Mary A. Hood

Effects of temperature, ph, salinity, and inorganic nitrogen on the rate of ammonium oxidation by nitrifiers isolated from wetland environments

Ammonium-oxidizing bacteria were examined in two wetland environments, a freshwater marsh and an estuarine bay, during a 2-year period. Two predominant types were consistently isolated, one from each environment. Both isolates were identified as species ofNitrosomonas. Using a closed culture, high cell density assay, the effects of temperature, pH, salinity, Na+, K+, nitrite, nitrate, and ammonium concentrations on ammonium oxidation were determined. Maximum activity was observed for the freshwater isolate at 35°C, pH 8.5, salinities of 0.3 to 0.5% Na+ and K+, and ammonium concentrations greater than 0.5 g/l. For the estuarine isolate, maximum activity was observed at 40°C, pH 8.0, salinities of 0.5 to 1.0%, 1.0% Na+ and K+, and 0.2 g/l ammonium. The estuarine isolate had a Na+ requirement which could be partially substituted by the K+, suggesting that the organism is a true estuarine bacterium. Nitrite inhibited both isolates at concentrations greater than 5 mg/l, whereas nitrate had no significant effect on either isolate.

Interaction between an ammonium-oxidizer,Nitrosomonas sp., and two heterotrophic bacteria,Nocardia atlantica andPseudomonas sp.: A note

Closed culture experiments showed that the ability of an estuarine chemolithotrophic ammonium-oxidizer,Nitrosomonas sp., to convert ammonium to nitrite was substantially increased by as much as 150% and 50% when grown in the presence of two heterotrophs isolated from the same environment. The heterotrophs were identified asNocardia atlantica andPseudomonas sp. The growth of the heterotrophs was stimulated by as much as one order of magnitude in the presence of the ammonium-oxidizer.

Distribution of Ultramicrobacteria in a Gulf Coast Estuary and Induction of Ultramicrobacteria

The abundance of ultramicrobacteria (i.e., bacteria that pass through a 0.2μm filter) in a subtropical Alabama estuary was determined during a 1-year period. Although phenotypic and molecular characterization indicated that the population of ultramicrobacteria was dominated byVibrio species, species ofListonella andPseudomonas were also abundant. Vibrios occurred with the greatest frequency in waters whose salinities were less than 14‰, and were the most abundant species of the total ultramicrobacterial population year-round, whilePseudomonas species were absent or considerably reduced during the winter months. The total number of ultramicrobacteria showed an inverse relationship to total heterotrophic bacteria as measured by colony-forming units (CFU)/ml and to water quality as measured by several parameters. Analysis by generic composition indicated that both salinity and temperature significantly affected the distribution of these organisms. Laboratory studies revealed that strains of vibrios under starvation in both static and continuous-flow microcosms could be induced to form cells that passed through 0.2 and/or 0.4μm filters. Cells exposed to low nutrients became very small; some grew on both oligotrophic (5.5 mg carbon/liter) and eutrophic (5.5 g carbon/liter) media; and some few cells grew only on oligotrophic media. By passing selected vibrio strains on progressively diluted nutrient media, cells were also obtained that were small, that passed through 0.4μm filters, and that could grow in oligotrophic media. These results suggest that ultramicrobacteria in estuaries (at least some portion of the population) may be nutrientstarved or low nutrient-induced forms of certain heterotrophic, eutrophic, autochthonous, estuarine bacteria.

Rates of chitin degradation in an estuarine environment

In vitro chitin degradation rates in pure cultures and in mixed natural cultures have been determined and compared with those of other workers.In situ studies in the salt marsh shrimp nursery grounds along the southeastern Louisiana coast showed that chitin degradation was most rapid (118 mg d−1 g−1 chitin) when water temperature averaged 30‡C. Maximum degradation rates were noted at the water-sediment interface and when substrate particles were reduced in size (0.25 cm2). Of the several types of chitin tested, including treated and untreated, native chitin was most rapidly solubilized. Microbial populations on this substrate developed more rapidly, suggesting that chitin degradationin situ is a function of initial colonization. This is further supported by the observation that degradation rates were most rapid when total bacteria, chitinoclastic bacteria, and the ratio of chitinoclasts to total bacterial biomass (15.6 %) was greatest.

Distribution ofVibrio cholerae in two Florida estuaries.

The distribution ofVibrio cholerae was examined in 2 Florida estuaries, Apalachicola and Tampa Bay.Vibrio cholerae serotype non-01 was the most abundant serotype, being isolated from 45% of the oyster samples, 30% of the sediments, 50% of the waters, and 75% of the blue crabs.Vibrio cholerae serotype 01 was isolated from only one oyster sample. Strong linear correlations betweenV. cholerae and temperature, salinity, or the other physical/chemical parameters measured,Escherichia coli, or fecal coliforms were not observed, but a range of temperatures and salinities appeared relevant to the distribution of the organism. The organism was present in the highest concentrations when salinities were 10‰–25‰ and temperatures were 20‡C–35‡C.In vitro growth curves of 95V. cholerae environmental isolates further supported that 10‰–25‰ was an ideal salinity range for the organisms. The results suggest thatV. cholerae is a widely distributed organism in the nutrient-rich warm waters of the Gulf Coast estuaries.

