Growth of Aeromonas hydrophila at Low Concentrations of Substrates Added to Tap Water

The ability of an Aeromonas hydrophila isolate obtained from filtered river water to grow at low substrate concentrations was studied in batch experiments with tap water supplied with low concentrations of substrates. Growth was assessed by colony count determinations. The isolate only multiplied in the used tap water (2 to 3 mg of dissolved organic carbon per liter) after the addition of a small amount of an assimilable carbon compound. d-Glucose especially caused growth of the organism even at initial concentrations below 10 mug of C per liter. At initial glucose concentrations below the K(s) value (12 mug of C per liter), generation times and yield (colony-forming units per milligram of substrate-C) were nonlinear with 1/initial glucose concentrations and initial glucose concentrations, respectively. From these observations, the maintenance coefficient m was calculated (m = 0.015 mg of glucose per mg [dry wt] per h at 12 degrees C). At initial concentrations below the K(s) value of starch (73 mug of C per liter), no growth was observed, but complete use of starch occurred in these situations after the addition of 10 mug of glucose-C per liter. The results of this study show that information of ecological significance may be obtained by very simple batch experiments. Moreover, the isolate studied may be used in growth experiments to assess the maximum concentration of glucose which might be present in water, particularly tap water.     

Determination of the concentration of maltose- and starch-like compounds in drinking water by growth measurements with a well-defined strain of a Flavobacterium species

The growth kinetics of Flavobacterium sp. strain S12 specialized in the utilization of glycerol, and a number of oligo- and polysaccharides were determined in batch-culture experiments at 15 degrees C in pasteurized tap water supplied with very low amounts of substrates. Kss for the growth on maltotriose, maltotetraose, maltopentaose, and maltohexaose were 0.03 microM or less and below those for glucose (1.5 microM) and maltose (0.16 microM). Kss for starch, amylose, and amylopectin were 8.4, 25.6, and 11.0 micrograms of C per liter, respectively. A yield of 2.3 X 10(7) CFU/micrograms of C on the oligo- and polysaccharides was calculated from the linear relationships observed between maximum colony counts in pasteurized tap water and the concentrations (usually below 25 micrograms of C per liter) of supplied compounds. The maximum colony counts of strain S12 grown in various types of raw water and tap water revealed that raw water contained only a few micrograms of maltose- and starch-like compounds per liter; in tap water the concentrations were all below 1 microgram of C and usually below 0.1 microgram of C per liter. The application of starch-based coagulant aids gave increased concentrations of maltose- and starch-like compounds in the water during treatment, but these concentrations were greatly reduced by coagulation and sedimentation, rapid sand filtration, and slow sand filtration.     

Determination of the concentration of maltose- and starch-like compounds in drinking water by growth measurements with a well-defined strain of a Flavobacterium species.

The growth kinetics of Flavobacterium sp. strain S12 specialized in the utilization of glycerol, and a number of oligo- and polysaccharides were determined in batch-culture experiments at 15 degrees C in pasteurized tap water supplied with very low amounts of substrates. Kss for the growth on maltotriose, maltotetraose, maltopentaose, and maltohexaose were 0.03 microM or less and below those for glucose (1.5 microM) and maltose (0.16 microM). Kss for starch, amylose, and amylopectin were 8.4, 25.6, and 11.0 micrograms of C per liter, respectively. A yield of 2.3 X 10(7) CFU/micrograms of C on the oligo- and polysaccharides was calculated from the linear relationships observed between maximum colony counts in pasteurized tap water and the concentrations (usually below 25 micrograms of C per liter) of supplied compounds. The maximum colony counts of strain S12 grown in various types of raw water and tap water revealed that raw water contained only a few micrograms of maltose- and starch-like compounds per liter; in tap water the concentrations were all below 1 microgram of C and usually below 0.1 microgram of C per liter. The application of starch-based coagulant aids gave increased concentrations of maltose- and starch-like compounds in the water during treatment, but these concentrations were greatly reduced by coagulation and sedimentation, rapid sand filtration, and slow sand filtration.     

