Showing papers in "Applied and Environmental Microbiology in 1976"

New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressureized atmosphere.

Abstract: The sensitivity of the requirement of Methanobacterium ruminantium strain M1 to a new coenzyme, 2-mercaptoethanesulfonic acid (HS-CoM) was examined by use of new techniques that were developed for rapid and efficient handling of large numbers of cultures of methanogenic bacteria. The system uses sealed tubes that contain a gas mixture of 80% hydrogen and 20% carbon dioxide under a pressure of 2 to 3 atm. This modification of the Hungate technique reduces variability among replicate cultures and simplifies the dispensing, sterilization, and storage of liquid media as well as the transfer and maintenance of methanogenic bacteria. Results indicate a limit of sensitivity of the assay at 5 nM HS-CoM, with half-maximal growth at 25 nM HS-CoM. Coenzyme activity could be replaced by 2,2'-dithiodiethanesulfonic acid at a half-molar equivalent of the HS-CoM concentration, or by 2-(methylthio)ethanesulfonic acid on an equimolar basis. These data reveal a very sensitive and precise requirement for HS-CoM in the nutrition of this fastidious anaerobe.

Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress.

Abstract: Data are presented on the distribution of 101 bacterial species and subspecies among 1,442 isolates from 25 fecal specimens from three men on: (i) their normal diet and normal living conditions, (ii) normal living conditions but eating the controlled metabolic diet designed for use in the Skylab simulation and missions, and (iii) the Skylab diet in simulated Skylab (isolation) conditions. These bacteria represent the most numerous kinds in the fecal flora. Analyses of the kinds of bacteria from each astronaut during the 5-month period showed more variation in the composition of the flora among the individual astronauts than among the eight or nine samples from each person. This observation indicates that the variations in fecal flora reported previously, but based on the study of only one specimen from each person, more certainly reflect real differences (and not daily variation) in the types of bacteria maintained by individual people. The proportions of the predominant fecal species in the astronauts were similar to those reported earlier from a Japanese-Hawaiian population and were generally insensitive to changes from the normal North American diet to the Skylab diet; only two of the most common species were affected by changes in diet. However, one of the predominant species (Bacteroides fragilis subsp. thetaiotaomicron) appeared to be affected during confinement of the men in the Skylab test chamber. Evidence is presented suggesting that an anger stress situation may have been responsible for the increase of this species simultaneously in all of the subjects studied. Phenotypic characteristics of some of the less common isolates are given. The statistical analyses used in interpretation of the results are discussed.

Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds.

Abstract: A variety of nitroaromatic compounds, including 2,4,6-trinitrotoluene (TNT), were reduced by hydrogen in the presence of enzyme preparations from Veillonella alkalescens. Consistent with the proposed reduction pathway, R-NO2 H2 leads to R-NO H2 leads to R-NHOH H2 leads to R-NH2, 3 mol of H2 was utilized per mol of nitro group. The rates of reduction of 40 mono-, di-, and trinitroaromatic compounds by V. alkalescens extract were determined. The reactivity of the nitro groups depended on other substituents and on the position of the nitro groups relative to these substituents. In the case of the nitrotoluenes, the para-nitro group was the most readily reduced, the 4-nitro position of 2,4-dinitrotulene being reduced first. The pattern of reduction of TNT (disappearance of TNT and reduction products formed) depended on the type of preparation (cell-free extract, resting cells, or growing culture), on the species, and on the atmosphere (air or H2). The "nitro-reductase" activity of V. alkalescens extracts was associated with protein fractions, one having some ferredoxin-like properties and the other possessing hydrogenase activity. Efforts to eliminate hydrogenase from the reaction have thus far been unsuccessful. The question of whether ferredoxin acts as a nonspecific reductase for nitroaromatic compounds remains unresolved.

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus

Abstract: Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene In the presence of 001 atmospheres of acetylene, however, nitrous oxide was not reduced Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene Cells exposed to 001 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed

Temperature limitation of methanogenesis in aquatic sediments.

