Seasonal variation of psychrotrophic bacteria in sediment from Lake Ontario

Bacteria were isolated from sediment samples obtained from the Bay of Quinte, Lake Ontario, at four different seasons. Isolation of the bacteria was accomplished at 0°C and 20°C and the growth of randomly-selected isolates at each of these temperatures was assessed. The bacteria were identified using miniaturized identification methods and Pseudomonas and Flavobacterium were the most frequently isolated genera. Significant differences were found in the numbers of bacteria in the sediment samples taken at different sampling times and incubated at different temperatures. The numbers isolated with incubation at 20°C were always the same or higher than those isolated from the same sample at 0°C. Almost all bacteria isolated at 0°C were capable of growth at 20°C. The greatest population diversity occurred in the sediment samples collected when the in situ sediment temperatures were low. Distinct changes in population composition were noted at the different sampling times. The majority of the bacteria isolated were capable of growth at 0°C, so that a large psychrotrophic population was present throughout the year in the sediment.     

Thermal tolerance of zooplankton

First immature instar Daphnia pulex Leydig acclimated at 5, 10, 15, 20, 25 and 30°C, and Daphnia magna Straus acclimated at 10, 15, 20, 25 and 30°C, were instantaneously immersed at the specified temperatures which differed from acclimation temperatures by 10°C or more. Observations for mortality were made at regular intervals for 48 h or longer until at least one molting had occurred. Organisms acclimated at the same temperatures were also instantaneously immersed at 35°C, an ultimately lethal temperature, and followed to 95% mortality. Daphnia pulex acclimated at 20°C were stepped over varying rates of temperature change to 35°C and observed for mortality over a 48-h period. Thirty first immature instar organisms were used in each test, and tests were carried out in temperature controlled water baths and incubators. Filtered pond water was used for culture and testing.Both species survived instantaneous temperature changes over the entire normal tolerance ranges tested. Animals succumbed more rapidly upon instantaneous immersion at 35°C as the temperature at which they were acclimated decreased, with D. magna succumbing more rapidly than D. pulex from all acclimation temperatures. Daphnia pulex acclimated at 20°C and stepped to 35°C at varying rates of temperature change exhibited a decreasing 48-h mortality percentage as rates decreased from 6°C h⁻¹ to 1.33°C h⁻¹.Shortcomings in methodologies of previous thermal tolerance studies on zooplankton were discussed, and recommendations were made as to how these methods can be improved.     

Delayed incubation as an alternative method to sample storage for enumeration of E. coli and culturable bacteria in water

The effects of sample storage on enumeration of Escherichia coli in marine bathing water and culturable bacteria in drinking water were evaluated. Results showed that overnight storage at 0-5 degrees C significantly reduced the counts of E. coli in bathing water (p = 0.0001) with a mean reduction of 25%. A similar effect of sample storage was observed for the enumeration of culturable bacteria in drinking water at 22 +/- 2 degrees C for 66 +/- 4 h (p = 0.0074; mean reduction = 25%) or at 36 +/- 2 degrees C for 44 +/- 4h (p = 0.0353; mean reduction = 6%). The use of a delayed incubation method, i.e. overnight storage at 0-5 degrees C of inoculated agar plates prior to incubation, did not significantly affect the counts of culturable bacteria when plates were incubated at 22 + 2 degrees C for 66 +/- 4 h, whereas it resulted in a significant increase of the bacterial numbers when plates were incubated at 36 +/- 2 degrees C for 44 +/- 4 h (p = 0.0002; mean increase = 32%). Based on these results, it is suggested to avoid the use of overnight or longer sample storage for the enumeration of E. coli in bathing water samples, as well as for the enumeration of culturable bacteria in drinking water. The delayed incubation method appears to be a reliable procedure for the enumeration of culturable bacteria and could represent a valid alternative to sample storage in order to overcome problems associated with the performance of bacteriological counts during weekends or statutory holidays. However, a multi-laboratory study is needed to evaluate the reproducibility of the delayed incubation method for the enumeration of culturable bacteria and its possible use for the enumeration of E. coli by membrane filtration.     