Substrate Utilization by an Oxalate-Consuming Spirillum Species in Relation to Its Growth in Ozonated Water

The nutritional versatility of a vibrio-shaped, oxalate-utilizing isolate, strain NOX, obtained from tap water supplied with low concentrations of formate, glyoxylate, and oxalate, was determined by growth experiments with low-molecular-weight carbon compounds at high (grams per liter) and very low (micrograms per liter) concentrations. The organism, which was identified as a Spirillum species, appeared to be specialized in the utilization of a number of carboxylic acids. Yields of 2.9 × 10⁶ CFU/μg of oxalate C and 1.2 × 10⁷ CFU/μg of acetate C were obtained from growth experiments in tap water supplied with various low amounts of either oxalate or acetate. A substrate saturation constant of 0.64 μM oxalate was calculated for strain NOX from the relationship between growth rate and concentration of added oxalate. Maximum colony counts of strain NOX grown in ozonated water (dosages of 2.0 to 3.2 mg of O₃ per liter) were 15 to 20 times larger than the maximum colony counts of strain NOX grown in water before ozonation. Based on the nutritional requirements of strain NOX, it was concluded that carboxylic acids were produced by ozonation. Oxalate concentrations were calculated from the maximum colony counts of strain NOX grown in samples of ozonated water in which a non-oxalate-utilizing strain of Pseudomonas fluorescens had already reached maximum growth. The oxalate concentrations obtained by this procedure ranged from 130 to 220 μg of C/liter.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water.

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Multiplication of fluorescent pseudomonads at low substrate concentrations in tap water

Two fluorescent pseudomonads, strains P17 and P500, belonging to different biotypes were tested for growth in tap water supplied with different concentrations of acetate and glutamate, low concentrations (10 and 20 g of C per liter) of various other substrates and mixtures of related substrates, the latter being present in amounts of 1 g of C per liter each. Amino acids appeared to be excellent substrates for both isolates, but many other substrates were utilized at very low concentrations as well. Saturation constants (K_s) of P17 with acetate, arginine, aspartate, glutamate, lactate, succinate, malonate, p-hydroxybenzoate and glucose were all below 1 m. The K_s values of strain P500 were about 5 times larger than those of P17. Since especially P17 is able to use a large number of different substrates at low concentrations, assessment of maximal colony counts of this organism by growth experiments in various types of tap water may give information about the concentrations of easily assimilable organic carbon.     

Nutritional versatility of a starch-utilizing Flavobacterium at low substrate concentrations.

A starch-utilizing yellow-pigmented bacterium, isolated from tap water, was tested for the utilization of 64 natural compounds at a concentration of 1 g/liter by measuring colony growth on agar media. Only 12 carbohydrates and glycerol promoted growth. Growth experiments with the organism in pasteurized tap water supplied with mixtures of substrates at concentrations of 1 or 10 micrograms of C of each substrate per liter, followed by separate experiments with a number of carbohydrates at 10 micrograms of C per liter showed that of these 64 natural compounds only sucrose, maltose, raffinose, starch, and glycerol promoted growth at very low concentrations. Also maltotriose, -tetraose, -pentaose, -hexaose, and stachyose, which were not included in the mixtures, enhanced growth, and generation times of 3 to 5 h at 10 micrograms of C per liter were observed. The organism, which was tentatively identified as a Flavobacterium species, thus appeared to be highly specialized in the utilization of glycerol and a number of oligo- and polysaccharides at very low concentrations.     

Utilization of oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater by Flavobacterium johnsoniae

Aims: To obtain a bacterial strain that can be used to quantify biodegradable polysaccharides at concentrations of a few micrograms per litre in freshwater. Methods and Results: Flavobacterium johnsoniae strain A3 was isolated from tap water supplemented with laminarin, pectin or amylopectin at 100 μg C l^?1 and river Rhine water. The organism utilized 14 of 23 oligo‐ and polysaccharides, and 1 of 9 monosaccharides, but none of the sugar acids, sugar alcohols, carboxylic acids or aromatic acids tested at 10 μg C l^?1. Amino acids promoted growth of strain A3, but not in coculture with assimilable organic carbon (AOC) test strain Pseudomonas fluorescens P17, which utilized these compounds more rapidly than strain A3. Compounds released by strain P17 and AOC test strain Spirillum sp. NOX grown on acetate promoted the growth of strain A3 at N_max values of ≥ 2 × 10⁵ CFU ml^?1 of strain P17 and ≥ 5 × 10⁵ CFU ml^?1 of strain NOX. Significant growth of strain A3 was observed in surface water and in tap water in the presence of strain P17 (N_max P17 < 2 × 10⁵ CFU ml^?1). Conclusions: Strain A3 utilizes oligo‐ and polysaccharides at microgram‐per‐litre levels. In surface water and in tap water, the organism was able to utilize compounds that were not utilized by strain P17. These compounds may include oligo‐ and/or polysaccharides. Significance and Impact of the Study: Phytoplanktonic and bacterial polysaccharides can constitute an important biodegradable fraction of natural organic matter in water and may promote growth of heterotrophic bacteria during water treatment and drinking water distribution. Strain A3 can be used to quantify a group of compounds that includes oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater.     

