Effect of Medium Composition on the Denitrification of Nitrate by Paracoccus denitrificans

The course of denitrification of nitrate in static cultures of Paracoccus denitrificans was studied. Reduction of nitrate to gaseous nitrogen without accumulation of nitrite because of parallel and balanced activities of nitrate and nitrite reductases was observed in nutrient broth. In minimal liquid cultures supplemented with either methanol, acetate, or ethanol as a sole carbon source, substantial amounts of nitrite (up to 70%) accumulated. The reduction in nitrite concentration began just after the transformation of nitrate to nitrite was completed. The addition of some growth factors to minimal media shortened the bacterial biomass doubling time. A correlation coefficient of 0.71 between the doubling time and the amount of accumulated nitrite in cultures was found. My results indicated that the type of denitrification carried out by P. denitrificans is not stable and depends on the nutritional composition of the culture medium.     

Atypical Polyphosphate Accumulation by the Denitrifying Bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Effect of oxygen on ubiquinone-10 production by Paracoccus denitrificans

Summary Ubiquinone-10 (Q₁₀) production was measured in batch cultures of Paracoccus denitrificans grown for 8 h at increasing oxygen concentrations (0–21 % O₂ in the sparging gas). Whereas the cellular level of Q₁₀ decreased monotonically from 1.2 to 0.5 mol/g d.w., the total yield of Q₁₀ was maximal at 2.5 % O₂ and amounted to 350 nmol (0.3 mg) per L of culture.     

The influence of pH on the kinetics of dissimilatory nitrite reduction in Paracoccus denitrificans

An almost stoichiometric conversion of nitrite to nitrous oxide was observed during the nitrite reduction by Paracoccus denitrificans cells in a medium of pH 6.4. The N₂O accumulated in the reaction medium and was decomposed only after nitrite had been consumed; when the pH of the medium was higher than 7.3–7.4, nitrous oxide did not accumulate. The activity of N₂O reductase was, in the whole range of pH 6.4–9.2, higher than the activity of NO⁻₂ reductase, both activities showing the maximum at the pH higher than 8.0. Using an artificial donor, TMPD plus ascorbate, the maximum activity of NO⁻₂ reductase, but not N₂O reductase was shifted by about two pH units to acidic region. The activity of nitrite reductase declined in the presence of N₂O only at higher pH values. Cytochrome c, as a common electron donor for both N₂O and NO⁻₂ reductase, was more oxidized at pH < 7.3 in the presence of NO⁻₂ than in the presence of N₂O, the opposite being true at pH > 7.3. The increased flux of electrons to cytochrome c has for a constant pH value (6.4) no effect on their distribution over NO⁻₂ and N₂O. The results indicate that the distribution of electrons in the terminal part is determined by the different pH optima for NO⁻₂ reductase and N₂O reductase, and by a mutual dependence of activities of the two reductases due to the competition for redox equivalents from a substrate.     

Respiratory Rate as a Regulatory Factor in the Biosynthesis of the Denitrification Pathway of the Bacterium Paracoccus denitrificans

The decrease in the electron flow of the aerobic respiratory chain of the bacterium Paracoccus denitrificans, owing to either the drop in the saturation of terminal oxidases by oxygen or to the inhibition of the rate of respiration by azide or nitrite, resulted in the synthesis of dissimilatory nitrate reductase and nitrite reductase. The dependence of the resulting activities of the two enzymes (after a three-hour adaptation) on the initial value of the parameter V_max/k_La (oxidase activity of the volume unit of the culture divided by the volumetric oxygen transfer coefficient) or on the concentrations of the inhibitors had a similar form, characterized by the appearance of a maximum. The increasing parts of the obtained curves reflect the synthesis of enzymes, probably initiated by the increase in the intracellular degree of reduction, the subsequent drop being evidently in connection with the lack of metabolic energy for biosynthesis. The possible mechanisms of the effect of nitrogenous terminal acceptors (NO^-₃ and NO^-₂) on the formation of the denitrification pathway are discussed.     

Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Production of superoxide anions in Paracoccus denitrificans

Like mitochondria, plasma membranes of the free-living bacterium Paracoccus denitrificans are able to produce superoxide ions. The production of superoxide ions was observed during the initial stages of electron transfer from the respiratory substrates to oxygen, even when the bacteria had been grown anaerobically on nitrate as oxidant. Generation of Superoxide anions was supported by NADH or succinate and occurred before the antimycin-sensitive site of the respiratory chain, presumably at the level of a low potential redox component. Superoxide anion formation was pH and substrate dependent; it was inhibited by cyanide and by exogenous superoxide dismutase.     

山梨糖脱氢酶在脱氮副球菌中的表达

目的:在脱氮副球菌PD1222中表达山梨糖脱氢酶(SDH)。方法:从质粒pMD-18T上复制氨苄西林抗性基因Ampr,从酮古龙酸菌中复制SDH基因sdh,先后酶切连接到pIND4质粒上,构建pIND4-Ampr-sdh穿梭质粒;再把pIND4-Ampr-sdh电转入大肠杆菌S17-1λpir作为供体菌,脱氮副球菌PD1222为受体菌进行双亲本接合转移;挑取壮观霉素和氨苄西林双抗平板上的接合子进行培养,菌液PCR复筛接合子,测序鉴定,通过DCIP法和非变性聚丙烯酰胺凝胶电泳法检测阳性克隆的SDH活性。结果:构建的质粒pIND4-Ampr-sdh成功转入脱氮副球菌PD1222中,SDH获得表达并检测到其蛋白活性。结论:实现了SDH在脱氮副球菌中的表达,为在脱氮副球菌中研究SDH的下游电子传递链奠定了基础。     

Biodegradation of Isopropanol by a Solvent-Tolerant Paracoccus denitrificans Strain

