Influence of phosphate ions and alkaline phosphatase activity of cells on survival ofEscherichia coli in seawater

When grown in a minimal medium and suspended for 2 hours in distilled water, seawater, phosphate buffer or a polyphosphate solution,E. coli MC4100 cells with high alkaline phosphatase activity survived in seawater for longer periods than cells with low or no activity. However, mutant cells totally deprived of alkaline phosphatase activity held in phosphate-containing media before transfer to seawater showed survival almost as high as the wild type strain, indicating that alkaline phosphatase activity is not the only factor influencing survival. Alkaline phosphatase activity also increased the protection of cells provided by glycine betaine. Survival was enhanced when cells were preincubated in the presence of phosphate or polyphosphate. Thus, the transfer of cells in wastewater could influence their subsequent survival in seawater.     

Effects of long-term incubation in seawater on the ability of Escherichia coli cells to incorporate histidine and maltose

The aim of this study was to compare the capacity of Escherichia coli resting cells suspended for a long period in seawater, to transport maltose and histidine. Histidine accumulation was constant throughout the observation and was probably due to energy-independent mechanisms. Maltose transport ability, on the other hand, increased during the first 2 weeks of the experiment and then slowly decreased, which suggested that maltose transport could be partly restored, improving survival of enterobacterial cells in seawater.     

Sperm-derived sperm motility-initiating substance from amphioxus Branchiostoma belcheri

The sperm of amphioxus, Branchiostoma belcheri, were immotile when excised from the testis and suspended in seawater. The sperm became motile upon spawning in natural seawater, suggesting mechanisms that triggered sperm motility during spawning. When a male amphioxus that underwent spawning was transferred to a cup containing a small amount of natural seawater, and then the seawater containing the spawned sperm was centrifuged, the supernatant caused motility initiation in the immotile sperm from the testis. This sperm motility-initiating activity was also found in the supernatant of seawater in which immotile sperm from the testis were incubated overnight. These suggest that in the amphioxus, a sperm motility-initiating substance resides in the sperm, and upon spawning, the substance is transformed into a free and active form to activate the sperm. Partial purification of the substance revealed it as a small and heat-stable substance with maximum UV absorbance at 234 nm.     

Nitrate deficiency shortens the circadian period in gonyaulax

The circadian rhythms in bioluminescence and photosynthesis in Gonyaulax polyedra suspended in unsupplemented sea water have been compared to the same rhythms in f/2, an enriched seawater medium. Cells suspended in sea water for 2 days in continuous light (450 microwatts per square centimeter) showed significantly shorter circadian periods and lower amplitudes than did cells in f/2 medium (a period of 22.2 hours as compared to 23.5 hours). Both period and amplitude changes could be completely reversed by the addition of nitrate at one-fourth or more of the concentration in f/2 medium (0.88 millimolar). The addition to autoclaved seawater of phosphate, vitamins, minerals, or soil extract in concentrations present in f/2 medium had no effect. Thus, the shortening of the circadian period is the consequence of reduced nitrogen supply. Since both the rhythms in bioluminescence and photosynthesis showed similarly shortened circadian periods and lower amplitudes, it is probable that the depletion of nitrate directly affects the circadian clock.     

Ecology ofVibrio vulnificus in Galveston Bay oysters,suspended particulate matter,sediment and seawater: Detection by monoclonal antibody — immunoassay — most probable number procedures

Summary Oysters, suspended particulate matter (SPM), sediment and seawater samples were collected from West Galveston Bay, Texas over a 16-month period and analyzed for the presence ofVibrio vulnificus, a naturally-occurring human marine pathogen. Detection and enumeration ofV. vulnificus was performed using a species-specific monoclonal antibody (mAb FRBT37) in an enzyme immunoassay (EIA)-most probable number (MPN) procedure capable of detecting as few as 2000 target organisms.V. vulnificus was not detected in seawater, oyster or SPM samples during the cold weather months, but was detected at low levels in several sediment samples during this time period. Increased levels of the organism were first observed in early spring in the sediment, and then in SPM and oysters. The major increase inV. vulnificus occurred only after the seawater temperature had increased above 20°C and the winter-spring rainfall had lowered the salinity below 16. The highestV. vulnificus levels at each site were associated with suspended particulate matter. These results are consistent with the hypothesis that (1)V. vulnificus over-winters in a floc zone present at the sediment-water interface, (2) is resuspended into the water column in early spring following changes in climatic conditions, (3) colonizes the surfaces of zooplankton which are also blooming during early spring and (4) are ingested by oysters during their normal feeding process.     

