Chloramphenicol Inhibition of Denitrifying Enzyme Activity in Two Agricultural Soils

Chloramphenicol, at concentrations greater than 0.1 g/liter (0.3 mM), inhibited the denitrifying enzyme activity (DEA) of slurries of humisol and sandy loam soils by disrupting the activity of existing nitrate reductase enzymes. When the concentration of chloramphenicol was increased from 0.1 to 2.0 g/liter (6.0 mM), the rate of nitrite production from nitrate decreased by 25 to 46%. The rate of NO production from nitrate decreased by 20 to 39%, and the rate of N₂O production from nitrate, in the presence of acetylene (DEA), decreased by 21 to 61%. The predicted values of DEA at 0 g of chloramphenicol/liter computed from linear regressions of DEA versus chloramphenicol concentration were 18 to 43% lower than DEA measurements made in the absence of chloramphenicol and within a few per cent of DEA rates measured in the presence of 0.1 g of chloramphenicol/liter. We conclude that DEA assays should be carried out with a single (0.1-g/liter) chloramphenicol concentration. Chloramphenicol at concentrations greater than 0.1 g/liter inhibits the activity of existing denitrifying enzymes and should not be used in DEA assays.     

Inhibition of existing denitrification enzyme activity by chloramphenicol.

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (greater than 50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log-phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.     

Inhibition of existing denitrification enzyme activity by chloramphenicol.

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (greater than 50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log-phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.     

Effect of Chloramphenicol on Denitrification in Flexibacter canadensis and "Pseudomonas denitrificans"

It was recently reported that chloramphenicol inhibits existing denitrification enzyme activity in sediments and carbon-starved cultures of "Pseudomonas denitrificans." Therefore, we studied the effect of chloramphenicol on denitrification by Flexibacter canadensis and "P. denitrificans." Production of N(inf2)O from nitrate by F. canadensis cells decreased as the concentration of chloramphenicol was increased, and 10.0 mM chloramphenicol completely inhibited N(inf2)O production. "P. denitrificans" was less sensitive to chloramphenicol, and production of N(inf2)O from nitrate was inhibited by only about 50% even in the presence of 10.0 mM chloramphenicol. These results suggested that inhibition of denitrification enzyme activity depended on the concentration of chloramphenicol. Increasing the concentration of chloramphenicol decreased the rate of production of nitrite from nitrate by F. canadensis cells, and the concentration of chloramphenicol which resulted in 50% inhibition of production of nitrite from nitrate was 2.5 mM. In contrast, the rates of production of nitrite from nitrate by intact cells and cell extracts of "P. denitrificans" were inhibited by only 58 and 54%, respectively, at a chloramphenicol concentration of 10.0 mM. Chloramphenicol caused accumulation of NO from nitrite but not from nitrate and inhibited NO consumption in F. canadensis; however, it had neither effect in "P. denitrificans." Chloramphenicol did not affect N(inf2)O consumption by these organisms. We concluded that chloramphenicol inhibits denitrification at the level of nitrate reduction and, in F. canadensis, also at the level of NO reduction.     

Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions

A strategy for sequential hydrocarbon bioremediation is proposed. The initial O(2)-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation. The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O(2)-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO(2)(-)-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO(2)(-)-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO(3)(-)-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation by P. aeruginosa was then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N(2)). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.     

Degradation of n-Hexadecane and Its Metabolites by Pseudomonas aeruginosa under Microaerobic and Anaerobic Denitrifying Conditions

A strategy for sequential hydrocarbon bioremediation is proposed. The initial O₂-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation. The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O₂-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO₂⁻-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO₂⁻-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO₃⁻-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation by P. aeruginosa was then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N₂). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.     

