Secondary substrate utilization of methylene chloride by an isolated strain of Pseudomonas sp.

Secondary substrate utilization of methylene chloride was analyzed by using Pseudomonas sp. strain LP. Both batch and continuously fed reactors demonstrated that this strain was capable of simultaneously consuming two substrates at different concentrations: the primary substrate at the higher concentration (milligrams per liter) and the secondary substrate at the lower concentration (micrograms per liter). The rate of methylene chloride utilization at trace concentrations was greater in the presence of the primary substrate, acetate, than without it. However, when the substrate roles were changed, the acetate secondary substrate utilization rate was less when methylene chloride was present. Thus, substrate interactions are important in the kinetics of secondary substrate utilization. Pseudomonas sp. strain LP showed a preference toward degrading methylene chloride over acetate, whether it was the primary or secondary substrate, providing it was below an inhibitory concentration of ca. 10 mg/liter.     

Solid-state 13C nuclear magnetic resonance spectroscopy of simultaneously metabolized acetate and phenol in a soil Pseudomonas sp.

We investigated concentration-dependent primary and secondary substrate relationships in the simultaneous metabolism of the ubiquitous pollutant phenol and the naturally occurring substrate acetate by a Pseudomonas sp. soil isolate capable of utilizing either substance as a sole source of carbon and energy. In addition to conventional analytical techniques, solid-state 13C nuclear magnetic resonance spectroscopy was used to follow the cellular distribution of [1-13C]acetate in the presence of unlabeled phenol. With 5 mM acetate as the primary substrate, Pseudomonas sp. 9S8D2 removed 1 mM phenol (secondary substrate) at a rate of 2 nmol/mg of total cell protein. Although extensive acetate metabolism was indicated by a significant redistribution of the carboxyl label, this redistribution was not affected by the presence of phenol as a secondary substrate. When the primary and secondary substrate roles were reversed, however, the presence of 1 mM phenol altered the metabolism of 0.1 mM acetate, as evidenced by both the two- to fourfold increases in carboxyl label that appeared in terminal methyl and acyl chain methylene carbon resonances and the decrease in label that occurred in the carbohydrate spectral region. These results suggest that, when phenol is present as the primary substrate, acetate is preferentially shuttled into fatty acyl chain synthesis, whereas phenol carbon is funnelled into the tricarboxylic acid cycle. Thus, simultaneous use of a xenobiotic compound and a natural substrate apparently does occur, and the relative concentrations of the two substrates do influence the rate and manner in which the compounds are utilized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-state 13C nuclear magnetic resonance spectroscopy of simultaneously metabolized acetate and phenol in a soil Pseudomonas sp

We investigated concentration-dependent primary and secondary substrate relationships in the simultaneous metabolism of the ubiquitous pollutant phenol and the naturally occurring substrate acetate by a Pseudomonas sp. soil isolate capable of utilizing either substance as a sole source of carbon and energy. In addition to conventional analytical techniques, solid-state 13C nuclear magnetic resonance spectroscopy was used to follow the cellular distribution of [1-13C]acetate in the presence of unlabeled phenol. With 5 mM acetate as the primary substrate, Pseudomonas sp. 9S8D2 removed 1 mM phenol (secondary substrate) at a rate of 2 nmol/mg of total cell protein. Although extensive acetate metabolism was indicated by a significant redistribution of the carboxyl label, this redistribution was not affected by the presence of phenol as a secondary substrate. When the primary and secondary substrate roles were reversed, however, the presence of 1 mM phenol altered the metabolism of 0.1 mM acetate, as evidenced by both the two- to fourfold increases in carboxyl label that appeared in terminal methyl and acyl chain methylene carbon resonances and the decrease in label that occurred in the carbohydrate spectral region. These results suggest that, when phenol is present as the primary substrate, acetate is preferentially shuttled into fatty acyl chain synthesis, whereas phenol carbon is funnelled into the tricarboxylic acid cycle. Thus, simultaneous use of a xenobiotic compound and a natural substrate apparently does occur, and the relative concentrations of the two substrates do influence the rate and manner in which the compounds are utilized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations.

