, and spiral bacteria constituted a higher proportion of the bacteria associated with the particles than were found among bacteria in the surrounding water. Protozoan populations were dominated numerically by heterotrophic microflagellates, but ciliates and amoebas were also observed. Macroaggregates are highly enriched heterotrophic microenvironments in the oceans and may be significant for the cycling of particulate organic matter in planktonic food chains.

Dissolved organic matter and heterotrophic microneuston in the surface microlayers of the north atlantic.

Dissolved organic carbon, carbohydrates, and adenosine triphosphate in the size fractions 0.2 to 3 micrometers and 3 to 1000 micrometers are significantly enriched in the upper 150-micrometer surface layer compared to subsurface water, mean enrichment factors being 1.6, 2.0, 2.5, and 3.1, respectively. When calculated as a 0.1-micrometer microlayer of wet surfactants, the mean concentration of organic matter was 2.9 grams per liter, of which carbohydrates accounted for 28 percent. The data for plant pigments and particulate adenosine triphosphate indicated that bacterioneuston was enriched at seven of nine stations while phagotrophic protists were enriched at five stations. Instances of enrichment and inhibition were verified by cultural data for bacteria and amoebas. The observations indicate that the surface microlayers are largely heterotrophic microcosms, which can be as rich as laboratory cultures, and that an appreciable part of the dissolved organic carbon is carbohydrate of phytoplankton origin, released and brought to the surface by migrating and excreting phagotrophic protists.

Cristispira from oyster styles: complex morphology of large symbiotic spirochetes.

Crystalline styles (digestive organs) of bivalve mollusks provide the habitat for highly motile bacteria. Styles from freshly-collected oysters, Crassostrea virginica, were studied by electron microscopy; Cristispira spirochetes were abundant in these organs. Detailed study reveals these spirochetes to be among the most complex prokaryotic cells known. More than 600 periplasmic flagella and an adhering outer lipoprotein membrane (e.g., a 270 degrees sillon) form the ultrastructural basis for the "crista," first described by light microscopy. Unique rosette structures corresponding to the "chambers" or "ovoid inclusions" of light microscopy were detected at the periphery of all protoplasmic cylinders. Polar organelles and linearly aligned flagellar insertions are conspicuous. In size and complexity, Cristispira more resembles Pillotina, Diplocalyx, Clevelandina and Hollandina (large spirochetes symbiotic in termites) than it does Treponema. Cristispira pectinis (Gross, 1910), the type species; Spirillum ostrea (Noguchi, 1921); and another, less frequent bacterial symbiont are the predominant inhabitants of the dense style matrix. The ultrastructure of the spirillum and an electron micrograph of the third bacterium are shown.

Inhibition and agglutination of arthrobacters by psuedomonads.

The bacterial flora of water in Narragansett Bay, R.I., was observed semimonthly from 1962 to 1964. Dominant isolates were keyed to genus, and the isolates for each genus were expressed as percentage of total isolates. There was a consistent inverse relationship between arthrobacters and the dominant pseudomonads. Pseudomonad growth on agar plates markedly inhibited arthrobacter cross-streaks. Agar from inhibition zones as well as supernatant fluids from pseudomonad broth cultures inhibited arthrobacter motility and caused the cells to agglutinate. Gummy pseudomonad residues from vacuum-evaporated broth cultures readily passed a G-25 Sephadex column. This material agglutinated arthrobacter cells, but failed to cause arthrobacter inhibition in filter-pad assays. In contrast, sterile medium inside a dialysis sac, inoculated externally with a pseudomonad, was inhibitory to arthrobacters in pad assay but failed to agglutinate arthrobacter cells. Pseudomonad isolates from soil showed similar inhibiting and agglutinating activities for both soil and seawater arthrobacter isolates. The inhibitory and agglutinating activities of pseudomonad isolates appeared to diminish on prolonged laboratory cultivation.

Cultural estimation of yeasts on seaweeds.

Five per cent suspensions of freshly harvested seaweeds were used as an inoculum to develop a selective medium for epiphytic yeasts. Conditions for satisfactory yeast growth and visualization as red colonies on membrane filters were obtained by supplementing a basal glucose-Trypticase-yeast extract-agar at pH 7.0 with 100 mg each of chloramphenicol and 2,3,5-triphenyl tetrazolium chloride per liter. Maximal counts were obtained by triturating the algae in prechilled (4 C) seawater with a blender for 2 to 5 min. Inhibitory phenolic materials released from phaeophytes during this process were removed with a modified Cholodny filtration. A preliminary survey indicated that yeasts were epiphytic on all nine species of seaweeds and that maximal populations occurred on the chlorophytes and rhodophytes especially during the periods of warmer water.

In situ morphology of nitrifying-like bacteria in aquaculture systems.