Abstract: Microbial methanogenesis was examined in sediments collected from Lake Mendota, Wisconsin, at water depths of 5, 10, and 18 m. The rate of sediment methanogenesis was shown to vary with respect to sediment site and depth, sampling date, in situ temperature, and number of methanogens. Increased numbers of methanogenic bacteria and rates of methanogenesis correlated with increased sediment temperature during seasonal change. The greatest methanogenic activity was observed for 18-m sediments throughout the sampling year. As compared with shallower sediments, 18-m sediment was removed from oxygenation effects and contained higher amounts of ammonia, carbonate, and methanogenic bacteria, and the population density of methanogens fluctuated less during seasonal change. Rates of methanogenesis in 18-m sediment cores decreased with increasing sediment depth. The optimum temperature, 35 to 42 C, for sediment methanogenesis was considerably higher than the maximum observed in situ temperature of 23 C. The conversion of H2 and [14C]carbonate to [14C]methane displayed the same temperature optimum when these substrates were added to sediments. The predominant methanogenic population had simple nutritional requirements and were metabolically active at 4 to 45 C. Hydrogen oxidizers were the major nutritional type of sediment methanogens; formate and methanol fermentors were present, but acetate fermentors were not observed. Methanobacterium species were most abundant in sediments although Methanosarcina, Methanococcus, and Methanospirillum species were observed in enrichment cultures. A chemolithotropic species of Methanosarcina and Methanobacterium was isolated in pure culture that displayed temperature optima above 30 C and had simple nutritional requirements.

Toxicity and mutagenicity of 2,4,-6-trinitrotoluene and its microbial metabolites.

Abstract: TNT (2,4,6-trinitrotoluene) of explosive grade is highly toxic to marine forms that included fresh water unicellular green algae (Selenastrum capricornutum), tidepool copepods (Tigriopus californicus), and oyster larvae (Crassostrea gigas), and mutagenic to Salmonella typhimurium. On the basis of mutagenic assays carried out with a set of histidine-requiring strains of the bacterium, TNT was detected as a frameshift mutagen that significantly accelerates the reversion rate of a frameshift tester, TA-98. In contrast, the major microbial metabolites of TNT appeared to be nontoxic and nonmutagenic. Images

Effect of sediments on the survival of Escherichia coli in marine waters.

Abstract: Escherichia coli, a fecal coliform, was found to survive for longer periods of time in unsterile natural seawater when sediment material was present than in seawater alone, and at least on one occasion growth was observed to occur This enteric bacterium was found to increase rapidly in number in autoclaved natural seawater and autoclaved sediment taken from areas receiving domestic wastes, even when the seawater had salinities as high as 34 g/kg However, in autoclaved seawater, growth was always more gradual and never reached numbers as high as those observed when sediment was present It was found that nutrients were easily eluted from the sediment after autoclaving or upon addition to artificial seawater, but little elution occured during mixing of the sediments with unsterile natural seawater The longer survival of E coli in the sediment is attributed to the greater content of organic matter present in the sediment than the sweater These laboratory results, in part, could explain why on a volume basis larger numbers of coliforms and fecal coliforms and fecal coliforms were found in estuarine sediments than the overlaying water at field sites

Organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water.

Abstract: A method is described for second-step concentration of viruses from water. This method, combined with an adsorption-elution method, yields a mean recovery of about 75%

Toxicity of ammonia to algae in sewage oxidation ponds.