Rotavirus and reovirus stability in microorganism-free distilled and wastewaters

Survival of calf rotavirus and reovirus under controlled laboratory conditions in microorganism-free, distilled and wastewater at 8 and 26°C was examined by periodic measurement of cytopathic effects (CPE) and indirect fluorescent antibody (IFA) assays. Five samples of both water-types were collected and inoculated with the two viruses. Three samples of each type of water were divided into two bottles, one per virus, for incubation at 8°C. Two samples were used at 26°C, one per trial. In the absence of light and shaking at 26°C, 7–13 days were required for a loss of 90% infectivity for rotavirus and reovirus, while at 8°C, averages were 80 days for rotavirus and 260 days for reovirus. Virus infectivity remained for more than 30 days at 26°C and 400 days at 8°C. Rates of decline were 10–100 times greater at 26 than at 8°C, but at both temperatures, the MPN log₁₀ rate of decline of infectivity was linear.     

Influence of temperature on lake bacterial activity

Oxygen utilization rates of isolates of Flavobacterium from Lake Ontario and Lake Superior, and an E. coli from the St. Lawrence River were observed at 4 and 20°C. Data presented indicate that the oxygen utilization rate of the lake bacteria at 4°C is similar to that of the river bacteria at 20°C. The observation is also extended to explain the seemingly satisfactory biodegradation of nutrients discharged into water bodies in temperate climates.     

Comparative study of membrane filtration methods for the isolation of group D streptococci from water

An evaluation of two membrane filtration media (KF-streptococcus agar and bile esculin azide agar) incubated at two temperatures (37 and 44°C) was undertaken as a collaborative study involving nine laboratories. A wide variety of samples: sewage, secondary effluent, chlorinated effluent, surface water, stored water and almost fully treated drinking water, were used. Bacteria from all the media studied, were subcultured and classified as group D streptococci if they were catalase negative and able to grow at 45°C. It was demonstrated that the latter characteristic showed a very good correlation with the presence or absence of the group D antigen.There was a marked difference in selectivity between the two media tested. On bile esculin azide agar significantly fewer typical colonies (81 vs 94%) were formed by group D streptococci than on KF. In addition, the suppression of the background flora was insufficient. Selectivity of the two media was not improved by incubation at 44°C instead of the usual 37°C, but the recovery at the higher temperature was significantly less, especially if chlorine-damaged bacteria were present. Therefore, the use of KF-streptococcus agar at 37°C is preferred for membrane filtration.     

The effect of photochemical treatment of water on algal growth

A new method for the destruction of algae in surface waters by dye-sensitized photooxidation is described. The algae (Peridinium, Pediastrum and Cosmarium) used as test organisms were inoculated in an artificial culture medium, containing a dye-sensitizer, with subsequent incubation in a controlled environment (20 ± 2°C, 14 h light, 10 h darkness). Water samples from Peridinium bloom in Lake Kinneret underwent similar treatment. The algicidal effect of various sensitizer concentrations and of different sunlight exposure times was investigated. Complete destruction of algae was obtained in about 2 weeks of incubation after exposure to solar radiation for 30–60 min in the presence of 0.25, 0.15 and 0.75 mg 1⁻¹ methylene blue or 0.5, 0.8 and 0.8 mg 1⁻¹ rose bengal in Peridinium, Pediastrum and Cosmarium cultures, respectively.     

Temperature and oxygen-nitrogen gas ratios affect fish survival in air-supersaturated water

Juvenile steelhead trout and juvenile chinook, coho and sockeye salmon were tested at different temperatures (8°, 9°, 10°, 12°, 15°, 18° and 20°C) at the same concentration of air-supersaturated water. Supersaturated water concentrations in different tests were 115, 116, 117, 118 and 120% saturation. Increased temperatures caused a significant (P< 0.005) increase in steelhead mortality, a significant increase (P < 0.025) in chinook deaths, but no significant effect on coho or sockeye mortality. Regression model data for steelhead indicate that a 10°C increase in temperature will decrease the time to 50% death by a factor of 2.7, e.g. from 190h at 8°C to 70h at 18°C, when tested at the same total dissolved gas pressure. Effects of different oxygen-nitrogen gas ratios on fish mortality at the same total dissolved gas pressure in supersaturated water were demonstrated with juvenile steelhead trout. Mortality was rapid (time to 50% death in 1–6 h) at 140, 135 and 130% saturation, with fish dying more rapidly as the ratio of oxygen-nitrogen decreased (decrease in O₂, increase in N₂) Mortality patterns were similar at 125%; time to 50% death occurred in 5–20 h, with more rapid deaths occurring as oxygen (O₂/N₂ ratio) was decreased.     