Effect of oxygen on denitrification in continuous chemostat culture withComamonas sp SGLY2

Continuous cultures ofComamonas sp SGLY2 were grown anaerobically prior to establishing steady states at different oxygen flow rates. At a low oxygen transfer rate, no dissolved oxygen accumulated in the medium and all nitrate was reduced to dinitrogen. Concurrently with the increase of dissolved oxygen concentration in the liquid phase, the rate of denitrification decreased. However, at a dissolved oxygen concentration near saturation (33 mg L^–1), a part of the electron flow always diverted to nitrate with production of dinitrogen: the aerobic denitrification rate was equivalent to 35% of that calculated under anaerobic conditions. These experiments reflected the co-utilization of oxygen and N-oxides and the production of dinitrogen, up to saturated conditions, which implied synthesis and activity of the four denitrifying enzymes under various aeration conditions.     

太湖流域上游竹林河岸带土壤反硝化酶活性及其影响因素

本研究以太湖流域上游竹林河岸带为对象,采用乙炔抑制法分析了夏季河岸带土壤反硝化酶活性(DEA)及其影响因素,以期为竹林河岸带在减少河流氮污染方面的生态功能评估提供数据支持。结果表明: 河岸带土壤DEA为6.32～23.22 μg N·kg^-1·h^-1,平均值为14.65 μg N·kg^-1·h^-1。河岸带土壤有机碳(SOC)、总氮、硝态氮含量、含水量和碳氮水解酶活性共同影响着DEA的垂直分布,使DEA随土壤深度(0～40 cm)的增加而递减;而DEA在水平方向上(同一土壤深度离水距离不同)的变化主要受SOC含量的影响。太湖流域上游竹林河岸带土壤在夏季可能会由于缺乏相对充足的可溶性有机碳而对DEA产生一定的限制。     

A Denitrifying Activity in an Aerobic Photosynthetic Bacterium, Erythrobacter sp. Strain OCh 114

An aerobic photosynthetic bacterium, Erythrobacter sp. strainOCh 114, was capable of growth under anaerobic conditions inthe dark with nitrate as a terminal electron acceptor. The optimalnitrate concentration was about 6 mM for anaerobic growth, althougha wide range of concentrations from 1 to 400 mM were effective.A large amount of N₂O gas was released during this anaerobicgrowth, indicating a denitrifying activity in this bacterium.Light had no stimulating or inhibiting effect on the rates ofanaerobic growth and gas release. The enzymes responsible forthe denitrifying activity, dissimilatory nitrate and nitritereductases, were present in aerobically grown cells. (Received February 19, 1988; Accepted May 16, 1988)     

Effect of monensin on the specific activity of ammonia production by ruminal bacteria and disappearance of amino nitrogen from the rumen.

When unadapted mixed ruminal bacteria (312 mg of protein per liter) were treated with monensin (5 mM) in vitro, the rates of ammonia production from enzymatic digests of casein, gelatin, and soy protein (0.5 g of N per liter) were decreased from 46 +/- 2 to 24 +/- 1, 20 +/- 1 to 7 +/- 1, and 40 +/- 2 to 18 +/- 2 nmol/mg of protein per min, respectively. Monensin also caused a decrease in ammonia production in vivo. Nonlactating dairy cows which were fed 0.56 kg of timothy hay 12 times per day had a steady-state ruminal ammonia concentration of 2.7 +/- 0.1 mM, and the ammonia concentration decreased to 1.2 +/- 0.2 mM when monensin (350 mg/day) was added to the diet. The decrease in ammonia production was associated with a 10-fold reduction (4.1 x 10(6) versus 4.2 x 10(5)/ml) in the most probable number of ammonia-producing ruminal bacteria that could use protein hydrolysate as an energy source. Monensin had little effect on the most probable number of carbohydrate-utilizing ruminal bacteria (6.5 versus 7.0 x 10(8)/ml). The addition of protein hydrolysates (560 g) to the rumen caused a rapid increase in the ammonia concentration, but this increase was at least 30% lower when the animals were fed monensin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO(2) gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h(-1) and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 muM Cu, 80 muM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h(-1). Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 muM, falling sharply at 20 muM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. (c) 1992 John Wiley & Sons, Inc.     