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation of Low Concentrations of 3-Chlorobenzoate by Pseudomonas sp. Strain B13: Kinetics and Residual Concentrations

The transformation of 3-chlorobenzoate (3CB) and acetate at initial concentrations in the wide range of 10 nM to 16 mM was studied in batch experiments with Pseudomonas sp. strain B13. Transformation rates of 3CB at millimolar concentrations could be described by Michaelis-Menten kinetics (K(infm), 0.13 mM; V(infmax), 24 nmol (middot) mg of protein(sup-1) (middot) min(sup-1)). Experiments with nanomolar and low micromolar concentrations of 3CB indicated the possible existence of two different transformation systems for 3CB. The first transformation system operated above 1 (mu)M 3CB, with an apparent threshold concentration of 0.50 (plusmn) 0.11 (mu)M. A second transformation system operated below 1 (mu)M 3CB and showed first-order kinetics (rate constant, 0.076 liter (middot) g of protein(sup-1) (middot) min(sup-1)), with no threshold concentration in the nanomolar range. A residual substrate concentration, as has been reported for some other Pseudomonas strains, could not be detected for 3CB (detection limit, 1.0 nM) in batch incubations with Pseudomonas sp. strain B13. The addition of various concentrations of acetate as a second, easily degradable substrate neither affected the transformation kinetics of 3CB nor induced a detectable residual substrate concentration. Acetate alone also showed no residual concentration (detection limit, 0.5 nM). The results presented indicate that the concentration limits for substrate conversion obtained by extrapolation from kinetic data at higher substrate concentrations may underestimate the true conversion capacity of a microbial culture.     

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.     

Batch- and Continuous-Culture Transients for Two Substrate Systems

Batch growth of Escherichia coli in the presence of equal initial concentrations of glucose and a secondary substrate (xylose) is characterized by sequential utilization of the substrates, whereas continuous-culture systems with equal concentrations of the two substrates in the feed are characterized by complete utilization of both substrates at both high and low dilution rates. Such systems at steady state at a low dilution rate, when suddenly shifted to a higher dilution rate, experience a transient drop in population density accompanied by accumulation of the secondary substrate but virtually no accumulation of glucose. Systems at steady state with 200 mg of glucose per liter were found to undergo a transient population decrease and eventual recovery when switched to feed containing 200 mg of a secondary substrate per liter.     

Growth of Pseudomonas aeruginosa in tap water in relation to utilization of substrates at concentrations of a few micrograms per liter.

Five Pseudomonas aeruginosa strains were tested for the utilization of 47 low-molecular-weight compounds as their sole sources of carbon and energy for growth at a concentration of 2.5 g/liter. Of these compounds, 31 to 35 were consumed. Growth experiments in tap water at 15 degrees C were carried out with one particular strain (P1525) isolated from drinking water. This strain was tested for the utilization of 30 compounds supplied at a concentration of 25 microgram of C per liter. The growth rate (number of generations per hour) of strain P1525 in this tap water was approximately 0.005 h-1, and with 10 compounds it was larger than 0.03 h-1. An average yield of 6.2 x 10(9) colony-forming units per mg of C was obtained from the maximum colony counts (colony-forming units per milliliter). The average yield and maximum colony count of strain P1525 grown in tap water supplied with a mixture of 45 compounds, each at a concentration of 1 microgram of C per liter, enabled us to calculate that 28 compounds were utilized. Growth rates of two P. aeruginosa strains (including P1525) in various types of water at 15 degrees C were half of those of a fluorescent pseudomonad. The concentrations of assimilable organic carbon calculated from maximum colony counts and average yield values amounted to 0.1 to 0.7% of the total organic carbon concentrations in five types of tap water. The assimilable organic carbon percentages were about 10 times larger in river water and in water after ozonation.     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Fate and effects of methylene chloride in activated sludge.

Activated sludge obtained from a municipal wastewater treatment plant was acclimated to methylene chloride at concentrations between 1 and 100 mg/liter by continuous exposure to the compound for 9 to 11 days. Acclimated cultures were shown to mineralize methylene chloride to carbon dioxide and chloride. Rates of methylene chloride degradation were 0.14, 2.3, and 7.4 mg of CH2Cl2 consumed per h per g of mixed-liquor suspended solids for cultures incubated in the presence of 1, 10, and 100 mg/liter, respectively. Concentrations of methylene chloride between 10 and 1,000 mg/liter had no significant effect on O2 consumption or glucose metabolism by activated sludge. A hypothetical model was developed to examine the significance of volatilization and biodegradation for the removal of methylene chloride from an activated sludge reactor. Application of the model indicated that the rate of biodegradation was approximately 12 times greater than the rate of volatilization. Thus, biodegradation may be the predominant process determining the fate of methylene chloride in activated sludge systems continuously exposed to the compound.     