A comparison of thermal loading effects on bacterial populations in polluted and non-polluted aquatic systems

Water from two aquatic systems including a new, non-polluted lake, and a lake receiving municipal and industrial wastes was studied for alterations in bacterial populations following temperature increases of 5–10°C above ambient. Lake water was studied under laboratory conditions utilizing 50 1. tank batteries to permit controlled temperature maintenance.Plate count agar was used for counts of total culturable heterotroph and chromagenic colonies. Selected limiting nutritional media were used to determine these types. Increases in total counts were observed in all systems during initial periods at temperatures 3–5°C above ambient; however, at 5–10°C above ambient. total counts and the number of different genera isolated were reduced. Effects on nutritional types and percentage of chromagenic types present were analyzed.     

Rapid enumeration of water bacteria

The research reported in this paper is devoted towards the development of a rapid bacteriological test for water. The proposed technique consists in measuring “Cold T.C.A. insoluble portion” of metabolized phosphorus in multiplying bacterial cells with recourse to radioactive phosphorus-32 as a tracer and enumerates bacteria in 1 h incubation time. A linear correlation (coefficient of correlation = +0·989) has been shown to exist between this portion of phosphorus and cell population for bacterial species of public-health significance, thereby providing a point d'appui for this approach of enumerating water bacteria.The data reported in the paper reveal a promise for the differential enumeration of Grams positive and negative cells by incorporating suitable chemical inhibitors in appropriate concentrations in the test portions for rapid test.The technique has been exhibited to be applicable to field samples drawn from surface and underground sources of water. Its reproducibility and economics are comparable with “Standard Method” procedures.     

The effect of acclimation temperature on the interactions of chlorine, ΔT and exposure duration to eggs, prolarvae and larvae of striped bass, Morone saxatilis

The effect of five acclimation temperatures on the interactions of total residual chlorine (TRC) (0.00, 0.15 and 0.30 mg l⁻¹, ΔT (2, 6, 10°C above acclimation temperature) and exposure duration (0.08, 2.0, 4.0 h) conditions were evaluated for striped bass, Morone saxatilis, eggs, prolarvae and larvae. The range of acclimation temperatures (12.5, 15.0, 17.5, 20.0, 22.5°C for eggs and prolarvae; 15.0, 17.5, 20.0, 22.5 and 25.0°C for larvae) tested with each life stage included the lower, optimum and upper environmental temperatures that exist when these life stages may be subjected to power plant chlorination conditions. All organisms were tested using a fractional composite design in a 3 × 3 × 3 factorial matrix. Mortality of the eggs was highest at temperatures below 17.5°C; overall mortality decreased with increasing acclimation temperature. The factor causing the greatest effect with this developmental stage was ΔT, followed by TRC and exposure time. Mortality of striped bass prolarvae increased with acclimation temperature after exposure to interacting treatment factors. Interactions involving TRC were most important from 12.5 to 17.5°C; ΔT interactions were prominent at acclimation temperatures of 20.0 and 22.5°C. Larvae mortality was highest at 20°C with a gradual decrease at 25.0°C. Interactions involving ΔT, TRC and exposure time all contributed to the mortality of this life stage; TRC and ΔT caused the main effects. Striped bass eggs were the most resistant life stage; mean mortality averaged over all acclimation temperatures was approximately the same for prolarvae and larvae.     

Comparative studies of methods for the enumeration of coli aerogenes bacteria and E. coli in surface water

In a comparative study of four enrichment media for the detection of bacteria of the coli aerogenes group, formate glutamate medium was found to be the best one. No significant differences were assessed between the three confirmation media used in this study.For the detection of E. coli, the use of Eijkman lactose broth, incubated in an air incubator at 44°C, was found to be better than the use of Eijkman glucose broth. When this method was applied, no significant differences could be observed between the results obtained upon immediate testing of the samples, and when the same samples were examined after 24 h storage at 4–6°C, as is often inevitable in practice.For the combined detection of E. coli and the coli aerogenes group, which may be required in practice, the formate glutamate medium proved again to be the best one. No evidence was found that use of this procedure may result in a low E. coli counts. On the contrary, this technique gave even slightly higher results than the direct Eijkman 44°C-procedure. Suitable confirmation tests on gassing tubes are required when this 37°C-procedure is used.     