The in situ microbiota from several aquaculture facilities with active nitrification was examined by transmission electron microscopy of thin sections for the presence of bacteria that contained intracytoplasmic membranes characteristic of the nitrifying bacteria. Colonies of bacteria with the cellular morphology of a species of Nitrosomonas were found to be present in both the culture water and in the biological filter slime of a freshwater chinook salmon (Oncorhynchus tshawytscha) culture system. bacteria in the water possessed the normal nitrosomonas type of ultrastructure, whereas similar bacteria in the slime had an aberrant morphology due to multiple invaginations of the cell wall and cyto-membranes and a significantly greater number of ribosomes. These nitrosomonas-like bacteria lysed during enrichment in commonly used media. Bacteria with the morphology of species of Nitrosomonas and Nitrosococcus were also observed in colonies in the surface slimes of marine culture systems for striped bass (Morone saxatilis) and quahaug (Mercenaria mercenaria).

Nitrate removal in closed-system aquaculture by columnar denitrification.

The columnar denitrification method of nitrate-nitrogen removal from high-density, closed system, salmonid aquaculture was investigated and found to be feasible. However, adequate chemical monitoring was found to be necessary for the optimization and quality control of this method. When methanol-carbon was not balanced with inlet nitrate-nitrogen, the column effluent became unsatisfactory for closed-system fish culture due to the presence of excess amounts of nitrite, ammonia, sulfide, and dissolved organic carbon. Sulfide production was also influenced by column maturity and residence time. Methane-carbon was found to be unsatisfactory as an exogenous carbon source. Endogenous carbon could not support high removal efficiencies. Freshwater columns adpated readily to an artificial seawater with a salinity of 18% without observable inhibition. Scanning electron microscopy revealed that the bacterial flora was mainly rod forms with the Peritricha (protozoa) dominating as the primary consumers. Denitrifying bacteria isolated from freshwater columns were tentatively identified as species of Pseudomonas and Alcaligenes. A pilot plant column was found to behave in a manner similar to the laboratory columns except that nitrite production was never observed.

Effect of processing on the microflora of Norwegian seaweed meal, with observations on Sporendonema minutum (Hoye) Frank and Hess.

Meal from the brown seaweed Ascophyllum nodosum (L.) Le Jol. is mainly used as an animal feed supplement. Since moist weed often develops a marked mold growth and since little was known about the microflora of seaweed meal, a cultural procedure was developed to enumerate the populations of bacteria, yeasts, and molds of seaweed meals manufactured by different drying processes. The microflora could be supported by a variety of media varying in levels of nutrition and in the source and concentration of salts. Fresh weed contained less than 10(3) bacteria and less than 10(2) yeasts and molds per g (dry weight). The type and extent of microbial populations in seaweed meal appeared to be dependent upon the method of seaweed drying. Rotary drum-drying at temperatures decreasing from 800 to 80 C maintained or reduced the microbial populations to 10(3) organisms per g (dry weight). Although meals with high nutritional quality can be obtained with warm air- or rock-dried weed, these conditions can also permit bacterial and mold development. Extended rock-drying in variable weather conditions and prolonged storage of moist weed, both of which decrease the nutritional quality, also lead to high bacterial numbers and to a marked development of the halophilic brown mold Sporendonema minutum which attained populations of 10(8) viable spores per g of dried weed. A poultry diet containing 5% badly molded weed had no apparent toxic or growth-depressing effect when fed to chicks.

Survival of natural sewage populations of enteric bacteria in diffusion and batch chambers in the marine environment.

The survival of natural populations of Escherichia coli and enterococci in sewage was measured in large-volume diffusion chambers in an estuary and a salt marsh. The 5-liter chambers, with polycarbonate membrane sidewalls, were found to be suitable for up to week-long experiments. Decay rates, measured monthly from February to August 1978, ranged from 0.042 to 0.088 h(-1) (time for 90% of the population to die = 25 to 55 h) for E. coli and 0.019 to 0.083 h(-1) (time for 90% of the population to die = 29 to 122 h) for enterococci and were significantly correlated with temperature. In contrast to the diffusion culture experiments, the decay of E. coli in batch culture did not correlate with temperature. Enterococci survived longer than E. coli in the Narragansett Bay (estuary) experiments, but survived less well in the more eutrophic salt marsh. The effect of light on survival was examined with light/dark experiments and sampling at frequent intervals over the diel cycle. Diel changes in survival were not evident in the Narragansett Bay experiments. E. coli, however, exhibited a diel pattern of growth during the day and death at night in the salt marsh. There was no significant difference in decay rates between light and dark diffusion chambers, nor were decay rates correlated with light intensity. In concurrent batch experiments, survival was significantly greater in the dark for both organisms. These results suggest that the effect of light is complex and that conditions in batch culture may modify the survival of enteric bacteria. Observations made in diffusion chambers may more closely follow the in situ survival of enteric microorganisms.

