Bacteria In Aquatic Environments Research Articles

Analysis of bacteria in aquatic environments using sedimentation field-flow fractionation: (II) physical characterization of cells

This paper describes the application of sedimentation field-flow fractionation (SdFFF) for the physical characterization of bacteria cells found in natural aquatic environments. This technique separates bacteria on the basis of the buoyant mass of the cells and fractions can be collected for determination of cell numbers and volume. This enables the cell density and size distribution of the sample to be calculated. SdFFF is also capable of generating very detailed cell size and specific gravity distributions which may be useful data in assessing the bacterial growth status of natural waters. Data for samples from seawater, two lakes and a sewer drain are given. The specific gravity of cells varies both within and between samples and falls in the range 1.03–1.2 g cm −3. This contrasts with previous work which shows little variation within a given cultured bacteria sample and indicates that natural populations contain a range of different bacteria types.

Phylogenetic Diversity of Natural Populations of Ammonia Oxidizers Investigated by Specific PCR Amplification

The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels.

Use of green fluorescent protein to monitor survival of genetically engineered bacteria in aquatic environments

Many methods for detecting model genetically engineered microorganisms (GEMs) in experimental ecosystems rely on cultivation of introduced cells. In this study, survival of Escherichia coli was monitored with the green fluorescent protein (GFP) gene. This approach allowed enumeration of GEMs by both plating and microscopy. Use of the GFP-marked GEMs revealed that E. coli persisted in stream water at higher densities as determined microscopically than as determined by CFU enumeration. The GFP gene did not negatively impact the fitness of the host strain.

Survival and Detection of Viable but non-culturable Escherichia coli in lake water

Three different methods were used for the detection of Escherichia coli survival in filtered-autoclaved or untreated lake water at different temperatures. It was shown that E.coli could be detected from lake water environments by means of respiration activity when exposed to starvation stress, but not by using the traditional plate count method. Thu number of bacteria detected by plate counts was always lower than by direct microscopic counts. E.coli disappeared from the lake water or lost its abilitiy to form colonies under the effect of starvation stress and higher temperatures, even though the bacteria were still metabolically active. It is implied that some E.coli cells probably entered a viable but non-culturable state, i. e., dormant state. Thus it is very important to detected the actual number of coliform bacteria in aquatic environments in terms of the human health aspects involved.

Survival ofEscherichia coli andYersinia enterocolitica in stream water: Comparison of field and laboratory exposure

Experiments were done to compare the influence of three aquatic exposure methods on the behavior of pathogenic and nonpathogenic enteric bacteria (Yersinia enterocolitica andEscherichia coli). Bacterial suspensions were exposed to stream water in membrane diffusion chambers in situ as well as in the laboratory using a large vessel of stream water and in enclosed bottles. The persistence of culturability of the bacterial suspensions was dependent upon the method of aquatic exposure. This difference was most apparent during the initial six days of each experiment. A steady decline in colony forming units was seen after a short stationary period in chambers in situ, while there was an abrupt increase in bacteria within chambers exposed in the laboratory. A rapid initial decrease was observed in the experimental variation using bottles, accompanied by higher levels of injury inE. coli and reduced expression of plasmid-borne virulence phenotypes inY. enterocolitica. However, there were no changes in the plasmid profiles of either organism throughout the 21-day duration of the experiments. In addition, the survival and injury of pathogenic and nonpathogenic strains of both test bacteria was very similar with aquatic exposure. These results suggest that the response of enteric bacteria in aquatic environments is influenced by experimental design as well as other factors and that the comparison of survival data should only be attempted when similar methods are used.

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)

Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy

Bacteria and virus particles were harvested from water samples by ultracentrifugation directly onto Formvar-coated electron microscopy grids and counted in a transmission electron microscope. With this technique, we have counted and sized bacteria and viruses in marine water samples and during laboratory incubations. By X-ray microanalysis, we could determine the elemental composition and dry-matter content of individual bacteria. The dry weight/volume ratio for the bacteria was 600 fg of dry weight microns-3. The potassium content of the bacteria was normal compared with previous estimates from other bacterial assemblages; thus, this harvesting procedure did not disrupt the bacterial cells. Virus particles were, by an order of magnitude, more abundant than bacteria in marine coastal waters. During the first 5 to 7 days of incubation, the total number of viruses increased exponentially at a rate of 0.4 day-1 and thereafter declined. The high proliferation rate suggests that viral parasitism may affect mortality of bacteria in aquatic environments.