Resistance of Pseudomonas to Quaternary Ammonium Compounds. I. Growth in Benzalkonium Chloride Solution

Resistant cells of Pseudomonas aeruginosa and a waterborne Pseudomonas sp. (strain Z-R) were able to multiply in nitrogen-free minimal salts solution containing various concentrations of commercially prepared, ammonium acetate-buffered benzalkonium chloride (CBC), a potent antimicrobial agent. As the CBC concentration increased, growth increased until a point was reached at which the extent of growth leveled off or was completely depressed. Minimal salts solutions of pure benzalkonium chloride (PBC) containing no ammonium acetate did not support bacterial growth. When ammonium acetate was added to PBC solutions in the same concentrations found in CBC solutions, growth patterns developed that were comparable to those found with CBC. Likewise, (NH(4))(2)SO(4) added to PBC solutions supported growth of both organisms. P. aeruginosa was initially resistant to CBC levels of 0.02% and it was adapted to tolerate levels as high as 0.36%. Strain Z-R was naturally resistant to 0.4% CBC. Since ammonium acetate, carried over by the CBC used in drug formulations and disinfectant solutions, has the potential to support the growth of resistant bacteria and thus make possible the risk of serious infection, it is suggested that regulations allowing the presence of ammonium acetate in CBC solution be reconsidered.     

Methanogenesis in a Thermophilic (58 degrees C) Anaerobic Digestor: Methanothrix sp. as an Important Aceticlastic Methanogen

Aceticlastic methanogens and other microbial groups were enumerated in a 58 degrees C laboratory-scale (3 liter) anaerobic digestor which was fed air-classified municipal refuse, a lignocellulosic waste (loading rate = 1.8 to 2.7 g of volatile solids per liter per day; retention time = 10 days). Two weeks after start-up, Methanosarcina sp. was present in high numbers (10 to 10 CFU/ml) and autofluorescent Methanosarcina-like clumps were abundant in sludge examined by using epifluorescence microscopy. After about 4 months of digestor operation, numbers of Methanosarcina sp. dropped 2 to 3 orders of magnitude and large numbers (most probable number = 10 to 10/ml) of a thermophilic aceticlastic methanogen morphologically resembing Methanothrix sp. were found. Methanothrix sp. had apparently displaced Methanosarcina sp. as the dominant aceticlastic methanogen in the digestor. During the period when Methanothrix sp. was apparently dominant, acetate concentrations varied between 0.3 and 1.5 mumol/ml during the daily feeding cycle, and acetate was the precursor of 63 to 66% of the methane produced during peak digestor methanogenesis. The apparent K(m) value obtained for methanogenesis from acetate, 0.3 mumol/ml, indicated that the aceticlastic methanogens were nearly saturated for substrate during most of the digestor cycle. CO(2)-reducing methanogens were capable of methanogenesis at rates more than 12 times greater than those usually found in the digestor. Added propionate (4.5 mumol/ml) was metabolized slowly by the digestor populations and slightly inhibited methanogenesis. Added n-butyrate, isobutyrate, or n-valerate (4.5 mumol/ml each) were broken down within 24 h. Isobutyrate was oxidized to acetate, a novel reaction possibly involving isomerization to n-butyrate. The rapid growth rate and versatile metabolism of Methanosarcina sp. make it a likely organism to be involved in start-up, whereas the low K(m) value of Methanothrix sp. for acetate may cause it to be favored in stable digestors operated with long retention times.     

Kinetics of Acetate Utilization by Two Thermophilic Acetotrophic Methanogens: Methanosarcina sp. Strain CALS-1 and Methanothrix sp. Strain CALS-1

The kinetics of acetate utilization were examined for washed concentrated cell suspensions of two thermophilic acetotrophic methanogens isolated from a 58°C anaerobic digestor. Progress curves for acetate utilization by cells of Methanosarcina sp. strain CALS-1 showed that the utilization rate was concentration independent (zero order) above concentrations near 3 mM and that acetate utilization ceased when a threshold concentration near 1 mM was reached. Acetate utilization by cells of Methanothrix sp. strain CALS-1 was concentration independent down to 0.1 to 0.2 mM, and threshold values of 12 to 21 μM were observed. Typical utilization rates in the concentration-independent stage were 210 and 130 nmol min⁻¹ mg of protein⁻¹ for the methanosarcina and the methanothrix, respectively. These results are in agreement with a general model in which high acetate concentrations favor Methanosarcina spp., while low concentrations favor Methanothrix spp. However, acetate utilization by these two strains did not follow simple Michaelis-Menton kinetics.     

Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations.