The influence of nitrate concentration upon the end-products of nitrate dissimilation by bacteria in anaerobic salt marsh sediment

Abstract Experiments were carried out with slurries of saltmarsh sediment to which varying concentrations of nitrate were added. The acetylene blocking technique was used to measure denitrification by accumulation of nitrous oxide, while reduction of nitrate to nitrite and ammonium was also measured. There was good recovery of reduced nitrate and at the smallest concentration of nitrate used (250 μM) there was approximately equal reduction to either ammonium or nitrous oxide (denitrification). Nitrite was only a minor end-product of nitrate reduction. As the nitrate concentration was increased the proportion of the nitrate which was denitrified to nitrous oxide increased, to 83% at the greatest nitrate concentration used (2 mM), while reduction to ammonium correspondingly decreased. This change was attributed either to a greater competitiveness by the denitrifiers for nitrate as the ratio of electron donor to electron acceptor decreased; or to the increased production of nitrite rather than ammonium by fermentative bacteria under high nitrate, the nitrite then being reduced to nitrous oxide by denitrifying bacteria.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

Flocculant production from kerosene

Cultivation of Corynebacterium hydrocarboclastus, which is capable of synthesizing an extracellular polymer and utilized hydrocarbons, has been reported. Growth studies in shake flasks and fermenters were made to obtain maximum polymer production. Polymer formation was found to be growth associated. The highest level of polymer accumulation was attained after 50–60 hr cultivation in the fermenter and it amounted to approximately 5.5–6 g/liter of fermentation broth. The medium contained initially 2% (v/v) kerosene as a carbon source. The maximum yield obtained corresponds to 37–40% (w/w) of kerosene supplied. At the same time the cell concentration was 10–13 g/liter which represents the yield of 67–87% (w/w). The rate of polymer production in the exponential phase was 0.25 g/liter hr and cell production rate was 0.27 g/liter hr. Sodium nitrate, 0.5%, and yeast extract, 0.3%, (w/w) were the best nigrogen sources for polymer formation. The highest level of polymer produced in broth was 6 g/liter.     

Anaerobic and aerobic degradation of pyridine by a newly isolated denitrifying bacterium.

New denitrifying bacteria that could degrade pyridine under both aerobic and anaerobic conditions were isolated from industrial wastewater. The successful enrichment and isolation of these strains required selenite as a trace element. These isolates appeared to be closely related to Azoarcus species according to the results of 16S rRNA sequence analysis. An isolated strain, pF6, metabolized pyridine through the same pathway under both aerobic and anaerobic conditions. Since pyridine induced NAD-linked glutarate-dialdehyde dehydrogenase and isocitratase activities, it is likely that the mechanism of pyridine degradation in strain pF6 involves N-C-2 ring cleavage. Strain pF6 could degrade pyridine in the presence of nitrate, nitrite, and nitrous oxide as electron acceptors. In a batch culture with 6 mM nitrate, degradation of pyridine and denitrification were not sensitively affected by the redox potential, which gradually decreased from 150 to -200 mV. In a batch culture with the nitrate concentration higher than 6 mM, nitrite transiently accumulated during denitrification significantly inhibited cell growth and pyridine degradation. Growth yield on pyridine decreased slightly under denitrifying conditions from that under aerobic conditions. Furthermore, when the pyridine concentration used was above 12 mM, the specific growth rate under denitrifying conditions was higher than that under aerobic conditions. Considering these characteristics, a newly isolated denitrifying bacterium, strain pF6, has advantages over strictly aerobic bacteria in field applications.     

