Paracoccus Denitrificans Strain Research Articles

Genome Sequence and Description of Paracoccus denitrificans Strain R-1, Isolated from Activated Sludge

ABSTRACTParacoccus denitrificans strain R-1 was isolated from an activated sludge sample from a sewage treatment plant in Taiwan. The complete genome, which was sequenced on the NovaSeq 6000 and PacBio Sequel platforms, consists of one chromosome with 4.05 Mb and one plasmid with 689 kb. Genome annotation predicts 4,167 protein-coding genes, 49 tRNAs, and 8 rRNAs.

Genome Sequence of a Heterotrophic Nitrifier and Aerobic Denitrifier, Paracoccus denitrificans Strain ISTOD1, Isolated from Wastewater

ABSTRACTWe report here the draft genome sequence of Paracoccus denitrificans strain ISTOD1 of 4.9 Mb, isolated from wastewater. It has been identified as a heterotrophic nitrifying and aerobic denitrifying bacterium. Genomic analysis revealed genes related to nitrogen and phosphorus removal, showing that the strain holds potential for bioremediation and biorefinery uses.

Biological Nitrous Oxide Abatement by Paracoccus denitrificans in Bubble Column and Airlift Reactors

Nitrous oxide (N2O), with a global warming potential 300 times higher than that of CO2, represents 6.2 % of the total greenhouse gas emission inventory worldwide. Furthermore, N2O is considered the most critical O3- depleting substance emitted in this XXI century. In spite of the environmental relevance of this pollutant, very little research on biotechnologies for the treatment of N2O emissions has been conducted. In this study, the potential of a bubble column (BCR) and an internal loop airlift (ALR) bioreactors of 2.3 L was evaluated for the abatement of N2O from industrial emissions from nitric acid plants along 62 days of operation. The systems were inoculated with a methylotrophic Paracoccus denitrificans strain (DSM 413) and continuously supplied with methanol as a carbon and electron donor source for the anoxic reduction of N2O. The simulated waste gas consisted of a N2 gas stream containing 1 ± 0.1 % of O2 and 3377 ± 312 ppmv of N2O at the inlet of theBCR and 1 ± 0.1 % of O2 with N2O concentration of 3617 ± 342 ppmv at the inlet of the ALR. This N2-laden stream was supplied at a constant flow rate of 110 ml min-1 in each reactor. The performance of the BCR was characterized by a steady state N2O removal efficiency (RE) of 87 ± 3 % with CO2 productions of 308 ± 36 gm-3 d-1 and total suspended solid (TSS) concentrations of 867 ± 109 mg L-1. On the other hand, the ALR showed a N2O RE of 88 ± 2 % with productions of CO2 of 346 ± 28 g m-3 d-1 and TSS concentrations of 874 ±88 mg L-1. This work constitutes, to the best of our knowledge, the first systematic study of a biotechnology for the continuous abatement of N2O from nitric acid plants.

ANAMMOX-like performances for nitrogen removal from ammonium-sulfate-rich wastewater in an anaerobic sequencing batch reactor

ABSTRACTAmmonium removal by the ANaerobic AMonium OXidation (ANAMMOX) process was observed through the Sulfate-Reducing Ammonium Oxidation (SRAO) process. The same concentration of ammonium (100 mg N L−1) was applied to two anaerobic sequencing batch reactors (AnSBRs) that were inoculated with the same activated sludge from the Vermicelli wastewater treatment process, while nitrite was fed in ANAMMOX and sulfate in SRAO reactors. In SRAO-AnSBR, in substrates that were fed with a ratio of NH4+/SO42− at 1:0.4 ± 0.03, a hydraulic retention time (HRT) of 48 h and without sludge draining, the Ammonium Removal Rate (ARR) was 0.02 ± 0.01 kg N m−3.d−1. Adding specific ANAMMOX substrates to SRAO-AnSBR sludge in batch tests results in specific ammonium and nitrite removal rates of 0.198 and 0.139 g N g−1 VSS.d, respectively, indicating that the ANAMMOX activity contributes to the removal of ammonium in the SRAO process using the nitrite that is produced from SRAO. Nevertheless, the inability of ANAMMOX to utilize sulfate to oxidize ammonium was also investigated in batch tests by augmenting enriched ANAMMOX culture in SRAO-AnSBR sludge and without nitrite supply. The time course of sulfate in a 24-hour cycle of SRAO-AnSBR showed an increase in sulfate after 6 h. For enriched SRAO culture, the uptake molar ratio of NH4+/SO42− at 8 hours in a batch test was 1:0.82 lower than the value of 1:0.20 ± 0.09 as obtained in an SRAO-AnSBR effluent, while the stoichiometric ratio of 1:0.5 that includes the ANAMMOX reaction was in this range. After a longer operation of more than 2 years without sludge draining, the accumulation of sulfate and the reduction of ammonium removal were observed, probably due to the gradual increase in the sulfur denitrification rate and the competitive use of nitrite with ANAMMOX. The 16S rRNA gene PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) and PCR cloning analyses resulted in the detection of the ANAMMOX bacterium (Candidatus Brocadia sinica JPN1) Desulfacinum subterraneum belonging to the genus Desulfacinum and bacteria that are involved in sulfur metabolism (Pseudomonas aeruginosa strain SBTPe-001 and Paracoccus denitrificans strain IAM12479) in SRAO-AnSBR.