Growth of psychrophilic and mesophilic, aquatic bacterial isolates at the environmental temperature

The majority (80.4%) of the bacteria isolated from the sediment-water interface of Bethel Lake, a highly productive lake which experiences extremes of temperature due to season, when the in situ temperature was 9°C were mesophiles. The remaining 19.6% of the bacteria were facultative psychrophiles, but the absence of obligate psychrophiles was peculiar to this sampling. A number of the facultative psychrophiles were capable of growth at 37°C. Nine distinct mesophilic “working types” and 11 distinct psychrophilic “working types” were obvious according to the criteria of colony morphology and colour, cell morphology and arrangement, Gram reaction, gelatin liquefaction, starch hydrolysis, cellulose digestion, chitin decomposition, and Tween 80 hydrolysis. The psychrophilic isolates were more versatile than the mesophilic isolates with regard to utilization of the substrates. Comparison of the growth of the psychrophilic and mesophilic types at the in situ temperature at the time of sampling did not reveal a direct relationship between numerical predominance and growth rate at the environmental temperature.     

Effect of acidification, metals and metalloids on sediment microorganisms

The mineralization of [¹⁴C]glucose was reduced in sediments at pH 4 and 5 compared to pH 7. A large reduction in glucose mineralization was also observed at low temperature (0°C). The majority of bacterial isolates recovered at incubation temperatures of 0 and 20°C were psychrotrophic and the majority of both populations was resistant to lead and selenium while a smaller proportion was resistant to mercury and arsenic. Many of the bacteria were resistant to more than one of the elements. Lead and mercury were more toxic to bacterial growth at pH 4.5 than at 7.5, while selenium and arsenic were slightly more toxic at the higher pH values.     

The mathematic-thermodynamic analysis of the anomalies of water and the temperature range of life

Water between 0 and 15°C consists of a vacuum component and of 3 substantial components: a quasi-crystalline component (K), a fluid component (F) and ice-relicts (R). Together they form a colloid system. (K) forms the dispersing milieu in which (R) and (F) are included as two dispersed phases. As the temperature rises from 0 to 15°C, the (R)-component diminishes in amount while the (K)-component increases proportionally. Between 19 and 30°C (F) remains the only dispersed phase. Between 30 and 45°C water consists of two phases (F) and (K), dispersed in the vacuum component. Between 45 and 60°C (F) is the dispersing medium with (K) the dispersed phase. After 60°C, water becomes a structurally homogeneous fluid, (F).All anomalies of water, both the “continuous” ones, occurring over large temperature and pressure ranges, and the “discrete” ones appearing at 15, 30, 45 and 60°C, are the results of these changes between the (R), (K), and (F) components.The biological evolution of warm-blooded animals has resulted in forms whose life is restricted to a narrow range of temperature with an optimum at approx. 37.5°C. At this temperature water possesses its minimum of heat capacity, a low coefficient of compressibility and the maximum of structural possibilities based on combinations of its (K) and (F) components.     

Psychrotrophic bacteria in sediments from the great lakes

The in vitro growth rates at 0°C of 8 representative psychrotrophs, isolated from Great Lake's sediment, ranged from k values of 0.047-0.022 h⁻¹ and generation times of 14.46-30.69 h. Faster rates were obtained at 15, 20 and 25°C, ranging from k values of 0.205-0.055 h⁻¹ and generation times of 3.38-12.48 h, with the optimum growth rate occurring at either 20°C or higher. Measurement of in situ microbial growth at 0°C in sediment columns by direct microscopy and protein-labelling fluorescent staining showed increases in microbial numbers of 10-fold when the sediment columns were amended with glucose, and 23-fold when amendment was with a more natural supplement, specifically, algal detritus. Determination of the number of viable microorganisms in the sediment columns again showed that considerable increases occurred at 0°C when glucose and detritus were added as substrates. Examination of the types of microbes occurring, during the growth conditions at 0°C when the substrates were added, by microscopy, and isolation of representative microbial types by replica plating showed that different populations responded to the two amendments.     