Abstract: Ammonia, at concentrations over 2.0 mM and at pH values over 8.0, inhibits photosynthesis and growth of Scenedesmus obliquus, a dominant species in high-rate sewage oxidation ponds. Photosynthesis of Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum is also susceptible to ammonia inhibition. Dark respiration and cell morphology were unaffected by any combination of pH and ammonia concentrations tested, thus limiting the apparent effect to inhibition of the normal function of the chloroplasts. Methylamine had the same effect as ammonia, and its penetration into the cells was found to be pH dependent. Therefore, the dependence of toxicity of amines to algae on pH apparently results from the inability to penetrate the cell membrane in the ionized form. When operated at 120-h detention time of raw wastewater, the high-rate oxidation pond maintained a steady state with respect to algal growth and oxygen concentration, and the concentration of ammonia did not exceed 1.0 mM. Shifting the pond to 48-h detention time caused an increase in ammonia concentration in the pond water to 2.5 mM, and the pond gradually turned anaerobic. Photosynthesis, which usually elevates the pH of the pond water to 9.0 to 10.0, could not proceed beyond pH 7.9 because of the high concentration of ammonia, and the algal population was washed out and reduced to a concentration that could maintain a doubling time of 48 h without photosynthesis bringing the pH to inhibitory levels. Under these conditions, the pH of the bond becomes a factor that limits the operational efficiency of the oxidation pond.

Requirement for a growth substrate during lignin decomposition by two wood-rotting fungi.

Abstract: Decomposition of 14C-labeled lignin to 14CO2 by the lignin-decomposing fungi Phanerochaete chrysosporium and Coriolus versicolor required a growth substrate such as cellulose or glucose. Growth with lignin as sole carbon addition to an otherwise complete medium was negligible.

Morphological characterization of small cells resulting from nutrient starvation of a psychrophilic marine vibrio.

Abstract: Upon starvation, Ant-300, a psychrophilic marine vibrio, was observed to decrease in size and change in shape from a rod to a coccus. After 3 weeks of starvation 50% of the starved population was able to pass through a filter with a pore size of 0.4 mum. Electron microscopy of thin sections of the small cells revealed normal cell structure except for an enlarged periplasmic space. When inoculated into a fresh medium, starved cells growth without a significant lag and regained "normal" size and shape within 48 h.

Ferric iron reduction by sulfur- and iron-oxidizing bacteria.

Abstract: Acidophilic bacteria of the genera Thiobacillus and Sulfolobus are able to reduce ferric iron when growing on elemental sulfur as an energy source. It has been previously thought that ferric iron serves as a nonbiological oxidant in the formation of acid mine drainage and in the leaching of ores, but these results suggest that bacterial catalysis may play a significant role in the reactivity of ferric iron.

Bacteria within ovules and seeds.

Abstract: Surface-sterilized ovules and seeds of 27 species of plants were cultured in the water of syneresis of a nutrient medium low in agar content. Bacteria were obtained from 30% of the ovules, 15% of the seeds of herbaceous plants, 16% of the seeds of woody plants, 5.4% of the overwintered noncereal seeds, and 13.5% of overwintered cereal seeds. In no instance did every ovule or seed of a plant species contain bacteria. No bacteria were obtained from the hard, waxy seeds of mimosa or yellowwood. They were not obtained from ovules with unbroken coats or from seeds with coats that were not ruptured during the swelling of the seed. Only one species of bacteria was recovered in 93% of the instances in which bacteria were obtained. Bacteria were obtained from seeds that were embedded in the acidic parenchyma of the lemon or surrounded by the thickened flesh of the cucurbits. The bacteria were distributed among 19 genera and 46 species. The species isolated in greatest numbers were Bacillus megaterium, B. cereus, Erwinia herbicola, Flavobacterium devorans, and Pseudomonas fluorescens. Bacteria recovered less frequently were in the genera Achromobacter, Acinetobacter, Alcaligenes, Brevibacterium, Corynebacterium, Cytophaga, Leuconostoc, Micrococcus, Nocardia, Proteus, Streptococcus, Streptomyces, and Xanthomonas. Members of 11 genera and 15 species of bacteria were isolated once.

Natural occurrence of Fusarium toxins in feedstuff.