Use of Genetically Marked Minicells as a Probe in Measurement of Predation on Bacteria in Aquatic Environments

Minicells produced by Escherichia coli M2141 were used as probes to measure predation on pelagic bacteria in situ. The minicells, labeled with [S]methionine in one specific protein, were shown to disappear in the presence of a microflagellate (Ochromonas sp.), as seen by a decrease in the amount of labeled marker protein with time. Incubation in filtered (pore size, 0.2 mum) and autoclaved seawater did not affect the amount of labeled marker protein in the minicell. The generation time of flagellates feeding on minicells was determined to be similar to that found for flagellates grown on seawater bacteria or living E. coli NC3. Data indicate that minicells are seen as true food particles by the flagellates. The minicell probe was used in recapture experiments, in which predation in situ on pelagic bacteria was demonstrated. The rate of bacterial production showed a clear covariation with the rate of predation, both in different sea areas and in depth profiles. The obtained results (11 field experiments) showed that the rate of predation, on average, accounts for the consumption of 62% of the bacteria produced.

Distribution of Mannan-degrading bacteria in aquatic environments.

Mannan-degrading bacteria were isolated by the surface inoculation method from various natural habitats. The Mannan employed was prepared from a green seaweed Codium fragile, and the colonies surrounded with a clear zone on mannan-containing agar media were judged to be mannan-degrading bacteria. In total, 117 isolates were obtained from 433 samples collected from sea, freshwater, and land environments. Identification according to Bergey's Manual of Determinative Bacteriology permitted assignment of most isolates to eight genera: Pseudomonas, Alcaligenes, Klebsiella, Enterobacter, Vibrio, Aeromonas, Moraxella, and Bacillus. Simultaneously, an enumeration of mannan-degrading bacteria was carried out by the pour plate method. The population density of the bacteria ranged from 0-1.0×lO3cells/ml in coastal sea water, 0-4.0×105cells/g in sea bottom mud, 0-5.5×105cells/g in seaweed, 0-1.8×105cells/ml in river and pond waters, 1.0×105-8.7×107cells/g in the intestinal contents of marine and freshwater fish, and 3.0×104-7.7×1O5cells/g in land soil.

Influence of Dilution Rate on Enzymes of Intermediary Metabolism in Two Freshwater Bacteria Grown in Continuous Culture

Two freshwater bacteria, a Pseudomonas sp. and a Spirillum sp., were grown in continuous culture under steady-state conditions in L-lactate-, succinate-, ammonium- or phosphate-limited media. In Pseudomonas sp., NAD-independent and NAD-dependent L-lactate dehydrogenases, aconitase, isocitrate dehydrogenase and glucose 6-phosphate dehydrogenase activities increased up to 10-fold as the dilution rate (D) was decreased from 0.5 to 0.02 h-1, regardless of whether the growth-limiting nutrient was carbon, ammonium or phosphate. In contrast, 2-oxoglutarate dehydrogenase and succinate dehydrogenase activities were not influenced by D, and NADH oxidase activity increased with D. Spirillum sp. gave different results in some respects, but it also exhibited an increase in the activity of several enzymes at low D values. Such increases may emanate from release of catabolite repression, and catabolite repressors for the five enzymes in Pseudomonas sp. showing such increases are probably compounds of carbon, nitrogen and phosphorus. It is likely that increased enzyme syntheses in low D cultures represent the normal physiological state for bacteria in aquatic environments where growth occurs slowly under nutrient limitations. Such increases probably permit a more effective utilization of nutrients present at sub-saturating concentrations.

Acridine Orange-Epifluorescence Technique for Counting Bacteria in Natural Waters

FRANCISCO, D. E., MAH, R. A. & RABIN, A. C. 1973. Acridine orange-epifluorescence technique for counting bacteria in natural waters. Trans. Amer. Micros. Soc., 92: 416-421. Total direct counts of aquatic bacteria from natural samples were estimated by epifluorescence microscopy after acridine orange staining. Cells were concentrated by membrane filtration on black membrane filters and counted directly. Using this method, bacterial counts of pure cultures corresponded to those obtained by the Petroff-Hauser and Coulter counter methods. Epifluorescence counting is the method of choice for ecological studies of the natural distribution of bacteria in aquatic environments since it permits ready discrimination of bacteria from detritus and does not rely on the adequacy of culture methods to elicit growth of all viable organisms. Total counts obtained in this way were related to glucose and acetate uptake by resident microbial populations of lake water sample. Viable counts of the same samples were not related.