Proteomic responses to a methyl viologen-induced oxidative stress in the wild type and FerB mutant strains of Paracoccus denitrificans

FerB is a cytoplasmic flavoprotein from the soil bacterium Paracoccus denitrificans with a putative role in defense against oxidative stress. To further explore this hypothesis, we compared protein variations upon methyl viologen treatment in wild-type and FerB mutant strains by a quantitative proteomic analysis based on iTRAQ-3DLC–MS/MS analysis. The proteins showing the most prominent increase in abundance were assigned to carbon fixation and sulfur assimilatory pathways. By employing these proteins as indirect markers, oxidative stress was found to be 15% less severe in the wild-type than in the FerB-deficient mutant cells. Oxidative stress altered the levels of proteins whose expression is dependent on the transcriptional factor FnrP. The observed down-regulation of the fnrP regulon members, most notably that of nitrous oxide reductase, was tentatively explained by an oxidative degradation of the [4Fe–4S] center of FnrP leading to a protein form which no longer activates transcription. While the level of FerB remained relatively constant, two proteins homologous to FerB accumulated during oxidative stress. When their genes were expressed in Escherichia coli, neither of the protein products contained a bound flavin, whereas they both had a high activity of flavin reductase, one preferentially utilizing NADH and the other NADPH.

Anodic biofilms in microbial fuel cells harbor low numbers of higher-power-producing bacteria than abundant genera

Microbial fuel cell (MFC) anode communities often reveal just a few genera, but it is not known to what extent less abundant bacteria could be important for improving performance. We examined the microbial community in an MFC fed with formic acid for more than 1 year and determined using 16S rRNA gene cloning and fluorescent in situ hybridization that members of the Paracoccus genus comprised most (approximately 30%) of the anode community. A Paracoccus isolate obtained from this biofilm (Paracoccus denitrificans strain PS-1) produced only 5.6 mW/m(2), whereas the original mixed culture produced up to 10 mW/m(2). Despite the absence of any Shewanella species in the clone library, we isolated a strain of Shewanella putrefaciens (strain PS-2) from the same biofilm capable of producing a higher-power density (17.4 mW/m(2)) than the mixed culture, although voltage generation was variable. Our results suggest that the numerical abundance of microorganisms in biofilms cannot be assumed a priori to correlate to capacities of these predominant species for high-power production. Detailed screening of bacterial biofilms may therefore be needed to identify important strains capable of high-power generation for specific substrates.

Electron transfer in Paracoccus denitrificans with the modified fbc operon

Membrane fragments of two mutant strains of Paracoccus denitrificans genetically modified in the bc(1) complex have been studied for comparison of enzymic activities of succinate-cytochrome-c reductase and its components, viz. succinate dehydrogenase (Complex II) and ubiquinol-cytochrome-c reductase (Complex III) and their response to changes in concentration of succinate, cytochrome c, ionic strength, pH, temperature and sensitivity to antimycin A. The mutants synthesized and assembled the b and c hemes in the ratio characteristic for the wild type strain. The mutant strain M 71 expressing the truncated copy of cytochrome c(1) (devoid of a stretch of 150 mainly acidic amino acids) was less sensitive to increasing concentration of cytochrome c and changes in ionic strength of the medium, but maintained the original affinity to succinate and sensitivity to antimycin A. The mutant strain M 36 with an overexpressed bc(1) content showed the highest response to changes in ionic strength and physical parameters, exhibited the lowest turnover number values with succinate-cytochrome-c reductase, but positively affected the succinate dehydrogenase. In view of the interaction of the redox components in native membranes the functional analyses of separated Complexes II and III should be regarded with caution.

Electrostatic attraction between cytochrome bc 1 and cytochrome c affects kinetics of cytochrome c reduction

The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.

Redefining Paracoccus denitrificans and Paracoccus pantotrophus and the case for a reassessment of the strains held by international culture collections

An outline of the current taxonomic diversity of the genus Paracoccus is presented. A definitive summary is given of the valid type strains of Paracoccus denitrificans and Paracoccus pantotrophus and of culture collection strains that can be assigned to these species. The case is established for a critical reassessment of the P. denitrificans strains held by international culture collections, to ensure that they are assigned to the correct species.

