Paracoccus denitrificans for the effluent recycling during continuous denitrification of liquid food

Nitrate is an undesirable component of several foods. A typical case of contamination with high nitrate contents is whey concentrate, containing nitrate in concentrations up to 25 l. The microbiological removal of nitrate by Paracoccus denitrificans under formation of harmless nitrogen in combination with a cell retention reactor is described here. Focus lies on the resource‐conserving design of a microbal denitrification process. Two methods are compared. The application of polyvinyl alcohol‐immobilized cells, which can be applied several times in whey feed, is compared with the implementation of a two step denitrification system. First, the whey concentrate's nitrate is removed by ion exchange and subsequently the eluent regenerated by microorganisms under their retention by crossflow filtration. Nitrite and nitrate concentrations were determined by reflectometric color measurement with a commercially available Reflectoquant® device. Correction factors for these media had to be determined. During the pilot development, bioreactors from 4 to 250 mg·L^?1 and crossflow units with membrane areas from 0.02 to 0.80 m² were examined. Based on the results of the pilot plants, a scaling for the exemplary process of denitrifying 1,000 tons per day is discussed. © 2010 American Institute of Chemical Engineers Biotechnol. Prog. 2010     

Energy transduction in the mitochondrionlike bacterium Paracoccus denitrificans during carbon- or sulphate-limited aerobic growth in continuous culture.

Paracoccus denitrificans was grown in carbon-limited aerobic continuous culture (critical dilution rate (Dc) = 0.48 h-1). The molar growth yield for carbon (succinate or malate) was constant at about 60 over a broad dilution range (growth rate) from 0.10 to 0.48 h-1. Measurements of the stoichiometry of proton translocation associated with the oxidation of endogenous substrates yielded a ratio of protons ejected from the cell per atom of oxygen consumed(leads to H+:O) of 8.55 which decreased to 5.85 in the presence of piericidin A (PA), a specific inhibitor of NADH dehydrogenase (EC 1.6.99.3). With starved cells, the observed leads to H+:O associated with the oxidation of added succinate in the presence of PA was 5.61. These observed leads to H:O's represent an underestimation since no correction was made for proton backflow during the short interval of respiratory activity. Aerobic growth of Pc. denitrificans in the chemostat becomes sulphate limited at entering concentrations of sulphate less than 300 is microM. Neither the maximum specific growth rate (measured at Dc) nor the observed molar growth yield for succinate decreased under sulphate limitation. The NADH oxidase in electron transport particles prepared from sulphate-limited cells was completely inhibited by PA. The stoichiometry of proton translocation associated with malate oxidation was similarly unaffected by sulphate limitation. It is concluded that (a) the respiratory chain of aerobic, heterotrophically grown Pc. denitrificans possesses three sites of energy conservation, including site III, (b) the number of protons ejected during the transfer of one pair of reducing equivalents along a region of the electron transport chain equivalent to a single energy-coupling site is 3, and (c) that sulphate limitation does not lead to a loss of proton translocation associated with the cytochrome-independent region of the respiratory chain.     

The effect of oxygen on denitrification in Paracoccus denitrificans and Pseudomonas aeruginosa

Denitrification by Paracoccus denitrificans and Pseudomonas aeruginosa was studied using quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously dissolved gases. Evidence was provided for aerobic denitrification by both species: in the presence of O2, N2O production increased in Pa. denitrificans, while that of N2 decreased; with Ps. aeruginosa, the concentrations of both N2 and N2O increased on introducing O2 into the gas phase. Disappearance of NO-3 was monitored in anaerobically and aerobically grown cells which were maintained either anaerobically or aerobically: the rate and extent of NO-3 utilization by both species depended on growth and maintenance conditions. The initial rate of disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred when cells were grown anaerobically and maintained aerobically. In nitrogen balance experiments both species converted over 87% of the added NO-3 to N2 and N2O under both anaerobic and aerobic maintenance conditions.     

Effects of electron transport inhibitors and uncouplers on denitrification in Paracoccus denitrificans

Abstract Gaschromatographic analysis shows that whole cells of Paracoccus denitrificans produce dinitrogen in the absence and nitrous oxide in the presence of thiocyanate during nitrate reduction. NADH nitrate reductase activity in vesicles is much more sensitive to thiocyanate than either NADH oxidase activity in vesicles or reduction of nitrate by endogenous substrates in whole cells. NADH nitrate reductase activity is not inhibited and NADH oxidase activity is partially inhibited by antimycin A in vesicles. Production of nitrous oxide from nitrate in cells is completely inhibited by the simultaneous presence of thiocyanate and Triton X-100. Carbonylcyanide m -chlorophenylhydrazone does not cause a lag phase in reduction of nitrate by NADH in vesicles, in contrast to the situation in cells.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Energy conservation during aerobic growth in Paracoccus denitrificans