Utilization of cellulose from waste paper by Myrothecium verrucaria

Extensive screening studies on cellulolytic bacteria and fungi led to the selection of Myrothecium verrucaria as the organism producing the maximum rate of protein biosynthesis from ball-milled newspaper. Studies in aerated stirred-jar fermentors were carried out to determine the conditions for maximum protein synthesis rate and maximum final protein concentration. The optimum aeration rate was 250 to 374 mM of oxygen at 300 to 400 rpm stirring rate. The pH optimum was broad, from 3.9 to 6.5. Urea at 0.03% and yeast autolysate at 0.1% stimulated growth rate and protein production. The maximum rate of protein biosynthesis and the maximum protein yield were 0.3 g/liter/day and 1.42 g/liter, respectively, from medium G3 with 4% ball-milled newspaper. The final product, obtained by evaporation of the total culture, was 33.7 g from one liter of medium which originally contained 40 g of ball-milled newspaper and 11.3 g of other dissolved materials. The protein content of this final product was 3.3 g, calculated from total organic N × 6.25 or 1.42 g calculated from the biuret method. Both the synthesis rate and the final cell yield are below those obtainable by growing Fungi Imperfecti, yeasts or bacteria on soluble materials such as glucose.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

Relationship between state of aggregation and catalytic activity for cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The detergent 1-O-n-octyl-beta-D-glucopyranoside (octylglucoside) was found to replace the phospholipid requirement in the demethylation of benzphetamine by cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase purified from phenobarbital-treated rabbit liver. At low enzyme concentration (0.1 microM) in the absence of glycerol and phosphate, the maximum rate of benzphetamine-specific NADPH oxidation was approximately 35% of that observed in the presence of dilauroylglyceryl-3-phosphoryl choline. At higher enzyme concentration (2.5 microM) and in the presence of 0.15 M phosphate, 20% glycerol, octylglucoside was as effective as phospholipid in stimulating the production of formaldehyde from benzphetamine. The detergent concentration required for maximal enzymatic activity was 2.5-4.0 g/liter, depending on the cytochrome preparation used. At higher octylglucoside concentrations (5-7 g/liter), activity decreased to zero, although neither enzyme appeared to be irreversibly denatured at these detergent concentrations. Sedimentation equilibrium experiments with P-450LM2 alone or in the presence of equimolar reductase showed that increasing octylglucoside levels promoted disaggregation of the cytochrome. Pentamers and hexamers predominated at detergent concentrations where maximal activity was observed, while higher levels of detergent where activity was absent produced cytochrome dimers and, ultimately, monomers. The reductase was monomeric at detergent levels between at least 3 and 7 g/liter. Moreover, both gel filtration and sedimentation equilibrium experiments demonstrated that a stable complex between P-450LM2 and its reductase was not formed at octylglucoside concentrations where high activity was evident. These results are consistent with a model of P-450/reductase interaction in which functional aggregates of three to six cytochrome polypeptides move laterally in the microsomal membrane and interact with the reductase by random collision.     

Dynamics of Soil Denitrifier Populations: Relationships between Enzyme Activity, Most-Probable-Number Counts, and Actual N Gas Loss

To better understand temporal variability in soil denitrification, denitrifying enzyme activity (DEA) and denitrifier populations (as determined by most-probable-number [MPN] counts) were measured in field and laboratory experiments. Measurements of DEA and MPN provided highly contradictory indications of denitrifier dynamics. In laboratory incubations, under conditions favoring active denitrification, the synthesis of new denitrifying enzymes and the actual amount of denitrification were closely related. In other experiments, however, both DEA and MPN counts were poor indicators of actual denitrification. In some cases, we found significant increases in DEA but no significant production of N gas. Except with unnaturally high substrate amendments, changes in DEA were small relative both to the persistently high DEA background and to changes in MPN. As estimated by MPN counts, denitrifier populations increased significantly during denitrification events. It was apparent that only a small fraction of the denitrifiers were included in the MPN counts, but it appeared that this isolatable fraction increased during periods of active denitrifier growth. Use of DEA as an index of biomass of cells which have synthesized denitrifying enzymes suggested that denitrifier populations were persistent, stable, and much larger than indicated by MPN procedures.