High pH-induced acrosome reaction and Ca2+ uptake in sea urchin sperm suspended in Na+-free seawater

The egg jelly-induced acrosome reaction of sea urchin sperm requires the presence of Ca2+ and Na+ in seawater at its normal pH 8. Sperm suspended in seawater at pH 9 undergo the acrosome reaction in the absence of jelly. We have attempted to understand the role of external Na+ in this reaction. Sperm were suspended in Na+-free seawater and the percentage of acrosome reaction and the amount of Ca2+ uptake were determined as a function of external pH. High pH (9.0) in Na+-free medium without jelly triggered a high percentage (above 65%) of sperm acrosome reactions and a two to fourfold increase in Ca2+ uptake. Both the percentage of acrosome reactions and the amount of Ca2+ uptake were similar to those induced by either jelly or pH 9 in Na+-containing seawater. On the other hand, the absence of Na+ in seawater inhibits jelly from inducing Ca2+ uptake and acrosome reactions at pH 8.0 and even at pH 8.5. These results indicate that the Na+ requirement for the acrosome reaction induced by jelly is lost when triggering is by high pH. In contrast, Ca2+ was strictly required since sperm did not react in Ca2+-free seawater at pH 9. We also found that like the jelly-induced acrosome reaction the high-pH-induced acrosome reaction and Ca2+ uptake in complete and Na+-free seawater were inhibited by D600. This finding suggests that the same transport system for Ca2+ uptake associated with the acrosome reaction operates at both triggering conditions, i.e., jelly or pH 9. Although D600 is not now considered a specific blocker, its effect has suggested the involvement of Ca2+ channels in the acrosome reaction. This proposal is supported by our results with nisoldipine, a highly specific inhibitor of calcium channels. The drug inhibited both the sperm acrosome reaction and Ca2+ uptake induced by jelly or pH 9 in complete seawater.     

Viability and virulençe of Escherichia coli suspended by membrane chamber in semitropical ocean water

Abstract Escherichia coli H10407 was suspended in seawater (38.5‰ salinity) contained in membrane chambers (0.4-μm polycarbonate membrane) incubated in situ at 25°C in Nixon's Harbor, South Bimini, Bahamas. Although colonies of E. coli could not be cultured after 13 h post chamber inoculation, the number of fluorescent-antibody staining cells remained constant. Direct viable counts revealed that viable cells were present, even though the cell suspension was not culturable on the media tested. After exposure to seawater for 112 h, cells were concentrated by centrifugation and introduced into ligated rabbit ileal loops. E. coli H10407 proved viable for recovery from inoculated loops and was confirmed by detection of characteristic plasmid bands. Results indicate that enteric pathogens remain viable in seawater long after they cease to be cultivable on laboratory media.     

Physiological and morphological changes during short term starvation of marine bacterial islates

Three marine bacteria were examined for physiological and morphological changes in the initial phase of starvation. It was found that the starvation process was induced in a similar way irrespective of whether the cells were suspended in nutrient and energy free artificial seawater (NSS) or NSS supplemented with nitrogen and phosphorus. An initial phase of increased activity was consistent with a decreased response to added nutrients. Recovery from starvation exhibited the same response in both these starvation regimes, measured throughout the starvation period. Cells in nitrogen or phosphorus deprived starvation regimes, showed a high and rapid increased activity, followed by a delayed and more pronounced decline in respiratory activity. The initial phase of starvation also included a loss of poly--hydroybutyrate as observed by transmission electron microscopy (TEM). Two bacterial strains showed formation of small vesicles on the outer cell layer when examined by TEM. This formation and release of vesicles was related to the continuous size reduction during starvation survival. The results are discussed in terms of defining the mechanisms of initial cellular responses to nutrient deprivation.Abbreviation NSS nine salt solution     

Visible light damage to Escherichia coli in seawater: oxidative stress hypothesis