Paracoccus denitrificans was aerobically grown in chemostat culture with succinate or gluconate as carbon source. Due to the presence of two phosphorylation sites in the respiratory chain and the absence of branching, theoretical P/O ratios of 1.71 and 1.82 were calculated for cells growing respectively with succinate and gluconate as carbon source. Using these data, 95% confidence intervals for the P/O ratio were determined, via a mathematical model, at 0.91–1.15 and 1.00–1.37 for sulphate-limited cultures, with respectively succinate and gluconate as carbon source.These results and measurements of P/O ratios in membrane particles and of proton translocation in whole cells have led to the conclusion that site I phosphorylation is affected under sulphate-limited conditions.Under conditions of carbon source-limitation the endogenous H⁺/O ratio is about 7–8. Average values of 3.40 and 4.78 were respectively found for sulphate-limited succinate- and gluconate grown cells. For starved cells, oxidizing succinate as exogenous substrate, the H⁺/O ratios were determined at about 3–4, independent of the growth limiting factor. It is concluded that the number of protons ejected per pair of electrons per energy-conserving site (H⁺/site ratio) is about 3–4, instead of 2 as postulated by the chemiosmotic hypothesis.     

Physiological regulation of Paracoccus denitrificans methanol dehydrogenase synthesis and activity.

An enzyme-linked immunosorbent assay and a whole-cell activity assay were developed which allowed detection of methanol dehydrogenase (MDH) of Paracoccus denitrificans with increased sensitivity. By these methods, it was shown that MDH was not induced by its natural substrate, methanol. Relief from a catabolite repression-like mechanism seemed responsible for low-level MDH synthesis, while product induction was the hypothesized mechanism for synthesis of high amounts of MDH. In the latter process, formaldehyde may play an important role as effector. For a variety of culture conditions, inconsistencies were observed in the relation between amounts of MDH protein synthesized and enzyme activities measured in vitro. Regulation of pyrrolo-quinoline-quinone biosynthesis or a modulation of its incorporation and stability in MDH may constitute an overriding mechanism to ensure a correct tuning between metabolic rates of methanol consumption and the required methanol oxidation rates.     

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.     

Energy conservation during nitrate respiration in Paracoccus denitrificans

P/2e ratios were calculated from anaerobic chemostat cultures of Paracoccus denitrificans with nitrogenous oxides as electron acceptor. P/2e ratios were calculated, using the Y _ATP ^max values determined for aerobic cultures. When succinate was the carbon and energy source the average P/2e values of the sulphate-and succinate-limited cultures with nitrate as electron acceptor were 0.5 and 0.7, respectively, and of the nitrite-limited culture 0.9. With gluconate as carbon and energy source the average P/2e values of the gluconate-limited with nitrate as electron acceptor and nitrate limited cultures were 0.9 and 1.1, respectively.H⁺/O ratios measured in cells obtained from sulphate-, succinate, nitrite-, gluconate-and nitratelimited cultures yielded respective average values of 3.4, 4.5, 3.5, 4.8 and 6.2 for endogenous substrates. From our data we conclude that sulphate-and nitritelimitation causes the loss of site I phosphorylation. Nitrite has no influence on the maximum growth yield on ATP. We propose that metabolism in heterotrophically grown cells of Paracoccus dentrificans is regulated on the level of phosphorylation in the site I region of the electron transport chain.     

Cytochrome c' of Paracoccus denitrificans.

Cytochrome c' was identified in periplasmic extracts of the Paracoccus denitrificans strains LMD 22.21 and LMD 52.44. The cytochrome c' was purified from the latter using the device of sequential molecular exclusion chromatography in the dimeric and monomeric states. Although showing the overall spectroscopic features of the cytochrome c' family, the Paracoccus cytochrome c' is unusual in having a red-shifted oxidised Soret band at 407 nm. Also unusual is the midpoint potential of 202 mV, well above the known cytochrome c' range. The amino-acid composition of Pa. denitrificans cytochrome c' showed the high alanine and low proline content characteristic of the group and reflecting the predominantly alpha-helical character of the protein. Comparison of the amino-acid compositions suggests some similarity to the cytochromes c' of Chromatium vinosum and halotolerant Paracoccus.     

Dimeric porin from Paracoccus denitrificans.

Paracoccus denitrificans was shown to contain a 33,000-dalton porin, which produced pores of large (1.6 to 1.8 nm) diameter. Cross-linking studies showed that the porin existed as dimers in the outer membrane.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Genetics of Paracoccus denitrificans

Abstract In bioenergetic research Paracoccus denitrificans has been used as an interesting model to elucidate the mechanisms of bacterial energy transduction. Genes for protein complexes of the respiratory chain and for proteins which are involved in periplasmic electron transport have been cloned and sequenced. Conjugational gene transfer has allowed the construction of site-specific mutant strains. Complementation experiments did not only open the field for site-directed mutagenesis and investigation of the structure/function relationship of the various electron-transport proteins, but also allowed first insights into processes like oxygen-dependent gene regulation or the assembly of electron-transport complexes. Also data will be presented that characterize two restriction-/modification systems, the codon usage and the promoter sequences of Paracoccus . Details will be given about the extrachromosomal localization of a duplicated cytochrome oxidase subunit I gene on one of the Paracoccus megaplasmids.     