Abstract: The mycotoxins diacetoxyscirpenol, deoxynivalenol, and zearalenone, produced by Fusarium roseum, were found naturally occuring in mixed feed samples. In all cases analyzed, deoxynivalenol occurred together with zearalenone. The natural occurrence of zearalenone in sesame seed is reported for the first time. Strains of F. roseum isolated in various parts of the world form feed implicated in animal mycotoxicosis produced monoacetoxyscirpenol, diacetoxyscirpenol, deoxynivalenol, and zearalenone.

Influence of the rate of ethanol production and accumulation on the viability of Saccharomyces cerevisiae in "rapid fermentation".

Abstract: Whereas "rapid fermentation" of diluted clover honey (25 degrees Brix) fortified with yeast nutrients using 8 X 10(8) brewers' yeast cells per ml resulted in an ethanol content of 9.5% (wt/vol; 12% vol/vol) in 3 h at 30 C, death rate of the yeast cells during this period was essentially logarithmic. Whereas 6 h was required to reach the same ethanol content at 15 C, the yeast cells retained their viability. Using a lower cell population (6 X 10(7) cells/ml), a level at which the fermentation was no longer "rapid," the yeast cells also retained their viability at 30 C. Ethanol added to the medium was much less lethal than the same or less quantities of ethanol produced by the cell in "rapid fermentation." It was considered possible that ethanol was produced so rapidly at 30 C that it could not diffuse out of the cell as rapidly as it was formed. The hypothesis was postulated that ethanol accumulating in the cell was contributing to the high death rate at 30 C. It was found that the intracellular ethanol concentration reached a level of approximately 2 X 10(11) ethanol molecules/cell in the first 30 min of fermentation at 30 C. At 15 C, with the same cell count, intracellular ethanol concentration reached a level of approximately 4 X 10(10) ethanol molecules/cell and viability remained high. Also, at 30 C with a lower cell population (6 X 10(7) cells/ml), under which conditions fermentation was no longer "rapid," intracellular ethanol concentration reached a similar level (4 X 10(10) molecules ethanol/cell) and the cells retained their viability. Alcohol dehydrogenase (ADH) lost its activity in brewers' yeast under conditions of "rapid fermentation" at 30 C but retained its activity in cells under similar conditions at 15 C. ADH activity was also retained in fermentations at 30 C with cell populations of 6 X 10(7)/ml. It would appear that an intracellular level of about 5 X 10(10) ethanol molecules/cell is normal and that this level does not damage either cell viability or ADH activity. Higher intracellular ethanol concentrations, such as 2 X 10(11) molecules ethanol/cell (a fourfold increase in intracellular ethanol concentration), are accompanied by inactivation of ADH and loss of cell viability.

Oil degradation in soil.

Abstract: The environmental effects of adding certain selected petroleum products to field soils at widely separated geographical locations under optimum conditions for biodegradation were studied. The locations selected for study of soil biodegradation of six oils (used crankcase oil from cars, used crankcase oil from trucks, an Arabian Heavy crude oil, a Coastal Mix crude oil, a home heating oil no. 2, and a residual fuel oil no. 6) were Marcus Hook, Pennsylvania, Tulsa, Oklahoma, and Corpus Christi, Texas. The investigative process, covering a period of 1 year at each location, was conducted in 14 fields plots (1.7 by 3.0 m) to which the oils were added in a single application at a rate of 11.9 m3/4 X 10(3) m2. One-half of the plots at each location were fertilized, and the incorporation of the oils and fertilizers was accomplished with rototillers to a depth of 10 to 15 cm. Concentrations of all oils decreased significantly at all locations. The average reduction ranged from 48.5 to 90.0% depending upon the type of oil and location. Rates of degradation did not exceed 2.4 m3/4 X 10(3) m2 per month. Compositional changes in the oil with time were investigated using silica gel fractionation, gas chromatography, and ultraviolet absorbance. With the possible exception of the two fuel oils, the compositional changes were generally in the same direction for all of the oils. The silica gel fractionation and gravimetric data on residual oils show that all classes of compounds were degraded, but the more polar type degrade more slowly. Analysis of runoff water, leachate, and soils indicated that at the concentration applied no oil less was observed from these plots via water movement. No significant movement of lead compounds added to the soils in the used crankcase oils was observed. Significant increases in hydrocarbon-utilizing microorganisms were demonstrated in all treated plots using either the pure hydrocarbon, n-hexadecane, or the applied oils as the growth substrate. These increases were usually sustained throughout the year. Significant increases in hydrocarbon-utilizing fungi were not demonstrated by the plating technique used. The concentrations of residual oils or their oxidation products were of sufficient magnitude in the treated plots, 9 months after application, to cause significant inhibition of plant growth. From the data obtained, it was not possible to determine the type of compounds causing this inhibition or their long-term environmental effects.