Disruption of the primary fouling sequence on fiber glass-reinforced plastic submerged in the marine environment.

Fiber glass-reinforced plastic immersed in an experimental estuarine mesocosm fouled at estimated rates of 0.5, 5.5, and 18.8 ng (wet weight) mm day over days 0 to 2, 2 to 6, and 6 to 14, respectively. Protists, dominated by diatoms, which developed between days 3 and 6 and covered 90% of the undisturbed surface in 2 weeks, were effectively removed by twice-weekly brushing of the surface to maintain an immature 3-day bacterial film which covered 12% or less of the surface and had a biomass 3 orders of magnitude smaller than surfaces with 2 weeks' unrestricted fouling. Direct brushing of the fiber glass-reinforced plastic tank walls of experimental estuarine mesocosms minimized the "wall effect" by keeping a surface that maintained a low biomass of a slowly accumulating bacterial film rather than a surface which supported the more rapid accumulation of protists which in turn may induce the settlement of invertebrates and macrophytes.

Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations.

Two marine bacteria, an Acinetobacter sp. (strain GO1) and a vibrio sp. (strain G1), were isolated by extinction dilution and maintained in natural seawater supplemented with nitrogen, phosphorus, and glucose at 0.01 and 10 mg of glucose carbon per liter above ambient monosaccharide concentrations, respectively. After 3 days in unsupplemented natural seawater, growth in batch culture with glucose supplements was determined by changes in cell numbers and glucose concentration. The exponential growth of the Acinetobacter strain with added glucose was indistinguishable from that in natural seawater alone, whereas that of the Vibrio strain was more rapid in the presence of glucose supplements, suggesting that the Acinetobacter strain preferred the natural organic matter in seawater as a carbon source. The ultrastructure for both isolates was unaffected by glucose supplements during exponential growth, but there were marked changes in stationary-phase cells. The Vibrio strain formed polyphosphate at 10 mg of glucose carbon per liter, whereas poly-beta-hydroxybutyrate formation occurred at 100 mg and became excessive at 1,000 mg, disrupting the cells. In contrast, the Acinetobacter strain elongated at 100 and 1,000 mg of glucose carbon per liter but failed to show poly-beta-hydroxybutyrate formation. The diversity of responses shown here would not have been detected with a single concentration of substrate, often used in the literature to characterize both pure and natural populations of marine bacteria.

The detection and characterization of bacteria-sized protists in "Protist-free" filtrates and their potential impact on experimental marine ecology.

Nuclepore filters of 0.6-1.0µm pore size have been used to prepare "protist-free" water for a number of studies in microbial ecology. This procedure has been called into question by a recent study claiming that a significant portion of bacterial loss in filtrates could be due to uncharacterized predators passing through 0.6µm filters. We were unable to directly observe protists in 0.6µm filtrates using phase contrast, epifluorescence, or transmission electron microscopy. Using the culture techniques of rice grain enrichment and most probable number, however, we were able to observe and quantify several species of bacterivorous nanoflagellates that developed not only in 0.6µm, but also in 0.4µm seawater filtrates. The ability of predacious nanoflagellates to squeeze through bacteria-sized pores questions studies of bacterial production and chemical cycling that have assumed protist-free filtrates.

Factors responsible for the differences in cultural estimates and direct microscopical counts of populations of bacterivorous nanoflagellates.

Bacterivorous nanoflagellates (microflagellates) have been routinely enumerated in marine and freshwater samples using either a Most Probable Number (MPN) culture method or by a direct microscopical counting method (DC). These two techniques typically yield highly disparate estimates of the density of nanoflagellates in natural samples. We compared these methods with seawater and marine snow (macroscopic detrital aggregate) samples collected from surface waters throughout the North Atlantic and in freshwater samples collected at three stations in Lake Ontario. Densities of nanoflagellates determined by the two methods differed by as much as four orders of magnitude; the MPN estimate rarely exceeded 10% of the microscopical count, and averaged ≈ 1% of this count. The MPN estimate constituted a higher percentage of the DC value in environments with high concentrations of nanoflagellates relative to environments with low concentrations of nanoflagellates. The ratio of the culture count to the microscopical count (MPN∶DC) increased along an environmental gradient from oligotrophy to eutrophy, and was positively correlated with the density of bacteria in the samples. In laboratory experiments with two species of bacterivorous nanoflagellates, the MPN count constituted a much greater percentage of the DC count during the exponential growth phase of the nanoflagellate than during the stationary growth phase. Differences in the estimates of nanoflagellate density obtained with these two techniques probably can be explained by the trophic mode of these protozoa, their growth stage, and the amenability of these species to laboratory culture.

Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy.

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.