Proton translocation during denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Proton translocation during the reduction of NO ₃ ^- , NO ₂ ^- , N₂O and O₂, with endogenous substrates, in washed cells of Rhodopseudomonas sphaeroides f. denitrificans was investigated by an oxidant pulse method. On adding NO ₂ ^- to washed cells, anaerobically in the dark, an alkalinization occurred in the reaction mixture followed by acidification. When NO ₃ ^- , N₂O or O₂ was added to cells in the dark or with these compounds and NO ₂ ^- in light an acidification only was observed. Proton translocation was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone.Valinomycin treated cells produced acid in response to the addition of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. The proton extrusion stoichiometry ( ratios) in illuminated cells were as follows: NO ₃ ^- 0.5N₂, 4.82; NO ₂ ^- 0.5N₂, 5.43; N₂ON₂, 6.20; and O₂H₂O, 6.43. In the dark the comparable values were 3.99, 4.10, 4.17 and 3.95. Thus, illuminated cells produced higher values than those in the dark, indicating a close link between photosynthesis and denitrification in the generation of proton gradients across the bacterial cell membranes.When reduced benzyl viologen was the electron donor in the presence of 1 mM N-ethylmaleimide and 0.5 mM 2-n-heptyl-4-hydroxyquinoline-N-oxide in the dark, the addition of either NO ₃ ^- , NO ₂ ^- or N₂O to washed cells resulted in a rapid alkalinization of the reaction mixture. The stoichiometries for proton consumption, ratios without a permeant ion were NO ₃ ^- NO ₂ ^- ,-1.95; NO ₂ ^- 0.5 N₂O,-3.03 and N₂ON₂,-2.02. The data indicate that these reductions occur on the periplasmic side of the cytoplasmic membrane.Abbreviations BVH reduced benzyl viologen - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DIECA N, N-diethyl-dithiocarbamate - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NEM N-ethylmaleimide     

Demonstration of ferric L-parabactin-binding activity in the outer membrane of Paracoccus denitrificans.

Under low-iron conditions, Paracoccus denitrificans excretes a catecholamine siderophore, L-parabactin, to sequester and utilize iron. In this report, we demonstrate the presence of stereospecific high-affinity ferric L-parabactin-binding activity associated with P. denitrificans membranes grown in low-iron medium. Isolated outer membrane components were shown to be three to four times higher in specific activity for ferric L-parabactin. The same amount of binding activity existed whether or not the radiolabel was present in the metal (55Fe) or the ligand (3H) portion of ferric parabactin chelate, suggesting that binding was to the intact complex. Ion-exchange chromatography of a Triton X-100-solubilized outer membrane mixture on DEAE-cellulose resulted in a 10-fold increase in binding activity relative to that present in whole membranes. Polypeptide profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of each stage of the purification showed that binding activity copurified with one or more of the low-iron-induced outer membrane proteins in the 80-kilodalton (kDa) region. Membrane proteins and [55Fe]ferric L-parabactin electrophoresed in nondenaturing gels demonstrated the presence of membrane component(s) which stereo-specifically bound ferric L-parabactin, thus providing independent confirmation of the binding assay results. Moreover, when the band labeled by [55Fe]ferric L-parabactin was excised and profiled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 80-kDa polypeptides were the major components present. These results demonstrate the presence of a high-affinity ferric L-parabactin receptor in P. denitrificans membranes and suggest that one or more of the 80-kDa low-iron-induced polypeptides are components of the ferric L-parabactin receptor.     

Effects induced by rotenone during aerobic growth ofParacoccus denitrificans in continuous culture

Paracoccus denitrificans was grown aerobically in chemostat culture in the presence of rotenone. After 6 to 10 generation times, cells showed an oxygen uptake which was completely rotenone-insensitive after removal of rotenone by washing with bovine serum albumin containing medium.The H⁺/O ratio of these cells for endogenous substrates decreased from about 7.50 to 3.95. The latter ratio was similar to the value obtained for starved cells oxidizing exogenous succinate, indicating that site I phosphorylation was absent in these rotenone-insensitive cells.Membrane particles prepared from these cells showed an 80% decrease in activity of reduced nicotinamide adenine dinucleotide oxidase and reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase, while also the kinetic behaviour of the reduced nicotinamide adenine dinucleotide dehydrogenase in the reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase assay was changed. Moreover the reduced nicotinamide adenine dinucleotide oxidase activity was practically rotenone-insensitive.Electron paramagnetic resonance spectroscopy on membrane particles from rotenone-insensitive cells at 15 K revealed that the resonance lines atg _z 2.05 andg _y g _x 1.92 arising from iron-sulfur center 2 were undetectable. The intensities of the other electron paramagnetic resonance signals originating from reduced nicotinamide adenine dinucleotide dehydrogenase linked iron-sulfur centers were only slightly diminished.These observations confirm our previous suggestion that site I phosphorylation, rotenone sensitivity and the presence of iron-sulfur center 2 are correlated.Abbreviations EPR electron paramagnetic resonance - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate - ATP adenosine triphosphate