Growth and nitrite and nitrous oxide accumulation of Paracoccus denitrificans ATCC 19367 in the presence of selected pesticides.

The effects of the application of eight pesticides (aldrin, lindane, dimetoate, methylparathion, methidation, atrazine, simazine, and captan) on growth, respiratory activity (as CO2 production), denitrifying activity (as N2O released), and nitrite accumulation in the culture medium by Paracoccus denitrificans strain ATCC 19367 were studied. The fungicide captan totally inhibited growth and biological activity of P. denitrificans, while the rest of the tested pesticides delayed the growth and CO2 release of P. denitrificans but did not drastically affect the bacterial growth or respiratory capacity after 96 h of culture. The denitrifying activity of P. denitrificans ATCC 19367 (as N2O released) was negatively affected by all tested pesticides. The release of N2O was strongly inhibited by several organochlorinated and organophosphorated insecticides (aldrin, lindane, dimetoate, and methidation), which led to high accumulation of nitrite in the surrounding medium. Atrazine decreased N2O release after 48 h of culture because of negative effects on growth, and methylparathion and simazine delayed the onset of N2O release by P. denitrificans. These three pesticides reduced the accumulation of NO2- compared to unamended control cultures.

Microbial decomposition of 3,4-dichloroaniline, adsorbed by activated charcoal

The accessibility of 3,4-dichloroaniline (DCA) sorbed by activated carbon to degradative microorganisms was studied. A Paracoccus denitrificans strain capable of growing on medium with DCA as the only source of energy, carbon, and nitrogen was used in the experiment. The high sorption capacity of all the carbons studied (powdered RS and SKT-6A and granular AG-3) in relation to DCA (350 to 360, 480 to 520, and 540 to 580 mg/g, respectively) was demonstrated. The sorption capacity correlated positively with the specific surface area and the total volume of the sorbent micropores. The bulk of the DCA was reversibly sorbed and amenable to microbial decomposition; however, the decomposition rates significantly differed. When RS, SKT, and Agrosorb preliminarily saturated with DCA were incubated in a culture of P. denitrificans, the bulk of the reversibly sorbed DCA was decomposed (in the absence of the other carbon sources) in 2, 5, and 10 weeks, respectively, after which the process slowed down. At the end of the experiment (29 weeks), 81 to 87% of the DCA underwent full mineralization, which was accompanied by the release of chlorine ions; a small fraction of the xenobiotic (0.8 to 1.9%) remained a reversibly sorbed fraction (extractable with acetone), and 12 to 17% of the initial DCA seemed to have been chemically transformed and bound by carbon. The studied carbons may be used in biological decontamination of chloroaniline-polluted soils to decrease the toxic effect of chloroanilines on microorganisms.

A re-evaluation of the taxonomy of Paracoccus denitrificans and a proposal for the combination Paracoccus pantotrophus comb. nov.

Comparison of both 16S rRNA coding sequences and DNA-DNA hybridization of ten strains of alpha-subclass of Proteobacteria currently classified as strains of Paracoccus denitrificans has shown that they fall into two groups which are distinct from each other at the species level. Comparison with published data on the cytochrome c profiles and other 16S rRNA coding sequences in the literature has confirmed these observations and enabled several other strains also to be assigned to these two groups. Group A comprises strains ATCC 17741T (the type strain of P. denitrificans), LMD 22.21T, DSM 413T, ATCC 19367, ATCC 13543, DSM 1404, DSM 1405, Pd 1222 (a genetic modification of DSM 413T) and NCIMB 8944. Group B comprises ATCC 35512T (the original type strain of Thiosphaera pantotropha), LMD 82.5T, LMD 92.63, DSM 65, LMG 4218, IAM 12479, JCM 6892, DSM 11072, DSM 11073 and DSM 11104. In light of these findings, it is proposed that: (1) strains of group A are retained as P. denitrificans, with ATCC 17741T as the type strain of the type species; and (2) all strains of group B are assigned to the new species combination Paracoccus pantotrophus comb. nov., with strain ATCC 35512T as the type strain. Comparative 16S rRNA sequence analysis and DNA-DNA hybridization of strains of Paracoccus versutus confirm that this species is distinct from both P. denitrificans and P. pantotrophus, but that its nearest phylogenetic neighbour is P. pantotrophus.

The surface-charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans--implications for the interaction with cytochrome c peroxidase.