Enzymatic hydrolysis of organophosphate insecticides, a possible pesticide disposal method.

Abstract: A crude cell extract from a mixed bacterial culture growing on parathion, an organophosphate insecticide, hydrolyzed parathion (21 C) at a rate of 416 nmol/min per mg of protein. This rate of enzymatic hydrolysis, when compared with chemical hydrolysis by 0.1 N sodium hydroxide at 40 C, was 2, 450 times faster. Eight of 12 commonly used organophosphate insecticides were enzymatically hydrolyzed with this enzyme preparation at rates ranging from 12 to 1,360 nmol/min per mg of protein. Seven pesticides were hydrolyzed at rates significantly higher (40 to 1,005 times faster) than chemical hydrolysis. The pH optimum for enzymatic hydrolysis of the eight pesticides ranged from 8.5 to 9.5, with less than 50% of maximal activity expressed at pH 7.0. Maximal enzyme activity occurred at 35 C. The crude extract lost its activity at the rate of only 0.75%/day when stored at 6 C. Eight organic solvents, ranging from methanol to hexane, at low concentrations stimulated enzymatic hydrolysis by 3 to 20%, whereas at higher concentrations (1,000 mg/liter) they inhibited the reaction (9 to 50%). Parathion metabolites p-nitrophenol, hydroquinone, and diethylthiophosphoric acid, at up to 100-mg/liter concentrations, did not significantly influence enzyme activity.

Beta-glucosidase of Trichoderma: its biosynthesis and role in saccharification of cellulose.

Abstract: The extracellular beta-glucosidase of Trichoderma viride generally is present in low levels when the organism is cultured on cellulose because it is inactivated under the acid conditions which develop in the medium while the other enzymes of the cellulase complex are more stable. With the appropriate pH control, inactivation of beta-glucosidase is prevented and the activity of this enzyme increases during growth. In the saccharification of crystalline cellulose, or of cellulose at low concentrations, much of the glucose produced is the result of the cleavage of cellobiose by beta-glucosidase. However when high concentrations (10%) of pretreated cellulose are saccharified, significant quantities of glucose are produced by action of enzymes other than beta-glucosidase.

Oxidation of arsenite to arsenate by Alcaligenes faecalis.

Abstract: Alcaligenes faecalis, resistant to the toxic effects of 0.01 M sodium arsenite, was isolated from raw sewage and shown to be capable of oxidizing arsenite to arsenate. When the organisms were grown in chemically defined medium, this conversion was due to the appearance at stationary phase of an intracellular, oxygen-sensitive, inducible enzyme and/or component of the electron transport system; when the organisms were grown in a nutrient broth-yeast extract medium, the enzyme appeared in the late exponential phase of growth. The presence of 0.02 M arsenite in the culture medium affected neither growth rate nor final cell yield.