Isolation and identification of a morpholine-degrading bacterium

A gram-positive, slowly growing rod effectively utilizing morpholine as the sole source of organic carbon, nitrogen, and energy was isolated from a mixed culture in a laboratory reactor. The strain was tentatively identified as Mycobacterium aurum. Its growth characteristics at 20 degrees C and pH 6.5 were as follows: maximum specific growth rate, 0.052 h-1; half-velocity constant, 1.3 mg/liter; and yield, 0.37 g/g. The optimum temperature and pH were 31 degrees C and 6.0, respectively.     

Isolation and identification of a morpholine-degrading bacterium.

A gram-positive, slowly growing rod effectively utilizing morpholine as the sole source of organic carbon, nitrogen, and energy was isolated from a mixed culture in a laboratory reactor. The strain was tentatively identified as Mycobacterium aurum. Its growth characteristics at 20 degrees C and pH 6.5 were as follows: maximum specific growth rate, 0.052 h-1; half-velocity constant, 1.3 mg/liter; and yield, 0.37 g/g. The optimum temperature and pH were 31 degrees C and 6.0, respectively.     

Bacterial nutrients in drinking water

Regrowth of coliform bacteria in distribution systems has been a problem for a number of water utilities. Efforts to solve the regrowth problem have not been totally successful. The current project, which was conducted at the New Jersey American Water Co.-Swimming River Treatment Plant, showed that the occurrence of coliform bacteria in the distribution system could be associated with rainfall, water temperatures greater than 15 degrees C, total organic carbon levels greater than 2.4 mg/liter, and assimilable organic carbon levels greater than 50 micrograms of acetate carbon equivalents per liter. A multiple linear regression model based on free chlorine residuals present in dead-end sections of the distribution system and temperature predicted 83.8% of the heterotrophic plate count bacterial variation. To limit the growth of coliform bacteria in drinking water, the study concludes that assimilable organic carbon levels should be reduced to less than 50 micrograms/liter.     

Bacterial nutrients in drinking water.

Regrowth of coliform bacteria in distribution systems has been a problem for a number of water utilities. Efforts to solve the regrowth problem have not been totally successful. The current project, which was conducted at the New Jersey American Water Co.-Swimming River Treatment Plant, showed that the occurrence of coliform bacteria in the distribution system could be associated with rainfall, water temperatures greater than 15 degrees C, total organic carbon levels greater than 2.4 mg/liter, and assimilable organic carbon levels greater than 50 micrograms of acetate carbon equivalents per liter. A multiple linear regression model based on free chlorine residuals present in dead-end sections of the distribution system and temperature predicted 83.8% of the heterotrophic plate count bacterial variation. To limit the growth of coliform bacteria in drinking water, the study concludes that assimilable organic carbon levels should be reduced to less than 50 micrograms/liter.     

Growth of Bacteria in Inorganic Medium at Different Levels of Airborne Organic Substances

Invasion rates of airborne organic substances into sterile mineral medium were compared by using flasks closed with cotton stoppers, silicone stoppers, and screw caps with Teflon gaskets. The resulting increases of dissolved organic carbon were 0.5, 0.2, and 0 mg/liter per week, respectively. The compounds supported the growth of lake water bacteria and a strain of Pseudomonas fluorescens. Growth rates were correlated to the permeability of the stoppers used. The measured input of organic carbon in the sterile mineral medium is considered to be a minimum value for the actual contribution of organic compounds by the air. Multiplication rates of the bacteria suggest that the organisms prevent the escape of volatile organic substances from the medium by rapid utilization. The steady nutrient supply through the air should be considered in growth experiments with bacteria at low concentrations of nutrients.     