The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated. The amino acid sequence of cytochrome c550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21. Based on the X-ray structure of the latter, a structure for the cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge. The behaviour of the cytochrome on molecular-exclusion chromatography is indicative of an ionic strength-dependent monomer (15 kDa)/dimer (30 kDa) equilibrium that can also be detected by 1H-NMR spectroscopy. The apparent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer. This was confirmed by cross-linking studies, which showed that a cross-linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular-exclusion chromatography. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other. The resultant dipole moment of the dimer is therefore small. Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1H-NMR spectroscopy shows that it is the monomer that binds.

Expression of the Escherichia coli cyo operon in Paracoccus denitrificans results in a fully active quinol oxidase of unexpected heme composition

The cyo operon coding for the membrane-bound bo 3-type quinol oxidase of Escherichia coli has been expressed in a Paracoccus denitrificans strain deleted in its endogenous ba 3 quinol oxidase. Using the P. denitrificans qox promoter, the His tagged protein complex is synthesized to a level comparable to that in E. coli and the enzyme purified in a single step on a metal-chelating column. Whereas the activity of the isolated complex matches that of the oxidase purified directly from E. coli, the heterologously expressed oxidase does not show the characteristic heme composition but now carries heme a in its binuclear site.

Potential nitrosamine formation and its prevention during biological denitrification of red beet juice

High nitrate intake has been shown to result in an increased risk of endogenous formation of N-nitroso compounds. Certain vegetables and vegetable juices contain high concentrations of nitrate. Biological denitrification using strains of Paracoccus denitrificans (P.d.) has been proposed as effective means to reduce nitrate contents in such vegetable juices. During this bacterial denitrification process, substantial nitrite concentrations are transiently formed. This study investigated whether N-nitrosation reactions might occur. The easily nitrosatable amine morpholine was added to red beet juice at high concentration (100 ppm) during denitrification 10 different batches of red beet juice served as raw material. Each batch was submitted to denitrification in the presence and absence of ascorbic acid. In the absence of ascorbic acid, formation of N-nitrosomorpholine (NMOR) was observed in the low ppb range (0.5–8 ppb). Addition of ascorbic acid (500 mg/litre) inhibited the formation of NMOR, except for those instances where the pH was less than 6 and/or nitrate turnover was low ( < 200 mg NO 3 −/litre/hr). Under conditions leading to high rates of nitrate turnover ( > 200 mg NO 3 −/litre/hr), nitrosamine formation can reliably be prevented by ascorbic acid. The results show that bacterial denitrification of red beet juice high in nitrate can be accomplished without the risk of nitrosamine formation.

Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient in nitrate respiration

A Paracoccus denitrificans strain (M6Omega) unable to use nitrate as a terminal electron acceptor was constructed by insertional inactivation of the periplasmic and membrane-bound nitrate reductases. The mutant strain was able to grow aerobically with nitrate as the sole nitrogen source. It also grew anaerobically with nitrate as sole nitrogen source when nitrous oxide was provided as a respiratory electron acceptor. These growth characteristics are attributed to the presence of a third, assimilatory nitrate reductase. Nitrate reductase activity was detectable in intact cells and soluble fractions using nonphysiological electron donors. The enzyme activity was not detectable when ammonium was included in the growth medium. The results provide an unequivocal demonstration that P. denitrificans can express an assimilatory nitrate reductase in addition to the well-characterised periplasmic and membrane-bound nitrate reductases.

Aerobic and anaerobic degradation of furan-3-carboxylate by Paracoccus denitrificans strain MK33

An organism identified as Paracoccus denitrificans was isolated from an enrichment culture with furan-3-carboxylate as its sole source of carbon and energy. The organism degraded furan-3-carboxylate under aerobic and — in the presence of nitrate - under anaerobic conditions. The aerobic degradation was initiated by an oxygenase reaction to form probably 2-hydroxy-3-furoate. Under anaerobic conditions the first intermediate was 3-furoyl-CoA which was reduced by NADPH to probably 4,5-dihydro-furoyl-CoA. Succinic semialdehyde was an intermediate in both aerobic and anaerobic mechanism of furan-3-carboxylate.

Genetic tools forParacoccus denitrificans

Paracoccus denitrificans strain PD1222 (from N. Harms, Vrije Universiteit, Amsterdam, The Netherlands), which is deficient in a host-specific restriction system, could be successfully used for (1) transposon Tn5 mutagenesis, (2) phage P1-mediated generalized transduction and (3) construction of Hfr strains. Experimental protocols and some properties of an ammonium transport-deficient mutant are described.

Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol (td=2.7–6.4 h) and on ethanol (td=15–50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth (td=3–9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme.

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 μmol/min·mg protein. Lactate dehydrogenase was not detected. Six strains tested reacted almost alike.