The effect of visible light on Escherichia coli H10407 in seawater microcosms was investigated. Light damage was estimated by loss of colony-forming ability. Illumination of E. coli suspended in oligotrophic seawater with visible light at an intensity of about 40 klux caused a drastic decrease of culturable bacteria which turned to a viable but non-culturable state. In seawater E. coli exhibited weak metabolic activity as estimated by ³H methyl-thymidine incorporation in the cell. Visible light did not significantly alter this metabolic activity and did not involve detectable oxidation of lipid membranes as evaluated by gas chromatography analysis of fatty acids. The involvement of oxygen and reactive oxygen species in phototoxicity was studied. A decrease of the toxic effect was observed when E. coli was exposed to visible light under anaerobic conditions. Scavengers of reactive oxygen species exhibited variable protective effects. β-Carotene, a singlet oxygen scavenger, and superoxide dismutase were equally ineffective. On the other hand, catalase, which eliminates hydrogen peroxide and thiourea, a hydroxyl radical scavenger, showed a net protection. In addition desferrioxamine B, an iron chelator, was also effective in reducing phototoxicity, probably by preventing hydroxyl radical generation by decomposition of hydrogen peroxide in the presence of iron (Fenton reaction). Therefore, hydrogen peroxide and hydroxyl radical seem to be reactive intermediates of oxygen-dependent (type II) photosensitized reactions.     

Influence of osmoregulation processes on starvation survival of Escherichia coli in seawater.

The adaptation of enteric bacteria in seawater has previously been described in terms of nutrient starvation. In the present paper, we bring experimental arguments suggesting that survival of these microorganisms could also depend on their ability to overcome the effects of osmotic stress. We analyzed the influence of osmoregulatory mechanisms (potassium transport, transport and accumulation of organic osmolytes) on the survival of Escherichia coli in seawater microcosms by using mutants lacking components of the osmotic stress response. Long-term protection was afforded to cells by growth in a medium whose osmotic pressure was increased by either NaCl, LiCl, or saccharose. Achievement of the protection state depended at least partly on osmoregulatory mechanisms, but differed when these were activated or induced during prior growth or in resting cells suspended in phosphate buffer or in seawater. When achieved during growth, K+ transport, glycine-betaine (GBT) synthesis or transport, and trehalose synthesis helped increase the ability to survive in seawater. Protection by GBT was also obtained with resting cells in a phosphate buffer at high osmotic pressure. However, when added only to the seawater, GBT did not change the survival ability of cells no matter what their osmoregulation potential. These results showed that the survival of E. coli cells in seawater depends, at least partly, on whether they possess certain genes which enable them to regulate osmotic pressure and whether they can be stimulated to express those genes before or after their release into the environment. This expression requires nutrients as the substrates from which the corresponding gene products are made.     

Production of a Capsular Polysaccharide by a Marine Filamentous Fungus

The spent seawater medium of 4-day-old-cultures of the filamentous marine fungus Leptosphaeria albopunctata had a high viscosity after the fungus was collected by high-speed centrifugation. Microscopic examination of uncentrifuged mycelium suspended in India ink revealed that the viscosity resulted from capsular material. These capsules became disassociated from the mycelium during centrifugation. Precipitation of the medium of centrifuged cultures with 95% ethyl alcohol yielded a highly anthrone-positive polysaccharide material, composed of large amounts of glucose and minute amounts of mannose. Time course studies of the nutritional requirements for capsular polysaccharide production revealed that the capsular material was produced in large amounts, and on a wide variety of sugars, during the period of rapid growth, but was quickly degraded and presumably remetabolized in older cultures. The amount of capsular material produced was enhanced by NaCl concentrations above that of artificial seawater, and KCl could be substituted for NaCl. The salts MgCl(2) and CaCl(2) were also required for capsule production by L. albopunctata, although growth was obtained in cultures without added amounts of these constituents. The possible role of these salts in the metabolism of the fungus is discussed.     

Radioactive contamination in the upper part of the Techa river: stirring-up of bottom sediments and precipitation of suspended particles

A hydrodynamic model of the upper part of the Techa river was developed on the basis of the river valley geometry as well as data of hydrological conditions and of the granulometric composition of bottom sediments. The model describes the transport of radioactivity by suspended sediments with different granulometric compositions (clay, silt) in the early 1950s. It includes the stirring-up of bottom sediments and the precipitation of suspended sediments as a function of water discharge rate and water level in the investigated part of the river. The results allow to specify the development of the river system contamination as a result of inflow of suspended sediments contaminated with radionuclides. In the period of liquid radioactive waste (LRW) discharges, the water of the Techa river contained a large fraction of finely dispersed particles of less than 5 micro m diameter. At the site of LRW discharge 80% of the discharged activity was adsorbed to these particles. Depending on the water flow, 40-80% of the suspensions precipitated at the bottom of subsequent sedimentation reservoirs. A total of about 1.6 MCi adsorbed to the suspended particles entered the open hydrographic system of the Techa river. The conclusion that the largest part of the activity was adsorbed on the suspended particles contradicts the assumption in the Techa river dosimetry system, TRDS-2000, that most of the released activity entered the Techa river in soluble form. For a correct reconstruction of the doses received by the Techa river population it is, therefore, essential to consider hydrodynamic models that take into account the transport of radionuclides adsorbed on the suspended particles.     