Comparison between the evaluation of bacterial regrowth capability in a turbidimeter and biodegradable dissolved organic carbon bioreactor measurements in water

In recent years, two different approaches to the study of biodegradable organic matter in distribution systems have been followed. The assimilable organic carbon (AOC) indicates the portion of the dissolved organic matter used by bacteria and converted to biomass, which is directly measured as total bacteria, active bacteria or colony-forming units and indirectly as ATP or increase in turbidity. In contrast, the biodegradable dissolved organic carbon (BDOC) is the portion of the dissolved organic carbon that can be mineralized by heterotrophic micro-organisms, and it is measured as the difference between the inflow and the outflow of a bioreactor. In this study, at different steps in a water treatment plant, the bacterial regrowth capability was determined by the AOC method that measures the maximum growth rate by using a computerized Monitek turbidimeter. The BDOC was determined using a plug flow bioreactor. Measurements of colony-forming units and total organic carbon (TOC) evolution in a turbidimeter and of colony-forming units at the inflow/outflow of the bioreactor were also performed, calculating at all sampling points the coefficient yield ( Y = cfu/ΔTOC) in both systems. The correlations between the results from the bioreactor and turbidimeter have been calculated ; a high correlation level was observed between BDOC values and all the other parameters, except for Y calculated from bacterial suspension measured in the turbidimeter.     

Fungal growth on C1 compounds: quantitative aspects of growth of a methanol-utilizing strain of Trichoderma lignorum in batch culture.

A study was made of some salient parameters that influence growth of the methanol-utilizing fungus Trichoderma lignorum growing in batch culture on a minimal medium containing methanol as the sole source of carbon. Maximum cell yield was recorded at the expense of 1.58 g of methanol per liter. Inhibition was observed with methanol concentrations in excess of 4.7 g/liter. The optimum temperature for fungal growth was 23 degrees C. Growth of the fungus was directly proportional to an inorganic nitrogen concentration up to 0.2 g of NH4NO3 per liter. No inhibition of growth occurred at any concentration of NH4NO3 up to 11 g/liter. The pH of the growth medium decreased from 7.0 to 3.5 during growth of the fungus on methanol, which may have been due, in part, to the accumulation of trace amounts of organic acids in the growth medium. An analysis of the commercial potential of the fungus, as a source of edible protein, indicated that the strain of methanol-utilizing T. lignorum used was uneconomical in terms of the yield and the specific growth rate.     

Fungal growth on C1 compounds: quantitative aspects of growth of a methanol-utilizing strain of Trichoderma lignorum in batch culture.

A study was made of some salient parameters that influence growth of the methanol-utilizing fungus Trichoderma lignorum growing in batch culture on a minimal medium containing methanol as the sole source of carbon. Maximum cell yield was recorded at the expense of 1.58 g of methanol per liter. Inhibition was observed with methanol concentrations in excess of 4.7 g/liter. The optimum temperature for fungal growth was 23 degrees C. Growth of the fungus was directly proportional to an inorganic nitrogen concentration up to 0.2 g of NH4NO3 per liter. No inhibition of growth occurred at any concentration of NH4NO3 up to 11 g/liter. The pH of the growth medium decreased from 7.0 to 3.5 during growth of the fungus on methanol, which may have been due, in part, to the accumulation of trace amounts of organic acids in the growth medium. An analysis of the commercial potential of the fungus, as a source of edible protein, indicated that the strain of methanol-utilizing T. lignorum used was uneconomical in terms of the yield and the specific growth rate.     

Microbiological Assay for Organic Compounds in Seawater

A method for the quantitative identification of organic compounds in seawater has been developed. When auxotrophic mutants of Serratia marinorubra were incubated at 21 to 24 C for 72 hr with constant agitation, standard bioassay reference curves were obtained. Sodium glycerophosphate (400 mg per liter), ammonium dibasic citrate (5 g per liter), and glycerol (25 ml per liter) supplied the needed nutrients for maximal growth with a limited concentration of the required metabolite. Data are presented for the microbiological assay for biotin in waters of the Gulf of Mexico and adjacent bays. The range of sensitivity for the biotin mutant A101V is 5 to 12 mmug per liter in seawater, with a growth response from 2 to 16 mmug per liter of seawater. The possible ecological and chemical significance of biotin occurrence in spring-summer off-shore water is discussed.     

Evaluation of yield and maintenance coefficients, expressed in carbon units, for Pseudomonas fluorescens and P. aeruginosa.

The maximum cell yield (YMc - g biomass carbon per gram substrate carbon) and the rate of maintenance metabolism (mc - g substrate carbon/g biomass carbon per hour) have been determined for substrate limited continuous cultures of Pseudomonas fluorescens and P. aeruginosa. The metabolism of the organic substrates was monitored by measuring the COD-removal1) rates at different dilution rates. The advantages of expressing yield and maintenance coefficients in carbon units is discussed.     

Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.