Microbial Degradation of Lignocellulose: the Lignin Component

Abstract: A new procedure was developed for the study of lignin biodegradation by pure or mixed cultures of microorganisms. Natural lignocelluloses were prepared containing C in primarily their lignin components by feeding plants l-[U-C]phenylalanine through their cut stems. Lignin degradation was observed in numerous soils by monitoring evolution of CO(2) from [C]lignin-labeled oak (Quercus albus), maple (Acer rubrum), and cattail (Typha latifola). An organism (Thermonospora fusca ATCC 27730) that is known to degrade cellulose but not lignin was shown to grow on lignocellulose in the presence of [C]lignocelluloses without evolution of CO(2). A known lignin degrader (a white-rot fungus, Polyporus versicolor) was shown to readily evolve CO(2) from damp C-labeled cattail and C-labeled maple.

Adsorption of bacteria to roots as related to host specificity in the Rhizobium-clover symbiosis.

Abstract: Quantitative microscope techniques were utilized to examine the adsorption of rhizobial cells to clover root hairs. Adsorption of cells of noninfective strains of Rhizobium trifolii or infective R. meliloti strains to clover root hairs was four to five times less than that of the infective R. trifolii strains. Attachment of the rod-shaped bacteria to clover root cells occurred in a polar, end-on fashion. Viable or heat-killed R. trifolii cells precoated with a clover lectin having 2-deoxyglucose specificity had increased adsorption to clover roots. Adsorption of bacteria to roots was not increased if the clover lectin was inactivated by heat or 2-deoxyglucose treatment prior to incubation with R. trifolii. Adsorption of R. trifolii to clover root hairs was inhibited by 2-deoxyglucose (30 mM) but not by 2-deoxygalactose or alpha-D-glucose. Adsorption of R. meliloti cells to alfalfa root hairs was not affected by 2-deoxyglucose at that concentration. These results suggest that expression of host specificity in the Rhizobium-clover symbiosis involves a preferential adsorption of infective cells to clover root hairs through a 2-deoxyglucose-sensitive receptor site.

Teratogenicity of patulin and patulin adducts formed with cysteine.

Abstract: The mean lethal dose of patulin for the chicken embryo injected in the air cell before incubation was determined to be 68.7 mug and that for the 4-day-old embryo was 2.35 mug. Both patulin (1 to 2 mug/egg) and the reaction mixture between patulin and cysteine (15 to 150 mug of patulin equivalents) were teratogenic to the chicken embryo. At least two ninhydrin-negative and four ninhydrin-positive products were formed during the latter reaction. Our explanation of the reaction mechanism remains to be elaborated.

Catalase: its effect on microbial enumeration.

Abstract: The addition of catalase to the surface of selective medium plates permitted increased enumeration of physically or chemically injured (stressed) microorganisms. Catalase acted by preventing the accumulation of hydrogen peroxide in, or around, injured cells. Heat-injured Staphylococcus aureus cells had decreased catalase activity, and heat-inactivated catalase had no effect on enumeration.

Co-identity of the refusal and emetic principle from Fusarium-infected corn.

Abstract: The structure of vomitoxin isolated from Fusarium-contaminated corn was proved to be 3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one. This same toxin is responsible for the "refusal phenomenon" exhibited by swine fed contaminated corn. In addition, two new substances believed to be trichothecenes were isolated from naturally infected corn. Vomitoxin was also isolated from rice inoculated with F. graminearium NRRL 5883.

Inhibitory effects of H2 on growth of Clostridium cellobioparum.

Abstract: Hydrogen inhibits the growth of hydrogen-producing Clostridium cellobioparum, but not of Escherichia coli or Bacteroides ruminicola. The inhibition is reversible. When hydrogen was removed either by palladium black or by gassing out the tube, glucose utilization increased as did optical density and hydrogen production of C. cellobioparum. Removal of the H2 by methanogenic bacteria favors the growth of C. cellobioparum. Grown with Methanobacterium ruminantium in various concentrations of glucose, the Clostridium reaches a higher optical density and produces more H2 and a higher viable cell count. The cell yield is also higher than in pure culture. In mixed culture, C. cellobioparum produces more acetic acid and less lactic acid, ethanol, and butyric acid than in pure culture. The significance of this metabolic shift and hydrogen utilization in methanogenesis is discussed.