The effects of temperature on the performance of experimental percolating filters with and without mixed macro-invertebrate populations

The performance and biology of small percolating filters (capacity 16·7 dm³) treating a synthetic sewage at a BOD loading of 0.15 kg m⁻³ d with and without psychodid flies and enchytraeid worms and maintained at constant temperatures of 7, 10, and 13°C for 280 days were compared. The performance of the filters inoculated with flies and worms was substantially better than that of the controls. In the filters with animals nitrification increased with temperature, the temperature coefficient being much higher than that for the growth of nitrifying bacteria. There appeared to be no nitrification in the filters not inoculated with animals.In most cases the peaks in numbers of flies and worms occurred at intervals predicted by their thermal characteristics. Flies were not successful at 7°C. Psychoda severini was the dominant fly at 7 and 10°C but both P. severini and P. alternata appeared to develop equally well at 13°C. Enchytraeid worms were always more numerous than the fly larvae, the highest densities of worms occurring at the two lower temperatures.Protozoan and fungal populations were studied and the application of published expressions for the effect of temperature on the rate coefficient of BOD removal is discussed.     

Quantitative determination of the population biological activity of biological sludges used in the activated sludge waste water treatment process

The population biological activity of biological sludges used in the activated sludge waste water treatment process is defined as that minimum amount of biological sludge, still able to reduce in a 21 h contact period, the soluble COD of a waste water by one half the value equivalent to the BOD_5d. This A_mg^BOD50% 21 h value was determined by incubating hundred fold serial dilutions of a biological sludge with a previously sterilized waste water and by plotting the residual soluble COD remaining after a 21 h incubation period vs the sludge dilution. The method allows any given biological sludge to be readily assayed on any given waste water. Biological sludges were found stable for periods up to three months in the frozen state or when stored at 4°C. Lyophilization of biological sludges resulted in a severe loss of population biological activity. Biological sludges of trade waste water treatment plants were followed for periods up to 70 days. A correlation was found between the population biological activity and changes in environmental factors such as reduced N and P nutrient feeding, reduced aeration or changes in pH. Minor changes resulted in a decrease in population biological activity. Drastic changes resulted in a modified shape of the soluble COD vs biological sludge dilution curves, presumably reflecting deep modifications of the waste water-treating biological community.     

Experimental simulation of biodegradation in rivers: Oxygen, organic matter and biomass concentration changes

Dissolved oxygen (DO) and biochemical oxygen demand (BOD) concentration changes after an organic matter discharge into a river have been studied in the absence of oxygen transfer. According to these laboratory experiments, biodegradation of various organic compounds (glucose, glutamic acid, starch, ovalbumin and ethanol) in Seine river samples incubated at 15 30°C follow a biphasic behaviour. During a lag-phase of 10–20 h, DO decreases linearly (0.12 ppm h⁻¹ at 20°C), whereas BOD is constant. During a subsequent aerobic exponential phase, DO and BOD uptake are proportional and increase exponentially with time (0.13 h⁻¹ at 20°C). Using cell ATP as biomass indicator, the latter phase was shown to correspond to a cell division step. A kinetic model was developed for stimulating DO and BOD concentration changes after a waste water discharge at temperatures ranging between 15 and 30°C.     

Effects of temperature and pH on the growth of heterotrophic bacteria in waste stabilization ponds

This paper presents the effects of temperature and pH on the growth of heterotrophic bacteria in Chlorella vulgaris-heterotrophic bacteria culture. The growth of heterotrophic bacteria was studied at 10, 15, 20 and 30°C, and pH was controlled from 3.0 to 11.5 in a series of fed-batch chemostat reactors supplied with glucose as the sole source of carbon. Samples were analyzed for heterotrophic bacteria by tryptone glucose extract agar in triplicate. The agar pH of 7.0 was the best for enumeration of heterotrophic bacteria. The bacteria grown at pH near neutral were more sensitive to the variation of agar pH than those grown at very alkaline pH. No significant difference in the number of cells capable of forming colonies was noted for incubation temperature of 20 and 35°C, but the lag time for colony formation was longer at 20°C. Samples for enumeration of heterotrophic bacteria collected from algal-bacterial systems such as waste stabilization ponds are recommended to be incubated at 35°C for 72 h. Depending on the pH of the culture, about 86–98% of the cells capable of forming colonies will be visible to the naked eyes after incubation at 35°C for 72 h. At the steady state conditions, heterotrophic bacteria were not sensitive to temperature in the range of 10–20°C. However, at 30°C, a notable competition for glucose between Chlorella vulgaris and heterotrophic bacteria was observed. This competition was responsible for the low bacterial density near neutral pH. No evidence was found to support the view that the discharge of bactericidal substances from Chlorella vulgaris was responsible for reduction of heterotrophic bacteria at high pH.