Two marine bacteria, an Acinetobacter sp. (strain GO1) and a vibrio sp. (strain G1), were isolated by extinction dilution and maintained in natural seawater supplemented with nitrogen, phosphorus, and glucose at 0.01 and 10 mg of glucose carbon per liter above ambient monosaccharide concentrations, respectively. After 3 days in unsupplemented natural seawater, growth in batch culture with glucose supplements was determined by changes in cell numbers and glucose concentration. The exponential growth of the Acinetobacter strain with added glucose was indistinguishable from that in natural seawater alone, whereas that of the Vibrio strain was more rapid in the presence of glucose supplements, suggesting that the Acinetobacter strain preferred the natural organic matter in seawater as a carbon source. The ultrastructure for both isolates was unaffected by glucose supplements during exponential growth, but there were marked changes in stationary-phase cells. The Vibrio strain formed polyphosphate at 10 mg of glucose carbon per liter, whereas poly-beta-hydroxybutyrate formation occurred at 100 mg and became excessive at 1,000 mg, disrupting the cells. In contrast, the Acinetobacter strain elongated at 100 and 1,000 mg of glucose carbon per liter but failed to show poly-beta-hydroxybutyrate formation. The diversity of responses shown here would not have been detected with a single concentration of substrate, often used in the literature to characterize both pure and natural populations of marine bacteria.     

Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations

Two marine bacteria, an Acinetobacter sp. (strain GO1) and a vibrio sp. (strain G1), were isolated by extinction dilution and maintained in natural seawater supplemented with nitrogen, phosphorus, and glucose at 0.01 and 10 mg of glucose carbon per liter above ambient monosaccharide concentrations, respectively. After 3 days in unsupplemented natural seawater, growth in batch culture with glucose supplements was determined by changes in cell numbers and glucose concentration. The exponential growth of the Acinetobacter strain with added glucose was indistinguishable from that in natural seawater alone, whereas that of the Vibrio strain was more rapid in the presence of glucose supplements, suggesting that the Acinetobacter strain preferred the natural organic matter in seawater as a carbon source. The ultrastructure for both isolates was unaffected by glucose supplements during exponential growth, but there were marked changes in stationary-phase cells. The Vibrio strain formed polyphosphate at 10 mg of glucose carbon per liter, whereas poly-beta-hydroxybutyrate formation occurred at 100 mg and became excessive at 1,000 mg, disrupting the cells. In contrast, the Acinetobacter strain elongated at 100 and 1,000 mg of glucose carbon per liter but failed to show poly-beta-hydroxybutyrate formation. The diversity of responses shown here would not have been detected with a single concentration of substrate, often used in the literature to characterize both pure and natural populations of marine bacteria.     

Utilization of Halogenated Benzenes, Phenols, and Benzoates by Rhodococcus opacus GM-14

Strain GM-14 was isolated by selective enrichment from contaminated soil with chlorobenzene as the sole source of carbon and energy. It utilizes an exceptionally wide spectrum of haloaromatic substrates. It is a gram-positive, weakly acid-fast actinomycete, with a morphological cycle from cocci and short rods to long rods and branched filaments; it grew optimally at 28(deg)C; and it tolerated 5% NaCl in rich medium. The chemotaxonomic characteristics, the diagnostic biochemical tests, the whole-cell fatty acid composition, and 16S rDNA analysis were consistent with Rhodococcus opacus. R. opacus GM-14 grew on 48 of 117 different aromatic and haloaromatic compounds. It utilized phenol at concentrations up to 1.2 g/liter, 3- and 4-methylphenols up to 0.5 g/liter, 2- and 4-chlorophenols up to 0.25 g/liter, and 3-chlorophenol up to 0.1 g/liter. It grew in saturated aqueous solutions of benzene, chlorobenzene, and 1,3- and 1,4-dichlorobenzene (up to 13, 3, 0.5, and 0.5 g/liter, respectively). The specific growth rate of strain GM-14 on phenol and 3- and 4-chlorophenols in batch culture was 0.27 to 0.29 h(sup-1), and that on benzene and chlorobenzene was similar to the rate on fructose, i.e., 0.2 h(sup-1). The growth yield on benzene and on chlorobenzene (<=0.4 g liter(sup-1)) was 40 to 50 g (dry weight) per mol of substrate consumed, equalling 8 g of dry weight biomass per mol of substrate carbon, similar to that obtained on acetate. During growth of strain GM-14 on chlorobenzene, 1,3-dichlorobenzene, and all isomers of monochlorophenol, stoichiometric amounts of chloride were released, and 50% of the stoichiometric amount was released from 1,4-dichlorobenzene.