The biodegradation of high concentration isopropanol (2-propanol, IPA) at 16 g/L was investigated by a solvent-tolerant strain of bacteria identified as Paracoccus denitrificans for the first time by 16S rDNA gene sequencing. The strain P. denitrificans GH3 was able to utilize the high concentration of IPA as the sole carbon source within a minimal salts medium with a cell density of 1.5 × 10⁸ cells/mL. The optimal conditions were found as follows: initial pH 7.0, incubation temperature 30°C, with IPA concentration 8 g/L. Under the optimal conditions, strain GH3 utilized 90.3% of IPA in 7 days. Acetone, the major intermediate of aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Both IPA and acetone were completely removed from the medium following 216 hr and 240 hr, respectively. The growth of strain GH3 on IPA as a sole carbon and energy source was well described by the Andrews model with a maximum growth rate (μ _max ) = 0.0277/hr, a saturation constant (K _S ) = 0.7333 g/L, and an inhibition concentration (Ki) = 8.9887 g/L. Paracoccus denitrificans GH3 is considered to be well used in degrading IPA in wastewater.     

Inhibition of denitrification activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH

The influence of pH on the denitrification activity of a continuous culture of Paracoccus denitrificans was studied in relation to the presence of nitrite. After a transition from aerobic to anaerobic conditions at the suboptimal pH of 6.8, P. denitrificans was not able to build up a functional denitrification pathway. Nitrite accumulated in the medium as the predominant denitrification product. Although the nitrite reductase gene was induced properly, the enzyme could not be detected at sufficient amounts in the culture. These observations indicate that either translation was somehow inhibited, or once synthesized nitrite reductase was inactivated, possibly by the high concentrations of nitrous acid (HNO₂. Interestingly, when a P. denitrificans culture which was grown to steady-state under anaerobic conditions was then exposed to suboptimal pHs, cells exhibited a reduced overall denitrification activity, but neither nitrite nor any other denitrification intermediate accumulated.     

Fermentation enzymes in strains of Paracoccus denitrificans

Various strains of Paracoccus denitrificans grown under conditions of unrestricted oxygen supply contained low but measurable activities of fermentation enzymes such as ethanol dehydrogenase and 2,3-butanediol dehydrogenase. However, when the bacteria were subsequently incubated for up to 22 h under restricted aeration conditions permitting respiration rates of only 10 or 6% of the maximum value to occur, the above enzymes increased in specific activities by 5- or 10-fold to 0.14 mol/min·mg protein. Lactate dehydrogenase was not detected. Six strains tested reacted almost alike.Cells grown anaerobically on fructose in the presence of limiting concentrations of KNO₃ contained specific activities of up to 0.41 (in case of ethanol dehydrogenase) and 0.56 (butanediol dehydrogenase) mol/min·mg protein. Lactate dehydrogenase was only formed at low activity (0.012 mol/min·mg protein) after a long period of incubation.Cells of P. denitrificans strain Stanier 381 grown anaerobically in the chemostat on fructose+KNO₃ with either fructose or nitrate as the limiting factor differed with respect to the specific enzyme activities, too. Ethanol dehydrogenase was high under conditions of nitrate limitation and low under fructose limitation. 2,3-Butanediol dehydrogenase, but not lactate dehydrogenase, was formed in moderate activities.     

Proteolysis of Paracoccus denitrificans cytochrome oxidase by trypsin and chymotrypsin

Paracoccus oxidase containing only two subunits was subjected to proteolysis by trypsin and chymotrypsin. Both subunits of the purified enzyme were cleaved at only a few sites and enzymatic activity was not inhibited. The cleavage sites were identified by protein sequencing. Subunit I was cleaved near the amino-terminus and subunit II in the loop connecting the two predicted trans-membrane helices. In native membrane fragments, but not in intact spheroplasts, this loop was accessible to both proteases. These results provide experimental evidence for the folding of subunit II in the membrane.     

Removal of Paracoccus denitrificans outer membrane material by sodium chloride

The effects of water washing and NaCl treatment on the cell surface of P. denitrificans were studied. Both treatments caused a release of material from cells. Chemical studies showed that NaCl treatment released material containing components characteristic of outer membrane. This treatment also increased the susceptibility of the organism to lysozyme. Scanning electron microscopy was used to monitor the effects of water washing and NaCl treatment on the cell surface. Both treatments were shown to alter the appearance of the cell surface. The disruptive effects of these procedures were found to be dependent upon the age of the culture.Non-Standard Abbreviations WW water wash - SE saline extract     

Intermediates of denitrification in the chemoautotroph Thiobacillus denitrificans

Summary Intact cells obtained from Thiobacillus denitrificans grown autotrophically with thiosulfate as the oxidizable substrate and nitrate as the final electron acceptor catalyzed the reduction of nitrate, nitrite and nitric oxide stoichiometrically to nitrogen gas with the concomitant oxidation of thiosulfate. In addition, nitrous oxide was also capable of acting as the terminal oxidant of the respiratory chain with thiosulfate as the reductant. The anaerobic oxidation of thiosulfate by NO₃ ^-, NO, and N₂O was sensitive to the flavoprotein inhibitors, antimycin A or NHQNO, and cyanide or azide thus, implicating the participation of flavins, and cytochromes of b-, c-, and a-types in the denitrification process. The nitrite reductase system, however, was not markedly affected by the electron transport chain inhibitors. The experimental observations suggest that the dissimilatory nitrate reduction in the chemoautotroph T. denitrificans involves nitrite, nitric oxide, and nitrous oxide as theintermediates with nitrogen gas as the final reduction product.Non-Standard Abbreviations TTFA Thenoyltrifluoroacetone - NHQNO 2-n-nonyl-4-hydroxyquinoline N-oxide