Inhibition of methanogenesis in salt marsh sediments and whole-cell suspensions of methanogenic bacteria by nitrogen oxides.

Abstract: Hydrogen-dependent evolution of methane from salt marsh sediments and whole-cell suspensions of Methanobacterium thermoautotrophicum and Methanobacterium fornicicum ceased or decreased after the introduction of nitrate, nitrite, nitric oxide, or nitrous oxide. Sulfite had a similar effect on methanogenesis in the whole-cell suspensions. In salt marsh sediments, nitrous oxide was the strongest inhibitor, followed by nitric oxide, nitrite, and nitrate in decreasing order of inhibition. In whole-cell suspensions, nitric oxide was the strongest inhibitor, followed by nitrous oxide, nitrite, and nitrate. Consideration of the results from experiments using an indicator of oxidation potential, along with the reversed order of effectiveness of the nitrogen oxides in relation to their degree of reduction ,suggests that the inhibitory effect observed was not due to a redox change. Evidence is also presented that suggests that the decrease in the rate of methane production in the presence of oxides of nitrogen was not attributable to competition for methane-producing substrates.

Role of bacteria and protozoa in the removal of Escherichia coli from estuarine waters.

Abstract: The removal of Escherichia coli from estuarine water was investigated The survival of E coli was dependent on the presence of protozoan predators and not on the presence of lytic bacteria When indigenous protozoa were removed from estuarine water by filtration, the destruction of coliform populations was negligible In studies designed to prevent the growth of indigenous bacterial populations without affecting protozoan populations, coliform destruction was significant

Differences in destruction of cysts of pathogenic and nonpathogenic Naegleria and Acanthamoeba by chlorine.

Abstract: The destructive action of chlorine on the pathogenic Naegleria fowleri and Acanthamoeba culbertsoni, the nonpathogenic N. gruberi, and an avirulent Acanthamoeba isolate was investigated. N fowleri is somewhat more sensitive to chlorine than N. gruberi, whereas the two Acanthamoeba strains are very resistant. This study yields information needed for the destruction of amoebic cysts in drinking water and swimming pools. It also gives some explanation for the occurence of Acanthamoeba strains in these waters.

Demonstration of solids-associated virus in wastewater and sludge.

Abstract: Data presented demonstrate the relatively high multiplicity of solids-associated virus in field samples, i.e., wastewater, sludge, and soils. Influent, effluent, and chlorinated effluent samples showed 16.1 to 100% of the total virus demonstrated in samples to be solids associated. Three techniques for freeing solids-associated virus are described and compared. Using sonication of solids and polyethylene glycol concentration, virus was demonstrated in fully digested sludge (60 days at 34 C), sand at the site of a sewer leak, and dried sludge cake and mud 900 m downstream from a sewage disposal site. These data emphasize the inadequacy of virus concentration techniques that do not include the processing of solids. In situ elution failed to free solids-associated virus.

Concentration of viruses from large volumes of tap water using pleated membrane filters.

Abstract: A method is described for the efficient concentration of viruses from large volumes of tap water in relatively short time periods Virus in acidified tap water in the presence of aluminum chloride is adsorbed to a 10-inch (ca 254 cm) fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in series at flow rates of up to 378 liters/min (10 gallons/min) This filter series is capable of efficiently adsorbing virus from greater than 19,000 liters (5,000 gallons) of treated tap water Adsorbed viruses are eluted from the filters with glycine buffer (pH 105) and the eluate is reconcentrated using an aluminum flocculation process Viruses are eluted from the aluminum floc with glycine buffer (pH 115) Using this procedure, viruses in 1,900 liters (500 gallons) of tap water can be concentrated 100,000-fold in 3 h with an average recovery of 40 to 50%