Flocculation of harmful algal cells using modified clay: effects of the properties of the clay suspension

Large differences occur between the removal efficiencies (RE) of polyaluminum chloride modified clay (PAC-MC) suspensions prepared using deionized water (DW) and seawater (SW). This showed that physicochemical properties of PAC-MC suspension influenced its ability to remove algal cells. The efficiency at which a PAC-MC suspension removed Aureococcus anophagefferens (CCMP 1984) and Phaeocystis globosa from water was determined using diluted seawater (DSW), dissolved ion species, ion concentrations, pH, temperatures, and suspension aging times. The RE decreased generally as the percentage of seawater increased, and the RE was higher for PAC-MC prepared using DW than that using SW. The ion species present and the ion concentrations in the suspension influenced the RE, with sulfate negatively affecting the RE more than the other ions tested. Sulfate in the PAC-MC suspension showed a “V”-shaped effect which can be partially explained by charge neutralization and “patch coagulation” that played different parts in the flocculation process. The effects of pH of PAC-MC suspension on the RE were greatly influenced by the dispersion medium. The RE of the PAC-MC suspended in DW decreased gradually, whereas RE of the PAC-MC suspended in SW followed a V-shaped trend, as the pH was increased. Surface charge is an important aspect in the removal mechanism of PAC-MC, but the flocculation behavior at low suspension pH conditions is less clear. Temperature and suspension aging time had no obvious effects on the RE. Appropriate explanation and mechanism is also proposed in the discussion. These studies showed that the RE could be further improved given proper attention to the properties of PAC-MC suspension.     

Distribution of rare earth elements in seaweed: implication of two different sources of rare earth elements and silicon in seaweed

Rare earth elements (REEs) and Si in five species of seaweed, ambient surface seawaters, and suspended solid particles in the seawaters were determined separately. Inductively coupled plasma mass spectrometry (ICP-MS) was used for REEs and inductively coupled plasma emission spectrometry (ICP-ES) was used for Si in order to evaluate REEs as a tracer in seaweeds and to understand the source of inorganic elements, especially Si, in seaweeds. Two different REE patterns, one similar to that of the seawater solution and another resembling that of suspended particles, were observed in seaweeds, and the variation of REE patterns seems to show a clear dependence on the abundance of Si. The REE pattern and Si concentration seem to vary depending on the division: green and red algae showed REE patterns similar to that of suspended particles, but brown algae showed patterns closer to that of seawater solutions and relatively lower Si concentration. The possibility of contamination from silicate particles on the surface of seaweeds was ruled out for several reasons. Silicate particles, not dissolved silicate, have been identified as the direct source of REEs and Si in plants ( Fu et al. 1998 ), and seaweeds are no exception. We have to consider that seaweeds can take up Si from suspended particles through their blade or branches. From the appearance of tetrad-effect-like variation of REEs, Si is assumed to enter a dissolved state just before the particles are taken up. From the results of a sonication experiment, REEs, once taken up as silicate particles, seem to be separated from Si in the thallus.     

Influence of estuarine sediment on virus survival under field conditions

The survival of poliovirus 1 (LSc) and echovirus 1 (Farouk) in estuarine water and sediment was studied in Galveston Bay, Texas. Viruses were suspended in estuarine water and sediment both in dialysis tubing and in chambers constructed with polycarbonate membrane walls. Virus inactivation rates in seawater were similar in both types of chambers. Virus adsorption to sediment greatly increased survival time. The time required to inactivate 99% (T-99) of poliovirus increased from 1.4 days in seawater alone to 6.0 days for virus adsorbed to sediment at a relatively nonpolluted site. At a more polluted site, poliovirus T-99 was increased from approximately 1 h to 4925 days by virus adsorption to sediment. This study demonstrates that under field conditions virus association with estuarine sediment acts to prolong its survival in the marine environment.     