Microbiological and chitinoclastic activities associated with Penaeus setiferus

Analysis of microbiological and chitinase activities relative to the white shrimpPenaeus setiferus, and its chitinoclastic endosymbionts, have demonstrated the relevance of total chitinolytic processes in penaeid biology. Microorganisms may serve as a direct source of nutrients for the animal as well as in the elaboration of extracellularin situ chitinase enzymes. The enzyme produced by the predominant gut bacteria,Beneckea neptuna, is a moderately actively inducible chitinase while the shrimp has an indigenous constitutive chitinase and chitobiase system. Factors of temperature, pH, ion inhibition and reducing sugar ratios have been compared for the bacterial and animal enzymes. This dual enzyme system suggests that metabolic chitin transformation may play a vital role in crustacean metabolism.

Factors Affecting Attachment of Enterobacter cloacae to Germinating Cotton Seed

A bstractAttachment of Enterobacter cloacae EcCT-50,—a biological seed protectant used to control the seed-rotting fungi, Pythium ultimum—to cotton seed was examined using conventional fluorescent microscopy (CFM), scanning electron microscopy (SEM), and laser scanning microscopy (LSM). In sand microcosms, E. cloacae quickly attached to the seed coat, with maximum attachment, 3 to 5 h after inoculation at 24°C. In contrast, initial attachment of non-bacterized seed by Pythium ultimum was not observed until 6 h (and not until 8 h on bacterized seeds). Comparison of the movement of E. cloacae and P. ultimum in seed exudate gradient semi-soft agar showed faster movement by the bacterium within the first 6 h, and reduction of P. ultimum hyphal and germ tube growth in the presence of the bacterium. Microscopic observation of the seed coat revealed an early, intimate association, mediated, in part, by fimbriae, and confirmed a loose association of E. cloacae with the seed coat previously reported. Spatially, the attached E. cloacae cells were distributed over the entire surface of the seed coat, but were especially abundant in the groves and near cracks where water imbibition and seed exudate release may occur. As the seed germinated and exposed various seed tissues, the bacterium rapidly attached to these tissues. Attachment of the bacterium to the surface of intact germinating seeds, excised seed coat, polystyrene, and glass was 300, 110, 51, and <1 cell field−1 3 h−1, respectively, suggesting that attachment is enhanced by seed germination. Attachment of E. cloacae to the seed coat was optimum in sands with high water concentrations, at temperatures of 18 to 30°C, and at times that corresponded with optimum water imbibition during germination. Using several assays, attachment was shown to be enhanced by seed exudate, and compounds such as methanol, fructose, and calcium. The results suggest that the release of certain nutrients and water imbibition during germination may play a role in the rapid attachment to the seed by E. cloacae. The ability of E. cloacae to rapidly move and attach to the seed coat may be related to its ability to function as a biocontrol agent.

The examination of Seliberia stellata exopolymers using lectin assays

Holdfast exopolymers of the dimorphic oligotrophic bacterium Seliberia stellata were examined using fluorescent lectins under light microscopy and colloidal gold lectins using transmission electron microscopy. Examination using fluorescent-labeled lectins revealed that lectins specific for polysaccharides and monosaccharides such as glucose and/or mannose, galactose, N-acetylgalactosamine, and N-acetylglucosamine (and its dimer) adhered to holdfast structure. Colloidal gold-labeled lectin assays also suggested the presence of these sugars. Both the holdfast that mediates swarmer cell adhesion and the holdfast that facilitates rosette formation gave similar results, suggesting the structures may be the same. Another exopolymer produced later in the growth cycle was observed using transmission electron microscopy. It appeared as an amorphous glycocalyx-like material very different from holdfast exopolymers. Retention of the gold lectin Wheat Germ Agglutinin (WGA), suggested the presence of N-acetylglucosamine, but fluorescent analyses were unsuccessful. The data suggest that S. stellata produces at least two different exopolymers: (a) the exopolymer of the swarmer cell and rosette holdfast whose function is adhesion and whose composition is (but may not be limited to) polysaccharides and (b) a slime-like exopolymer whose composition and function remain unknown.

Design of microcosms to provide data reflecting field trials of GEMS

The issue of releasing genetically engineered micro-organisms into the environment is not one of either we do or we do not. It has already been done [19]. There have been more than 1,000 applications for testing genetically engineered organisms in the environment, most of which have or will occur in the USA [28].