Influence of estuarine sediment on virus survival under field conditions.

The survival of poliovirus 1 (LSc) and echovirus 1 (Farouk) in estuarine water and sediment was studied in Galveston Bay, Texas. Viruses were suspended in estuarine water and sediment both in dialysis tubing and in chambers constructed with polycarbonate membrane walls. Virus inactivation rates in seawater were similar in both types of chambers. Virus adsorption to sediment greatly increased survival time. The time required to inactivate 99% (T-99) of poliovirus increased from 1.4 days in seawater alone to 6.0 days for virus adsorbed to sediment at a relatively nonpolluted site. At a more polluted site, poliovirus T-99 was increased from approximately 1 h to 4925 days by virus adsorption to sediment. This study demonstrates that under field conditions virus association with estuarine sediment acts to prolong its survival in the marine environment.     

High pH-induced acrosome reaction and Ca uptake in sea urchin sperm suspended in Na-free seawater

The egg jelly-induced acrosome reaction of sea urchin sperm requires the presence of Ca²⁺ and Na⁺ in seawater at its normal pH 8. Sperm suspended in seawater at pH 9 undergo the acrosome reaction in the absence of jelly. We have attempted to understand the role of external Na⁺ in this reaction. Sperm were suspended in Na⁺-free seawater and the percentage of acrosome reaction and the amount of Ca²⁺ uptake were determined as a function of external pH. High pH (9.0) in Na⁺-free medium without jelly triggered a high percentage (above 65%) of sperm acrosome reactions and a two to fourfold increase in Ca²⁺ uptake. Both the percentage of acrosome reactions and the amount of Ca²⁺ uptake were similar to those induced by either jelly or pH 9 in Na⁺-containing seawater. On the other hand, the absence of Na⁺ in seawater inhibits jelly from inducing Ca²⁺ uptake and acrosome reactions at pH 8.0 and even at pH 8.5. These results indicate that the Na⁺ requirement for the acrosome reaction induced by jelly is lost when triggering is by high pH. In contrast, Ca²⁺ was strictly required since sperm did not react in Ca²⁺-free seawater at pH 9. We also found that like the jelly-induced acrosome reaction the high-pH-induced acrosome reaction and Ca²⁺ uptake in complete and Na⁺-free seawater were inhibited by D600. This finding suggests that the same transport system for Ca²⁺ uptake associated with the acrosome reaction operates at both triggering conditions, i.e., jelly or pH 9. Although D600 is not now considered a specific blocker, its effect has suggested the involvement of Ca²⁺ channels in the acrosome reaction. This proposal is supported by our results with nisoldipine, a highly specific inhibitor of calcium channels. The drug inhibited both the sperm acrosome reaction and Ca²⁺ uptake induced by jelly or pH 9 in complete seawater.     

Histamine-producing bacteria in decomposing skipjack tuna (Katsuwonus pelamis).

Spoilage in skipjack tuna (Katsuwonus pelamis) was studied under controlled conditions by incubating whole, fresh fish in seawater at 38 degrees C, the optimum temperature for histamine formation. Bacterial isolates were obtained from the loin tissue of a decomposing tuna containing 134 mg of histamine per 100 g and a total anaerobic count of 3.5 x 10(5)/g after incubation for 24 h. Over 92% of the 134 isolates obtained were facultatively or obligately anaerobic bacteria. Eighteen isolates produced histamine in culture media containing histidine, and these were identified as Clostridium perfringens, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, and Vibrio alginolyticus. Histidine decarboxylase activity of several isolates was measured in a tuna broth medium and with resting cells suspended in a buffered histidine solution.     

Histamine-producing bacteria in decomposing skipjack tuna (Katsuwonus pelamis)

Spoilage in skipjack tuna (Katsuwonus pelamis) was studied under controlled conditions by incubating whole, fresh fish in seawater at 38 degrees C, the optimum temperature for histamine formation. Bacterial isolates were obtained from the loin tissue of a decomposing tuna containing 134 mg of histamine per 100 g and a total anaerobic count of 3.5 x 10(5)/g after incubation for 24 h. Over 92% of the 134 isolates obtained were facultatively or obligately anaerobic bacteria. Eighteen isolates produced histamine in culture media containing histidine, and these were identified as Clostridium perfringens, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, and Vibrio alginolyticus. Histidine decarboxylase activity of several isolates was measured in a tuna broth medium and with resting cells suspended in a buffered histidine solution.